WELCOME TO THE WEDNESDAY
AFTERNOON LECTURE SERIES.
I'M SUZANNE BUCHANAN, THE DEPUTY
SCIENTIFIC DIRECTOR FOR THE
NATIONAL INSTITUTED OF DIABETES
AND DIGESTIVE AND KIDNEY
DISEASES.
AND I'M ALSO THE CHIEF OF THE
LABORATORY OF MOLECULAR BIOLOGY
WHERE OUR SPEAKER HAILS FROM.
SO IT'S A REAL PLEASURE FOR ME
TO BE GIVING THIS INTRODUCTION.
AND I'M EXCITED TODAY TO
INTRODUCE OUR SPEAKER, DR. ERIC
GREENE WHO WILL BE DISCUSSING
SINGLE MOLECULE STUDIES OF THE
HOMOLOGOUS RECOMBINATION.
THAT'S ERIC C. GREENE WITH
A FINAL E AND NOT TO BE CONFUSED
WITH THE NHGRI DIRECTOR.
SO, OUR SPEAKER FOR TODAY IS
ERIC GREENE, WHO IS A PROFESSOR
IN THE DEPARTMENT OF
BIOCHEMISTRY AND MOLECULAR
BIOPHYSICS AT COLUMBIA
UNIVERSITY IN NEW YORK CITY.
HIS LAB USES SINGLE-MOLECULE
OPTICAL MICROSCOPY TO STUDY
FUNDAMENTAL INTERACTIONS BETWEEN
PROTEINS AND NUCLEIC ACIDS.
TO OBSERVE INDIVIDUAL PROTEIN
MOLECULES OR PROTEIN COMPLEXES
AS THEY INTERACT WITH DNA
SUBSTRATES.
ERIC'S LAB HAS PIONEERED THE
DEVELOPMENT OF DNA CURTAINS FOR
THIS DIRECT VISUALIZATION OF
HUNDREDS TO THOUSANDS OF
INDIVIDUAL MOLECULES IN REALTIME
BY OPTICAL MICROSCOPY.
TECHNYNIQUE IS BASED ON THE
TECHNIQUE OF NANOFABRICATED
SURFACES, ALLOWING THEM TO BE
VIEWED AS A SINGLE MOLECULE
LEVEL BY TOTAL INTERNAL
REFLECTION FLUORESCENCE
MICROSCOPY.
NOW MANY OF YOU MAY REMEMBER
ERIC WAS A POSTDOC HERE IN THE
LABORATORY OF MOLECULAR BIOLOGY
AT NIDDK FROM ROUGHLY 1999-2003.
I WAS HERE THEN TOO.
AND THAT'S HARD TO BELIEVE IT'S
BEEN 20 YEARS PLUS.
ERIC RECEIVED HIS UNDERGRADUATE
DEGREE IN BIOCHEMISTRY FROM THE
UNIVERSITY OF ILLINOIS.
HE FOLLOWED THAT WITH A PH.D. IN
BIOCHEMISTRY FROM TEXAS A&M THEN
A POSTDOC AT THE NIH.
AFTER LEAVING THE NIH, HE TOOK A
POSITION AT COLUMBIA UNIVERSITY
IN 2004 AS AN ASSISTANT
PROFESSOR WHERE HE HAS BEEN EVER
SINCE BEING PROMOTED TO
ASSOCIATE PROFESSOR IN 2010 AND
FULL PROFESSOR IN 2015.
HE HAS HAD MANY HONORS.
SOME OF THEM ARE A CAREER AWARD
IN 2006, A PRESIDENTIAL
ENGINEERING PKAS AWARD IN 2007.
I SHOULD KNOW, I HAD ONE TOO.
A HOWARD HUGHES MEDICAL
INSTITUTE AWARD FROM 2009-2015.
THE DOCTOR HAROLD RESEARCH AWARD
IN 2009, AND MULTIPLE
INVITATIONS TO BE A KEYNOTE
SPEAKER, NOTABLY RECENTLY AT
GORDON CONFERENCE AT KEYSTONE
SYMPOSIUM.
HE GIVES AN ENGAGING LECTURE.
