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WELCOME TO THE WEDNESDAY

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AFTERNOON LECTURE SERIES.

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I'M SUZANNE BUCHANAN, THE DEPUTY

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SCIENTIFIC DIRECTOR FOR THE

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NATIONAL INSTITUTED OF DIABETES

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AND DIGESTIVE AND KIDNEY

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DISEASES.

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AND I'M ALSO THE CHIEF OF THE

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LABORATORY OF MOLECULAR BIOLOGY

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WHERE OUR SPEAKER HAILS FROM.

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SO IT'S A REAL PLEASURE FOR ME

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TO BE GIVING THIS INTRODUCTION.

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AND I'M EXCITED TODAY TO

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INTRODUCE OUR SPEAKER, DR. ERIC

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GREENE WHO WILL BE DISCUSSING

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SINGLE MOLECULE STUDIES OF THE

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HOMOLOGOUS RECOMBINATION.

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THAT'S ERIC C. GREENE WITH

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A FINAL E AND NOT TO BE CONFUSED

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WITH THE NHGRI DIRECTOR.

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SO, OUR SPEAKER FOR TODAY IS

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ERIC GREENE, WHO IS A PROFESSOR

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IN THE DEPARTMENT OF

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BIOCHEMISTRY AND MOLECULAR

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BIOPHYSICS AT COLUMBIA

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UNIVERSITY IN NEW YORK CITY.

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HIS LAB USES SINGLE-MOLECULE

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OPTICAL MICROSCOPY TO STUDY

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FUNDAMENTAL INTERACTIONS BETWEEN

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PROTEINS AND NUCLEIC ACIDS.

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TO OBSERVE INDIVIDUAL PROTEIN

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MOLECULES OR PROTEIN COMPLEXES

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AS THEY INTERACT WITH DNA

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SUBSTRATES.

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ERIC'S LAB HAS PIONEERED THE

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DEVELOPMENT OF DNA CURTAINS FOR

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THIS DIRECT VISUALIZATION OF

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HUNDREDS TO THOUSANDS OF

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INDIVIDUAL MOLECULES IN REALTIME

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BY OPTICAL MICROSCOPY.

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TECHNYNIQUE IS BASED ON THE

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TECHNIQUE OF NANOFABRICATED

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SURFACES, ALLOWING THEM TO BE

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VIEWED AS A SINGLE MOLECULE

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LEVEL BY TOTAL INTERNAL

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REFLECTION FLUORESCENCE

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MICROSCOPY.

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NOW MANY OF YOU MAY REMEMBER

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ERIC WAS A POSTDOC HERE IN THE

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LABORATORY OF MOLECULAR BIOLOGY

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AT NIDDK FROM ROUGHLY 1999-2003.

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I WAS HERE THEN TOO.

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AND THAT'S HARD TO BELIEVE IT'S

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BEEN 20 YEARS PLUS.

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ERIC RECEIVED HIS UNDERGRADUATE

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DEGREE IN BIOCHEMISTRY FROM THE

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UNIVERSITY OF ILLINOIS.

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HE FOLLOWED THAT WITH A PH.D. IN

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BIOCHEMISTRY FROM TEXAS A&M THEN

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A POSTDOC AT THE NIH.

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AFTER LEAVING THE NIH, HE TOOK A

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POSITION AT COLUMBIA UNIVERSITY

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IN 2004 AS AN ASSISTANT

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PROFESSOR WHERE HE HAS BEEN EVER

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SINCE BEING PROMOTED TO

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ASSOCIATE PROFESSOR IN 2010 AND

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FULL PROFESSOR IN 2015.

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HE HAS HAD MANY HONORS.

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SOME OF THEM ARE A CAREER AWARD

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IN 2006, A PRESIDENTIAL

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ENGINEERING PKAS AWARD IN 2007.

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I SHOULD KNOW, I HAD ONE TOO.

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A HOWARD HUGHES MEDICAL

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INSTITUTE AWARD FROM 2009-2015.

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THE DOCTOR HAROLD RESEARCH AWARD

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IN 2009, AND MULTIPLE

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INVITATIONS TO BE A KEYNOTE

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SPEAKER, NOTABLY RECENTLY AT

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GORDON CONFERENCE AT KEYSTONE

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SYMPOSIUM.

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HE GIVES AN ENGAGING LECTURE.

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THE TITLE OF HIS TALK IS SINGLE

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MOLECULE STUDIES OF HOMOLOGOUS

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RECULMINATION.

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WELCOME BACK TO THE NIH, WE WISH

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YOU WERE HERE IN PERSON, BUT

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THANK YOU VERY MUCH FOR BEING

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WITH US TODAY.

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>> PERFECT.

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THANK YOU, SUSAN.

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I ALSO WISH THAT I WAS THERE.

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I HAD AGREED TO DO THIS ASSUMING

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I WOULD BE ABLE TO COME DOWN AND

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VISIT WITH SO MANY FRIENDS.

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SO THAT'S A LITTLE BIT OF A

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BUMMER.

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IT'S ALSO FUNNY THAT YOU BROUGHT

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UP THE FACT THAT THERE IS

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ANOTHER ERIC GREEN AT NIH AND IT

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REMINDED ME OF A STORY WHEN I

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WAS A POSTDOC AND SOME LAWYER

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CALLED ME AND STARTED TALKING

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ABOUT SOME THINGS THAT OBVIOUSLY

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HAD NO NEED TO KNOW.

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AND I REALIZED I WAS THE WRONG

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ERIC GREEN.

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SO THAT WAS FUN.

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SO, TODAY, I WAS THINKING ABOUT

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WHAT IT IS I SHOULD TALK TO YOU

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ABOUT AND I DECIDED TO TALK TO

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YOU ABOUT SOMETHING THAT IS NOT

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PUBLISHED.

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SO THE TALK IS A LITTLE ROUGH.

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I WAS ORIGINALLY GOING TO SAY

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SOMETHING ABOUT OUR RECENT WORK

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ON HOMOLOGY RESEARCH BUT I

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DECIDED TO TALK ABOUT HOW DNA IS

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A PROCESS OR AT LEAST OUR SMALL

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PORTION OF THE PUZZLE IN TERMS

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OF DNA END PROCESSING.

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AND YOU CAN SEE I CHANGED THE

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TITLE TO SOMETHING A LITTLE MORE

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SPIFFY TALKING ABOUT BLUE

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HELICASE.

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AND WHAT I'M GOING TO TRY TO

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TELL YOU ABOUT IS HOW BLOOM

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HELICASE OR SEX DNA ENDS AND I'M

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GOING TO TRY TO CONVINCE YOU

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IT'S DOING SOMETHING TO THOSE

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ENDS THAT LOOKS LIKE IT'S MAKING

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SOME KIND OF A LOOP WITH THE

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SINGLE STRANDED OVER HANG THAT

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IS BEING PRODUCED.

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SO WITH THAT, I WILL JUMP RIGHT

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IN.

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AND I'M GOING TO POINT OUT THE

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FOLKS WHO HAVE CONTRIBUTED TO

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THIS.

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REALLY FROM MY LABORATORY A

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FORMER POSTDOC AND HAS HIS OWN

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LAB IN CHINA, WILL HAVE DONE ALL

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OF THE SINGLE MOLECULE WORK THAT

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I'M GOING TO TALK TO YOU ABOUT.

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WE HAVE BEEN WORKING CLOSELY

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WITH UT SAN ANTONIO AND THEY

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HAVE BEEN A MAIN DRIVER OF THE

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WORK IN HIS LAB.

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ANY TIME I'M SHOWING SOMETHING

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THAT IS A LIVE-CELL ASSAY, THAT

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HAS NOTHING TO DO WITH US.

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AND THEN I SHOULD ALSO MENTION

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THOSE WHO CAME IN AT THE END AND

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HELPED US WITH STUFF WE ALSO HAD

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TO GET DONE IN TERMS OF MAKING

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REVISIONS.

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AND I'LL TRY TO POINT OUT WHO

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AMONG THESE FOLKS HAVE DONE WHAT

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AS WE HAVE GONE ALONG.

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AND I HAVE TO SAY THANK YOU TO

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OUR FUNDING, MOST OF WHICH IS

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NIH.

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SO THIS IS CHOW, SORT OF IN THE

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PANDEMIC AS PREPARING HIS FAMILY

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TO MOVE BACK TO CHINA.

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AND HE PUBLISHED A NUMBER OF

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PAPERS WITH US, AND SOME OF

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THESE I'LL BE REFERRING BACK

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FROM TIME TO TIME.

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SO IF ANYONE WANTS TO SEE THE

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FULL REFERENCES, THEY ARE GOING

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TO BE LISTED IN THIS SLIDE.

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THE KEY ONES HERE ARE GOING TO

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BE A STUDY THAT HE DID ON END

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RESECTION IN SACCHAROMYCES SIR

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VIS YEAH IN A MOLECULAR CELL

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PAPER, AS WELL AS A STUDY HE DID

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ON HUMAN BLOOM HELICASE, WHICH

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I'LL TALK ABOUT IN A MINUTE THAT

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WE PUBLISHED IN NUCLEIC ACIDS

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RESEARCH.

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SO THIS IS HIM AND HE WAS GREAT.

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I REALLY MISS HIM.

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SO, I'M GOING TO KEEP THE

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INTRODUCTION TO THIS FAIRLY

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SIMPLE.

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I REALLY ONLY HAVE TWO

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INTRODUCTORY SLIDES AND THEN

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WE'RE GOING TO GET RIGHT INTO

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DATA.

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AND THEN AT THE END I'LL TRY TO

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TELL YOU WHAT WE THINK THE DATA

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MEANS IF I HAVEN'T CONVINCED YOU

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AS WE GO ALONG.

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SO, OVER THE YEARS, MY LAB HAS

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WORKED ON A LOT OF STUFF.

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I'LL SHOW YOU THE TOOL WE USED

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IN THE NEXT SLIDE.

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BUT WE HAVE BEEN COMING MORE AND

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MORE TOWARDS FOCUSING ON DNA

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HOMOLOGOUS RECOMBINATION.

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WHEN I STARTED MY LAB, THIS IS

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WHAT WAS WE WERE GOING TO BE

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WORKING ON.

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AS WE DEVELOPED TECHNIQUES AND

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REALIZED WE CAN APPLY THESE TO

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LOTS OF SYSTEMS, THAT'S WHAT WE

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DID.

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BUT WE HAD SOME BREAKTHROUGHS

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THAT ALLOWED US TO COME BACK TO

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RECOMBINATION IN WAYS THAT I

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THINK HAVE BEEN VERY EXCITING

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FOR THE GROUP.

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SO THIS IS SORT OF THE

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BIOPHYSICS OVERVIEW OF DNA

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RECOMBINATION MY GENETICS

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COLLEAGUES LOVE THIS.

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SO WE HAVE A DOUBLE STRAND DNA

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MOLECULE, A LITTLE LIGHTNING

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BOLT COMES DOWN AND DAMAGES IT

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AND THAT CAN DO ALL KINDS OF

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THINGS THAT WILL DO THIS IT TO

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GENERATE A DOUBLE-STRANDED

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BREAK.

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AND FOR HOMOLOGOUS

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RECOMBINATION, THAT BREAK HAS TO

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BE PROCESSED.

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IN FACT, THE PROCESSING IS WHAT

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WE ARE GOING TO BE TALKING ABOUT

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TODAY.

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SO WHAT HAPPENS IS YOU GENERATE

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A LONG 3-PRIME OVERHANG AND THIS

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OVERHANG SERVES AT PLATFORM FOR

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THE FORMATION OF A PRESYNAPTIC

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COMPLEX AND THAT WILL BE IN

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HUMANS, A FILAMENT OF A PROTEIN

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CALLED Rad51.

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SAME PROCESS HAPPENS IN EVEN

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BACTERIA.

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THE DIFFERENCE IS Rad51 IS NOT

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THERE.

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IT'S THE PROTEIN CALLED -- AND

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THEY ARE CLOSELY RELATED

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PROTEINS.

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THE PROCESS IS HIGHLY CONSERVED

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ACROSS BIOLOGY.

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NOW ONCE YOU HAVE RESECTED THESE

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ENDS AND YOU ASSEMBLE YOUR

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RECOMBINASE FILAMENT, THAT

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FILAMENT THEN HAS TO SEARCH THE

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GENOME TO FIND ANOTHER PIECE OF

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DNA DEPICTED AS A RED MOLECULE,

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THAT IT CAN PAIR THE BLACK DNA

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WITH.

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ONCE THAT PAIRING TAKES PLACE,

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YOU CAN THEN START TO DO DNA

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SYNTHESIS AND FILL IN

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INFORMATION THAT WAS LOST DURING

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THESE STAGES AND THEN THIS

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INTERMEDIATE CAN BE CHANNELED

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THROUGH A NUMBER OF INTERRELATED

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PATHWAYS, THE MOST SIMPLE OF

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WHICH IS TO HAVE THIS

263
00:09:38,280 --> 00:09:39,480
INTERMEDIATE DISRUPTED.

264
00:09:39,480 --> 00:09:41,880
THIS REALLY SYNTHESIZED STRAND

265
00:09:41,880 --> 00:09:44,480
SIMPLY PAIRS WITH THE SECOND

266
00:09:44,480 --> 00:09:47,800
OVERHANG AND YOU GET

267
00:09:47,800 --> 00:09:49,200
RELYINGATION OF A STRAND AFTER

268
00:09:49,200 --> 00:09:49,520
THIS PROCESS.

269
00:09:49,520 --> 00:09:50,480
THAT'S THE SIMPLEST.

270
00:09:50,480 --> 00:09:52,680
BUT THERE ARE OTHER WAYS TO DO

271
00:09:52,680 --> 00:09:53,000
THIS AS WELL.

272
00:09:53,000 --> 00:09:54,080
I'M NOT GOING TO GO OVER THOSE

273
00:09:54,080 --> 00:09:55,480
BECAUSE WE ARE NOT GOING TO BE

274
00:09:55,480 --> 00:09:59,440
TALKING ABOUT THEM IN DEPTH.

275
00:09:59,440 --> 00:10:02,160
SO NOW RECOMBINATION IN AND OF

276
00:10:02,160 --> 00:10:04,480
ITSELF IS IMPORTANT FOR

277
00:10:04,480 --> 00:10:07,480
MAINTAINING GENOME INTEGRITY AND

278
00:10:07,480 --> 00:10:08,760
ESSENTIALLY EVERY ORGANISM.

279
00:10:08,760 --> 00:10:10,360
IT'S INTIMATELY INVOLVED IN THE

280
00:10:10,360 --> 00:10:13,880
REPAIR OF DNA DAMAGE AND OF

281
00:10:13,880 --> 00:10:16,880
COURSE IN CANCER BIOLOGY.

282
00:10:16,880 --> 00:10:18,680
SO I'M NOT REALLY SURE WHAT THE

283
00:10:18,680 --> 00:10:19,560
MAKEUP OF THE AUDIENCE IS RIGHT

284
00:10:19,560 --> 00:10:21,360
NOW BUT MANY OF YOU WILL HAVE

285
00:10:21,360 --> 00:10:24,760
HEARD OF THE PROTEINS BRCA1 AND

286
00:10:24,760 --> 00:10:28,080
BRCA2, CANCER-ASSOCIATED

287
00:10:28,080 --> 00:10:29,440
ONCOGENES.

288
00:10:29,440 --> 00:10:31,640
AND THESE GUYS, WHEN THEY ARE

289
00:10:31,640 --> 00:10:33,760
MUTATED, YOU HAVE A HIGH

290
00:10:33,760 --> 00:10:36,560
PROPENSITY TO HAVE HEREDITARY

291
00:10:36,560 --> 00:10:38,160
BREAST OR OVARIAN CANCERS.

292
00:10:38,160 --> 00:10:40,000
YOU'RE MESSING UP THIS PORTION

293
00:10:40,000 --> 00:10:40,920
OF THE REACTION WHERE YOU'RE

294
00:10:40,920 --> 00:10:42,920
TRYING TO ASSEMBLE A FILAMENT.

295
00:10:42,920 --> 00:10:45,440
SO THERE IS AN INTIMATE

296
00:10:45,440 --> 00:10:46,920
RELATIONSHIP BETWEEN CANCER

297
00:10:46,920 --> 00:10:48,640
BIOLOGY AND HOMOLOGOUS

298
00:10:48,640 --> 00:10:48,960
RECOMBINATION.

299
00:10:48,960 --> 00:10:51,080
AND IN FACT, MUTATIONS AND

300
00:10:51,080 --> 00:10:52,640
PROTEINS THAT HAPPEN ALONG THIS

301
00:10:52,640 --> 00:10:54,920
PATHWAY ARE OFTEN ASSOCIATED

302
00:10:54,920 --> 00:10:57,000
WITH CANCER-PRONE SYNDROMES.

303
00:10:57,000 --> 00:10:58,280
ONE OF THE PROTEINS WE WILL TALK

304
00:10:58,280 --> 00:11:00,200
ABOUT TODAY IS JUST LIKE THIS.

305
00:11:00,200 --> 00:11:02,080
AND OF COURSE, DEFECTS CAN LEAD

306
00:11:02,080 --> 00:11:04,160
TO CHROMOSOMAL REARRANGEMENTS,

307
00:11:04,160 --> 00:11:07,680
CHANGES IN COPY NUMBER, LOTS OF

308
00:11:07,680 --> 00:11:09,000
BAD THINGS.

309
00:11:09,000 --> 00:11:11,680
AND I SHOULD NOTE ALSO THAT

310
00:11:11,680 --> 00:11:14,080
RECOMBINATION IS INTIMATELY

311
00:11:14,080 --> 00:11:16,880
INVOLVED IN THE RESCUE OF

312
00:11:16,880 --> 00:11:21,080
STALLED OR DAMAGED OR COLLAPSED

313
00:11:21,080 --> 00:11:21,480
REPLICATION FORKS.

314
00:11:21,480 --> 00:11:23,480
SO THIS IS REALLY ESSENTIAL FOR

315
00:11:23,480 --> 00:11:23,680
THAT.

316
00:11:23,680 --> 00:11:26,360
IT'S ALSO INVOLVED IN HORIZONTAL

317
00:11:26,360 --> 00:11:27,280
GENE TRANSFER.

318
00:11:27,280 --> 00:11:28,680
SO IF YOU'RE DOING SOME SORT OF

319
00:11:28,680 --> 00:11:30,600
CHRIS PER TYPE OF THINGS,

320
00:11:30,600 --> 00:11:31,680
CHANCES ARE YOU'RE RELYING ON

321
00:11:31,680 --> 00:11:35,240
THE COMBINATION TO INSERT A GENE

322
00:11:35,240 --> 00:11:35,880
SOMEWHERE.

323
00:11:35,880 --> 00:11:38,000
AND THEN OF COURSE FOR THE FOLKS

324
00:11:38,000 --> 00:11:40,840
INTERESTED IN MEIOSIS, THIS IS

325
00:11:40,840 --> 00:11:44,320
THE PROCESS BY WHICH GENETIC

326
00:11:44,320 --> 00:11:46,160
DIVERSITY IS ESTABLISHED IN

327
00:11:46,160 --> 00:11:48,560
MEIOSIS THROUGH RECOMBINATION

328
00:11:48,560 --> 00:11:51,040
BETWEEN DIFFERENT DNAs.

329
00:11:51,040 --> 00:11:55,160
NOW, WE HAVE STUDIED REACTIONS

330
00:11:55,160 --> 00:11:59,280
ALONG THIS PATHWAY IN A NUMBER

331
00:11:59,280 --> 00:12:00,280
OF DIFFERENT REACTIONS.

332
00:12:00,280 --> 00:12:02,080
A LOT OF WHICH HAVE FOCUSED ON

333
00:12:02,080 --> 00:12:04,760
THE ASSEMBLY OF THESE

334
00:12:04,760 --> 00:12:06,440
NUCLEOPROTEIN FILAMENTS ON THESE

335
00:12:06,440 --> 00:12:07,840
PROCESSED DNA ENDS AS WELL AS

336
00:12:07,840 --> 00:12:10,840
THE PAIRING PROCESSES THAT TAKE

337
00:12:10,840 --> 00:12:13,320
PLACE TO GET YOUR BLACK DNA

338
00:12:13,320 --> 00:12:15,760
PAIRED WITH YOUR RED DNA.

339
00:12:15,760 --> 00:12:16,520
ED THAT I'M GOING TO TALK ABOUT

340
00:12:16,520 --> 00:12:19,240
IS HOW YOU GO FROM THIS DOUBLE

341
00:12:19,240 --> 00:12:21,120
STRAND BREAK TO A PROCESSED

342
00:12:21,120 --> 00:12:22,840
DOUBLE STRAND BREAK WHERE YOU

343
00:12:22,840 --> 00:12:23,680
RESECTED THESE ENDS BACK.

344
00:12:23,680 --> 00:12:25,280
AND I SHOULD POINT OUT THAT I'M

345
00:12:25,280 --> 00:12:29,080
GOING TO TALK ABOUT THREE

346
00:12:29,080 --> 00:12:29,320
PROTEINS.

347
00:12:29,320 --> 00:12:32,640
BLOOM HELICASE, DNA2 AND RPA.

348
00:12:32,640 --> 00:12:34,640
I'LL TELL YOU WHAT THEY ARE IN A

349
00:12:34,640 --> 00:12:34,880
MOMENT.

350
00:12:34,880 --> 00:12:36,880
THE UNDERSTANDING WE ARE USING A

351
00:12:36,880 --> 00:12:38,320
VERY SIMPLIFIED SYSTEM TO TRY TO

352
00:12:38,320 --> 00:12:40,320
LOOK AT WHAT WE THINK ARE SORT

353
00:12:40,320 --> 00:12:42,800
OF KEY ASPECTS OF THE REACTION

354
00:12:42,800 --> 00:12:45,400
AND IN REALITY, INSIDE OF CELLS,

355
00:12:45,400 --> 00:12:47,280
THIS IS GOING TO INVOLVE A

356
00:12:47,280 --> 00:12:49,480
LARGER NUMBER OF PROTEINS THAT

357
00:12:49,480 --> 00:12:50,760
WE HOPE TO EVENTUALLY BE ABLE TO

358
00:12:50,760 --> 00:12:53,080
LOOK AT IN OUR RECONSTITUTED

359
00:12:53,080 --> 00:12:53,280
SYSTEM.

360
00:12:53,280 --> 00:12:55,160
BUT FOR THE TIME BEING, WE ARE

361
00:12:55,160 --> 00:12:57,960
LOOKING AT A MINIMAL SYSTEM.

362
00:12:57,960 --> 00:13:00,080
SO NOW, BLOOM HELICASE, THE

363
00:13:00,080 --> 00:13:01,760
FIRST OF THESE GUYS, AND YOU'LL

364
00:13:01,760 --> 00:13:04,960
HEAR THESE THREE NAMES

365
00:13:04,960 --> 00:13:06,560
THROUGHOUT -- IS A HELICASE,

366
00:13:06,560 --> 00:13:07,880
WHEN HAVE YOU DEFECTS IN IT, YOU

367
00:13:07,880 --> 00:13:11,640
END UP WITH A CANCER-PRONE

368
00:13:11,640 --> 00:13:12,760
SYNDROME, BLOOM SYNDROME, THAT

369
00:13:12,760 --> 00:13:16,160
IS CHARACTERIZED BY REALLY

370
00:13:16,160 --> 00:13:18,080
PROFOUND DNA REPAIRED DEFECTS

371
00:13:18,080 --> 00:13:20,680
AND CHROMOSOMAL REARRANGEMENTS.