THE TITLE OF HIS TALK IS SINGLE
MOLECULE STUDIES OF HOMOLOGOUS
RECULMINATION.
WELCOME BACK TO THE NIH, WE WISH
YOU WERE HERE IN PERSON, BUT
THANK YOU VERY MUCH FOR BEING
WITH US TODAY.
>> PERFECT.
THANK YOU, SUSAN.
I ALSO WISH THAT I WAS THERE.
I HAD AGREED TO DO THIS ASSUMING
I WOULD BE ABLE TO COME DOWN AND
VISIT WITH SO MANY FRIENDS.
SO THAT'S A LITTLE BIT OF A
BUMMER.
IT'S ALSO FUNNY THAT YOU BROUGHT
UP THE FACT THAT THERE IS
ANOTHER ERIC GREEN AT NIH AND IT
REMINDED ME OF A STORY WHEN I
WAS A POSTDOC AND SOME LAWYER
CALLED ME AND STARTED TALKING
ABOUT SOME THINGS THAT OBVIOUSLY
HAD NO NEED TO KNOW.
AND I REALIZED I WAS THE WRONG
ERIC GREEN.
SO THAT WAS FUN.
SO, TODAY, I WAS THINKING ABOUT
WHAT IT IS I SHOULD TALK TO YOU
ABOUT AND I DECIDED TO TALK TO
YOU ABOUT SOMETHING THAT IS NOT
PUBLISHED.
SO THE TALK IS A LITTLE ROUGH.
I WAS ORIGINALLY GOING TO SAY
SOMETHING ABOUT OUR RECENT WORK
ON HOMOLOGY RESEARCH BUT I
DECIDED TO TALK ABOUT HOW DNA IS
A PROCESS OR AT LEAST OUR SMALL
PORTION OF THE PUZZLE IN TERMS
OF DNA END PROCESSING.
AND YOU CAN SEE I CHANGED THE
TITLE TO SOMETHING A LITTLE MORE
SPIFFY TALKING ABOUT BLUE
HELICASE.
AND WHAT I'M GOING TO TRY TO
TELL YOU ABOUT IS HOW BLOOM
HELICASE OR SEX DNA ENDS AND I'M
GOING TO TRY TO CONVINCE YOU
IT'S DOING SOMETHING TO THOSE
ENDS THAT LOOKS LIKE IT'S MAKING
SOME KIND OF A LOOP WITH THE
SINGLE STRANDED OVER HANG THAT
IS BEING PRODUCED.
SO WITH THAT, I WILL JUMP RIGHT
IN.
AND I'M GOING TO POINT OUT THE
FOLKS WHO HAVE CONTRIBUTED TO
THIS.
REALLY FROM MY LABORATORY A
FORMER POSTDOC AND HAS HIS OWN
LAB IN CHINA, WILL HAVE DONE ALL
OF THE SINGLE MOLECULE WORK THAT
I'M GOING TO TALK TO YOU ABOUT.
WE HAVE BEEN WORKING CLOSELY
WITH UT SAN ANTONIO AND THEY
HAVE BEEN A MAIN DRIVER OF THE
WORK IN HIS LAB.
ANY TIME I'M SHOWING SOMETHING
THAT IS A LIVE-CELL ASSAY, THAT
HAS NOTHING TO DO WITH US.
AND THEN I SHOULD ALSO MENTION
THOSE WHO CAME IN AT THE END AND
HELPED US WITH STUFF WE ALSO HAD
TO GET DONE IN TERMS OF MAKING
REVISIONS.
AND I'LL TRY TO POINT OUT WHO
AMONG THESE FOLKS HAVE DONE WHAT
AS WE HAVE GONE ALONG.
AND I HAVE TO SAY THANK YOU TO
OUR FUNDING, MOST OF WHICH IS
NIH.
SO THIS IS CHOW, SORT OF IN THE
PANDEMIC AS PREPARING HIS FAMILY
TO MOVE BACK TO CHINA.
AND HE PUBLISHED A NUMBER OF
PAPERS WITH US, AND SOME OF
THESE I'LL BE REFERRING BACK
FROM TIME TO TIME.
SO IF ANYONE WANTS TO SEE THE
FULL REFERENCES, THEY ARE GOING
TO BE LISTED IN THIS SLIDE.
THE KEY ONES HERE ARE GOING TO
BE A STUDY THAT HE DID ON END
RESECTION IN SACCHAROMYCES SIR
VIS YEAH IN A MOLECULAR CELL
PAPER, AS WELL AS A STUDY HE DID
ON HUMAN BLOOM HELICASE, WHICH
I'LL TALK ABOUT IN A MINUTE THAT
WE PUBLISHED IN NUCLEIC ACIDS
RESEARCH.
SO THIS IS HIM AND HE WAS GREAT.
I REALLY MISS HIM.
SO, I'M GOING TO KEEP THE
INTRODUCTION TO THIS FAIRLY
SIMPLE.
I REALLY ONLY HAVE TWO
INTRODUCTORY SLIDES AND THEN
WE'RE GOING TO GET RIGHT INTO
DATA.
AND THEN AT THE END I'LL TRY TO
TELL YOU WHAT WE THINK THE DATA
MEANS IF I HAVEN'T CONVINCED YOU
AS WE GO ALONG.
SO, OVER THE YEARS, MY LAB HAS
WORKED ON A LOT OF STUFF.
I'LL SHOW YOU THE TOOL WE USED
IN THE NEXT SLIDE.
BUT WE HAVE BEEN COMING MORE AND
MORE TOWARDS FOCUSING ON DNA
HOMOLOGOUS RECOMBINATION.
WHEN I STARTED MY LAB, THIS IS
WHAT WAS WE WERE GOING TO BE
WORKING ON.
AS WE DEVELOPED TECHNIQUES AND
REALIZED WE CAN APPLY THESE TO
LOTS OF SYSTEMS, THAT'S WHAT WE
DID.
BUT WE HAD SOME BREAKTHROUGHS
THAT ALLOWED US TO COME BACK TO
RECOMBINATION IN WAYS THAT I
THINK HAVE BEEN VERY EXCITING
FOR THE GROUP.
SO THIS IS SORT OF THE
BIOPHYSICS OVERVIEW OF DNA
RECOMBINATION MY GENETICS
COLLEAGUES LOVE THIS.
SO WE HAVE A DOUBLE STRAND DNA
MOLECULE, A LITTLE LIGHTNING
BOLT COMES DOWN AND DAMAGES IT
AND THAT CAN DO ALL KINDS OF
THINGS THAT WILL DO THIS IT TO
GENERATE A DOUBLE-STRANDED
BREAK.
AND FOR HOMOLOGOUS
RECOMBINATION, THAT BREAK HAS TO
BE PROCESSED.
IN FACT, THE PROCESSING IS WHAT
WE ARE GOING TO BE TALKING ABOUT
TODAY.
SO WHAT HAPPENS IS YOU GENERATE
A LONG 3-PRIME OVERHANG AND THIS
OVERHANG SERVES AT PLATFORM FOR
THE FORMATION OF A PRESYNAPTIC
COMPLEX AND THAT WILL BE IN
HUMANS, A FILAMENT OF A PROTEIN
CALLED Rad51.
SAME PROCESS HAPPENS IN EVEN
BACTERIA.
THE DIFFERENCE IS Rad51 IS NOT
THERE.
IT'S THE PROTEIN CALLED -- AND
THEY ARE CLOSELY RELATED
PROTEINS.
THE PROCESS IS HIGHLY CONSERVED
ACROSS BIOLOGY.
NOW ONCE YOU HAVE RESECTED THESE
ENDS AND YOU ASSEMBLE YOUR
RECOMBINASE FILAMENT, THAT
FILAMENT THEN HAS TO SEARCH THE
GENOME TO FIND ANOTHER PIECE OF
DNA DEPICTED AS A RED MOLECULE,
THAT IT CAN PAIR THE BLACK DNA
WITH.
ONCE THAT PAIRING TAKES PLACE,
YOU CAN THEN START TO DO DNA
SYNTHESIS AND FILL IN
INFORMATION THAT WAS LOST DURING
THESE STAGES AND THEN THIS
INTERMEDIATE CAN BE CHANNELED
THROUGH A NUMBER OF INTERRELATED
PATHWAYS, THE MOST SIMPLE OF
WHICH IS TO HAVE THIS
INTERMEDIATE DISRUPTED.