372
00:13:20,680 --> 00:13:23,280
SO THE FOLKS OR THE PATIENTS IN

373
00:13:23,280 --> 00:13:24,880
HAVE THIS END UP HAVING CANCER

374
00:13:24,880 --> 00:13:27,960
AT A VERY, VERY YOUNG AGE,

375
00:13:27,960 --> 00:13:28,280
UNFORTUNATELY.

376
00:13:28,280 --> 00:13:29,360
NOW THE SECOND PROTEIN THAT I'M

377
00:13:29,360 --> 00:13:32,080
GOING TO TALK ABOUT IS DNA2.

378
00:13:32,080 --> 00:13:34,480
IT IS ALSO A HELICASE.

379
00:13:34,480 --> 00:13:37,640
IT'S NOT QUITE AS ROBUST AS BLM

380
00:13:37,640 --> 00:13:40,000
BUT IT'S ALSO A NUCLEASE.

381
00:13:40,000 --> 00:13:43,640
DNA2 IS WHAT IS RESPONSIBLE FOR

382
00:13:43,640 --> 00:13:45,880
CUTTING OUT THIS OTHER STRAND OF

383
00:13:45,880 --> 00:13:50,080
DNA TO LEAVE A 3-PRIME OVERHANG.

384
00:13:50,080 --> 00:13:53,880
AND THEN RPA IS A HETEROTRIMERIC

385
00:13:53,880 --> 00:13:55,360
SINGLE STRANDED BINDING PROTEIN.

386
00:13:55,360 --> 00:13:58,480
THIS IS A PROTEIN THAT IS

387
00:13:58,480 --> 00:13:59,080
ESSENTIAL EVOLVED IN ANYTHING

388
00:13:59,080 --> 00:14:02,200
INVOLVING SINGLE STRANDED DNA

389
00:14:02,200 --> 00:14:04,280
INSIDE OF A EUKARYOTIC CELL.

390
00:14:04,280 --> 00:14:07,080
IT'S HIGHLY ABUNDANT PROTEIN AND

391
00:14:07,080 --> 00:14:07,400
UBIQUITOUS.

392
00:14:07,400 --> 00:14:09,080
IT'S INVOLVED IN EVERYTHING.

393
00:14:09,080 --> 00:14:10,400
SO THESE THREE PLAYERS ARE WHAT

394
00:14:10,400 --> 00:14:11,800
WE ARE GOING TO TALK ABOUT IN

395
00:14:11,800 --> 00:14:17,720
TERMS OF GENERATING THIS PROCESS

396
00:14:17,720 --> 00:14:18,800
DNA END.

397
00:14:18,800 --> 00:14:20,280
NOW, JUST SO EVERYBODY IS

398
00:14:20,280 --> 00:14:21,800
ONBOARD WITH THE TECHNOLOGY THAT

399
00:14:21,800 --> 00:14:26,080
WE WILL BE TALKING ABOUT, THIS

400
00:14:26,080 --> 00:14:28,400
IS WHAT WE CALL DNA CURTAINS.

401
00:14:28,400 --> 00:14:32,560
ESSENTIALLY WE HAVE A FLOW CELL

402
00:14:32,560 --> 00:14:35,480
THAT WE MAKE WITH AN IN LET AND

403
00:14:35,480 --> 00:14:38,760
OUT LET CHAMBER, A SAMPLE

404
00:14:38,760 --> 00:14:39,960
CHAMBER IN BETWEEN THAT IS MADE

405
00:14:39,960 --> 00:14:41,480
WITH FANCY DOUBLE SIDED STICKY

406
00:14:41,480 --> 00:14:42,680
TAPE, IF YOU CAN BELIEVE THAT.

407
00:14:42,680 --> 00:14:43,960
AND THEN WHAT IS REALLY SPECIAL

408
00:14:43,960 --> 00:14:45,880
ABOUT WHAT WE ARE DOING IS WE

409
00:14:45,880 --> 00:14:49,360
ARE MODIFYING THE SURFACE OF

410
00:14:49,360 --> 00:14:50,760
THIS CHAMBER.

411
00:14:50,760 --> 00:14:53,600
AND EXACTLY WHAT WE'RE DOING IS

412
00:14:53,600 --> 00:14:57,560
PUTTING DOWN PATTERNS OF METAL,

413
00:14:57,560 --> 00:14:57,960
METALLIC BARRIERS.

414
00:14:57,960 --> 00:15:01,040
I'M SHOWING YOU A BLOW UP,Y AND

415
00:15:01,040 --> 00:15:04,160
MAT TIESED BLOW UP OF A

416
00:15:04,160 --> 00:15:05,840
ZIGZAG-SHAPED PATTERN MADE OF

417
00:15:05,840 --> 00:15:07,960
CHROME THAT WE DEPOSIT ON TO

418
00:15:07,960 --> 00:15:08,720
THAT SURFACE.

419
00:15:08,720 --> 00:15:10,440
I'M SHOWING YOU ONE BUT WE PUT

420
00:15:10,440 --> 00:15:12,840
THESE ALL OVER THE PLACE IN

421
00:15:12,840 --> 00:15:13,040
HERE.

422
00:15:13,040 --> 00:15:17,240
AND WHAT WE THEN DO IS TAKE THIS

423
00:15:17,240 --> 00:15:19,720
SURFACE, THIS GLASS SURF AS AND

424
00:15:19,720 --> 00:15:24,080
COVER IT WITH A FLUID LIPID

425
00:15:24,080 --> 00:15:24,320
BILAYER.

426
00:15:24,320 --> 00:15:27,960
IT'S SERVING TWO PURPOSES.

427
00:15:27,960 --> 00:15:29,480
THE FIRST IT'S GOING TO ENSURE

428
00:15:29,480 --> 00:15:32,520
THAT THIS SURFACE IS AS INERT AS

429
00:15:32,520 --> 00:15:34,640
POSSIBLE IN TERMS OF US PUTTING

430
00:15:34,640 --> 00:15:37,320
PROTEINS INTO THESE CHAMBERS.

431
00:15:37,320 --> 00:15:41,280
SO, PROTEINS LOVE TO STICK TO

432
00:15:41,280 --> 00:15:41,480
GLASS.

433
00:15:41,480 --> 00:15:42,200
IF YOU WERE TO JUST INJECT ANY

434
00:15:42,200 --> 00:15:43,800
PROTEIN WE WORK WITH INTO A

435
00:15:43,800 --> 00:15:46,480
GLASS CHAMBER, IT WOULD SIMPLY

436
00:15:46,480 --> 00:15:47,840
COAT THE SURFACE AND THAT WOULD

437
00:15:47,840 --> 00:15:50,360
BE THE END OF YOUR EXPERIMENT.

438
00:15:50,360 --> 00:15:51,600
YOU WOULDN'T BE ABLE TO TELL

439
00:15:51,600 --> 00:15:52,080
ANYTHING.

440
00:15:52,080 --> 00:15:53,560
SO THE BILAYER IS OUR EFFORT TO

441
00:15:53,560 --> 00:15:56,600
TRY TO MAKE THIS CHAMBER SURFACE

442
00:15:56,600 --> 00:15:59,360
LOOK MORE LIKE THE INSIDE OF A

443
00:15:59,360 --> 00:16:06,680
LIVING CELL THAN GLASS.

444
00:16:06,680 --> 00:16:10,080
A SUBSET OF THE LIPIDS WITHIN

445
00:16:10,080 --> 00:16:12,480
THE BILAYER HAVE A BY TIN GROUP

446
00:16:12,480 --> 00:16:15,560
AND WE COUPLE DNA MOLECULES

447
00:16:15,560 --> 00:16:18,560
THROUGH THAT SURFACE.

448
00:16:18,560 --> 00:16:21,000
THE LIPID BILAYER IS FLUID IN

449
00:16:21,000 --> 00:16:22,160
TWO DIMENSIONS.

450
00:16:22,160 --> 00:16:24,360
WHEN WE ATTACH THINGS TO IT,

451
00:16:24,360 --> 00:16:25,400
THEY CAN DIFFUSE AROUND ATTACH

452
00:16:25,400 --> 00:16:26,480
TO THE SURFACE.

453
00:16:26,480 --> 00:16:30,680
WHAT WE DO THEN IS APPLY A FLOW

454
00:16:30,680 --> 00:16:31,960
FORCE, HYDRODYNAMIC FORCE THAT

455
00:16:31,960 --> 00:16:34,560
PUSHES THE DNA INTO THIS

456
00:16:34,560 --> 00:16:34,800
BARRIER.

457
00:16:34,800 --> 00:16:37,200
SO THE DNA IS ACTING ESSENTIALLY

458
00:16:37,200 --> 00:16:39,840
LIKE A SAIL ON A SHIP.

459
00:16:39,840 --> 00:16:42,240
IT'S PUSHING THEM INTO THE

460
00:16:42,240 --> 00:16:42,640
BARRIER.

461
00:16:42,640 --> 00:16:44,320
THEY THEN LAY DOWN PARALLEL TO

462
00:16:44,320 --> 00:16:47,400
THE SURFACE AND THEN WE'RE

463
00:16:47,400 --> 00:16:49,680
ILLUMINATING THEM WITH TOTAL

464
00:16:49,680 --> 00:16:50,600
INTERNAL REFLECTION,

465
00:16:50,600 --> 00:16:52,120
FLUORESCENCE MICROSCOPY.

466
00:16:52,120 --> 00:16:54,320
SO ILLUMINATING A SMALL

467
00:16:54,320 --> 00:16:55,640
EXCITATION VOLUME NEAR THE

468
00:16:55,640 --> 00:16:57,440
SURFACE WITHIN THE VOLUME WHERE

469
00:16:57,440 --> 00:16:59,120
THESE DNA MOLECULES RESIDE.

470
00:16:59,120 --> 00:17:01,320
WE HAVE MADE MANY FLAVORS OF

471
00:17:01,320 --> 00:17:02,920
THESE CURTAINS, MANY DIFFERENT

472
00:17:02,920 --> 00:17:03,280
STYLES.

473
00:17:03,280 --> 00:17:05,440
THE ONES I'M GOING TO BE TALKING

474
00:17:05,440 --> 00:17:07,440
ABOUT TODAY ARE AS SHOWN HERE,

475
00:17:07,440 --> 00:17:09,560
MEANING ONE END OF THE DNA IS

476
00:17:09,560 --> 00:17:11,520
ATTACHED AND ONE END IS FREE.

477
00:17:11,520 --> 00:17:13,600
THIS IS WHAT WE WOULD CALL A

478
00:17:13,600 --> 00:17:15,680
SINGLE AND TETHERED DNA CURTAIN.

479
00:17:15,680 --> 00:17:16,880
FOR ANYONE THAT WANTS ANY

480
00:17:16,880 --> 00:17:19,200
ADDITIONAL INFORMATION, WE

481
00:17:19,200 --> 00:17:21,200
PUBLISHED MANY, MANY METHODS,

482
00:17:21,200 --> 00:17:23,040
PAPERS, DESCRIBING THIS WORK.

483
00:17:23,040 --> 00:17:25,480
AND WE HAVE ALSO PUBLISHED A

484
00:17:25,480 --> 00:17:26,680
NUMBER OF PAPERS DESCRIBING

485
00:17:26,680 --> 00:17:28,080
STUDIES THAT HAVE COME OUT OF

486
00:17:28,080 --> 00:17:33,480
THE USE OF THESE TYPES OF

487
00:17:33,480 --> 00:17:33,720
THINGS.

488
00:17:33,720 --> 00:17:35,080
SO THAT IS IT FOR THE

489
00:17:35,080 --> 00:17:35,440
INTRODUCTION.

490
00:17:35,440 --> 00:17:37,280
I WANT TO MAKE ONE MORE POINT

491
00:17:37,280 --> 00:17:38,880
WHILE I'M THINKING ABOUT IT.

492
00:17:38,880 --> 00:17:40,200
IN THE SUBSEQUENT SLIDES I WILL

493
00:17:40,200 --> 00:17:43,480
BE SHOWING YOU CYMOGRAPHS.

494
00:17:43,480 --> 00:17:45,480
AND THEY ARE SINGLE -- SO THE

495
00:17:45,480 --> 00:17:47,280
DATA WE COLLECT IS VIDEOS.

496
00:17:47,280 --> 00:17:49,880
VIDEOS OF THESE MOLECULES

497
00:17:49,880 --> 00:17:50,160
INTERACTING.

498
00:17:50,160 --> 00:17:52,800
AND THE CYMOGRAPHS ARE SINGLE

499
00:17:52,800 --> 00:17:54,040
ONE PIXEL SLICES THROUGH

500
00:17:54,040 --> 00:17:55,160
INDIVIDUAL DNA MOLECULES THAT

501
00:17:55,160 --> 00:17:56,560
ARE THEN PROJECTED THROUGH TIME.

502
00:17:56,560 --> 00:17:57,800
AND I'LL TRY TO EXPLAIN THAT

503
00:17:57,800 --> 00:18:00,880
MORE CAREFULLY IN THE FIRST

504
00:18:00,880 --> 00:18:01,560
CYMEO GRAPH SO WE ARE ALL

505
00:18:01,560 --> 00:18:03,280
ONBOARD WITH UNDERSTANDING HOW

506
00:18:03,280 --> 00:18:05,960
THE EXPERIMENT WORKS.

507
00:18:05,960 --> 00:18:12,080
SO NOW THIS IS AN EXAMPLE

508
00:18:12,080 --> 00:18:15,880
SHOWING A SCHEMATIC, SORE

509
00:18:15,880 --> 00:18:16,760
ESHOWING THE SUBSTRATE WE WILL

510
00:18:16,760 --> 00:18:17,760
BE WORKING WITH.

511
00:18:17,760 --> 00:18:19,880
THIS IS OUR DNA CURTAIN FOCUSING

512
00:18:19,880 --> 00:18:22,040
ON THE END OF THE MOLECULE.

513
00:18:22,040 --> 00:18:23,960
WHAT WE HAVE DONE IS ENGINEERED

514
00:18:23,960 --> 00:18:26,880
THAT SO WE HAVE 30 NUCLEOTIDE

515
00:18:26,880 --> 00:18:28,880
SINGLE STRANDED DNA OVERHANG.

516
00:18:28,880 --> 00:18:32,720
THE INTENTION IS TO MIMIC A

517
00:18:32,720 --> 00:18:33,440
PARTIALLY-PROCESSED

518
00:18:33,440 --> 00:18:33,760
INTERMEDIATE.

519
00:18:33,760 --> 00:18:35,120
I MENTIONED EARLIER THERE WERE A

520
00:18:35,120 --> 00:18:36,520
NUMBER OF PROTEINS THAT COME

521
00:18:36,520 --> 00:18:37,920
INTO THESE REACTIONS THAT WE'RE

522
00:18:37,920 --> 00:18:39,040
NOT LOOKING AT.

523
00:18:39,040 --> 00:18:46,160
ONE IS CALLED THE MRN COMPLEX.

524
00:18:46,160 --> 00:18:48,880
MRE11, Rad50MBS1T GENERATES A

525
00:18:48,880 --> 00:18:50,320
SINGLE STRAND OVERHANG AND THEN

526
00:18:50,320 --> 00:18:52,320
WHAT HAPPENS ON THAT OVERHANG IS

527
00:18:52,320 --> 00:18:55,520
WE CAN LOAD OUR REACTIONS

528
00:18:55,520 --> 00:19:02,400
CONTAINING BLM, RadAND DNA2.

529
00:19:02,400 --> 00:19:04,680
AND I'LL POINT OUT WE PUBLISHED

530
00:19:04,680 --> 00:19:06,080
SIMILAR WORK WITH THE YEAST

531
00:19:06,080 --> 00:19:09,280
RESECTION SYSTEM USING THESE

532
00:19:09,280 --> 00:19:11,680
SAME SUBSTRATES.

533
00:19:11,680 --> 00:19:15,080
NOW THIS IS A WIDE FIELD IMAGE

534
00:19:15,080 --> 00:19:17,080
OF DNA CURTAIN.

535
00:19:17,080 --> 00:19:19,280
THE DNA IS NOT LABELED SO YOU DO

536
00:19:19,280 --> 00:19:20,200
NOT SEE IT.

537
00:19:20,200 --> 00:19:22,560
AND WHAT YOU DO SEE IS THIS B

538
00:19:22,560 --> 00:19:23,480
AND THIS E.

539
00:19:23,480 --> 00:19:25,280
SO THE B SILENT BARRIER.

540
00:19:25,280 --> 00:19:27,600
THAT'S WHERE WE ALIGNED OUR DNA

541
00:19:27,600 --> 00:19:27,880
MOLECULES.

542
00:19:27,880 --> 00:19:29,960
AND THE E IS THE END OF THE DNA.

543
00:19:29,960 --> 00:19:31,800
SO THE FREE END OF THE MOLECULE.

544
00:19:31,800 --> 00:19:36,720
AND WHAT WE ARE LOOKING AT THEN

545
00:19:36,720 --> 00:19:39,080
IS BLOOM HELICASE BOUND TO THAT

546
00:19:39,080 --> 00:19:39,280
END.

547
00:19:39,280 --> 00:19:40,320
SO BLOOM IS LABELED IN THIS CASE

548
00:19:40,320 --> 00:19:41,720
WITH GFP.

549
00:19:41,720 --> 00:19:43,680
WE HAVE BUFFER FLOWING FROM

550
00:19:43,680 --> 00:19:44,000
TOP-TO-BOTTOM.

551
00:19:44,000 --> 00:19:45,160
I'M SURE YOU CAN APPRECIATE JUST

552
00:19:45,160 --> 00:19:47,360
FROM THE IMAGE THAT YOU CAN SEE

553
00:19:47,360 --> 00:19:49,680
THESE GREEN SPOTS AND MOST OF

554
00:19:49,680 --> 00:19:51,080
THEM SEEM TO BE COALIGNED WITH

555
00:19:51,080 --> 00:19:53,160
WHAT WE SAY IS THE END OF THE

556
00:19:53,160 --> 00:19:53,320
DNA.

557
00:19:53,320 --> 00:19:55,160
AND THIS IS JUST TAKING A SINGLE

558
00:19:55,160 --> 00:19:55,360
IMAGE.

559
00:19:55,360 --> 00:19:57,680
THIS IS NOW LOOKING AT THAT DATA

560
00:19:57,680 --> 00:20:03,280
SORT OF IN AGGREGATE WHERE N IS

561
00:20:03,280 --> 00:20:04,680
176 MOLECULES AND THE BARS

562
00:20:04,680 --> 00:20:08,000
REPRESENT THE LOCATIONS OF THE

563
00:20:08,000 --> 00:20:08,560
GFP BLOOM COMPLEX.

564
00:20:08,560 --> 00:20:10,400
IN MOST OF THE REACTIONS WE DO,

565
00:20:10,400 --> 00:20:11,480
WITH SOME EXCEPTIONS AND I'LL

566
00:20:11,480 --> 00:20:13,240
SHOW YOU THESE LATER, THE BLOOM

567
00:20:13,240 --> 00:20:15,160
IS ACCUMULATING AT DNA END.

568
00:20:15,160 --> 00:20:17,280
OF COURSE WE DO SEE SOME BOUND

569
00:20:17,280 --> 00:20:17,560
INTERNALLY.

570
00:20:17,560 --> 00:20:19,640
I'M GOING TO MENTION, THEY

571
00:20:19,640 --> 00:20:20,080
BEHAVE DIFFERENTLY.

572
00:20:20,080 --> 00:20:22,160
I'LL TALK ABOUT THAT A LITTLE

573
00:20:22,160 --> 00:20:22,320
BIT.

574
00:20:22,320 --> 00:20:25,440
SO MOST OF THE PROTEINS GO TO

575
00:20:25,440 --> 00:20:26,160
THE END.

576
00:20:26,160 --> 00:20:27,680
THEY TEND TO STAYED THERE.

577
00:20:27,680 --> 00:20:30,280
SO ONCE THEY ARE BOUND, THEY

578
00:20:30,280 --> 00:20:31,680
HAVE HALF-LIVES THAT EXCEED OUR

579
00:20:31,680 --> 00:20:32,680
EXPERIMENTAL MEASUREMENTS UNDER

580
00:20:32,680 --> 00:20:35,440
ALL THE CONDITIONS THAT WE HAVE

581
00:20:35,440 --> 00:20:35,720
TESTED.

582
00:20:35,720 --> 00:20:38,640
AND I NOTE HERE THAT THEY ARE

583
00:20:38,640 --> 00:20:40,280
RESISTANT TO THIS PROTEIN, RPA

584
00:20:40,280 --> 00:20:41,680
AND I MAKE THAT NOTE BECAUSE WE

585
00:20:41,680 --> 00:20:43,600
DID A SIMILAR STUDY WITH THE

586
00:20:43,600 --> 00:20:44,800
YEAST SYSTEM AND PUT THE

587
00:20:44,800 --> 00:20:47,280
PROTEINS SGS1 HERE AS SOON AS WE

588
00:20:47,280 --> 00:20:51,680
INJECT RPA, IT KICKS SGS1 OFF.

589
00:20:51,680 --> 00:20:52,600
IT'S AN INTERESTING DIFFERENCE

590
00:20:52,600 --> 00:20:54,480
THAT WE DON'T FULLY UNDERSTAND

591
00:20:54,480 --> 00:20:54,720
YET.

592
00:20:54,720 --> 00:20:56,880
BUT WE DO KNOW NOW THAT BLOOM

593
00:20:56,880 --> 00:20:59,240
GOES TO THE DNA ENDS.

594
00:20:59,240 --> 00:21:01,440
SO NOW THIS IS OUR FIRST EXAMPLE

595
00:21:01,440 --> 00:21:03,080
OF A CYMEO GRAPH.

596
00:21:03,080 --> 00:21:04,680
AGAIN WHAT I'M SHOWING YOU TO

597
00:21:04,680 --> 00:21:06,400
JUMP BACK IS GOING TO BE A

598
00:21:06,400 --> 00:21:07,880
SINGLE ONE PIXEL SLICE THROUGH

599
00:21:07,880 --> 00:21:11,000
ONE OF THESE DNAs AND SHOWING

600
00:21:11,000 --> 00:21:12,800
THE END-BOUND COMPLEX.

601
00:21:12,800 --> 00:21:14,480
AND THEN PROJECTED OVER TIME.

602
00:21:14,480 --> 00:21:19,280
SO WHAT YOU CAN SEE IS THAT THAT

603
00:21:19,280 --> 00:21:21,160
MOLECULE OR THAT GREEN SPOT,

604
00:21:21,160 --> 00:21:21,560
SUPPORTS MOVE UP.

605
00:21:21,560 --> 00:21:24,480
SO IT'S MOVING UP AGAINST THE

606
00:21:24,480 --> 00:21:26,640
DIRECTION OF THE BUFFER FLOW

607
00:21:26,640 --> 00:21:28,040
FROM E TO NOW THE B DIRECTION.

608
00:21:28,040 --> 00:21:29,800
AND I'M GOING TO POINT OUT THERE

609
00:21:29,800 --> 00:21:33,560
IS LITTLE FLUCTUATIONS IN THIS

610
00:21:33,560 --> 00:21:34,880
MOLECULE, ESSENTIALLY WHAT IS

611
00:21:34,880 --> 00:21:36,280
HAPPENING HERE IS THAT DNA IS

612
00:21:36,280 --> 00:21:36,880
FREE.