THIS REALLY SYNTHESIZED STRAND
SIMPLY PAIRS WITH THE SECOND
OVERHANG AND YOU GET
RELYINGATION OF A STRAND AFTER
THIS PROCESS.
THAT'S THE SIMPLEST.
BUT THERE ARE OTHER WAYS TO DO
THIS AS WELL.
I'M NOT GOING TO GO OVER THOSE
BECAUSE WE ARE NOT GOING TO BE
TALKING ABOUT THEM IN DEPTH.
SO NOW RECOMBINATION IN AND OF
ITSELF IS IMPORTANT FOR
MAINTAINING GENOME INTEGRITY AND
ESSENTIALLY EVERY ORGANISM.
IT'S INTIMATELY INVOLVED IN THE
REPAIR OF DNA DAMAGE AND OF
COURSE IN CANCER BIOLOGY.
SO I'M NOT REALLY SURE WHAT THE
MAKEUP OF THE AUDIENCE IS RIGHT
NOW BUT MANY OF YOU WILL HAVE
HEARD OF THE PROTEINS BRCA1 AND
BRCA2, CANCER-ASSOCIATED
ONCOGENES.
AND THESE GUYS, WHEN THEY ARE
MUTATED, YOU HAVE A HIGH
PROPENSITY TO HAVE HEREDITARY
BREAST OR OVARIAN CANCERS.
YOU'RE MESSING UP THIS PORTION
OF THE REACTION WHERE YOU'RE
TRYING TO ASSEMBLE A FILAMENT.
SO THERE IS AN INTIMATE
RELATIONSHIP BETWEEN CANCER
BIOLOGY AND HOMOLOGOUS
RECOMBINATION.
AND IN FACT, MUTATIONS AND
PROTEINS THAT HAPPEN ALONG THIS
PATHWAY ARE OFTEN ASSOCIATED
WITH CANCER-PRONE SYNDROMES.
ONE OF THE PROTEINS WE WILL TALK
ABOUT TODAY IS JUST LIKE THIS.
AND OF COURSE, DEFECTS CAN LEAD
TO CHROMOSOMAL REARRANGEMENTS,
CHANGES IN COPY NUMBER, LOTS OF
BAD THINGS.
AND I SHOULD NOTE ALSO THAT
RECOMBINATION IS INTIMATELY
INVOLVED IN THE RESCUE OF
STALLED OR DAMAGED OR COLLAPSED
REPLICATION FORKS.
SO THIS IS REALLY ESSENTIAL FOR
THAT.
IT'S ALSO INVOLVED IN HORIZONTAL
GENE TRANSFER.
SO IF YOU'RE DOING SOME SORT OF
CHRIS PER TYPE OF THINGS,
CHANCES ARE YOU'RE RELYING ON
THE COMBINATION TO INSERT A GENE
SOMEWHERE.
AND THEN OF COURSE FOR THE FOLKS
INTERESTED IN MEIOSIS, THIS IS
THE PROCESS BY WHICH GENETIC
DIVERSITY IS ESTABLISHED IN
MEIOSIS THROUGH RECOMBINATION
BETWEEN DIFFERENT DNAs.
NOW, WE HAVE STUDIED REACTIONS
ALONG THIS PATHWAY IN A NUMBER
OF DIFFERENT REACTIONS.
A LOT OF WHICH HAVE FOCUSED ON
THE ASSEMBLY OF THESE
NUCLEOPROTEIN FILAMENTS ON THESE
PROCESSED DNA ENDS AS WELL AS
THE PAIRING PROCESSES THAT TAKE
PLACE TO GET YOUR BLACK DNA
PAIRED WITH YOUR RED DNA.
ED THAT I'M GOING TO TALK ABOUT
IS HOW YOU GO FROM THIS DOUBLE
STRAND BREAK TO A PROCESSED
DOUBLE STRAND BREAK WHERE YOU
RESECTED THESE ENDS BACK.
AND I SHOULD POINT OUT THAT I'M
GOING TO TALK ABOUT THREE
PROTEINS.
BLOOM HELICASE, DNA2 AND RPA.
I'LL TELL YOU WHAT THEY ARE IN A
MOMENT.
THE UNDERSTANDING WE ARE USING A
VERY SIMPLIFIED SYSTEM TO TRY TO
LOOK AT WHAT WE THINK ARE SORT
OF KEY ASPECTS OF THE REACTION
AND IN REALITY, INSIDE OF CELLS,
THIS IS GOING TO INVOLVE A
LARGER NUMBER OF PROTEINS THAT
WE HOPE TO EVENTUALLY BE ABLE TO
LOOK AT IN OUR RECONSTITUTED
SYSTEM.
BUT FOR THE TIME BEING, WE ARE
LOOKING AT A MINIMAL SYSTEM.
SO NOW, BLOOM HELICASE, THE
FIRST OF THESE GUYS, AND YOU'LL
HEAR THESE THREE NAMES
THROUGHOUT -- IS A HELICASE,
WHEN HAVE YOU DEFECTS IN IT, YOU
END UP WITH A CANCER-PRONE
SYNDROME, BLOOM SYNDROME, THAT
IS CHARACTERIZED BY REALLY
PROFOUND DNA REPAIRED DEFECTS
AND CHROMOSOMAL REARRANGEMENTS.
SO THE FOLKS OR THE PATIENTS IN
HAVE THIS END UP HAVING CANCER
AT A VERY, VERY YOUNG AGE,
UNFORTUNATELY.
NOW THE SECOND PROTEIN THAT I'M
GOING TO TALK ABOUT IS DNA2.
IT IS ALSO A HELICASE.
IT'S NOT QUITE AS ROBUST AS BLM
BUT IT'S ALSO A NUCLEASE.
DNA2 IS WHAT IS RESPONSIBLE FOR
CUTTING OUT THIS OTHER STRAND OF
DNA TO LEAVE A 3-PRIME OVERHANG.
AND THEN RPA IS A HETEROTRIMERIC
SINGLE STRANDED BINDING PROTEIN.
THIS IS A PROTEIN THAT IS
ESSENTIAL EVOLVED IN ANYTHING
INVOLVING SINGLE STRANDED DNA
INSIDE OF A EUKARYOTIC CELL.
IT'S HIGHLY ABUNDANT PROTEIN AND
UBIQUITOUS.
IT'S INVOLVED IN EVERYTHING.
SO THESE THREE PLAYERS ARE WHAT
WE ARE GOING TO TALK ABOUT IN
TERMS OF GENERATING THIS PROCESS
DNA END.
NOW, JUST SO EVERYBODY IS
ONBOARD WITH THE TECHNOLOGY THAT
WE WILL BE TALKING ABOUT, THIS
IS WHAT WE CALL DNA CURTAINS.
ESSENTIALLY WE HAVE A FLOW CELL
THAT WE MAKE WITH AN IN LET AND
OUT LET CHAMBER, A SAMPLE
CHAMBER IN BETWEEN THAT IS MADE
WITH FANCY DOUBLE SIDED STICKY
TAPE, IF YOU CAN BELIEVE THAT.
AND THEN WHAT IS REALLY SPECIAL
ABOUT WHAT WE ARE DOING IS WE
ARE MODIFYING THE SURFACE OF
THIS CHAMBER.
AND EXACTLY WHAT WE'RE DOING IS
PUTTING DOWN PATTERNS OF METAL,
METALLIC BARRIERS.
I'M SHOWING YOU A BLOW UP,Y AND
MAT TIESED BLOW UP OF A
ZIGZAG-SHAPED PATTERN MADE OF
CHROME THAT WE DEPOSIT ON TO
THAT SURFACE.
I'M SHOWING YOU ONE BUT WE PUT
THESE ALL OVER THE PLACE IN
HERE.
AND WHAT WE THEN DO IS TAKE THIS
SURFACE, THIS GLASS SURF AS AND
COVER IT WITH A FLUID LIPID
BILAYER.
IT'S SERVING TWO PURPOSES.