613
00:21:36,880 --> 00:21:39,440
WE DON'T HAVE A TREMENDOUS

614
00:21:39,440 --> 00:21:41,960
AMOUNT OF DYNAMIC FORCE EXERTOD

615
00:21:41,960 --> 00:21:44,720
IT SO IT'S SORT OF FLIPPING

616
00:21:44,720 --> 00:21:45,920
BACK-AND-FORTH IN THE BUFFER.

617
00:21:45,920 --> 00:21:47,440
NOT THINGS THE ENZYMES ARE

618
00:21:47,440 --> 00:21:49,040
DOING, IT'S SOMETHING THE DNA IS

619
00:21:49,040 --> 00:21:50,680
DOING ON A MACROSCOPIC SCALE.

620
00:21:50,680 --> 00:21:53,160
AND SO WE CAN MAKE MEASUREMENTS

621
00:21:53,160 --> 00:21:54,840
BASED ON, HERE IS WHERE IT

622
00:21:54,840 --> 00:21:56,120
STARTED, HERE IS WHERE IT ENDED.

623
00:21:56,120 --> 00:21:57,440
WE KNOW THE TIMEFRAME.

624
00:21:57,440 --> 00:21:58,320
THIS IS A SCALE.

625
00:21:58,320 --> 00:21:59,920
WE CAN MAKE MEASUREMENTS OF HOW

626
00:21:59,920 --> 00:22:01,840
FAR DID IT GO AND HOW FAST DID

627
00:22:01,840 --> 00:22:02,800
IT GO.

628
00:22:02,800 --> 00:22:04,320
I'M JUST GOING TO POINT OUT, WE

629
00:22:04,320 --> 00:22:07,040
DID A CONTROL HERE WHERE WE

630
00:22:07,040 --> 00:22:08,400
INJECTED YO-YO 1 AT THE END OF

631
00:22:08,400 --> 00:22:12,160
THE REACTION AND YO-YO 1 IS AN

632
00:22:12,160 --> 00:22:14,120
IN TECH LATING DYE THAT LET'S US

633
00:22:14,120 --> 00:22:15,480
SEE THE DNA.

634
00:22:15,480 --> 00:22:17,320
AND THIS COM FIRMS THE DNA IS

635
00:22:17,320 --> 00:22:19,920
THERE AND CONFIRMINGS THE DNA IS

636
00:22:19,920 --> 00:22:21,720
SHORTER THAN IT WAS WHEN IT

637
00:22:21,720 --> 00:22:23,280
STARTED SO WE KNOW RESECTION IS

638
00:22:23,280 --> 00:22:23,960
TAKING PLACE.

639
00:22:23,960 --> 00:22:25,960
WE ALSO KNOW THAT THIS RESECTION

640
00:22:25,960 --> 00:22:28,320
IS DEPEND ENTER ON DNA2 AND

641
00:22:28,320 --> 00:22:31,280
REMEMBER DNA2 IS THE NUCLEASE

642
00:22:31,280 --> 00:22:33,160
INVOLVED IN THIS REACTION.

643
00:22:33,160 --> 00:22:36,680
AND WE SEE NO MOVEMENT OR NO

644
00:22:36,680 --> 00:22:39,880
RESECTION IF WE USE A BLOOM

645
00:22:39,880 --> 00:22:41,360
MUTANT WHERE THE ACTIVE SITES

646
00:22:41,360 --> 00:22:43,880
INVOLVED IN ATP BINDING, THE

647
00:22:43,880 --> 00:22:47,360
ACTIVE SITE IS MUTATED FROM A

648
00:22:47,360 --> 00:22:48,440
LYSINE TO ANALLINE.

649
00:22:48,440 --> 00:22:51,880
SO I WANT TO POINT OUT A COUPLE

650
00:22:51,880 --> 00:22:54,480
OF DETAILS.

651
00:22:54,480 --> 00:22:55,680
WE CAN MEASURE VELOCITY AND

652
00:22:55,680 --> 00:22:57,880
DISTANCE, HOW FAST AND FAR FAR

653
00:22:57,880 --> 00:22:58,640
DOES IT GO?

654
00:22:58,640 --> 00:23:00,680
IN THE ABSENCE OF RPA, WE ARE

655
00:23:00,680 --> 00:23:03,080
GOING ABOUT 10 BASE PAIRS PER

656
00:23:03,080 --> 00:23:03,280
SECOND.

657
00:23:03,280 --> 00:23:05,760
OVER DISTANCES OF ABOUT 7KB.

658
00:23:05,760 --> 00:23:07,280
IT'S NOT TREMENDOUSLY FAST.

659
00:23:07,280 --> 00:23:09,880
WE WORK WITH OTHER MOTOR

660
00:23:09,880 --> 00:23:12,160
PROTEINS, OTHER HELICASES THAT

661
00:23:12,160 --> 00:23:15,800
REALLY DO GO FAST.

662
00:23:15,800 --> 00:23:16,400
1000 BASE PAIRS PER SECOND.

663
00:23:16,400 --> 00:23:18,480
SO THIS GUY IS PRETTY SLOW.

664
00:23:18,480 --> 00:23:20,760
IT'S NOT UNEXPECTED.

665
00:23:20,760 --> 00:23:24,680
THE ADDITION OF RPA ACTUALLY

666
00:23:24,680 --> 00:23:27,160
STIMULATES BOTH THE VELOCITY AND

667
00:23:27,160 --> 00:23:29,240
THE -- ON THE ORDER OF 50%.

668
00:23:29,240 --> 00:23:31,640
AND I'M GOING TO POINT OUT, I

669
00:23:31,640 --> 00:23:34,080
MENTIONED EARLIER THAT ROUGHLY

670
00:23:34,080 --> 00:23:35,040
60-70% OF THE PROTEINS ARE BOUND

671
00:23:35,040 --> 00:23:35,920
AT THE END.

672
00:23:35,920 --> 00:23:37,640
OTHERS ARE BOUND IN INTERNAL

673
00:23:37,640 --> 00:23:37,960
SITES.

674
00:23:37,960 --> 00:23:40,880
THE INTERNAL-BOUND BLM COMPLEXES

675
00:23:40,880 --> 00:23:41,680
ARE MUCH FASTER.

676
00:23:41,680 --> 00:23:44,840
THEY ARE MOVING AT ABOUT 100

677
00:23:44,840 --> 00:23:45,600
BASE PAIRS PER SECOND.

678
00:23:45,600 --> 00:23:47,080
SO SOMETHING ABOUT HAVING BLOOM

679
00:23:47,080 --> 00:23:48,120
AT THE END OF THE DNA IS

680
00:23:48,120 --> 00:23:52,280
ACTUALLY CAUSING IT TO GO MUCH

681
00:23:52,280 --> 00:23:53,320
SLOWER.

682
00:23:53,320 --> 00:23:57,920
AND I WANT TO COMMENT BRIEFLY ON

683
00:23:57,920 --> 00:23:59,480
WHAT FRACTION OF PROTEINS ARE

684
00:23:59,480 --> 00:24:01,880
ACTUALLY SHOWING THIS TYPE OF

685
00:24:01,880 --> 00:24:02,480
MOVEMENT BEHAVIOR.

686
00:24:02,480 --> 00:24:06,320
SO IF WE ONLY HAVE BLM IN THE

687
00:24:06,320 --> 00:24:08,480
REACTION, IT'S ABOUT 7.4%.

688
00:24:08,480 --> 00:24:11,400
AS SOON AS WE ADD DNA TWO, OR

689
00:24:11,400 --> 00:24:13,400
RPA, IT'S PARTNER PROTEINS, WE

690
00:24:13,400 --> 00:24:17,200
GO FROM ON THE ORDER OF 7% UP TO

691
00:24:17,200 --> 00:24:17,440
20-25%.

692
00:24:17,440 --> 00:24:20,080
SO WE GET A BIG JUMP IN THE

693
00:24:20,080 --> 00:24:21,680
EFFECTIVENESS OF THE REACTION OF

694
00:24:21,680 --> 00:24:23,440
THE FRACTION OF ACTIVE PROTEINS

695
00:24:23,440 --> 00:24:24,680
BY ADDING ITS PARTNERS.

696
00:24:24,680 --> 00:24:27,200
AND IF WE ADD BOTH OF THOSE GUYS

697
00:24:27,200 --> 00:24:29,000
TOGETHER, THAT'S WHEN WE

698
00:24:29,000 --> 00:24:30,480
ACTUALLY GET THE MOST ROBUST

699
00:24:30,480 --> 00:24:33,480
ACTIVITY WHEN WE ARE STARTING TO

700
00:24:33,480 --> 00:24:35,000
APPROACH 40% ACTIVE.

701
00:24:35,000 --> 00:24:36,280
I'M GOING TO POINT OUT THERE WAS

702
00:24:36,280 --> 00:24:39,680
A PAPER IN MOLECULAR CELL BY A

703
00:24:39,680 --> 00:24:43,080
GUY NAMED MICHAEL SONIA, AND

704
00:24:43,080 --> 00:24:45,600
THEY SEE VERY, VERY SIMILAR

705
00:24:45,600 --> 00:24:48,000
BEHAVIORS IN TERMS OF BLM AND

706
00:24:48,000 --> 00:24:50,680
ITS RESPONSE TO THE ADDICTION OF

707
00:24:50,680 --> 00:24:53,160
PARTNER PROTEINS.

708
00:24:53,160 --> 00:24:54,920
-- ADDITION OF PARTNER PROTEINS.

709
00:24:54,920 --> 00:24:56,640
WHAT WE WANTED TO KNOW IS, WHAT

710
00:24:56,640 --> 00:24:59,320
IS THE STATUS OF THE SINGLE

711
00:24:59,320 --> 00:25:00,440
STRANDED DNA THAT THIS THING

712
00:25:00,440 --> 00:25:01,520
SHOULD BE PRODUCING?

713
00:25:01,520 --> 00:25:03,480
SO AGAIN, BECAUSE WE ARE LOOKING

714
00:25:03,480 --> 00:25:06,440
AT GFP-BLM, WE ARE NOT ACTUALLY

715
00:25:06,440 --> 00:25:07,840
LOOKING AT THE DNA AND WE CAN'T

716
00:25:07,840 --> 00:25:09,560
SEE WHAT IS GOING ON.

717
00:25:09,560 --> 00:25:12,040
SO TO TRY TO DO THIS, WHAT WE IF

718
00:25:12,040 --> 00:25:14,160
WAS TAKE RPA, THE SINGLE

719
00:25:14,160 --> 00:25:16,320
STRANDED BINDING PROTEIN, AND

720
00:25:16,320 --> 00:25:18,560
FUSED IT TO mCherry SO WE

721
00:25:18,560 --> 00:25:20,760
COULD WATCH IT BINDING TO THE

722
00:25:20,760 --> 00:25:21,840
DNA END AS IT WAS PRODUCED.

723
00:25:21,840 --> 00:25:23,320
IN THESE REACTIONS WHAT WE TEND

724
00:25:23,320 --> 00:25:24,760
TO SEE IS AT THE BEGINNING WE

725
00:25:24,760 --> 00:25:26,920
DON'T GET RPA BINDING BECAUSE

726
00:25:26,920 --> 00:25:28,920
THERE ISN'T MUCH SINGLE STRANDED

727
00:25:28,920 --> 00:25:29,640
DNA THERE.

728
00:25:29,640 --> 00:25:32,320
AND THEN OVER TIME, THAT RPA

729
00:25:32,320 --> 00:25:33,720
MCherry SIGNAL STARTS TO

730
00:25:33,720 --> 00:25:36,160
INCREASE AND WE CAN SHOW THAT

731
00:25:36,160 --> 00:25:38,040
100% OF THE MOLECULES THAT ARE

732
00:25:38,040 --> 00:25:42,080
MOVING SHOW INCREASE IN RPA

733
00:25:42,080 --> 00:25:43,280
BINDING THAT SCALES

734
00:25:43,280 --> 00:25:46,840
PROPORTIONALLY WITH TIME AND

735
00:25:46,840 --> 00:25:47,080
DISTANCE.

736
00:25:47,080 --> 00:25:48,280
NOW WHEN WE FIRST STARTED DOING

737
00:25:48,280 --> 00:25:51,200
THIS, WE NOTED SOMETHING QUITE

738
00:25:51,200 --> 00:25:51,880
INTERESTING.

739
00:25:51,880 --> 00:25:53,480
AND THAT IS, WE THOUGHT WE WERE

740
00:25:53,480 --> 00:25:56,680
GOING TO BE MAKING A 3 PRIME

741
00:25:56,680 --> 00:25:58,280
OVERHANG THAT SHOULD BE

742
00:25:58,280 --> 00:25:58,560
EXTENDED.

743
00:25:58,560 --> 00:26:00,480
BUT WHAT WE SEE IS THE RPA KEEPS

744
00:26:00,480 --> 00:26:01,480
BUILDING AND BUILDING BUT

745
00:26:01,480 --> 00:26:03,920
INSTEAD OF SHOWING AN EXTENDED

746
00:26:03,920 --> 00:26:06,080
END, IT SEEMS TO BE TRACKING

747
00:26:06,080 --> 00:26:10,400
VERY CLOSELY IN SPACE WITH THE

748
00:26:10,400 --> 00:26:11,880
GPP-BLM, THAT IS SHOWN HERE IN

749
00:26:11,880 --> 00:26:13,680
THIS MERGED IMAGE WHERE YOU CAN

750
00:26:13,680 --> 00:26:17,920
SEE THE WHITE COLOR FROM THE

751
00:26:17,920 --> 00:26:18,880
BLOOM AND THE MAGENTA SORT OF

752
00:26:18,880 --> 00:26:21,760
TRACKING VERY, VERY CLOSELY WITH

753
00:26:21,760 --> 00:26:22,080
IT.

754
00:26:22,080 --> 00:26:24,760
AND WE SEE THIS IN 100% OF THE

755
00:26:24,760 --> 00:26:26,080
CASES THAT WE LOOKED AT.

756
00:26:26,080 --> 00:26:28,560
IN FACT, WE ALSO SAW THIS WITH

757
00:26:28,560 --> 00:26:29,880
THE YEAST SYSTEM AND I THINK

758
00:26:29,880 --> 00:26:33,320
THAT MICHAEL SAW THIS IN HIS

759
00:26:33,320 --> 00:26:35,080
MOLECULAR CELL PAPER AS WELL.

760
00:26:35,080 --> 00:26:37,640
NOW THE DIFFERENCE IS, IN THIS

761
00:26:37,640 --> 00:26:40,840
SYSTEM, WHAT WE ALSO SAW WAS

762
00:26:40,840 --> 00:26:44,160
OCCASIONALLY THESE THINGS WOULD

763
00:26:44,160 --> 00:26:46,720
GET MUCH, MUCH LONGER.

764
00:26:46,720 --> 00:26:48,480
AND THAT HAPPENED IN ABOUT 30%

765
00:26:48,480 --> 00:26:50,240
OF THE CASES.

766
00:26:50,240 --> 00:26:52,240
SO, WHEN WE SEE SOMETHING LIKE

767
00:26:52,240 --> 00:26:54,240
THIS WHERE A DNA MOLECULE GOES

768
00:26:54,240 --> 00:26:55,440
FROM A SHORTENED LIFE TO

769
00:26:55,440 --> 00:26:58,040
SOMETHING THAT IS MUCH, MUCH

770
00:26:58,040 --> 00:27:00,040
LONGER THAN IT WAS JUST IN THE

771
00:27:00,040 --> 00:27:02,120
FRAME PRIOR, THAT SAYS TO US

772
00:27:02,120 --> 00:27:04,280
THAT SOMEHOW THIS DNA WAS

773
00:27:04,280 --> 00:27:06,880
COMPACTED INTO A STRUCTURE THAT

774
00:27:06,880 --> 00:27:08,600
THEN OPENED UP VERY, VERY

775
00:27:08,600 --> 00:27:12,280
QUICKLY TO LEAD TO THIS LONGER

776
00:27:12,280 --> 00:27:12,560
OVERHANG.

777
00:27:12,560 --> 00:27:15,040
WE KNOW THIS IS SINGLE STRANDED

778
00:27:15,040 --> 00:27:17,280
BY VIRTUE OF THE BINDING OF

779
00:27:17,280 --> 00:27:19,680
SINGLE STRANDED BINDING SPECIFIC

780
00:27:19,680 --> 00:27:19,880
RPA.

781
00:27:19,880 --> 00:27:21,600
AND SO WHAT THIS TOLD US IS THAT

782
00:27:21,600 --> 00:27:24,200
MAYBE THIS DNA IS FORMING A LOOP

783
00:27:24,200 --> 00:27:27,480
THAT IS SOMEHOW TRACKING WITH

784
00:27:27,480 --> 00:27:30,200
GFP BLOOM AS THE PROCESS IS

785
00:27:30,200 --> 00:27:31,480
GOING ON.

786
00:27:31,480 --> 00:27:33,680
NOW, WHAT WE WANTED TO ASK NEXT

787
00:27:33,680 --> 00:27:35,360
WAS WHAT IS GOING ON WITH THE

788
00:27:35,360 --> 00:27:37,680
ACTUAL ENDS OF THE DNA

789
00:27:37,680 --> 00:27:39,640
THEMSELVES?

790
00:27:39,640 --> 00:27:42,240
AND SO WHAT THEY DID IS MAKE A

791
00:27:42,240 --> 00:27:46,920
SUBSTRATE WHERE WE PUT A SINGLE

792
00:27:46,920 --> 00:27:47,720
565 DYE EITHER ON THE 3 PRIME

793
00:27:47,720 --> 00:27:49,600
END OF OUR INITIAL SUBSTRATE OR

794
00:27:49,600 --> 00:27:51,120
ON THE 5 PRIME END.

795
00:27:51,120 --> 00:27:53,640
HE SHOWED THEN THAT BLM BINDS TO

796
00:27:53,640 --> 00:27:54,040
THESE ENDS.

797
00:27:54,040 --> 00:27:56,400
YOU CAN SEE IT HERE AND SEE IT

798
00:27:56,400 --> 00:27:58,760
IN THE DISTRIBUTION HIST GRAMS

799
00:27:58,760 --> 00:28:01,480
AND WE CAN SEE THE END OF THE

800
00:28:01,480 --> 00:28:02,720
DNA THEMSELVES NOW RATHER THAN

801
00:28:02,720 --> 00:28:04,320
RELYING UPON RPA TO SEE WHERE

802
00:28:04,320 --> 00:28:08,160
THINGS ARE GOING.

803
00:28:08,160 --> 00:28:09,840
WE KNOW NOW THIS SYSTEM WORKS.

804
00:28:09,840 --> 00:28:12,040
WHAT WE DID IS START TRACKING

805
00:28:12,040 --> 00:28:13,320
THE ENDS DURING THE PROCESSING

806
00:28:13,320 --> 00:28:13,840
REACTION.

807
00:28:13,840 --> 00:28:17,360
SO THIS IS A SCHEMATIC OF THE

808
00:28:17,360 --> 00:28:18,200
REACTION AS WE ANTICIPATE IT

809
00:28:18,200 --> 00:28:19,600
MIGHT BE TAKING PLACE.

810
00:28:19,600 --> 00:28:23,800
SO, THIS IS OUR SUBSTRATE.

811
00:28:23,800 --> 00:28:24,200
GFP-BLM.

812
00:28:24,200 --> 00:28:26,320
OUR DNA2 MOLECULE AND RPA.

813
00:28:26,320 --> 00:28:28,080
WE THOUGHT MAYBE IT WILL DO

814
00:28:28,080 --> 00:28:30,120
SOMETHING LIKE THIS WHERE THE 5

815
00:28:30,120 --> 00:28:30,640
PRIME END IS CLEAVED.

816
00:28:30,640 --> 00:28:32,680
THIS IS WHAT WE ANTICIPATED.

817
00:28:32,680 --> 00:28:35,160
THE 5 PRIME END SHOULD GET CUT

818
00:28:35,160 --> 00:28:35,360
OFF.

819
00:28:35,360 --> 00:28:38,320
OR IN PRINCIPLE IF IT WASN'T

820
00:28:38,320 --> 00:28:39,040
GETTING CLEAVED, MAYBE BOTH ENDS

821
00:28:39,040 --> 00:28:41,480
TRACK WITH THE COMPLEX AND

822
00:28:41,480 --> 00:28:42,880
SOMETHING FUNNY WAS GOING ON IN

823
00:28:42,880 --> 00:28:44,880
TERMS OF BOTH OF THEM FORMING A

824
00:28:44,880 --> 00:28:45,160
LOOP.

825
00:28:45,160 --> 00:28:47,240
I SHOULD EMPHASIZE IN THIS

826
00:28:47,240 --> 00:28:48,480
DIAGRAM, THIS IS WHAT WE THINK

827
00:28:48,480 --> 00:28:51,560
OF AS DNA LOOP THAT MIGHT BE

828
00:28:51,560 --> 00:28:52,360
FORMING.

829
00:28:52,360 --> 00:28:54,920
SO THIS IS WHAT HAPPENED IN

830
00:28:54,920 --> 00:28:56,960
ABOUT 87.5% OF THE CASES.

831
00:28:56,960 --> 00:29:01,200
SO THIS IS GFP-BLM MOVING.

832
00:29:01,200 --> 00:29:04,280
THIS FAINT SIGNAL IS NOW OUR

833
00:29:04,280 --> 00:29:05,760
5-PRIME ADD 0565.

834
00:29:05,760 --> 00:29:07,480
AND RIGHT HERE, RIGHT WHEN YOU

835
00:29:07,480 --> 00:29:09,480
GET READY TO START MOVING, IT

836
00:29:09,480 --> 00:29:11,120
GETS CUT OFF AND JUST SIMPLY

837
00:29:11,120 --> 00:29:12,080
DISAPPEARS FROM VIEW.

838
00:29:12,080 --> 00:29:14,360
SO THIS IS CONSISTENT WITH WHAT

839
00:29:14,360 --> 00:29:15,960
WE THOUGHT MIGHT BE TAKING

840
00:29:15,960 --> 00:29:16,160
PLACE.

841
00:29:16,160 --> 00:29:18,440
NOW I'M GOING TO POINT OUT THAT

842
00:29:18,440 --> 00:29:20,880
IN ABOUT 12 1/2% OF THE CASES WE

843
00:29:20,880 --> 00:29:21,600
SEE SOMETHING DIFFERENCE AND

844
00:29:21,600 --> 00:29:24,720
THAT IS THE GFP-BLM STARTS

845
00:29:24,720 --> 00:29:27,120
MOVING AND THE 5 PRIME DYE ALSO

846
00:29:27,120 --> 00:29:28,960
STARTS TRACKING WITH IT.

847
00:29:28,960 --> 00:29:34,120
AND SO THIS TYPE OF OBSERVATION

848
00:29:34,120 --> 00:29:34,840
IS CONSISTENT WITH BOTH ENDS OF

849
00:29:34,840 --> 00:29:36,440
THE MOLECULE BEING BOUND SOMEHOW

850
00:29:36,440 --> 00:29:37,480
AND FORMING A LOOP AS THIS THING

851
00:29:37,480 --> 00:29:40,120
IS PROGRESSING ALONG THE DNA.

852
00:29:40,120 --> 00:29:41,640
NOW ONE THING THAT WE CANNOT

853
00:29:41,640 --> 00:29:43,560
RULE OUT IN THIS, AND THIS IS

854
00:29:43,560 --> 00:29:45,840
WHAT I THINK IS PROBABLY

855
00:29:45,840 --> 00:29:46,840
HAPPENING -- I SUSPECT THAT WHEN

856
00:29:46,840 --> 00:29:49,800
WE SEE THE 5 PRIME END TRACKING

857
00:29:49,800 --> 00:29:51,640
WITH THE RESECTION MACHINERY, WE

858
00:29:51,640 --> 00:29:55,320
ARE PROBABLY MISSING THE DNA2

859
00:29:55,320 --> 00:29:55,560
ENZYME.

860
00:29:55,560 --> 00:29:57,440
SO I'M ASSUMING THAT IT'S NOT

861
00:29:57,440 --> 00:29:59,040
THERE AND THAT'S WHY THE DNA IS

862
00:29:59,040 --> 00:29:59,920
NOT GETTING CUT.

863
00:29:59,920 --> 00:30:01,680
I'M ASSUMING THAT BUT I DON'T

864
00:30:01,680 --> 00:30:02,320
KNOW IT YET.

865
00:30:02,320 --> 00:30:03,720
WHAT WE REALLY WANT TO DO IS

866
00:30:03,720 --> 00:30:07,040
MAKE A GFP TAG DNA2 AND START

867
00:30:07,040 --> 00:30:09,440
TRACKING THAT MOLECULE AS WELL.

868
00:30:09,440 --> 00:30:11,000
SO, THE VAST MAJORITY OF THE

869
00:30:11,000 --> 00:30:12,880
CASES, THIS IS WHAT WE SEE.

870
00:30:12,880 --> 00:30:14,240
5 PRIME END IS CUT OFF.

871
00:30:14,240 --> 00:30:16,480
3 PRIME END WE ASSUME NOW IS THE

872
00:30:16,480 --> 00:30:19,520
THING MAKING THE LOOP.

873
00:30:19,520 --> 00:30:21,480
NOW, THIS IS TO TEST THAT EVEN

874
00:30:21,480 --> 00:30:23,400
FURTHER N THIS CASE, WE ARE

875
00:30:23,400 --> 00:30:25,040
LABELING THE 3 PRIME END WITH

876
00:30:25,040 --> 00:30:26,880
OUR SINGLE DYE AND THIS IS WHAT

877
00:30:26,880 --> 00:30:28,680
WE THOUGHT MIGHT TAKE PLACE.

878
00:30:28,680 --> 00:30:30,400
IF THE 3 PRIME END IS TRACKING,

879
00:30:30,400 --> 00:30:34,240
WE SHOULD SEE IT FOLLOWING THE

880
00:30:34,240 --> 00:30:35,600
RESECTION WHICH WE ARE TRACKING

881
00:30:35,600 --> 00:30:37,920
BY VIRTUE OF THIS BLM AND IF THE

882
00:30:37,920 --> 00:30:40,040
LOOP IS RELEASED THEN WE SHOULD

883
00:30:40,040 --> 00:30:41,960
SEE THAT DYE SUDDENLY MOVE

884
00:30:41,960 --> 00:30:42,680
BACKWARDS.

885
00:30:42,680 --> 00:30:46,680
AND SO, I POINT OUT 100% OF OUR

886
00:30:46,680 --> 00:30:48,680
OBSERVATIONS THAT 3-PRIME END

887
00:30:48,680 --> 00:30:50,560
DOES TRACK WITH THE RESECTION

888
00:30:50,560 --> 00:30:50,960
MACHINE.

889
00:30:50,960 --> 00:30:54,480
SO THE MAGENTA DYE HERE, IT

890
00:30:54,480 --> 00:30:55,880
COLOCALIZES WITH THE GFP AND THE

891
00:30:55,880 --> 00:30:57,960
TWO OF THEM MOVE TOGETHER ALONG

892
00:30:57,960 --> 00:31:00,960
THE DNA MOLECULE AS THEY ARE

893
00:31:00,960 --> 00:31:01,160
GOING.

894
00:31:01,160 --> 00:31:02,480
NOW THIS IS DIFFERENT THAN WHAT

895
00:31:02,480 --> 00:31:06,480
I SHOWED YOU BEFORE.

896
00:31:06,480 --> 00:31:08,680
IN THIS EXPERIMENT, 99% OF THEM

897
00:31:08,680 --> 00:31:10,440
SHOWED NO EVIDENCE OF SUDDEN

898
00:31:10,440 --> 00:31:11,240
RELEASE.

899
00:31:11,240 --> 00:31:12,880
THIS IS THE ONE THAT DID.

900
00:31:12,880 --> 00:31:15,520
AND SO WE SEE THAT THE DYE GOES

901
00:31:15,520 --> 00:31:16,920
FROM THIS POSITION ALL OF A

902
00:31:16,920 --> 00:31:18,360
SUDDEN DOWN TO THIS POSITION AND

903
00:31:18,360 --> 00:31:22,240
THAT MOVEMENT COINCIDED WITH

904
00:31:22,240 --> 00:31:24,200
BLM, THE MOTION OF BLM STOPPING.

905
00:31:24,200 --> 00:31:26,240
WE DON'T KNOW IF THAT'S

906
00:31:26,240 --> 00:31:27,440
COINCIDENT OR NOT.

907
00:31:27,440 --> 00:31:28,320
IN THE PREVIOUS EXAMPLES I SAID

908
00:31:28,320 --> 00:31:30,600
SOMETHING LIKE 30% DID THIS.

909
00:31:30,600 --> 00:31:33,680
HERE WE ARE ONLY SEEING 1% OR

910
00:31:33,680 --> 00:31:35,120
LESS THAN 1%, I SHOULD SAY.

911
00:31:35,120 --> 00:31:36,920
AND I'M GOING TO SAY THE

912
00:31:36,920 --> 00:31:37,480
DIFFERENCE BETWEEN THIS

913
00:31:37,480 --> 00:31:39,880
EXPERIMENT AND WHAT I SHOWED YOU

914
00:31:39,880 --> 00:31:41,760
BEFORE IS THAT THESE REACTIONS

915
00:31:41,760 --> 00:31:44,080
ARE INTENTIONALLY DONE WITH

916
00:31:44,080 --> 00:31:45,600
UNLABELED WILDTYPE RPA.

917
00:31:45,600 --> 00:31:47,080
THAT'S WHY WE CAN SEE THE END OF

918
00:31:47,080 --> 00:31:48,600
THE DNA.

919
00:31:48,600 --> 00:31:52,280
IF WE TRY TO LABEL RMA, THE

920
00:31:52,280 --> 00:31:54,800
SIGNAL IS TOO BRIGHT TO SEE THE

921
00:31:54,800 --> 00:31:55,080
DNA ITSELF.

922
00:31:55,080 --> 00:31:56,280
I SUSPECT THE RELEASE OF THE

923
00:31:56,280 --> 00:31:58,200
LOOP WE ARE SEEING IN REACTIONS

924
00:31:58,200 --> 00:32:01,200
WITH FLUORESCENT RPA MIGHT BE

925
00:32:01,200 --> 00:32:05,040
CAUSED BY HAVING A GFP OR AN

926
00:32:05,040 --> 00:32:06,840
MCherry ON THE RPA ITSELF.

927
00:32:06,840 --> 00:32:11,080
SO IT'S SORT OF A FORTUITOUS

928
00:32:11,080 --> 00:32:12,360
DISCOVERY THAT WAS MADE IN TERMS

929
00:32:12,360 --> 00:32:14,800
OF THIS LOOPING JUST BECAUSE

930
00:32:14,800 --> 00:32:16,880
THERE IS SOME PARTIALITY DEFECT

931
00:32:16,880 --> 00:32:19,280
WITH HAVING A GFP TAG ON THE

932
00:32:19,280 --> 00:32:19,480
RPA.

933
00:32:19,480 --> 00:32:21,560
IF WE DON'T HAVE IT, MOST DON'T

934
00:32:21,560 --> 00:32:24,080
GET RELEASED.

935
00:32:24,080 --> 00:32:25,400
SO WE THINK IT IS LOOPING.

936
00:32:25,400 --> 00:32:27,680
WE THINK OUR DITA ARE MOST

937
00:32:27,680 --> 00:32:29,480
CONSISTENT WITH A MODEL, WITH A

938
00:32:29,480 --> 00:32:31,360
5 PRIME END THAT GETS CUT OFF,

939
00:32:31,360 --> 00:32:34,000
THE 3 PRIME END TRACKS THEM WITH

940
00:32:34,000 --> 00:32:35,560
THE RESECTION MACHINERY AS IT

941
00:32:35,560 --> 00:32:36,200
MOVES ALONG.

942
00:32:36,200 --> 00:32:38,560
SO NOW WE WANT TO ASK, WHAT

943
00:32:38,560 --> 00:32:40,040
PROTEIN COMPONENT MIGHT BE

944
00:32:40,040 --> 00:32:41,320
INVOLVED IN THIS?

945
00:32:41,320 --> 00:32:44,640
IS IT BLM OR MIGHT IT BE DNA2?

946
00:32:44,640 --> 00:32:48,240
AND SO WHAT WE DID TO TEST THIS,

947
00:32:48,240 --> 00:32:50,520
OR WHAT CHOW DID WAS PERFORM THE

948
00:32:50,520 --> 00:32:53,880
SAME REACTIONS IN THE ABSENCE OF

949
00:32:53,880 --> 00:32:54,080
DNA2.

950
00:32:54,080 --> 00:32:56,720
AND SO THIS IS AN EXAMPLE

951
00:32:56,720 --> 00:32:57,920
SHOWING NO THREE PRIME END

952
00:32:57,920 --> 00:32:58,240
RELEASE.

953
00:32:58,240 --> 00:33:00,120
THIS IS AN EXAMPLE SHOWING SOME

954
00:33:00,120 --> 00:33:01,400
3 PRIME END RELEASE.

955
00:33:01,400 --> 00:33:03,880
SO WHAT CAN YOU SEE IS THAT WITH

956
00:33:03,880 --> 00:33:08,040
JUST BLM AND RPA, THE RPA

957
00:33:08,040 --> 00:33:10,040
COLOCALIZES WITH THE BLM AS IT

958
00:33:10,040 --> 00:33:12,720
IS MOVING ALONG PROCESSING THE

959
00:33:12,720 --> 00:33:14,440
DNA END AND IN THIS CASE, YOU DO

960
00:33:14,440 --> 00:33:16,560
GET AN EXAMPLE OF SUDDEN

961
00:33:16,560 --> 00:33:17,280
RELEASE.

962
00:33:17,280 --> 00:33:19,920
NOW, AGAIN 100% OF THE SUB

963
00:33:19,920 --> 00:33:20,680
STRAITS THAT WE LOOK AT SHOW

964
00:33:20,680 --> 00:33:24,320
EVIDENCE FOR THIS TYPE OF LOOP

965
00:33:24,320 --> 00:33:25,400
FORMATION.

966
00:33:25,400 --> 00:33:27,840
42.5% IN THIS CASE REMAIN

967
00:33:27,840 --> 00:33:28,080
INTACT.

968
00:33:28,080 --> 00:33:29,720
THE REMAINDER SHOW EVIDENCE OF

969
00:33:29,720 --> 00:33:33,200
THIS SUDDEN RELEASE.

970
00:33:33,200 --> 00:33:35,280
I SHOULD POINT OUT THESE ALSO,

971
00:33:35,280 --> 00:33:38,240
WHEN WE EMIT DNA2, THE REACTIONS

972
00:33:38,240 --> 00:33:41,080
ARE GOING FASTER IN THIS CASE

973
00:33:41,080 --> 00:33:42,560
THAN THEY WERE BEFORE.

974
00:33:42,560 --> 00:33:44,680
AGAIN, WE THINK DNA2 IS ACTUALLY

975
00:33:44,680 --> 00:33:46,680
STARTING TO SLOW THINGS DOWN A

976
00:33:46,680 --> 00:33:49,960
BIT AS THE REACTIONS ARE GOING.

977
00:33:49,960 --> 00:33:52,280
SO WE THINK IT LOOPS.

978
00:33:52,280 --> 00:33:53,080
WE THINK BLOOM IS PRIMARILY

979
00:33:53,080 --> 00:33:54,480
RESPONSIBLE FOR THAT.

980
00:33:54,480 --> 00:33:57,080
SO WE WANTED TO TRY TO

981
00:33:57,080 --> 00:33:59,680
INVESTIGATE WHAT PART OF BLOOM

982
00:33:59,680 --> 00:34:00,080
MIGHT BE INVOLVED.

983
00:34:00,080 --> 00:34:03,080
SO THIS IS SORT OF MY SIMPLIFIED

984
00:34:03,080 --> 00:34:04,880
DIAGRAM OF WHAT BLOOM LOOKS

985
00:34:04,880 --> 00:34:05,080
LIKE.

986
00:34:05,080 --> 00:34:08,560
THAT IS WE HAVE AN ATP ACE

987
00:34:08,560 --> 00:34:09,080
HELICASE CORE.

988
00:34:09,080 --> 00:34:13,680
WE HAVE AN EXTENDED INTERNAL

989
00:34:13,680 --> 00:34:15,320
DOMAIN, A LIGGERRIZATION.

990
00:34:15,320 --> 00:34:16,960
IT'S ALSO INVOLVED IN A NUMBER

991
00:34:16,960 --> 00:34:19,640
OF PROTEIN-PROTEIN INTERACTIONS

992
00:34:19,640 --> 00:34:21,280
INCLUDING INTERACTIONS WITH RPA

993
00:34:21,280 --> 00:34:24,360
AND RAD 51 AND SO ON AND SO

994
00:34:24,360 --> 00:34:25,640
FORTH.

995
00:34:25,640 --> 00:34:29,520
WE ALSO HAVE A RETQC TERMINAL

996
00:34:29,520 --> 00:34:31,640
DOMAIN, A NUCLEAR LOCALIZATION

997
00:34:31,640 --> 00:34:36,440
SIGNAL AND A HELICASE RNA-LIKE

998
00:34:36,440 --> 00:34:38,640
DOMAIN AT THE C TERMINIS.

999
00:34:38,640 --> 00:34:41,800
SO WE INITIALLY FOCUSED ON THE

1000
00:34:41,800 --> 00:34:43,880
END TERMINAL DOMAIN AND HE MADE

1001
00:34:43,880 --> 00:34:47,240
A BUNCH OF TRUNCATIONS WHERE HE

1002
00:34:47,240 --> 00:34:49,480
CUT THE WHOLE THING OFF OR CUT

1003
00:34:49,480 --> 00:34:51,400
OFF MARCHING SEGMENTS GOING IT

1004
00:34:51,400 --> 00:34:52,400
FROM ONE END TO THE OTHER.

1005
00:34:52,400 --> 00:34:54,840
WHAT WE ARE GOING TO ASK IS, HOW

1006
00:34:54,840 --> 00:34:56,000
DO THESE PROTEINS BEHAVE IN

1007
00:34:56,000 --> 00:34:58,600
TERMS OF DNA END RECOGNITION?

1008
00:34:58,600 --> 00:35:02,000
HOW DO THEY BEHAVE IN TERMS OF

1009
00:35:02,000 --> 00:35:02,920
MOVEMENT?

1010
00:35:02,920 --> 00:35:03,480
DISTANCE?

1011
00:35:03,480 --> 00:35:03,760
VELOCITY?

1012
00:35:03,760 --> 00:35:04,680
AND LOOP FORMATION?

1013
00:35:04,680 --> 00:35:06,680
AND I'M JUST GOING TO TELL YOU

1014
00:35:06,680 --> 00:35:08,400
THAT ALL OF THESE GUYS WERE

1015
00:35:08,400 --> 00:35:10,080
VERY, VERY SIMILAR TO

1016
00:35:10,080 --> 00:35:11,880
FULL-LENGTH BLM EXCEPT FOR ONE

1017
00:35:11,880 --> 00:35:12,880
EXCEPTION AND YOU'LL SEE WHAT

1018
00:35:12,880 --> 00:35:15,400
THAT IS IN JUST A MOMENT.

1019
00:35:15,400 --> 00:35:16,800
SO HERE IS OUR DIAGRAM AGAIN

1020
00:35:16,800 --> 00:35:18,680
JUST SO WE ARE ALL ORIENTED.

1021
00:35:18,680 --> 00:35:21,200
AS WE ARE MAKING THESE

1022
00:35:21,200 --> 00:35:23,200
TRUNCATIONS, EVERY SINGLE

1023
00:35:23,200 --> 00:35:25,160
PROTEIN WITH AN END TERMINAL

1024
00:35:25,160 --> 00:35:26,200
TRUNCATION WAS STILL VERY

1025
00:35:26,200 --> 00:35:28,000
EFFICIENTLY TARGETED TO THE DNA

1026
00:35:28,000 --> 00:35:29,480
ENDS AND OUR ASSAYS.

1027
00:35:29,480 --> 00:35:30,880
SO THERE DOESN'T SEEM TO BE ANY

1028
00:35:30,880 --> 00:35:34,880
DEFECT IN DNA BINDING OR DNA

1029
00:35:34,880 --> 00:35:35,600
RECOGNITION.

1030
00:35:35,600 --> 00:35:37,360
WE ALSO LOOKED AT VELOCITY AND

1031
00:35:37,360 --> 00:35:40,040
I'M GOING TO POINT OUT, THESE

1032
00:35:40,040 --> 00:35:41,080
ARE VELOCILITY MEASURED IN

1033
00:35:41,080 --> 00:35:44,840
REACTIONS WITH ONLY BMLM AND

1034
00:35:44,840 --> 00:35:46,240
BOUND WHEREVER IT HAPPENED.

1035
00:35:46,240 --> 00:35:47,920
I THINK THE WAY THE REACTION WAS

1036
00:35:47,920 --> 00:35:49,160
DONE IN THIS CASE, WE DIDN'T

1037
00:35:49,160 --> 00:35:50,440
HAVE FREE DNA.

1038
00:35:50,440 --> 00:35:52,360
WE HAD BOTH ENDS TETHERED.

1039
00:35:52,360 --> 00:35:54,320
BUT THAT IS NEITHER HERE NOR

1040
00:35:54,320 --> 00:35:54,640
THERE.

1041
00:35:54,640 --> 00:35:55,840
THE MOST IMPORTANT PART IS THEY

1042
00:35:55,840 --> 00:35:58,880
ARE ALL GOING ROUGHLY THE SAME

1043
00:35:58,880 --> 00:36:00,320
SPEED EXEMPT THIS END TERMINAL

1044
00:36:00,320 --> 00:36:01,640
DELETION WHERE WE CUT THE ENTIRE

1045
00:36:01,640 --> 00:36:02,040
THING OFF.

1046
00:36:02,040 --> 00:36:04,720
AND I HAVE THIS AS 614.

1047
00:36:04,720 --> 00:36:05,720
IT SHOULD BE 641.

1048
00:36:05,720 --> 00:36:08,240
BUT IT'S THE ENTIRE END TERMINAL

1049
00:36:08,240 --> 00:36:08,480
DELETION.

1050
00:36:08,480 --> 00:36:10,920
IT'S GOING FASTER THAN THE

1051
00:36:10,920 --> 00:36:11,840
WILDTYPE FULL-LENGTH BLOOM.

1052
00:36:11,840 --> 00:36:14,240
AND IT'S GOING AHEC OF A LOT

1053
00:36:14,240 --> 00:36:15,960
FURTHER THAN THE FULL-LENGTH

1054
00:36:15,960 --> 00:36:16,920
BLOOM AS WELL.

1055
00:36:16,920 --> 00:36:18,320
WHEREAS THE OTHERS ALL SEEM TO

1056
00:36:18,320 --> 00:36:20,640
GO ROUGHLY THE SAME DISTANCE.

1057
00:36:20,640 --> 00:36:22,440
SO SOMETHING ABOUT CHOPPING THAT

1058
00:36:22,440 --> 00:36:24,400
WHOLE END TERMINAL DOMAIN OFF IS

1059
00:36:24,400 --> 00:36:25,880
MAKING US GO A LITTLE FASTER AND

1060
00:36:25,880 --> 00:36:27,480
A LITTLE FURTHER AS WE ARE

1061
00:36:27,480 --> 00:36:30,720
MARCHING ALONG THE DNA MOLECULE.

1062
00:36:30,720 --> 00:36:34,120
SO NOW, THIS IS SOMETHING ELSE

1063
00:36:34,120 --> 00:36:36,120
THAT,NOTICED RIGHT AWAY.

1064
00:36:36,120 --> 00:36:38,280
AND THAT IS IF YOU CAN PICTURE

1065
00:36:38,280 --> 00:36:40,880
IN YOUR MIND WHAT THOSE GFP FOCI

1066
00:36:40,880 --> 00:36:42,880
I SHOWED IN THE EARLY SLIDES

1067
00:36:42,880 --> 00:36:45,080
LOOKED LIKE, WE HAVE NO PROBLEM

1068
00:36:45,080 --> 00:36:46,080
DETECTING THAT SIGNAL.

1069
00:36:46,080 --> 00:36:47,760
IT'S BRIGHT.

1070
00:36:47,760 --> 00:36:48,440
IT'S MUCH BRIGHTER THAN ONE

1071
00:36:48,440 --> 00:36:50,080
WOULD EXPECT IF WE WERE SIMPLY

1072
00:36:50,080 --> 00:36:52,360
LOOKING AT A SINGLE GFP JUST

1073
00:36:52,360 --> 00:36:54,800
BASED ON OUR OWN EXPERIENCES.

1074
00:36:54,800 --> 00:36:56,960
BUT NOW WHEN HE IS LOOKING AT

1075
00:36:56,960 --> 00:36:58,600
THIS END TERMINAL DELETION

1076
00:36:58,600 --> 00:37:01,320
CONSTRUCT, ALL OF A SUDDEN IT'S

1077
00:37:01,320 --> 00:37:02,160
MUCH DIMMER THAN ALL OF THE

1078
00:37:02,160 --> 00:37:05,280
OTHER GUYS THAT WE LOOKED AT,

1079
00:37:05,280 --> 00:37:06,280
INCLUDING FULL LENGTH BLOOM AND

1080
00:37:06,280 --> 00:37:07,080
ANY OF THE OTHER TRUNCATIONS.

1081
00:37:07,080 --> 00:37:10,080
SO THERE IS A SORT OF LARGE

1082
00:37:10,080 --> 00:37:12,600
DECREASE, ABOUT 80% DECREASE, IN

1083
00:37:12,600 --> 00:37:15,480
TERMS OF INTEGRATED SIGNAL

1084
00:37:15,480 --> 00:37:15,920
INTENSITY.

1085
00:37:15,920 --> 00:37:17,160
WHEN WE HAVE THAT MUTANT.

1086
00:37:17,160 --> 00:37:18,680
SO EVEN THOUGH IT'S GOING FASTER

1087
00:37:18,680 --> 00:37:20,160
AND FURTHER, IT LOOKS LIKE THERE

1088
00:37:20,160 --> 00:37:22,960
IS LESS AT THE END.

1089
00:37:22,960 --> 00:37:25,080
AND HE DID A LITTLE BIT OF A

1090
00:37:25,080 --> 00:37:27,480
FANCIER MEASUREMENT WHERE HE

1091
00:37:27,480 --> 00:37:29,880
BOMBARDED THESE END-BOUND

1092
00:37:29,880 --> 00:37:32,160
PROTEINS WITH LASER LIGHT AND

1093
00:37:32,160 --> 00:37:33,680
ASKED, OKAY, HOW MANY

1094
00:37:33,680 --> 00:37:35,560
PHOTOBLEACHING STEPS DO WE SEE?