THE FIRST IT'S GOING TO ENSURE
THAT THIS SURFACE IS AS INERT AS
POSSIBLE IN TERMS OF US PUTTING
PROTEINS INTO THESE CHAMBERS.
SO, PROTEINS LOVE TO STICK TO
GLASS.
IF YOU WERE TO JUST INJECT ANY
PROTEIN WE WORK WITH INTO A
GLASS CHAMBER, IT WOULD SIMPLY
COAT THE SURFACE AND THAT WOULD
BE THE END OF YOUR EXPERIMENT.
YOU WOULDN'T BE ABLE TO TELL
ANYTHING.
SO THE BILAYER IS OUR EFFORT TO
TRY TO MAKE THIS CHAMBER SURFACE
LOOK MORE LIKE THE INSIDE OF A
LIVING CELL THAN GLASS.
A SUBSET OF THE LIPIDS WITHIN
THE BILAYER HAVE A BY TIN GROUP
AND WE COUPLE DNA MOLECULES
THROUGH THAT SURFACE.
THE LIPID BILAYER IS FLUID IN
TWO DIMENSIONS.
WHEN WE ATTACH THINGS TO IT,
THEY CAN DIFFUSE AROUND ATTACH
TO THE SURFACE.
WHAT WE DO THEN IS APPLY A FLOW
FORCE, HYDRODYNAMIC FORCE THAT
PUSHES THE DNA INTO THIS
BARRIER.
SO THE DNA IS ACTING ESSENTIALLY
LIKE A SAIL ON A SHIP.
IT'S PUSHING THEM INTO THE
BARRIER.
THEY THEN LAY DOWN PARALLEL TO
THE SURFACE AND THEN WE'RE
ILLUMINATING THEM WITH TOTAL
INTERNAL REFLECTION,
FLUORESCENCE MICROSCOPY.
SO ILLUMINATING A SMALL
EXCITATION VOLUME NEAR THE
SURFACE WITHIN THE VOLUME WHERE
THESE DNA MOLECULES RESIDE.
WE HAVE MADE MANY FLAVORS OF
THESE CURTAINS, MANY DIFFERENT
STYLES.
THE ONES I'M GOING TO BE TALKING
ABOUT TODAY ARE AS SHOWN HERE,
MEANING ONE END OF THE DNA IS
ATTACHED AND ONE END IS FREE.
THIS IS WHAT WE WOULD CALL A
SINGLE AND TETHERED DNA CURTAIN.
FOR ANYONE THAT WANTS ANY
ADDITIONAL INFORMATION, WE
PUBLISHED MANY, MANY METHODS,
PAPERS, DESCRIBING THIS WORK.
AND WE HAVE ALSO PUBLISHED A
NUMBER OF PAPERS DESCRIBING
STUDIES THAT HAVE COME OUT OF
THE USE OF THESE TYPES OF
THINGS.
SO THAT IS IT FOR THE
INTRODUCTION.
I WANT TO MAKE ONE MORE POINT
WHILE I'M THINKING ABOUT IT.
IN THE SUBSEQUENT SLIDES I WILL
BE SHOWING YOU CYMOGRAPHS.
AND THEY ARE SINGLE -- SO THE
DATA WE COLLECT IS VIDEOS.
VIDEOS OF THESE MOLECULES
INTERACTING.
AND THE CYMOGRAPHS ARE SINGLE
ONE PIXEL SLICES THROUGH
INDIVIDUAL DNA MOLECULES THAT
ARE THEN PROJECTED THROUGH TIME.
AND I'LL TRY TO EXPLAIN THAT
MORE CAREFULLY IN THE FIRST
CYMEO GRAPH SO WE ARE ALL
ONBOARD WITH UNDERSTANDING HOW
THE EXPERIMENT WORKS.
SO NOW THIS IS AN EXAMPLE
SHOWING A SCHEMATIC, SORE
ESHOWING THE SUBSTRATE WE WILL
BE WORKING WITH.
THIS IS OUR DNA CURTAIN FOCUSING
ON THE END OF THE MOLECULE.
WHAT WE HAVE DONE IS ENGINEERED
THAT SO WE HAVE 30 NUCLEOTIDE
SINGLE STRANDED DNA OVERHANG.
THE INTENTION IS TO MIMIC A
PARTIALLY-PROCESSED
INTERMEDIATE.
I MENTIONED EARLIER THERE WERE A
NUMBER OF PROTEINS THAT COME
INTO THESE REACTIONS THAT WE'RE
NOT LOOKING AT.
ONE IS CALLED THE MRN COMPLEX.
MRE11, Rad50MBS1T GENERATES A
SINGLE STRAND OVERHANG AND THEN
WHAT HAPPENS ON THAT OVERHANG IS
WE CAN LOAD OUR REACTIONS
CONTAINING BLM, RadAND DNA2.
AND I'LL POINT OUT WE PUBLISHED
SIMILAR WORK WITH THE YEAST
RESECTION SYSTEM USING THESE
SAME SUBSTRATES.
NOW THIS IS A WIDE FIELD IMAGE
OF DNA CURTAIN.
THE DNA IS NOT LABELED SO YOU DO
NOT SEE IT.
AND WHAT YOU DO SEE IS THIS B
AND THIS E.
SO THE B SILENT BARRIER.
THAT'S WHERE WE ALIGNED OUR DNA
MOLECULES.
AND THE E IS THE END OF THE DNA.
SO THE FREE END OF THE MOLECULE.
AND WHAT WE ARE LOOKING AT THEN
IS BLOOM HELICASE BOUND TO THAT
END.
SO BLOOM IS LABELED IN THIS CASE
WITH GFP.
WE HAVE BUFFER FLOWING FROM
TOP-TO-BOTTOM.
I'M SURE YOU CAN APPRECIATE JUST
FROM THE IMAGE THAT YOU CAN SEE
THESE GREEN SPOTS AND MOST OF
THEM SEEM TO BE COALIGNED WITH
WHAT WE SAY IS THE END OF THE
DNA.
AND THIS IS JUST TAKING A SINGLE
IMAGE.
THIS IS NOW LOOKING AT THAT DATA
SORT OF IN AGGREGATE WHERE N IS
176 MOLECULES AND THE BARS
REPRESENT THE LOCATIONS OF THE
GFP BLOOM COMPLEX.
IN MOST OF THE REACTIONS WE DO,
WITH SOME EXCEPTIONS AND I'LL
SHOW YOU THESE LATER, THE BLOOM
IS ACCUMULATING AT DNA END.
OF COURSE WE DO SEE SOME BOUND
INTERNALLY.
I'M GOING TO MENTION, THEY
BEHAVE DIFFERENTLY.
I'LL TALK ABOUT THAT A LITTLE
BIT.
SO MOST OF THE PROTEINS GO TO
THE END.
THEY TEND TO STAYED THERE.
SO ONCE THEY ARE BOUND, THEY
HAVE HALF-LIVES THAT EXCEED OUR
EXPERIMENTAL MEASUREMENTS UNDER
ALL THE CONDITIONS THAT WE HAVE
TESTED.
AND I NOTE HERE THAT THEY ARE
RESISTANT TO THIS PROTEIN, RPA
AND I MAKE THAT NOTE BECAUSE WE
DID A SIMILAR STUDY WITH THE
YEAST SYSTEM AND PUT THE
PROTEINS SGS1 HERE AS SOON AS WE
INJECT RPA, IT KICKS SGS1 OFF.
IT'S AN INTERESTING DIFFERENCE
THAT WE DON'T FULLY UNDERSTAND
YET.
BUT WE DO KNOW NOW THAT BLOOM
GOES TO THE DNA ENDS.
SO NOW THIS IS OUR FIRST EXAMPLE
OF A CYMEO GRAPH.
AGAIN WHAT I'M SHOWING YOU TO
JUMP BACK IS GOING TO BE A
SINGLE ONE PIXEL SLICE THROUGH
ONE OF THESE DNAs AND SHOWING
THE END-BOUND COMPLEX.
AND THEN PROJECTED OVER TIME.
SO WHAT YOU CAN SEE IS THAT THAT
MOLECULE OR THAT GREEN SPOT,
SUPPORTS MOVE UP.
SO IT'S MOVING UP AGAINST THE
DIRECTION OF THE BUFFER FLOW
FROM E TO NOW THE B DIRECTION.