1095
00:37:35,560 --> 00:37:37,360
THIS IS AN EXAMPLE WITH

1096
00:37:37,360 --> 00:37:40,680
FULL-LENGTH BLM, GFP-TAGGED BLM.

1097
00:37:40,680 --> 00:37:45,240
WE ARE SEEING FOUR STEPS.

1098
00:37:45,240 --> 00:37:46,800
AND THAT INDICATES TO US THAT

1099
00:37:46,800 --> 00:37:47,840
THERE SHOULD BE FOUR MOLECULES

1100
00:37:47,840 --> 00:37:49,280
OF GFP BOUND.

1101
00:37:49,280 --> 00:37:50,840
AND IF WE LOOK AT ONE EXAMPLE,

1102
00:37:50,840 --> 00:37:53,840
IF WE LOOK AT A POPULATION

1103
00:37:53,840 --> 00:37:54,480
DISTRIBUTION, THAT IS MORE OR

1104
00:37:54,480 --> 00:37:56,560
LESS WHAT WE ARE SEEING ON THE

1105
00:37:56,560 --> 00:37:59,920
ORDER OF 3.6 PLUS OR MINUS .5.

1106
00:37:59,920 --> 00:38:01,920
WE ALSO SEE WHAT WE THINK ARE

1107
00:38:01,920 --> 00:38:03,120
PROBABLY AGGREGATES WHERE WE

1108
00:38:03,120 --> 00:38:04,880
CAN'T ACTUALLY MEASURE THE

1109
00:38:04,880 --> 00:38:05,080
STEPS.

1110
00:38:05,080 --> 00:38:06,280
IT'S JUST A LOT OF PROTEIN.

1111
00:38:06,280 --> 00:38:08,440
WE ARE JUST CATEGORIZING THESE

1112
00:38:08,440 --> 00:38:10,040
THAT ARE GREATER THAN 6.

1113
00:38:10,040 --> 00:38:11,520
NOW WHEN WE LOOK AT OUR END

1114
00:38:11,520 --> 00:38:13,520
TERMINAL DELETION, AND THAT'S A

1115
00:38:13,520 --> 00:38:13,880
TYPO.

1116
00:38:13,880 --> 00:38:15,720
IT SHOULD BE 641.

1117
00:38:15,720 --> 00:38:17,120
PRIMARILY, WHAT WE ARE SEEING

1118
00:38:17,120 --> 00:38:21,240
ARE SINGLE-STEP PHOTOBLEACHING

1119
00:38:21,240 --> 00:38:21,480
EVENTS.

1120
00:38:21,480 --> 00:38:23,320
THE PROTEIN IS THERE AND THEN IT

1121
00:38:23,320 --> 00:38:25,440
ALL OF A SUDDEN DISAPPEARS.

1122
00:38:25,440 --> 00:38:26,480
THAT IS MOST CONSISTENT WITH THE

1123
00:38:26,480 --> 00:38:28,640
IDEA THAT WE HAVE A SINGLE GFP

1124
00:38:28,640 --> 00:38:30,640
BOUND THERE, MEANING A SINGLE

1125
00:38:30,640 --> 00:38:35,720
COMPLEX OF GFP-BLM PROTEIN AND

1126
00:38:35,720 --> 00:38:37,440
THEN THERE ARE EXAMPLES WHERE WE

1127
00:38:37,440 --> 00:38:40,840
HAVE A FEW MORE BUT NOT VERY

1128
00:38:40,840 --> 00:38:41,040
MANY.

1129
00:38:41,040 --> 00:38:42,560
SO WE THINK SOMEHOW CHOPPING OFF

1130
00:38:42,560 --> 00:38:44,400
THE END TERMINAL DOMAIN MADE US

1131
00:38:44,400 --> 00:38:50,120
GO FASTER AND FURTHER AND

1132
00:38:50,120 --> 00:38:52,880
REDISED OUR LIGMERRIC STATE.

1133
00:38:52,880 --> 00:38:54,480
AND HERE IS ONE OF THE THINGS WE

1134
00:38:54,480 --> 00:38:56,080
FOUND WAS INTERESTING.

1135
00:38:56,080 --> 00:38:57,880
AND THIS IS SHOWING BLM WHERE

1136
00:38:57,880 --> 00:38:59,720
THE DNA GETS LOOPED AND LIE

1137
00:38:59,720 --> 00:39:01,560
LOOPED, I MEAN THE 3 PRIME END

1138
00:39:01,560 --> 00:39:04,640
IN THIS CASE IS TRACKING WITH

1139
00:39:04,640 --> 00:39:06,320
THE RESECTION MACHINERY, WHICH

1140
00:39:06,320 --> 00:39:09,520
WE DEMARKED WITH RPA-GFP IN THIS

1141
00:39:09,520 --> 00:39:09,720
CASE.

1142
00:39:09,720 --> 00:39:11,240
YOU'LL SEE WHY IN JUST A MINUTE.

1143
00:39:11,240 --> 00:39:13,080
THIS IS WHEN WE CUT OFF THE END

1144
00:39:13,080 --> 00:39:14,360
TERMINAL DOMAIN.

1145
00:39:14,360 --> 00:39:15,960
INSTEAD OF SEEING THIS

1146
00:39:15,960 --> 00:39:17,120
COLOCALIZATION THROUGH TIME,

1147
00:39:17,120 --> 00:39:20,840
WHAT WE SEE IS THAT 3 PRIME END

1148
00:39:20,840 --> 00:39:21,920
STAYS PUT OVER TIME.

1149
00:39:21,920 --> 00:39:24,680
IT STARTS TO GET A LITTLE BIT

1150
00:39:24,680 --> 00:39:27,120
LONGER OUT HERE AND YOU START TO

1151
00:39:27,120 --> 00:39:29,120
GENERATE THIS LONG TRACK OF RPA.

1152
00:39:29,120 --> 00:39:31,040
SO WHAT WE THINK IS HAPPENING IS

1153
00:39:31,040 --> 00:39:33,320
THAT AT THE LEADING EDGE OF THIS

1154
00:39:33,320 --> 00:39:37,320
RPA, THAT IS WHERE OUR GFP END

1155
00:39:37,320 --> 00:39:38,560
TERMINAL DELETION WILL BE

1156
00:39:38,560 --> 00:39:38,800
SITTING.

1157
00:39:38,800 --> 00:39:39,440
B WILL.

1158
00:39:39,440 --> 00:39:41,760
M WILL BE SITTING, MOVING,

1159
00:39:41,760 --> 00:39:43,560
TRACKING DNA2 WITH IT, CHOPPING

1160
00:39:43,560 --> 00:39:45,480
UP ONE OF THE DNA STRANDS.

1161
00:39:45,480 --> 00:39:47,280
BUT THE 3 PRIME END IS

1162
00:39:47,280 --> 00:39:47,560
UNAFFECTED.

1163
00:39:47,560 --> 00:39:49,320
IT'S NOT GENERATING A LOOP SO WE

1164
00:39:49,320 --> 00:39:52,520
GET THIS LONG EXTENDED STRUCTURE

1165
00:39:52,520 --> 00:39:56,160
OF SINGLE STRANDED DNA LIT UP BY

1166
00:39:56,160 --> 00:39:56,440
GFP-RPA.

1167
00:39:56,440 --> 00:39:59,880
I'LL POINT OUT THAT MOST OF OUR

1168
00:39:59,880 --> 00:40:01,720
TRUNCATIONS LOOK COMPARABLE TO

1169
00:40:01,720 --> 00:40:04,120
FULL-LENGTH BLM IN TERMS OF THE

1170
00:40:04,120 --> 00:40:05,720
PERCENT THAT UNDERGO LOOPING AND

1171
00:40:05,720 --> 00:40:08,040
IT'S THIS SINGLE, THIS ONE

1172
00:40:08,040 --> 00:40:10,080
MUTANT WHERE WE CHOPPED OFF THE

1173
00:40:10,080 --> 00:40:12,280
ENTIRE END TERMINAL DOMAIN THAT

1174
00:40:12,280 --> 00:40:15,040
SEEMS TO GO FROM A LOOPING

1175
00:40:15,040 --> 00:40:15,720
PROFICIENT ENZYME NOW TO

1176
00:40:15,720 --> 00:40:18,120
SOMETHING THAT IS NO LONGER

1177
00:40:18,120 --> 00:40:21,600
GENERATING DNA LOOPS.

1178
00:40:21,600 --> 00:40:22,920
SO, WE FOUND THAT EXCITING.

1179
00:40:22,920 --> 00:40:26,000
NO YOU WE WANTED TO ASK WHAT

1180
00:40:26,000 --> 00:40:27,640
ABOUT THE END TERMINAL OR THE C

1181
00:40:27,640 --> 00:40:28,920
TERMINAL DOMAIN?

1182
00:40:28,920 --> 00:40:30,920
AND SO HE WENT ABOUT GENERATING

1183
00:40:30,920 --> 00:40:33,520
A NUMBER OF DIFFERENT CONSTRUCTS

1184
00:40:33,520 --> 00:40:35,480
HERE WHERE HE EITHER CHOPPED OFF

1185
00:40:35,480 --> 00:40:39,080
THE ENTIRE C TERMINAL DOMAIN UP

1186
00:40:39,080 --> 00:40:45,560
TO THE ROCQ LIKE INCLUDING THE

1187
00:40:45,560 --> 00:40:47,480
HRDC AND THE DARK BLUE AND

1188
00:40:47,480 --> 00:40:48,040
YELLOW REGIONS.

1189
00:40:48,040 --> 00:40:50,160
WE KNOW THEY ARE INVOLVED IN

1190
00:40:50,160 --> 00:40:51,080
PROTEIN-PROTEIN INTERACTIONS.

1191
00:40:51,080 --> 00:40:54,400
HE ALSO CUT OFF THE C TERMINAL

1192
00:40:54,400 --> 00:40:57,080
DEMAIN BUT INCLUDED HRDC.

1193
00:40:57,080 --> 00:40:58,760
HE MADE ANOTHER CONSTRUCT WHERE

1194
00:40:58,760 --> 00:41:00,280
HE DELIGHTED THE DARK BLUE AND

1195
00:41:00,280 --> 00:41:04,080
THE YELLOW BUT LEFT THE NUCLEAR

1196
00:41:04,080 --> 00:41:06,080
LOCALIZATION SIGNAL INTACT.

1197
00:41:06,080 --> 00:41:07,760
HE MADE ANOTHER GUY THAT SORT OF

1198
00:41:07,760 --> 00:41:09,560
THE TWO DARK AND A LIGHT BLUE

1199
00:41:09,560 --> 00:41:13,480
AND THEN ANOTHER LITTLE GUY THAT

1200
00:41:13,480 --> 00:41:16,760
IS 40 AMINO ACIDS OF THIS BLUE

1201
00:41:16,760 --> 00:41:17,000
REGION.

1202
00:41:17,000 --> 00:41:19,360
HE IS ASKING, HOW DO THESE GUYS

1203
00:41:19,360 --> 00:41:20,160
BEHAVE?

1204
00:41:20,160 --> 00:41:21,880
AND SO, THE FIRST THING THAT

1205
00:41:21,880 --> 00:41:23,320
STRUCK US WHEN HE STARTED

1206
00:41:23,320 --> 00:41:25,400
LOOKING AT THESE CONSTRUCTS IS

1207
00:41:25,400 --> 00:41:27,600
THAT THEY USE THE CAPACITY TO

1208
00:41:27,600 --> 00:41:29,080
RECOGNIZE DNA ENDS.

1209
00:41:29,080 --> 00:41:32,280
SO THIS IS GFP-BLM.

1210
00:41:32,280 --> 00:41:35,760
NICELY LINED UP IN OUR DNA ENDS.

1211
00:41:35,760 --> 00:41:37,320
THING THIS IS THE SAME SLIDE I

1212
00:41:37,320 --> 00:41:38,080
SHOWED AT THE BEGINNING.

1213
00:41:38,080 --> 00:41:41,320
THIS IS OUR C TERMINAL DELETION

1214
00:41:41,320 --> 00:41:41,600
CONSTRUCT.

1215
00:41:41,600 --> 00:41:43,520
WHAT IS HAPPENING IS IT IS GOING

1216
00:41:43,520 --> 00:41:43,840
EVERYWHERE.

1217
00:41:43,840 --> 00:41:45,640
SO IT'S NOT SIMPLY GOING TO

1218
00:41:45,640 --> 00:41:45,840
ENDS.

1219
00:41:45,840 --> 00:41:47,600
IT'S GOING ALL OVER THE PLACE.

1220
00:41:47,600 --> 00:41:49,520
AND THIS IS TRUE FOR EVERY

1221
00:41:49,520 --> 00:41:52,240
SINGLE C TERMINAL DOMAIN

1222
00:41:52,240 --> 00:41:53,400
TRUNCATION CONSTRUCT HE LOOKED

1223
00:41:53,400 --> 00:42:00,480
AT, EVEN THE SMALLEST GUY, DELTA

1224
00:42:00,480 --> 00:42:01,840
1291 TO 1330T GOES ALL OVER THE

1225
00:42:01,840 --> 00:42:02,080
DNA.

1226
00:42:02,080 --> 00:42:03,960
WE ARE NOT LOSING THE CAPACITY

1227
00:42:03,960 --> 00:42:08,160
TO BIND, WE ARE LOSING TARGET

1228
00:42:08,160 --> 00:42:08,640
SPECIFICILITY.

1229
00:42:08,640 --> 00:42:10,680
NOW HE ALSO TESTED THESE GUYS IN

1230
00:42:10,680 --> 00:42:13,680
TERMS OF BULK, ATPI DRAW

1231
00:42:13,680 --> 00:42:13,960
ACTIVITY.

1232
00:42:13,960 --> 00:42:15,880
I'M GOING TO POINT OUT WHERE WE

1233
00:42:15,880 --> 00:42:18,400
CHOP OFF THE WHOLE C TERMINAL

1234
00:42:18,400 --> 00:42:18,640
DOMAIN.

1235
00:42:18,640 --> 00:42:21,280
YOU END UP WITH A BETTER ATP

1236
00:42:21,280 --> 00:42:21,480
ACE.

1237
00:42:21,480 --> 00:42:22,800
THESE ARE ALL THE OTHER

1238
00:42:22,800 --> 00:42:25,280
CONSTRUCTS COMPARED TO

1239
00:42:25,280 --> 00:42:25,720
FULL-LENGTH BLOOM.

1240
00:42:25,720 --> 00:42:29,200
THIS IS ATP HIDEALIZED PER TIME.

1241
00:42:29,200 --> 00:42:31,800
AND HE MEASURED THE VELOCITIES

1242
00:42:31,800 --> 00:42:33,280
ON DNA.

1243
00:42:33,280 --> 00:42:34,880
THE FUN THING HERE IS THIS

1244
00:42:34,880 --> 00:42:36,000
PROTEIN, EVEN THOUGH IT'S GOING

1245
00:42:36,000 --> 00:42:37,880
MUCH, MUCH FASTER IN TERMS OF

1246
00:42:37,880 --> 00:42:39,400
TURNING OVER ATP, IT'S GOING

1247
00:42:39,400 --> 00:42:41,080
MUCH, MUCH SLOWER IN TERMS OF

1248
00:42:41,080 --> 00:42:42,880
ITS MOVEMENT ON THE DNA.

1249
00:42:42,880 --> 00:42:44,880
SO WHAT WE ARE DOING HERE WITH

1250
00:42:44,880 --> 00:42:47,800
THIS MUTATION OR THIS TRUNCATION

1251
00:42:47,800 --> 00:42:50,120
IS DECOUPLING ATP HYDROLYSIS

1252
00:42:50,120 --> 00:42:52,440
FROM THE PROTEIN'S MOTOR

1253
00:42:52,440 --> 00:42:52,680
ACTIVITY.

1254
00:42:52,680 --> 00:42:54,240
AND THEN IN THIS CASE, IT'S

1255
00:42:54,240 --> 00:42:55,960
ABOUT 64% SLOWER.

1256
00:42:55,960 --> 00:42:59,920
I'M GOING POINT OUT THIS PROTEIN

1257
00:42:59,920 --> 00:43:02,880
ACTUALLY WAS CHARACTERIZED IN

1258
00:43:02,880 --> 00:43:04,440
BOTH CHEMICAL ASSAYS A NUMBER OF

1259
00:43:04,440 --> 00:43:06,440
YEARS AGO BY TWO DIFFERENT LABS.

1260
00:43:06,440 --> 00:43:08,920
AND BOTH OF THEM SHOWED THAT IT

1261
00:43:08,920 --> 00:43:11,040
HAD DECREASED HELICASE ACTIVITY

1262
00:43:11,040 --> 00:43:13,040
BUT INCREASE ATP ACE ACTIVITY.

1263
00:43:13,040 --> 00:43:14,640
WE THINK OUR RESULT IS FULLY

1264
00:43:14,640 --> 00:43:15,320
CONSISTENT WITH THE LITERATURE

1265
00:43:15,320 --> 00:43:16,640
AND WHAT WE ARE ADDING TO THIS

1266
00:43:16,640 --> 00:43:18,640
IS SHOWING THAT IT IS ACTUALLY

1267
00:43:18,640 --> 00:43:19,920
GOING SLOWER.

1268
00:43:19,920 --> 00:43:22,920
SO YOU'RE DECOUPLING MOTOR FROM

1269
00:43:22,920 --> 00:43:27,720
ATP HYDROLYSIS.

1270
00:43:27,720 --> 00:43:31,680
NOW, SORRY, NEEDED ANGDRINK.

1271
00:43:31,680 --> 00:43:33,240
EVERYTHING I SHOWED YOU UPON UP

1272
00:43:33,240 --> 00:43:35,840
TO THIS POINT HAS BEEN WORK BY

1273
00:43:35,840 --> 00:43:36,440
CHOW YOU.

1274
00:43:36,440 --> 00:43:38,400
I'M GOING TO START TALKING ABOUT

1275
00:43:38,400 --> 00:43:40,400
EXPERIMENTS DONE IN PATRICK'S

1276
00:43:40,400 --> 00:43:40,560
LAB.

1277
00:43:40,560 --> 00:43:42,760
FIRST OF WHICH IS DONE BY HIS

1278
00:43:42,760 --> 00:43:44,400
LONG TIME ASSOCIATE WHO HAS BEEN

1279
00:43:44,400 --> 00:43:48,280
AMAZING TO WORK WITH OVER THE

1280
00:43:48,280 --> 00:43:48,560
YEARS.

1281
00:43:48,560 --> 00:43:50,960
HE DID SIMPLE BULK BIOCHEMICAL

1282
00:43:50,960 --> 00:43:53,000
ASSAYS WITH TWO MUTANTS.

1283
00:43:53,000 --> 00:43:55,360
WE SETTLED ON THESE TWO GUYS AS

1284
00:43:55,360 --> 00:43:57,600
THE MOST INTERESTING.

1285
00:43:57,600 --> 00:43:59,080
OUR END TERMINAL DELETION

1286
00:43:59,080 --> 00:44:00,560
CONSTRUCT WHICH NO LONGER

1287
00:44:00,560 --> 00:44:02,560
GENERATES DNA LOOPS AND OUR C

1288
00:44:02,560 --> 00:44:04,360
TERMINAL PROTEIN WHERE WE

1289
00:44:04,360 --> 00:44:05,960
DELETED THIS SHORT TRACK OF

1290
00:44:05,960 --> 00:44:09,160
AMINO ACIDS, 40 ROUGHLY.

1291
00:44:09,160 --> 00:44:12,080
AND IT NO LONGER RECOGNIZES DNA

1292
00:44:12,080 --> 00:44:12,800
ENDS VERY WELL.

1293
00:44:12,800 --> 00:44:14,880
THIS IS A SIMPLE MEASUREMENT OF

1294
00:44:14,880 --> 00:44:17,680
DNA UNWINDING TAKING A 2 1/2 KB

1295
00:44:17,680 --> 00:44:20,080
FRAGMENT AND ASKING HOW WELL DO

1296
00:44:20,080 --> 00:44:20,680
YOU UNWIND?

1297
00:44:20,680 --> 00:44:23,160
WE SEE END TERMINAL GUY UNWINDS

1298
00:44:23,160 --> 00:44:25,480
IT BETTER CONSISTENT WITH THE

1299
00:44:25,480 --> 00:44:26,560
MORE RAPID VELOCITY.

1300
00:44:26,560 --> 00:44:28,440
AND THIS GUY ALSO UNWINDS.

1301
00:44:28,440 --> 00:44:30,360
IT'S COMPARABLE TO WILDTYPE.

1302
00:44:30,360 --> 00:44:31,960
IT'S NOT SO BAD.

1303
00:44:31,960 --> 00:44:33,280
AND WE THINK EVEN THOUGH IT'S

1304
00:44:33,280 --> 00:44:35,880
NOT RECOGNIZING ENDS, WHEN YOU

1305
00:44:35,880 --> 00:44:37,560
HAVE EYE SHORT SUBSTRATE IN A

1306
00:44:37,560 --> 00:44:38,960
TEST TUBE, THAT DOESN'T REALLY

1307
00:44:38,960 --> 00:44:41,280
MATTER IT'S ENOUGH TO GET THE

1308
00:44:41,280 --> 00:44:42,120
JOB DONE.

1309
00:44:42,120 --> 00:44:43,800
AND SO IT'S STILL CAN UNWINE

1310
00:44:43,800 --> 00:44:46,320
THIS SUBSTRATE.

1311
00:44:46,320 --> 00:44:47,840
PUTTING ENOUGH ENZYME ON THE DNA

1312
00:44:47,840 --> 00:44:49,840
IT'S NOT A PROBLEM.

1313
00:44:49,840 --> 00:44:51,800
WE THEN ASK, WHAT ABOUT

1314
00:44:51,800 --> 00:44:52,200
PROCESSING?

1315
00:44:52,200 --> 00:44:54,520
WHEN WE ADD DNA2 TO THESE SAME

1316
00:44:54,520 --> 00:44:55,200
REACTIONS.

1317
00:44:55,200 --> 00:44:56,880
WHAT WE SEE HERE IS INTERESTING

1318
00:44:56,880 --> 00:44:58,680
IN THAT OUR END TERMINAL

1319
00:44:58,680 --> 00:45:00,880
DELETION, A BETTER HELICASE,

1320
00:45:00,880 --> 00:45:02,840
ACTUALLY SHOWS PROCESSING LEVELS

1321
00:45:02,840 --> 00:45:04,480
THAT ARE COMPARABLE TO WILDTYPE

1322
00:45:04,480 --> 00:45:05,800
AND WE THINK WHAT THIS IS

1323
00:45:05,800 --> 00:45:08,720
TELLING US IS SOMETHING ABOUT

1324
00:45:08,720 --> 00:45:09,280
DNA2.

1325
00:45:09,280 --> 00:45:11,840
IT'S LIKELY THAT DNA2,

1326
00:45:11,840 --> 00:45:12,680
DEGRADATION, THE NUCLEASE,

1327
00:45:12,680 --> 00:45:12,920
REMEMBER.

1328
00:45:12,920 --> 00:45:15,880
IT'S LIKELY RATE LIMITING

1329
00:45:15,880 --> 00:45:16,160
REACTIONS.

1330
00:45:16,160 --> 00:45:18,040
BUT NOW OUR END TERMINAL --

1331
00:45:18,040 --> 00:45:20,280
SORRY, OUR C TERMINAL DELETION

1332
00:45:20,280 --> 00:45:21,280
CONSTRUCT IS NOT DOING SUCH A

1333
00:45:21,280 --> 00:45:21,680
GOOD JOB.

1334
00:45:21,680 --> 00:45:22,840
AGAIN WE THINK THAT IS BECAUSE

1335
00:45:22,840 --> 00:45:24,080
IT'S NOT GETTING TO THE ENDS

1336
00:45:24,080 --> 00:45:25,280
VERY WELL.

1337
00:45:25,280 --> 00:45:27,800
AND THESE PROPERTIES IN GENERAL

1338
00:45:27,800 --> 00:45:29,080
ARE SCALING WITH WHAT WE ARE

1339
00:45:29,080 --> 00:45:31,680
SEEING IN OUR SINGLE MOLECULE

1340
00:45:31,680 --> 00:45:31,960
REACTIONS.

1341
00:45:31,960 --> 00:45:34,680
AND SO WHAT WE NOW WANTED TO DO

1342
00:45:34,680 --> 00:45:36,360
IS FUN EXPERIMENTS AND ASK, WHAT

1343
00:45:36,360 --> 00:45:38,160
HAPPENS WHEN WE PUT THESE

1344
00:45:38,160 --> 00:45:39,000
MUTANTS INTO CELLS?

1345
00:45:39,000 --> 00:45:40,760
AGAIN I'M TALKING ABOUT THE END

1346
00:45:40,760 --> 00:45:43,160
TERMINAL AND C TERMINAL

1347
00:45:43,160 --> 00:45:43,480
TRUNCATION.

1348
00:45:43,480 --> 00:45:46,960
AND SO, THIS IS ALL WORK DONE BY

1349
00:45:46,960 --> 00:45:50,360
SA MERE WHERE HE IS TAKING THESE

1350
00:45:50,360 --> 00:45:53,480
U20S CELLS AND KNOCKING DOWN

1351
00:45:53,480 --> 00:45:54,680
EITHER BLOOM OR XO1 OR IN EVERY

1352
00:45:54,680 --> 00:45:57,120
EXPERIMENT WE ARE KNOCKING DOWN

1353
00:45:57,120 --> 00:45:59,000
BOTH THAT I WILL SHOW YOU.

1354
00:45:59,000 --> 00:46:02,920
THE REASON WE ARE KNOCKING XO1

1355
00:46:02,920 --> 00:46:03,880
DOWN IT OFFERS ALTERNATIVE

1356
00:46:03,880 --> 00:46:06,080
PATHWAY TO END PROCESSING IN THE

1357
00:46:06,080 --> 00:46:06,680
EVENT THAT BLOOM IS NOT THERE.

1358
00:46:06,680 --> 00:46:08,520
SO WE WANT TO GET RID OF BOTH OF

1359
00:46:08,520 --> 00:46:10,240
THESE WHEN LOOKING AT OUR

1360
00:46:10,240 --> 00:46:10,640
MUTANTS.

1361
00:46:10,640 --> 00:46:12,120
SO WE ARE KNOCKING THEM BOTH

1362
00:46:12,120 --> 00:46:14,720
DOWN AND THEN PUTTING IN

1363
00:46:14,720 --> 00:46:16,320
FULL-LENGTH BLOOM, A VECTOR

1364
00:46:16,320 --> 00:46:18,040
CONTROL OR OUR, IN THIS CASE, C

1365
00:46:18,040 --> 00:46:22,120
AND END TERMINAL CONSTRUCTS.

1366
00:46:22,120 --> 00:46:23,680
NOW WE ARE THEN ASKING IF WE

1367
00:46:23,680 --> 00:46:26,040
TAKE THESE CELLS AND SIMPLY

1368
00:46:26,040 --> 00:46:28,920
TREAT THEM WITH AGENTS TO CAUSE

1369
00:46:28,920 --> 00:46:33,240
PROBLEMS, DNA DAMAGE, EITHER A

1370
00:46:33,240 --> 00:46:34,440
HOW DO THE CELLS DO?

1371
00:46:34,440 --> 00:46:36,720
SO THIS IS OUR VECTOR-ONLY

1372
00:46:36,720 --> 00:46:39,080
CONTROL AT THE BEGINNING.

1373
00:46:39,080 --> 00:46:39,760
CELLS DON'T SURVIVE VERY WELL.

1374
00:46:39,760 --> 00:46:43,320
IF WE PUT IN FULL-LEAPT BLOOM,

1375
00:46:43,320 --> 00:46:47,120
WE GET AN UPTICK IN SURVIVAL IN

1376
00:46:47,120 --> 00:46:51,000
THE CASE OF BOTH.

1377
00:46:51,000 --> 00:46:53,400
WHEREAS END TERMINAL AND C

1378
00:46:53,400 --> 00:46:55,000
TERMINAL DELETION CONSTRUCTS

1379
00:46:55,000 --> 00:46:56,280
SHOW POOR SURVIVAL.

1380
00:46:56,280 --> 00:46:58,040
SO WE NOW WANT TO ASK WHY IS

1381
00:46:58,040 --> 00:46:58,280
THAT?

1382
00:46:58,280 --> 00:46:59,760
WHY ARE THESE GUYS NOT SURVIVING

1383
00:46:59,760 --> 00:47:00,280
VERY WELL?

1384
00:47:00,280 --> 00:47:01,680
AND AGAIN, I REMIND YOU THIS IS

1385
00:47:01,680 --> 00:47:03,240
THE ONE THAT DOESN'T DO LOOPING.

1386
00:47:03,240 --> 00:47:04,880
THIS IS THE ONE THAT IS NOT

1387
00:47:04,880 --> 00:47:05,800
TARGETED TO ENDS.

1388
00:47:05,800 --> 00:47:07,680
IT'S PROBABLY PRETTY EASY TO

1389
00:47:07,680 --> 00:47:08,800
IMAGINE WHY THIS ONE IS NOT

1390
00:47:08,800 --> 00:47:11,560
DOING SO WELL.

1391
00:47:11,560 --> 00:47:14,760
SO NOW WHAT SA MERE DID WAS TO

1392
00:47:14,760 --> 00:47:16,360
ASK, WHAT HAPPENS WITH THESE

1393
00:47:16,360 --> 00:47:19,360
GUYS WHEN WE TREAT THESE CELLS

1394
00:47:19,360 --> 00:47:19,800
IONIZING RADIATION?

1395
00:47:19,800 --> 00:47:22,240
CAN WE SEE FOCI FORMING?

1396
00:47:22,240 --> 00:47:23,160
PROTEIN FOCI?

1397
00:47:23,160 --> 00:47:28,360
BOTH FOR BLM, RPA AND ALSO FOR

1398
00:47:28,360 --> 00:47:28,560
Rad51.

1399
00:47:28,560 --> 00:47:31,480
SO WE ARE USING Rad51 AS AN

1400
00:47:31,480 --> 00:47:32,480
INDICATORS HOMOLOGOUS

1401
00:47:32,480 --> 00:47:33,360
RECOMBINATION CAN TAKE PLACE

1402
00:47:33,360 --> 00:47:35,280
BECAUSE WE ARE MAKING A

1403
00:47:35,280 --> 00:47:36,960
PROCESSED END WHERE WE CAN

1404
00:47:36,960 --> 00:47:38,960
ASSEMBLE OUR RECOMBINASE

1405
00:47:38,960 --> 00:47:39,240
FILAMENT.

1406
00:47:39,240 --> 00:47:40,240
AND WHAT I'M GOING TO SHOW YOU

1407
00:47:40,240 --> 00:47:42,800
AS WE CAN SEE BLM FOCI IN ALL

1408
00:47:42,800 --> 00:47:44,840
THESE GUYS, THESE FOCI IN TERMS

1409
00:47:44,840 --> 00:47:46,320
OF THE INTERNAL DELETION LOOK A

1410
00:47:46,320 --> 00:47:47,040
LITTLE BIT BIG.

1411
00:47:47,040 --> 00:47:48,280
IT'S NOT UNUSUAL.

1412
00:47:48,280 --> 00:47:50,880
WE ARE OVEREXPRESSIONING A GFP

1413
00:47:50,880 --> 00:47:51,440
TAG PROTEIN.

1414
00:47:51,440 --> 00:47:53,920
IN ALL CASES WE ARE SEEING RPA,

1415
00:47:53,920 --> 00:47:55,240
TO SOME EXTENT.

1416
00:47:55,240 --> 00:47:56,720
AND THEN Rad51 WE ARE SEEING

1417
00:47:56,720 --> 00:47:58,640
IT AND IT VARIES QUITE A BIT.

1418
00:47:58,640 --> 00:47:59,880
I'LL SHOW YOU QUANTITATION OF

1419
00:47:59,880 --> 00:48:00,920
THIS DATA NOW.

1420
00:48:00,920 --> 00:48:03,320
SO FIRST THING WE LOOKED AT WAS

1421
00:48:03,320 --> 00:48:05,040
53BP1 WHICH WE ARE TAKING AS

1422
00:48:05,040 --> 00:48:07,240
EARLY INDICATOR OF DNA DAMAGE.

1423
00:48:07,240 --> 00:48:10,120
AND ALL THEE THERE ARE SOME

1424
00:48:10,120 --> 00:48:11,320
DIFFERENCES FROM CELL LINE TO

1425
00:48:11,320 --> 00:48:12,520
CELL LINE BACKGROUND TO

1426
00:48:12,520 --> 00:48:13,840
BACKGROUND, PRETTY MUCH THEY ARE

1427
00:48:13,840 --> 00:48:15,920
ALL MAKING DNA DOUBLE STRAND

1428
00:48:15,920 --> 00:48:16,160
BREAKS.

1429
00:48:16,160 --> 00:48:18,240
WE KNOW THAT WE ARE INDUCING DNA

1430
00:48:18,240 --> 00:48:19,640
DAMAGE WHEN WE EXPOSE THESE

1431
00:48:19,640 --> 00:48:23,600
CELLS TO IONIZING RADIATION.

1432
00:48:23,600 --> 00:48:25,520
NOW ALL CELLS ALSO SHOW EVIDENCE

1433
00:48:25,520 --> 00:48:30,040
OF GFP-BLM FOCI AND THERE ARE

1434
00:48:30,040 --> 00:48:31,960
DIFFERENCES FROM ONE CONSTRUCT

1435
00:48:31,960 --> 00:48:33,280
TO THE NEXT.

1436
00:48:33,280 --> 00:48:34,800
BUT THEY ARE STILL FORMING FOCI.

1437
00:48:34,800 --> 00:48:35,720
IT'S INTERESTING TO THINK ABOUT

1438
00:48:35,720 --> 00:48:37,800
THIS GUY, WHICH IS NOT TARGETED

1439
00:48:37,800 --> 00:48:40,560
TO DNA ENDS BUT IT IS STILL

1440
00:48:40,560 --> 00:48:43,040
FORMING FOCI CONSISTENT WITH IT

1441
00:48:43,040 --> 00:48:44,680
GOING TO WHERE THE DNA DAMAGE

1442
00:48:44,680 --> 00:48:44,880
IS.

1443
00:48:44,880 --> 00:48:47,280
I'LL BRING THAT UP AGAIN LATER,

1444
00:48:47,280 --> 00:48:48,880
AT THE END.

1445
00:48:48,880 --> 00:48:50,360
NOW, WHAT WE WANTED TO DO THEN

1446
00:48:50,360 --> 00:48:52,960
WAS LOOK AT RPA FOCI.

1447
00:48:52,960 --> 00:48:56,360
SO RPA FOCI ARE AN INDICATION

1448
00:48:56,360 --> 00:48:58,600
THAT END PROCESSING SELL

1449
00:48:58,600 --> 00:48:59,480
ACTUALLY TAKING PLACE.

1450
00:48:59,480 --> 00:49:01,880
NOW THIS IS OUR VECTOR-ONLY

1451
00:49:01,880 --> 00:49:02,080
CONTROL.

1452
00:49:02,080 --> 00:49:05,160
THIS IS WHEN WE PUT IN

1453
00:49:05,160 --> 00:49:06,160
FULL-LENGTH BLM.

1454
00:49:06,160 --> 00:49:08,280
SO GOING FROM HERE TO HERE,

1455
00:49:08,280 --> 00:49:10,400
STARTING TO SEE MORE FOCI SAYING

1456
00:49:10,400 --> 00:49:12,760
WE ARE GENERATING PROCESSED

1457
00:49:12,760 --> 00:49:13,200
BREAKS.

1458
00:49:13,200 --> 00:49:15,440
WE SEE A SIMILAR INCREASE WHEN

1459
00:49:15,440 --> 00:49:18,760
WE DELETE THE END TERMINIS OF

1460
00:49:18,760 --> 00:49:19,080
BLM.

1461
00:49:19,080 --> 00:49:21,360
WE STILL SEIZE RPA FOCI FORMING.

1462
00:49:21,360 --> 00:49:23,760
THAT'S TELLING US THAT THE

1463
00:49:23,760 --> 00:49:26,080
PROTEIN IS PROCESSING THE DNA

1464
00:49:26,080 --> 00:49:26,280
ENDS.

1465
00:49:26,280 --> 00:49:28,560
BUT IF WE TAKE OUR C TERMINAL

1466
00:49:28,560 --> 00:49:30,680
CONSTRUCT, NOW WE DON'T SEE THAT

1467
00:49:30,680 --> 00:49:32,680
INCREASE IN RPA FOCI.

1468
00:49:32,680 --> 00:49:34,880
SO WE THINK THIS GUY IS NO

1469
00:49:34,880 --> 00:49:36,960
LONGER MAKING RPA FOCI BECAUSE

1470
00:49:36,960 --> 00:49:39,880
IT'S NO LONGER SUPPORTING RPA

1471
00:49:39,880 --> 00:49:41,280
FOCI FORMATION BECAUSE IT'S NOT

1472
00:49:41,280 --> 00:49:42,680
PROCESSING THE DNA ENDS.

1473
00:49:42,680 --> 00:49:45,560
AND I'M JUST GOING TO POINT OUT

1474
00:49:45,560 --> 00:49:47,360
THAT WE ALSO HAVE PHYSICAL

1475
00:49:47,360 --> 00:49:49,560
ASSAYS WHERE WE ARE DOING PCR

1476
00:49:49,560 --> 00:49:51,360
NEAR THE BREAK TO DETECT THE

1477
00:49:51,360 --> 00:49:52,800
PRODUCTION OF SINGLE STRANDED

1478
00:49:52,800 --> 00:49:54,760
DNA AND IT ALSO SAYS THAT THIS

1479
00:49:54,760 --> 00:49:57,960
GUY IS NOT GENERATING SINGLE

1480
00:49:57,960 --> 00:50:00,080
STRANDED DNA WHEREAS THESE GUYS

1481
00:50:00,080 --> 00:50:00,280
ARE.

1482
00:50:00,280 --> 00:50:03,400
SO NOW IF WE LOOK AT Rad51

1483
00:50:03,400 --> 00:50:05,760
FOCI, WHAT WE SEE IS THAT WITH

1484
00:50:05,760 --> 00:50:08,200
BLM, WE GET AN UPTICK COMPARED

1485
00:50:08,200 --> 00:50:10,440
TO VECTOR-ONLY CONTROL.

1486
00:50:10,440 --> 00:50:12,400
BUT NOW NEITHER OF OUR DELETION

1487
00:50:12,400 --> 00:50:15,720
CONSTRUCTS ARE SHOWING EVIDENCE

1488
00:50:15,720 --> 00:50:16,800
OF STRONG UPTICKS IN Rad51

1489
00:50:16,800 --> 00:50:18,000
FOCI FORMATION.

1490
00:50:18,000 --> 00:50:20,200
SO, WE THINK THAT THE C TERMINAL

1491
00:50:20,200 --> 00:50:23,200
GUY IS NOT MAKING Rad51 FOCI

1492
00:50:23,200 --> 00:50:24,680
BECAUSE THE ENDS AREN'T GETTING

1493
00:50:24,680 --> 00:50:27,320
RESECTED SO NO RPA OR Rad51

1494
00:50:27,320 --> 00:50:27,760
LOADING.

1495
00:50:27,760 --> 00:50:30,080
WHEREAS THIS ONE, THE END

1496
00:50:30,080 --> 00:50:31,400
TERMINAL CONSTRUCT, WE THINK IT

1497
00:50:31,400 --> 00:50:32,680
IS RESECTING THE ENDS.

1498
00:50:32,680 --> 00:50:35,560
WE DO SEE RPA FOCI, BUT IT SEEMS

1499
00:50:35,560 --> 00:50:39,280
TO BE SPECIFICALLY DEFECTIVE IN

1500
00:50:39,280 --> 00:50:40,320
Rad51 FILAMENT FORMATION OR

1501
00:50:40,320 --> 00:50:41,440
FOCI FORMATION.

1502
00:50:41,440 --> 00:50:43,920
AND SO WE THINK THERE IS SOME

1503
00:50:43,920 --> 00:50:46,120
KIND OF DEFECT ASSOCIATED WITH

1504
00:50:46,120 --> 00:50:48,040
THIS GUY THAT DOESN'T LET

1505
00:50:48,040 --> 00:50:50,680
RAD51 NOW BIND TO THE DNA.

1506
00:50:50,680 --> 00:50:53,600
NOW I'M GOING TO GO THROUGH TWO

1507
00:50:53,600 --> 00:50:54,680
ASSAYS RELATIVELY QUICK AND GET

1508
00:50:54,680 --> 00:50:56,480
TO THE CONCLUSIONS.

1509
00:50:56,480 --> 00:50:58,200
IN THESE TWO ASSAYS, WHAT WE ARE

1510
00:50:58,200 --> 00:51:03,080
DOING ARE TAKING GFP CONSTRUCTS

1511
00:51:03,080 --> 00:51:05,800
WHERE WE HAVE A GFP GENE THAT IS

1512
00:51:05,800 --> 00:51:07,040
INACTIVE AND ONE THAT IS ACTIVE

1513
00:51:07,040 --> 00:51:11,760
AND THEN WE ARE USING DNA

1514
00:51:11,760 --> 00:51:13,000
BREAK-IN DUDES BY RESTRICTION IN

1515
00:51:13,000 --> 00:51:18,200
THE NUCLEASE, ICE1, TO INDUCE

1516
00:51:18,200 --> 00:51:19,600
REPAIR THAT RESULTS IN REPAIR OF

1517
00:51:19,600 --> 00:51:23,480
THE GFP GENE SO IT GOES FROM NOT

1518
00:51:23,480 --> 00:51:25,480
FLUORESCENT TO NOW FLUORESCENT.

1519
00:51:25,480 --> 00:51:27,480
AND WE CAN TUNE THESE OR I

1520
00:51:27,480 --> 00:51:30,000
SHOULD SAY, THE ASSAYS WE HAVE

1521
00:51:30,000 --> 00:51:33,080
ADAPTED, CAN ATTUNE THESE TO

1522
00:51:33,080 --> 00:51:34,640
DETECT HOMOLOGOUS RECOMBINATION

1523
00:51:34,640 --> 00:51:38,880
OR SYNTHESIS DEPENDENT STRAND

1524
00:51:38,880 --> 00:51:39,840
KNEELING.

1525
00:51:39,840 --> 00:51:40,000
SSA.

1526
00:51:40,000 --> 00:51:41,680
I SHOULD SAY THAT.

1527
00:51:41,680 --> 00:51:43,280
OKAY, SO LET ME WALK YOU THROUGH

1528
00:51:43,280 --> 00:51:44,880
THIS DATA REALLY QUICK.

1529
00:51:44,880 --> 00:51:46,360
THESE ARE CONTROLLED CELL WHERE

1530
00:51:46,360 --> 00:51:47,400
WE DO KNOWING.

1531
00:51:47,400 --> 00:51:49,080
THIS IS A CELL WHERE WE BREAK

1532
00:51:49,080 --> 00:51:53,160
THE DNA AND HAVE AN siRNA NOT

1533
00:51:53,160 --> 00:51:54,960
TARGETED AGAINST ANYTHING AND WE

1534
00:51:54,960 --> 00:51:56,440
SEE HOMOLOGOUS RECOMBINATION

1535
00:51:56,440 --> 00:51:56,920
TAKING PLACE.

1536
00:51:56,920 --> 00:51:59,840
THIS IS WHERE WE CUT THE DNA BUT

1537
00:51:59,840 --> 00:52:03,520
INHIBIT BRCA2 AND SO WE SEE A

1538
00:52:03,520 --> 00:52:05,880
DOWNTICK IN HR, MOM OL GUS

1539
00:52:05,880 --> 00:52:06,320
RECOMBINATION.

1540
00:52:06,320 --> 00:52:10,640
THIS IS WHERE WE KNOCKOUT B R&D

1541
00:52:10,640 --> 00:52:11,160
O1.

1542
00:52:11,160 --> 00:52:12,520
NOT VERY GOOD RECOMBINATION.

1543
00:52:12,520 --> 00:52:14,320
THIS IS OUR VECTOR-ONLY CONTROL.

1544
00:52:14,320 --> 00:52:16,320
THIS IS WHERE WE PUT THAT

1545
00:52:16,320 --> 00:52:17,320
FULL-LENGTH BLM.

1546
00:52:17,320 --> 00:52:19,560
SO WE DO GET A PARTIAL REGULARS

1547
00:52:19,560 --> 00:52:22,280
CUE OF HOMOLOGOUS RECOMBINATION

1548
00:52:22,280 --> 00:52:23,320
AS LONG AS WHEAT HAVE

1549
00:52:23,320 --> 00:52:23,640
FULL-LENGTH.

1550
00:52:23,640 --> 00:52:25,920
NOW NEITHER OF THESE GUYS ARE

1551
00:52:25,920 --> 00:52:27,640
SUPPORTING HOMOLOGOUS

1552
00:52:27,640 --> 00:52:28,640
RECOMBINATION IN THESE CELLS

1553
00:52:28,640 --> 00:52:30,440
COMPARED TO OUR VECTOR-ONLY

1554
00:52:30,440 --> 00:52:30,680
CONTROL.

1555
00:52:30,680 --> 00:52:32,920
AND SO THAT IS TELLING US THAT

1556
00:52:32,920 --> 00:52:34,840
CONSISTENT WITH OUR PRIOR

1557
00:52:34,840 --> 00:52:36,320
RESULTS, THERE IS SOME SORT OF

1558
00:52:36,320 --> 00:52:38,640
DEFECT ASSOCIATED WITH REPAIR.

1559
00:52:38,640 --> 00:52:42,000
NOW IF WE LOOK AT SINGLE STRAND

1560
00:52:42,000 --> 00:52:45,880
KNEELING WHICH IS NOT RadPOP

1561
00:52:45,880 --> 00:52:47,800
DEPENDENT -- Rad51 DEPENDENT,

1562
00:52:47,800 --> 00:52:51,480
WHAT WE SEE IS WHEN WE KNOCKOUT

1563
00:52:51,480 --> 00:52:53,600
BRCA2, WE GET INCREASE IN SINGLE

1564
00:52:53,600 --> 00:52:55,600
STRANDED KNEELING BECAUSE WE ARE

1565
00:52:55,600 --> 00:52:56,520
TAKING THINGS THAT NORMALLY

1566
00:52:56,520 --> 00:52:58,400
WOULD BE REPAIRED BY HR AND

1567
00:52:58,400 --> 00:52:59,840
CHANNELING THEM INTO THIS NEW

1568
00:52:59,840 --> 00:53:03,400
PATHWAY THAT IS Rad51

1569
00:53:03,400 --> 00:53:03,680
INDEPENDENT.

1570
00:53:03,680 --> 00:53:05,880
NOW IF WE LOOK AT OUR

1571
00:53:05,880 --> 00:53:07,080
VECTOR-ONLY CONTROL THIS IS WHAT

1572
00:53:07,080 --> 00:53:08,680
WE GET.

1573
00:53:08,680 --> 00:53:09,320
ADDING THE BLOOM DOESN'T REALLY

1574
00:53:09,320 --> 00:53:10,600
HAVE MUCH OF AN EFFECT.

1575
00:53:10,600 --> 00:53:12,800
OUR C TERMINAL DELETION ALSO

1576
00:53:12,800 --> 00:53:14,400
DOESN'T HAVE AN EFFECT.

1577
00:53:14,400 --> 00:53:16,080
BUT ADDING THIS END TERMINAL

1578
00:53:16,080 --> 00:53:17,680
DELETION NOW WE GET A

1579
00:53:17,680 --> 00:53:19,680
SUBSTANTIAL INCREASE IN SINGLE

1580
00:53:19,680 --> 00:53:20,080
STRANDED KNEELING.

1581
00:53:20,080 --> 00:53:21,360
AND WE THINK THAT IS BECAUSE

1582
00:53:21,360 --> 00:53:23,480
THIS GUY IS MAKING SINGLE

1583
00:53:23,480 --> 00:53:25,680
STRANDED ENDS THAT ARE LOADED BY

1584
00:53:25,680 --> 00:53:27,800
RPA BUT THEY ARE NOT LOADED

1585
00:53:27,800 --> 00:53:28,880
WITH Rad51.

1586
00:53:28,880 --> 00:53:31,480
SO Rad51 ACTUALLY IN HIBITS

1587
00:53:31,480 --> 00:53:31,680
PATHWAY.

1588
00:53:31,680 --> 00:53:33,680
AND SO WE THINK THAT THAT IS

1589
00:53:33,680 --> 00:53:34,960
FULLY CONSISTENT WITH THIS IDEA

1590
00:53:34,960 --> 00:53:37,880
THAT SOMEHOW THIS END TERMINAL

1591
00:53:37,880 --> 00:53:40,920
DELETION CONSTRUCT IS DEFICIENT

1592
00:53:40,920 --> 00:53:42,120
FOR Rad51 FILAMENT FORMATION.

1593
00:53:42,120 --> 00:53:46,120
SO NOW I'M GOING TO GO TO MY

1594
00:53:46,120 --> 00:53:46,640
SUMMARY SLIDE.

1595
00:53:46,640 --> 00:53:48,920
AND I HOPE THAT I HAVE CONVINCED

1596
00:53:48,920 --> 00:53:52,120
YOU THAT END RESECONDS IN

1597
00:53:52,120 --> 00:53:53,000
EUKARYOTES IS SOMEHOW COUPLED TO

1598
00:53:53,000 --> 00:53:55,840
THE FORMATION OF THIS LARGE

1599
00:53:55,840 --> 00:53:57,040
SINGLE STRANDED LOOP AND THAT

1600
00:53:57,040 --> 00:54:00,600
THE END TERMINAL DOMAIN OF BLM

1601
00:54:00,600 --> 00:54:01,720
HELICASE IS SOMEHOW CONTRIBUTING

1602
00:54:01,720 --> 00:54:04,280
TO THIS FORMATION.

1603
00:54:04,280 --> 00:54:05,840
AND CELLS EXPRESSING THIS MUTANT

1604
00:54:05,840 --> 00:54:08,320
ARE DEFICIENT FOR DNA REPAIR IN

1605
00:54:08,320 --> 00:54:11,680
THE PRESENCE OF DNA DAMAGING

1606
00:54:11,680 --> 00:54:11,920
DRUGS.

1607
00:54:11,920 --> 00:54:14,720
AND IT SEEMS AS THOUGH THIS

1608
00:54:14,720 --> 00:54:17,720
MUTANT CAN SUPPORT END RESECTION

1609
00:54:17,720 --> 00:54:20,000
IN-VIVO BUT Rad51 IS FAILING

1610
00:54:20,000 --> 00:54:21,240
TO FORM ON THIS.

1611
00:54:21,240 --> 00:54:23,280
AND SO OUR HYPOTHESES GOING

1612
00:54:23,280 --> 00:54:25,400
FORWARD IS THERE MAY BE SOME

1613
00:54:25,400 --> 00:54:27,000
KIND OF LINK BETWEEN LOOP

1614
00:54:27,000 --> 00:54:28,280
FORMATION DURING END RESECTION

1615
00:54:28,280 --> 00:54:32,040
AND THE ASSEMBLY OF THE Rad51

1616
00:54:32,040 --> 00:54:33,080
PRESYNAPTIC COMPLEX.

1617
00:54:33,080 --> 00:54:34,880
I COLORED THIS BLUE AND ADDED A

1618
00:54:34,880 --> 00:54:35,840
QUESTION MARK BECAUSE THIS IS

1619
00:54:35,840 --> 00:54:38,200
SPECULATIVE AT THIS POINT.

1620
00:54:38,200 --> 00:54:40,160
AND WE ARE CURIOUS WHAT IS

1621
00:54:40,160 --> 00:54:41,400
DRIVING THESE LOOPING

1622
00:54:41,400 --> 00:54:41,720
INTERACTIONS?

1623
00:54:41,720 --> 00:54:43,600
IS IT BLM INTERACTIONS WITH

1624
00:54:43,600 --> 00:54:46,360
SINGLE STRANDED DNA OR PERHAPS

1625
00:54:46,360 --> 00:54:48,080
BLM INTERACTIONS WITH RP!

1626
00:54:48,080 --> 00:54:48,680
WE DON'T KNOW.

1627
00:54:48,680 --> 00:54:50,760
AND WE ARE ALSO CURIOUS WHETHER

1628
00:54:50,760 --> 00:54:53,480
BLM ITSELF MAY BE SOMEHOW LOADED

1629
00:54:53,480 --> 00:54:55,800
OR INVOLVED IN LOADING Rad51

1630
00:54:55,800 --> 00:54:59,160
ON TO THE DNA ENDS DURING

1631
00:54:59,160 --> 00:54:59,440
RESECTION.

1632
00:54:59,440 --> 00:55:00,280
OF COURSE WE ALWAYS HAVE THE

1633
00:55:00,280 --> 00:55:02,680
QUESTION OF CAN WE SEPARATE OR

1634
00:55:02,680 --> 00:55:04,680
GENERATE MUTANTS THAT SEPARATE

1635
00:55:04,680 --> 00:55:05,960
LOOPING FROM Rad51 LOADING?

1636
00:55:05,960 --> 00:55:08,920
WE DON'T KNOW THE ANSWER TO THAT

1637
00:55:08,920 --> 00:55:09,080
YET.

1638
00:55:09,080 --> 00:55:13,080
I ALSO WANT TO MAKE A POINT

1639
00:55:13,080 --> 00:55:13,360
HERE.

1640
00:55:13,360 --> 00:55:16,520
THE KEY BACTERIAL SYSTEM, SO FOR

1641
00:55:16,520 --> 00:55:18,760
EXAMPLE, E.COLI, IT ALSO MAKES

1642
00:55:18,760 --> 00:55:20,680
DNA LOOPS DURING END RESECTION.

1643
00:55:20,680 --> 00:55:22,040
BUT WE DON'T KNOW WHY.

1644
00:55:22,040 --> 00:55:25,080
SO IT SEEMS AS THOUGH, AND I

1645
00:55:25,080 --> 00:55:28,240
WANT TO MAKE THIS CLEAR,

1646
00:55:28,240 --> 00:55:30,440
PROCESSING DNA ENDS IN E.COLI IS

1647
00:55:30,440 --> 00:55:33,160
COMPLETELY DIFFERENT FROM BLM,

1648
00:55:33,160 --> 00:55:35,360
DNA2, RPA, WHICH IS INVOLVED IN

1649
00:55:35,360 --> 00:55:37,720
PROCESSING IN EUKARYOTES.

1650
00:55:37,720 --> 00:55:40,080
BUT BOTH SYSTEMS ARE FORMING

1651
00:55:40,080 --> 00:55:40,280
LOOPS.

1652
00:55:40,280 --> 00:55:41,680
IS IT A COINCIDENCE OR SOMETHING

1653
00:55:41,680 --> 00:55:43,240
TO LOOP FORMATION THAT IS

1654
00:55:43,240 --> 00:55:44,680
IMPORTANT FOR RECOMBINATION?

1655
00:55:44,680 --> 00:55:47,440
WE DON'T KNOW THE ANSWER TO

1656
00:55:47,440 --> 00:55:47,640
THAT.

1657
00:55:47,640 --> 00:55:51,200
NOW IN TERMS OF OUR C TERMINAL

1658
00:55:51,200 --> 00:55:54,600
DOMAIN, WE KNOW THAT HDRC MOTIF

1659
00:55:54,600 --> 00:55:56,680
HELPS COUPLE ATP HYDROLYSIS TO

1660
00:55:56,680 --> 00:55:57,200
TRANSLOCATION.

1661
00:55:57,200 --> 00:55:59,280
WE HAVE NO IDEA HOW THAT TAKES

1662
00:55:59,280 --> 00:56:00,280
PLACE IT'S INTERESTING BECAUSE

1663
00:56:00,280 --> 00:56:01,680
WITHIN THE CRYSTAL STRUCTURE,

1664
00:56:01,680 --> 00:56:03,080
THIS DOMAIN SEEMS TO BE WELL

1665
00:56:03,080 --> 00:56:05,880
AWAY FROM ANY DNA BINDING MOTIF.

1666
00:56:05,880 --> 00:56:07,280
ALTHOUGH YOU CAN ALWAYS THINK

1667
00:56:07,280 --> 00:56:10,400
ABOUT NEW STRUCTURES THAT MIGHT

1668
00:56:10,400 --> 00:56:10,680
CHANGE THAT.

1669
00:56:10,680 --> 00:56:14,080
WE HAVE IDENTIFIED A NEW PEPTIDE

1670
00:56:14,080 --> 00:56:15,720
WITHIN BLM.

1671
00:56:15,720 --> 00:56:17,480
IT'S FRANKING HDRC BUT NOT

1672
00:56:17,480 --> 00:56:19,480
WITHIN HDRC ITSELF THAT SEEM TO

1673
00:56:19,480 --> 00:56:21,280
BE NECESSARY FOR END RESECTION.

1674
00:56:21,280 --> 00:56:24,600
EVEN THOUGH IT IS NOT MESSING UP

1675
00:56:24,600 --> 00:56:26,720
ANY OF THE OTHER PROPERTIES D-NA

1676
00:56:26,720 --> 00:56:29,200
BINDING OR HELICASE ACTIVITIES.

1677
00:56:29,200 --> 00:56:31,000
SPECIFICALLY AFFECTING

1678
00:56:31,000 --> 00:56:31,280
RECOGNITION.

1679
00:56:31,280 --> 00:56:33,480
AND WE CAN SHOW THAT THIS MULE

1680
00:56:33,480 --> 00:56:35,880
ANNUAL IS RECRUITED TO SITES OF

1681
00:56:35,880 --> 00:56:38,480
OF DNA REPAIR BUT IT'S NOT

1682
00:56:38,480 --> 00:56:40,600
TARGETED TO D-NA AND ENDS IT

1683
00:56:40,600 --> 00:56:41,680
SEEMS TO BE SERIOUSLY

1684
00:56:41,680 --> 00:56:45,160
COMPROMISED FOR END PROCESSING

1685
00:56:45,160 --> 00:56:46,800
BOTH, FOR FORMATION OF RPA AND

1686
00:56:46,800 --> 00:56:50,200
RAD 51 FOCI AND IT DOESN'T DO

1687
00:56:50,200 --> 00:56:50,360
HR.

1688
00:56:50,360 --> 00:56:51,760
AND SO ONE THING THAT WE ARE

1689
00:56:51,760 --> 00:56:54,680
THINKING THIS IS TELLING US, IS

1690
00:56:54,680 --> 00:56:57,120
THAT BECAUSE IT'S GOING TO FOCI,

1691
00:56:57,120 --> 00:56:59,080
ACTUAL RECRUITMENT TO THE REPAIR

1692
00:56:59,080 --> 00:57:03,160
FOCI MAY BE A SEPARATE PROCESS

1693
00:57:03,160 --> 00:57:04,200
FROM DIRECTORY CRUELTY TO THE

1694
00:57:04,200 --> 00:57:04,960
ACTUAL DNA ENDS.

1695
00:57:04,960 --> 00:57:07,360
AND AGAIN, THAT IS SORT OF A

1696
00:57:07,360 --> 00:57:08,880
SPECULATIVE THINKING WE HAVE TO

1697
00:57:08,880 --> 00:57:11,600
SORT OF WORK OUT IN THE FUTURE.

1698
00:57:11,600 --> 00:57:14,600
SO I'M HAPPY TO END THERE AND

1699
00:57:14,600 --> 00:57:16,640
ADDRESS ANY QUESTIONS THAT

1700
00:57:16,640 --> 00:57:18,160
ANYONE MIGHT HAVE.

1701
00:57:18,160 --> 00:57:20,480
NOT ENTIRELY SURE HOW THIS WORKS

1702
00:57:20,480 --> 00:57:24,160
BUT I THINK SUSAN WILL READ YOUR

1703
00:57:24,160 --> 00:57:25,400
QUESTIONS TO ME.

1704
00:57:25,400 --> 00:57:28,480
SO WE'LL SEE HOW THAT GOES.

1705
00:57:28,480 --> 00:57:29,520
AND I'LL JUST THANK EVERYBODY

1706
00:57:29,520 --> 00:57:31,280
FOR LISTENING TO ME.

1707
00:57:31,280 --> 00:57:33,320
>> SUSAN: THANK YOU, ERIC.

1708
00:57:33,320 --> 00:57:34,960
I AM SURE IF WE COULD SEE

1709
00:57:34,960 --> 00:57:36,120
PEOPLE, THERE WOULD BE A LOT OF

1710
00:57:36,120 --> 00:57:37,160
HANDS CLAPPING.

1711
00:57:37,160 --> 00:57:38,640
I'M NOT SURE HOW THIS WORKS

1712
00:57:38,640 --> 00:57:41,280
EITHER BUT I THINK WE'LL WORK IT

1713
00:57:41,280 --> 00:57:41,480
OUT.

1714
00:57:41,480 --> 00:57:42,640
IT WAS A BEAUTIFUL TALK AND

1715
00:57:42,640 --> 00:57:45,800
THERE ARE SOME QUESTIONS IN THE

1716
00:57:45,800 --> 00:57:46,600
CHAT.

1717
00:57:46,600 --> 00:57:47,600
PLEASE CONTINUE EVERYBODY TO ADD

1718
00:57:47,600 --> 00:57:50,800
YOUR QUESTIONS AND I WILL GO

1719
00:57:50,800 --> 00:57:52,560
THROUGH AS MANY AS WE CAN.

1720
00:57:52,560 --> 00:57:55,480
SO THE FIRST ONE IS FROM RACHEL

1721
00:57:55,480 --> 00:57:57,680
JOHNSTON AND IT'S MORE OF A

1722
00:57:57,680 --> 00:57:59,480
TECHNICAL SET OF QUESTIONS ABOUT

1723
00:57:59,480 --> 00:58:01,800
CONTROLS FOR YOUR CURTAIN ASSAY.

1724
00:58:01,800 --> 00:58:04,880
SHE ASKS, YOU NOTED THAT THE BLM

1725
00:58:04,880 --> 00:58:07,400
LOCALIZED TO THE DNA ENDS USING

1726
00:58:07,400 --> 00:58:07,880
GFP LABELING.

1727
00:58:07,880 --> 00:58:10,280
DID YOU HAVE A CONTROL FOR

1728
00:58:10,280 --> 00:58:11,920
LOCALIZATION FOR A PROTEIN?

1729
00:58:11,920 --> 00:58:13,400
WHICH WOULD LOCALIZE TO ANOTHER

1730
00:58:13,400 --> 00:58:15,400
LOCATION ON THE DNA?

1731
00:58:15,400 --> 00:58:16,720
SO RATHER THAN IT BEING BUFFER

1732
00:58:16,720 --> 00:58:20,960
FLOAT ISSUE, YOU KNOW IT IS TRUE

1733
00:58:20,960 --> 00:58:21,520
LOCALIZATION.

1734
00:58:21,520 --> 00:58:22,960
>> DR. GREENE: YES.

1735
00:58:22,960 --> 00:58:27,960
SO OVER THE YEARS WE PUBLISHED

1736
00:58:27,960 --> 00:58:29,760
100 -- NORTH OF 100 PAPERS NOW

1737
00:58:29,760 --> 00:58:32,880
AND IN SEVERAL OF THOSE WE HAVE

1738
00:58:32,880 --> 00:58:34,480
PUBLISHED PROTEINS THAT BIND TO

1739
00:58:34,480 --> 00:58:36,480
SPECIFIC SITES WITHIN THE DNA.

1740
00:58:36,480 --> 00:58:38,560
I THINK ONE OF THE MORE FUN ONES

1741
00:58:38,560 --> 00:58:43,040
WE DID WAS WITH CAS9 ACTUALLY.

1742
00:58:43,040 --> 00:58:44,520
AND WE COULD TARGET IT BASICALLY

1743
00:58:44,520 --> 00:58:46,840
WHEREVER WE WANTED WITHIN THE

1744
00:58:46,840 --> 00:58:49,280
DNA JUST BY CHANGING THE GUIDE

1745
00:58:49,280 --> 00:58:49,720
RNA.

1746
00:58:49,720 --> 00:58:51,320
WE HAVE ALSO DID STUDIES -- I

1747
00:58:51,320 --> 00:58:53,120
SHOULD HAVE MENTIONED THIS.

1748
00:58:53,120 --> 00:58:55,640
THIS IS LAMBDA PHAGE DNA WE ARE

1749
00:58:55,640 --> 00:58:55,840
USING.

1750
00:58:55,840 --> 00:58:58,080
IT'S NOTHING FANCY.

1751
00:58:58,080 --> 00:59:02,560
NICE THING ABOUT IT HAS RNA

1752
00:59:02,560 --> 00:59:03,720
POLYMERASE PROMOTORS AND IT GOES

1753
00:59:03,720 --> 00:59:05,480
TO ALL THE RIGHT PROMOTORS.

1754
00:59:05,480 --> 00:59:09,000
WE CAN PUT IN ALSO -- LET ME

1755
00:59:09,000 --> 00:59:13,000
THINK ABOUT THIS -- A NUCLEASE

1756
00:59:13,000 --> 00:59:16,320
DEFICIENT VERSION OF FOR1.

1757
00:59:16,320 --> 00:59:17,480
IT GOES TRITE THAT SITE.

1758
00:59:17,480 --> 00:59:19,680
AND WE CAN PUT IN A MISMATCH IN

1759
00:59:19,680 --> 00:59:22,480
THAT DNA AND WE CAN TARGET THE

1760
00:59:22,480 --> 00:59:26,080
MISMATCH RECOGNITION PROTEINS.

1761
00:59:26,080 --> 00:59:28,800
MSH26 RIGHT TO THE MISMATCH.

1762
00:59:28,800 --> 00:59:30,280
WE DON'T THINK FLOW IS CAUSING

1763
00:59:30,280 --> 00:59:34,080
IT TO GO TO THE END OF THE DNA.

1764
00:59:34,080 --> 00:59:36,040
I SHOULD ALSO NOTE WE ARE OFTEN

1765
00:59:36,040 --> 00:59:37,400
ASKED, HOW DO YOU KNOW FORCE IS

1766
00:59:37,400 --> 00:59:38,600
NOT INFLUENCING THIS?

1767
00:59:38,600 --> 00:59:41,200
AND I MEAN, I CAN'T SAY WITH

1768
00:59:41,200 --> 00:59:42,160
CERTAINTY THAT IT'S NOT, BUT

1769
00:59:42,160 --> 00:59:45,240
WHAT I CAN SAY IS THAT WE ARE

1770
00:59:45,240 --> 00:59:46,000
NOT EXERTING A TREMENDOUS AMOUNT

1771
00:59:46,000 --> 00:59:47,360
OF FORCE ON THESE MOLECULES.

1772
00:59:47,360 --> 00:59:48,880
THIS IS ONE OF THE REASONS THEY

1773
00:59:48,880 --> 00:59:50,280
SORT OF LOOK LIKE THEY ARE

1774
00:59:50,280 --> 00:59:51,480
FLIPPING BACK-AND-FORTH A LITTLE

1775
00:59:51,480 --> 00:59:51,640
BIT.

1776
00:59:51,640 --> 00:59:59,080
WE ARE SORT OF DOWN IN THE

1777
00:59:59,080 --> 01:00:02,280
SUBSINGLE PEEKA NEWTON DIGITS

1778
01:00:02,280 --> 01:00:04,720
JUST ENOUGH TO EXTEND THE DNA

1779
01:00:04,720 --> 01:00:07,480
ABOUT 70% OF ITS FULL CONTOUR

1780
01:00:07,480 --> 01:00:07,680
LENGTH.

1781
01:00:07,680 --> 01:00:08,880
SO WE DON'T THINK WE ARE DOING

1782
01:00:08,880 --> 01:00:09,160
THAT.

1783
01:00:09,160 --> 01:00:10,760
NOW I WILL NOTE WE HAVE SEEN

1784
01:00:10,760 --> 01:00:11,760
EXAMPLES OF ENZYMES WHERE WE

1785
01:00:11,760 --> 01:00:14,760
PUSH IT OFF THE D IN.

1786
01:00:14,760 --> 01:00:16,920
A, AT LEAST WE THINK WE DO, BUT

1787
01:00:16,920 --> 01:00:18,480
I DON'T THINK IT IS TAKING

1788
01:00:18,480 --> 01:00:20,280
PLAGUES IN THIS EXAMPLE.

1789
01:00:20,280 --> 01:00:20,600
>> GREAT.

1790
01:00:20,600 --> 01:00:21,280
THANK YOU.

1791
01:00:21,280 --> 01:00:22,960
SO TOM SNYDER HAS A COUPLE OF

1792
01:00:22,960 --> 01:00:24,520
QUESTIONLESS I'LL ASK ON HIS

1793
01:00:24,520 --> 01:00:24,800
BEHALF.

1794
01:00:24,800 --> 01:00:26,560
THE FIRST IS, DID YOU GET THE

1795
01:00:26,560 --> 01:00:29,680
NUMBER OF ATP HYDROL SIZED PER

1796
01:00:29,680 --> 01:00:36,400
STEP IN WILDTYPE BLM?

1797
01:00:36,400 --> 01:00:36,760
>> DR. GREENE:

1798
01:00:36,760 --> 01:00:37,080
[ LAUGHS ]

1799
01:00:37,080 --> 01:00:39,440
TOM, I LOVE THAT QUESTION.

1800
01:00:39,440 --> 01:00:41,520
I THOUGHT TOO.

1801
01:00:41,520 --> 01:00:41,800
[ LAUGHS ]

1802
01:00:41,800 --> 01:00:43,520
 I HATE IT TOO.

1803
01:00:43,520 --> 01:00:45,840
THIS IS OF COURSE -- HE MUST BE

1804
01:00:45,840 --> 01:00:47,720
A MOTOR PROTEIN AFFICIONADO.

1805
01:00:47,720 --> 01:00:49,040
THIS IS A QUESTION THAT OFTEN

1806
01:00:49,040 --> 01:00:50,320
COMES UP.

1807
01:00:50,320 --> 01:00:54,840
THAT IS HOW MUCH ATP YOU

1808
01:00:54,840 --> 01:00:56,040
HYDROLYZE PER STEP?

1809
01:00:56,040 --> 01:00:57,600
I ALWAYS MAKE THE SIMPLEST

1810
01:00:57,600 --> 01:00:59,800
ASSUMPTION AND I THINK THIS IS

1811
01:00:59,800 --> 01:01:01,200
CONSISTENT WITH STRUCTURAL DATA

1812
01:01:01,200 --> 01:01:03,400
FOR ALL HELICASES I KNOW OF OR

1813
01:01:03,400 --> 01:01:05,120
MOST OF THEM ANYWAY, THAT YOU'RE

1814
01:01:05,120 --> 01:01:07,320
TAKING A SINGLE NUCLEOTIDE STEP.

1815
01:01:07,320 --> 01:01:09,080
SO IT'S OF COURSE A GREAT

1816
01:01:09,080 --> 01:01:12,040
INTEREST TO KNOW, IS ONE STEP

1817
01:01:12,040 --> 01:01:13,600
CORRESPOND TO 1ATP?

1818
01:01:13,600 --> 01:01:15,360
AND YOU KNOW, TOM, I WOULD LOVE

1819
01:01:15,360 --> 01:01:17,480
TO BE ABLE TO ANSWER THAT.

1820
01:01:17,480 --> 01:01:18,720
I CAN'T.

1821
01:01:18,720 --> 01:01:21,200
WE HAVEN'T NADE MEASUREMENT YET.

1822
01:01:21,200 --> 01:01:25,320
IT IS NOT SUCH AN EASY

1823
01:01:25,320 --> 01:01:25,960
MEASUREMENT TO MAKE CORRECTLY.

1824
01:01:25,960 --> 01:01:29,520
AND REALLY THE ONLY WAY THAT WE

1825
01:01:29,520 --> 01:01:34,480
CAN DO THIS IS TO DO BULK

1826
01:01:34,480 --> 01:01:36,080
CHEMICAL MEASUREMENTS MEASURING

1827
01:01:36,080 --> 01:01:38,400
ATP HYDROLYSIS PER UNIT TIME AND

1828
01:01:38,400 --> 01:01:40,880
THEN COMPARE TO THE VELOCITY.

1829
01:01:40,880 --> 01:01:43,200
THE CHALLENGE WITH THAT IN THIS

1830
01:01:43,200 --> 01:01:44,000
PARTICULAR SYSTEM IS THAT WE

1831
01:01:44,000 --> 01:01:45,960
KNOW THAT BLM BINDS TO ENDS.

1832
01:01:45,960 --> 01:01:48,040
WE ALSO KNOW THAT IT BINDS

1833
01:01:48,040 --> 01:01:49,960
INTERNAL POSITIONS.

1834
01:01:49,960 --> 01:01:52,240
AND THE VELOCITY IT MOVES IN

1835
01:01:52,240 --> 01:01:53,840
THOSE TWO DIFFERENT

1836
01:01:53,840 --> 01:01:54,800
CONFIGURATIONS IS VERY

1837
01:01:54,800 --> 01:01:55,200
DIFFERENT.

1838
01:01:55,200 --> 01:01:56,440
IT MOVES MUCH FASTER WHEN IT'S

1839
01:01:56,440 --> 01:01:58,920
BOUND TO AN INTERNAL SITE.

1840
01:01:58,920 --> 01:02:01,440
SO ANY BULK BIOCHEMICAL ATP

1841
01:02:01,440 --> 01:02:03,840
HYDROLYSIS DATA IS GOING TO BE A

1842
01:02:03,840 --> 01:02:05,640
MIXTURE OF THINGS THAT ARE BOUND

1843
01:02:05,640 --> 01:02:06,720
TO ENDS AND THINGS THAT ARE

1844
01:02:06,720 --> 01:02:08,680
BOUND TO INTERNAL SITES THAT WE

1845
01:02:08,680 --> 01:02:13,040
JUST DON'T HAVE A GOOD WAY OF

1846
01:02:13,040 --> 01:02:13,280
KNOWING.

1847
01:02:13,280 --> 01:02:15,720
I DO APPRECIATE THE QUESTION,

1848
01:02:15,720 --> 01:02:18,880
TOM AND I WOULD LOVE TO KNOW THE

1849
01:02:18,880 --> 01:02:19,520
ANSWER MYSELF.

1850
01:02:19,520 --> 01:02:23,640
>> THAT'S A GOOD POINT.

1851
01:02:23,640 --> 01:02:24,560
BIOCHEMISTRY IS NOT REALLY GOING

1852
01:02:24,560 --> 01:02:25,720
TO ANSWER THAT WELL, I DON'T

1853
01:02:25,720 --> 01:02:26,440
THINK, EITHER.

1854
01:02:26,440 --> 01:02:27,920
HE ALSO ASKS, HAVE YOU LOOKED AT

1855
01:02:27,920 --> 01:02:30,520
THE LOOP USING CRYO-EM?

1856
01:02:30,520 --> 01:02:35,320
MAYBE IT'S A COIL HELICAL

1857
01:02:35,320 --> 01:02:35,600
STRUCTURE?

1858
01:02:35,600 --> 01:02:39,320
>> WE HAVE NOT DONE THAT YET.

1859
01:02:39,320 --> 01:02:43,680
YES, WE ARE REALLY -- I'LL

1860
01:02:43,680 --> 01:02:44,320
JUSTSADE A LOT OF THE PROJECTS

1861
01:02:44,320 --> 01:02:46,600
WE ARE WORKING ON, WE HAVE KIND

1862
01:02:46,600 --> 01:02:47,960
OF COME INTO THESE BARRIERS

1863
01:02:47,960 --> 01:02:49,280
WHERE IT'S LIKE, MAN I WOULD

1864
01:02:49,280 --> 01:02:50,640
REALLY LIKE TO HAVE A STRUCTURE

1865
01:02:50,640 --> 01:02:51,200
OF THAT.

1866
01:02:51,200 --> 01:02:53,680
THIS IS NOT JUST THIS PROJECT

1867
01:02:53,680 --> 01:02:56,360
BUT OF COURSE IT APPLIES HERE AS

1868
01:02:56,360 --> 01:02:56,560
WELL.

1869
01:02:56,560 --> 01:02:58,480
AND SO, WE REALLY NEED TO START

1870
01:02:58,480 --> 01:03:00,080
DOING SOME CRYO-EM ON SOME OF

1871
01:03:00,080 --> 01:03:01,680
THE THINGS WE ARE LOOKING AT.

1872
01:03:01,680 --> 01:03:04,280
AND TRY TO GET SOME STRUCTURAL

1873
01:03:04,280 --> 01:03:04,560
INSIGHT.

1874
01:03:04,560 --> 01:03:06,960
ANOTHER GREAT QUESTION, WHICH I

1875
01:03:06,960 --> 01:03:07,800
UNFORTUNATELY CAN'T ANSWER VERY

1876
01:03:07,800 --> 01:03:09,720
WELL RIGHT NOW.

1877
01:03:09,720 --> 01:03:10,040
SORRY, TOM.

1878
01:03:10,040 --> 01:03:12,560
>> YOU HAVE A LOT OF COLLEAGUES

1879
01:03:12,560 --> 01:03:14,960
AT COLUMBIA THAT COULD HELP WITH

1880
01:03:14,960 --> 01:03:15,160
THAT.

1881
01:03:15,160 --> 01:03:17,440
>> THEY ARE ALL TRYING TO SOLVE

1882
01:03:17,440 --> 01:03:19,440
A VIRUS STRUCTURE, ANTIBODY

1883
01:03:19,440 --> 01:03:20,080
STRUCTURE NOW.

1884
01:03:20,080 --> 01:03:20,520
>> NEXT QUESTION.

1885
01:03:20,520 --> 01:03:21,960
>> WE ARE GOING TO DO IT

1886
01:03:21,960 --> 01:03:22,360
OURSELVES.

1887
01:03:22,360 --> 01:03:23,160
>> GOOD FOR YOU!

1888
01:03:23,160 --> 01:03:26,360
YOU CAN.

1889
01:03:26,360 --> 01:03:27,680
ALSO INTERESTING TO NOTE THAT A

1890
01:03:27,680 --> 01:03:30,760
RECK A LOADING FUNCTION HAS BEEN

1891
01:03:30,760 --> 01:03:34,080
SUGGESTED FOR RECBCD.

1892
01:03:34,080 --> 01:03:36,080
>> SO IS THAT MICHAEL?

1893
01:03:36,080 --> 01:03:38,520
I WISH I COULD SEE YOU AND HEAR

1894
01:03:38,520 --> 01:03:38,680
YOU.

1895
01:03:38,680 --> 01:03:39,920
THIS IS ONE OF THE THINGS I

1896
01:03:39,920 --> 01:03:42,240
DON'T LIKE SO MUCH ABOUT ZOOM.

1897
01:03:42,240 --> 01:03:44,240
THAT'S A GREAT POINT, MICHAEL.

1898
01:03:44,240 --> 01:03:47,120
IT'S VERY CLEAR THAT RECB IS

1899
01:03:47,120 --> 01:03:49,240
SOMEHOW INVOLVED IN LOADING RECA

1900
01:03:49,240 --> 01:03:55,520
AND WE ALSO KNOW THAT BLM

1901
01:03:55,520 --> 01:03:57,640
INTERACTS WITH Rad51.

1902
01:03:57,640 --> 01:04:00,320
SO WE WOULD LOVE TO BE ABLE TO

1903
01:04:00,320 --> 01:04:03,600
SEE THIS, TO SEE COUPLED Rad51

1904
01:04:03,600 --> 01:04:05,240
LOADING IN OUR SYSTEM.

1905
01:04:05,240 --> 01:04:06,640
WE ARE NOT THERE.

1906
01:04:06,640 --> 01:04:10,400
WE ARE GOING TO MOVE IN THAT

1907
01:04:10,400 --> 01:04:10,680
DIRECTION.

1908
01:04:10,680 --> 01:04:12,720
THE CHALLENGE MICHAEL, IS THAT

1909
01:04:12,720 --> 01:04:13,840
AND YOU'RE PROBABLY WELL AWARE

1910
01:04:13,840 --> 01:04:17,680
OF THIS IN A TEST TUBE, BECAUSE

1911
01:04:17,680 --> 01:04:21,000
IT'S AN ARTIFICIAL SYSTEM, AN

1912
01:04:21,000 --> 01:04:22,200
IMPORTANT CAVEAT YOU HAVE TO

1913
01:04:22,200 --> 01:04:25,000
CONTEND WITH IS THAT Rad51

1914
01:04:25,000 --> 01:04:26,400
DOESN'T DISTINGUISH VERY WELL

1915
01:04:26,400 --> 01:04:29,000
BETWEEN DOUBLE AND SINGLE

1916
01:04:29,000 --> 01:04:29,320
STRANDED DNA.

1917
01:04:29,320 --> 01:04:31,400
SO I KNOW THAT IF WE JUST THROW

1918
01:04:31,400 --> 01:04:33,000
Rad51 IN THERE WILLY NILLY, IT

1919
01:04:33,000 --> 01:04:34,080
WILL GO EVERYWHERE.

1920
01:04:34,080 --> 01:04:35,480
IT WILL GET IN FRONT OF THIS

1921
01:04:35,480 --> 01:04:37,280
THING, BEHIND IT, IT WILL CAUSE

1922
01:04:37,280 --> 01:04:38,800
ALL KINDS OF CHAOS.

1923
01:04:38,800 --> 01:04:43,880
I SUSPECT WE CAN OVERCOME THAT

1924
01:04:43,880 --> 01:04:45,360
BY PERHAPS ONE OR TWO WAYS.

1925
01:04:45,360 --> 01:04:47,360
THE FIRST OF WHICH IS TO PERFORM

1926
01:04:47,360 --> 01:04:49,760
THE REACTIONS UNDER CONDITIONS

1927
01:04:49,760 --> 01:04:51,800
IN WHICH Rad51 LOADING ITSELF

1928
01:04:51,800 --> 01:04:53,760
ON ITS OWN IS COMPROMISED.

1929
01:04:53,760 --> 01:04:55,960
MEANING A LOWER CONCENTRATION

1930
01:04:55,960 --> 01:04:57,160
OF Rad51 THAT IS NECESSARY TO

1931
01:04:57,160 --> 01:05:00,160
GET ON WITH DNA ITSELF UNDER THE

1932
01:05:00,160 --> 01:05:01,560
ASSUMPTION THAT BLM MIGHT HELP

1933
01:05:01,560 --> 01:05:02,480
IT GET ON THERE.

1934
01:05:02,480 --> 01:05:03,680
SO WE ARE DEFINITELY THINKING

1935
01:05:03,680 --> 01:05:04,200
ABOUT THAT.

1936
01:05:04,200 --> 01:05:07,000
AND THEN THE OTHER WAY, AS YOU

1937
01:05:07,000 --> 01:05:08,280
KNOW, THERE IS A NUMBER OF

1938
01:05:08,280 --> 01:05:09,480
DIFFERENT PROTEINS THAT ARE

1939
01:05:09,480 --> 01:05:11,400
INVOLVED IN HELPING TO GET

1940
01:05:11,400 --> 01:05:14,520
RAD51 ON DNA.

1941
01:05:14,520 --> 01:05:17,560
SO MEDIATOR COMPLEXES LIKE

1942
01:05:17,560 --> 01:05:18,640
BRCA2, FOR EXAMPLE.

1943
01:05:18,640 --> 01:05:21,040
OR Rad51 PARALOGS POSSIBLY.

1944
01:05:21,040 --> 01:05:24,160
WE WOULD LOVE TO BE ABLE TO SEE

1945
01:05:24,160 --> 01:05:25,480
COUPLED REACTIONS AND WHERE WE

1946
01:05:25,480 --> 01:05:29,560
ARE DOING THIS PROCESSING.

1947
01:05:29,560 --> 01:05:30,080
PUTTH IN THE MEDIATORS AND

1948
01:05:30,080 --> 01:05:31,760
PUTTING IN Rad51 AND TRYING TO

1949
01:05:31,760 --> 01:05:34,280
SEE WHERE DOES EVERYONE FALL IN

1950
01:05:34,280 --> 01:05:34,480
PLACE.

1951
01:05:34,480 --> 01:05:36,200
WE ARE NOT THERE YET.

1952
01:05:36,200 --> 01:05:38,600
SORRY, MICHAEL.

1953
01:05:38,600 --> 01:05:39,960
>> WE ARE GOING TO TAKE ONE

1954
01:05:39,960 --> 01:05:40,960
FINAL QUESTION BUT BEFORE THAT,

1955
01:05:40,960 --> 01:05:43,320
LET ME REPEAT THE CME CODE FOR

1956
01:05:43,320 --> 01:05:47,440
TODAY FOR THOSE WHO NEED IT IT'S

1957
01:05:47,440 --> 01:05:48,040
37930.

1958
01:05:48,040 --> 01:05:48,920
SO I THOUGHT THE LAST QUESTION

1959
01:05:48,920 --> 01:05:52,600
SHOULD COME FROM A FELLOW LMB

1960
01:05:52,600 --> 01:05:52,840
PERSON.

1961
01:05:52,840 --> 01:05:54,440
ONE OF OUR NEWER INVESTIGATORS

1962
01:05:54,440 --> 01:05:57,040
AT LMB AND HE ASKS, FANTASTIC

1963
01:05:57,040 --> 01:05:58,720
WHAT HE SAYS.

1964
01:05:58,720 --> 01:05:59,440
FANTASTIC TALK, ERIC.

1965
01:05:59,440 --> 01:06:02,080
DO YOU THINK THE SINGLE STRANDED

1966
01:06:02,080 --> 01:06:04,720
DNA FORMS A GIANT SINGLE LOOM OR

1967
01:06:04,720 --> 01:06:05,680
MANY SMALLER LOOPS?

1968
01:06:05,680 --> 01:06:09,280
COULD IT BE FORMING NUMEROUS DNA

1969
01:06:09,280 --> 01:06:10,760
HAIR PINS AS ALONG SINGLE STRAND

1970
01:06:10,760 --> 01:06:13,240
RNA LOOP DOES?

1971
01:06:13,240 --> 01:06:15,240
>> YES, SO THAT'S A GREAT

1972
01:06:15,240 --> 01:06:15,480
QUESTION.

1973
01:06:15,480 --> 01:06:18,640
I DON'T KNOW IF WE MET.

1974
01:06:18,640 --> 01:06:21,320
I HOPE I'M NOT FORGETTING YOU.

1975
01:06:21,320 --> 01:06:22,520
IF NOT, I'M SORRY I COULDN'T

1976
01:06:22,520 --> 01:06:24,320
COME THERE AND MEET YOU.

1977
01:06:24,320 --> 01:06:27,440
SO THOSE ARE TWO DIFFERENT

1978
01:06:27,440 --> 01:06:29,000
THINGS, KIND OF.

1979
01:06:29,000 --> 01:06:30,320
SO EXACTLY AS YOU SAY, YOU COULD

1980
01:06:30,320 --> 01:06:32,240
BE FORMING MULTIPLE SORT OF HAIR

1981
01:06:32,240 --> 01:06:35,720
PINS THAT ARE NOT INVOLVED IN A

1982
01:06:35,720 --> 01:06:37,520
INTERACTION BUT ARE INVOLVED IN

1983
01:06:37,520 --> 01:06:40,720
A DNA-DNA PAIRING INTERACTION.

1984
01:06:40,720 --> 01:06:42,400
OR YOU COULD FORM MULTIPLE

1985
01:06:42,400 --> 01:06:43,400
CONTACTS PERHAPS WITH THE

1986
01:06:43,400 --> 01:06:46,280
RESECTION MACHINERY.

1987
01:06:46,280 --> 01:06:47,000
I DON'T THINK THAT EITHER OF

1988
01:06:47,000 --> 01:06:49,200
THOSE IS TAKING PLACE.

1989
01:06:49,200 --> 01:06:50,080
AND THE REASON I DON'T THINK SO

1990
01:06:50,080 --> 01:06:51,280
IS WHEN WE SEE, THERE IS A

1991
01:06:51,280 --> 01:06:52,080
COUPLE OF THINGS.

1992
01:06:52,080 --> 01:06:54,080
FIRST OF ALL YOU, WHEN WE SEE

1993
01:06:54,080 --> 01:06:55,880
THE LOOPS BREAK AND THAT DNA

1994
01:06:55,880 --> 01:06:58,480
GETS LONG AGAIN, IT TENDS TO

1995
01:06:58,480 --> 01:06:59,680
HAPPEN ALL IN ONE STEP.

1996
01:06:59,680 --> 01:07:02,920
SO THAT TELLS ME THAT WE HAVE

1997
01:07:02,920 --> 01:07:05,280
RUPTURED A SINGLE CONTACT.

1998
01:07:05,280 --> 01:07:06,360
OF COURSE I CAN'T SAY THAT MAYBE

1999
01:07:06,360 --> 01:07:07,800
THERE WERE MULTIPLE CONTACTS AND

2000
01:07:07,800 --> 01:07:09,600
IT'S THE LAST ONE YOU SEE.

2001
01:07:09,600 --> 01:07:12,200
THAT'S ALSO A POSSIBILITY.

2002
01:07:12,200 --> 01:07:14,960
I DON'T THINK HAIR PINS ARE

2003
01:07:14,960 --> 01:07:17,080
FORMING AT LEAST NOT TO AN

2004
01:07:17,080 --> 01:07:18,280
EXCESSIVE AMOUNT AND THE REASON

2005
01:07:18,280 --> 01:07:22,760
FOR THAT IS THAT WE KNOW RPA,

2006
01:07:22,760 --> 01:07:27,080
ONE OF THE PRIMARY ROLLS IN

2007
01:07:27,080 --> 01:07:28,480
THESE REACTIONS LIKE THIS IS TO

2008
01:07:28,480 --> 01:07:29,640
PREVENT HAIR PIN FORMATION.

2009
01:07:29,640 --> 01:07:31,440
SO I'M SURE IF WE DIDN'T HAVE

2010
01:07:31,440 --> 01:07:32,800
RPA THERE, THAT WHOLE THING

2011
01:07:32,800 --> 01:07:35,160
WOULD BE KNOTTED UP LIKE CRAZY

2012
01:07:35,160 --> 01:07:36,760
LIKE YOU WOULD ENVISION IN THE

2013
01:07:36,760 --> 01:07:37,520
RNA MOLECULE.

2014
01:07:37,520 --> 01:07:41,320
BUT WHEN WE HAVE RPA THERE, I

2015
01:07:41,320 --> 01:07:43,920
THINK IT'S PREVENTING THAT FROM

2016
01:07:43,920 --> 01:07:44,560
TAKING PLACE.

2017
01:07:44,560 --> 01:07:48,920
SO HOPEFULLY I ANSWERED YOUR

2018
01:07:48,920 --> 01:07:50,040
QUESTION AND HOPEFULLY I CAN

2019
01:07:50,040 --> 01:07:52,320
MEET YOU SOME DAY TOO.

2020
01:07:52,320 --> 01:07:53,680
>> WE ALL HAVE THAT.

2021
01:07:53,680 --> 01:07:55,720
THAT WOULD BE REALLY NICE.

2022
01:07:55,720 --> 01:07:57,240
SO I THINK IT'S PROBABLY UP TO

2023
01:07:57,240 --> 01:07:59,240
ME TO THANK ERIC AGAIN FOR A

2024
01:07:59,240 --> 01:07:59,720
LOVELY TALK.

2025
01:07:59,720 --> 01:08:01,360
I HAVE QUESTIONS TOO BUT WE

2026
01:08:01,360 --> 01:08:03,920
COULDN'T GET TO EVERYTHING AND

2027
01:08:03,920 --> 01:08:07,880
WE HAVE A FULL TRAINEE SCHEDULE.

2028
01:08:07,880 --> 01:08:08,840
WHICH WAS SUPPOSED TO GIVE ERIC

2029
01:08:08,840 --> 01:08:10,120
A LITTLE BIT OF A BREAK BUT

2030
01:08:10,120 --> 01:08:12,320
THERE IS NOT MUCH OF A BREAK

2031
01:08:12,320 --> 01:08:13,600
LEFT.

2032
01:08:13,600 --> 01:08:14,840
THIS IS A GORGEOUS CITIZEN.

2033
01:08:14,840 --> 01:08:15,680
IT'S SO ELEGANT.

2034
01:08:15,680 --> 01:08:17,440
SO MUCH YOU CAN DO WITH IT.

2035
01:08:17,440 --> 01:08:18,040
-- SYSTEM.

2036
01:08:18,040 --> 01:08:19,200
IT'S GOING TO BE VERY EXCITING

2037
01:08:19,200 --> 01:08:20,440
WHERE THIS GOES FROM HERE.

2038
01:08:20,440 --> 01:08:22,840
SO ON BEHALF OF AL ALL OF US AT

2039
01:08:22,840 --> 01:08:24,320
NIH, WE MISS YOU AND THANK YOU

2040
01:08:24,320 --> 01:08:26,840
VERY MUCH FOR GIVING THIS TALK

2041
01:08:26,840 --> 01:08:27,040
TODAY.

2042
01:08:27,040 --> 00:00:00,000
>> THANK YOU, SUSAN.