AND I'M GOING TO POINT OUT THERE
IS LITTLE FLUCTUATIONS IN THIS
MOLECULE, ESSENTIALLY WHAT IS
HAPPENING HERE IS THAT DNA IS
FREE.
WE DON'T HAVE A TREMENDOUS
AMOUNT OF DYNAMIC FORCE EXERTOD
IT SO IT'S SORT OF FLIPPING
BACK-AND-FORTH IN THE BUFFER.
NOT THINGS THE ENZYMES ARE
DOING, IT'S SOMETHING THE DNA IS
DOING ON A MACROSCOPIC SCALE.
AND SO WE CAN MAKE MEASUREMENTS
BASED ON, HERE IS WHERE IT
STARTED, HERE IS WHERE IT ENDED.
WE KNOW THE TIMEFRAME.
THIS IS A SCALE.
WE CAN MAKE MEASUREMENTS OF HOW
FAR DID IT GO AND HOW FAST DID
IT GO.
I'M JUST GOING TO POINT OUT, WE
DID A CONTROL HERE WHERE WE
INJECTED YO-YO 1 AT THE END OF
THE REACTION AND YO-YO 1 IS AN
IN TECH LATING DYE THAT LET'S US
SEE THE DNA.
AND THIS COM FIRMS THE DNA IS
THERE AND CONFIRMINGS THE DNA IS
SHORTER THAN IT WAS WHEN IT
STARTED SO WE KNOW RESECTION IS
TAKING PLACE.
WE ALSO KNOW THAT THIS RESECTION
IS DEPEND ENTER ON DNA2 AND
REMEMBER DNA2 IS THE NUCLEASE
INVOLVED IN THIS REACTION.
AND WE SEE NO MOVEMENT OR NO
RESECTION IF WE USE A BLOOM
MUTANT WHERE THE ACTIVE SITES
INVOLVED IN ATP BINDING, THE
ACTIVE SITE IS MUTATED FROM A
LYSINE TO ANALLINE.
SO I WANT TO POINT OUT A COUPLE
OF DETAILS.
WE CAN MEASURE VELOCITY AND
DISTANCE, HOW FAST AND FAR FAR
DOES IT GO?
IN THE ABSENCE OF RPA, WE ARE
GOING ABOUT 10 BASE PAIRS PER
SECOND.
OVER DISTANCES OF ABOUT 7KB.
IT'S NOT TREMENDOUSLY FAST.
WE WORK WITH OTHER MOTOR
PROTEINS, OTHER HELICASES THAT
REALLY DO GO FAST.
1000 BASE PAIRS PER SECOND.
SO THIS GUY IS PRETTY SLOW.
IT'S NOT UNEXPECTED.
THE ADDITION OF RPA ACTUALLY
STIMULATES BOTH THE VELOCITY AND
THE -- ON THE ORDER OF 50%.
AND I'M GOING TO POINT OUT, I
MENTIONED EARLIER THAT ROUGHLY
60-70% OF THE PROTEINS ARE BOUND
AT THE END.
OTHERS ARE BOUND IN INTERNAL
SITES.
THE INTERNAL-BOUND BLM COMPLEXES
ARE MUCH FASTER.
THEY ARE MOVING AT ABOUT 100
BASE PAIRS PER SECOND.
SO SOMETHING ABOUT HAVING BLOOM
AT THE END OF THE DNA IS
ACTUALLY CAUSING IT TO GO MUCH
SLOWER.
AND I WANT TO COMMENT BRIEFLY ON
WHAT FRACTION OF PROTEINS ARE
ACTUALLY SHOWING THIS TYPE OF
MOVEMENT BEHAVIOR.
SO IF WE ONLY HAVE BLM IN THE
REACTION, IT'S ABOUT 7.4%.
AS SOON AS WE ADD DNA TWO, OR
RPA, IT'S PARTNER PROTEINS, WE
GO FROM ON THE ORDER OF 7% UP TO
20-25%.
SO WE GET A BIG JUMP IN THE
EFFECTIVENESS OF THE REACTION OF
THE FRACTION OF ACTIVE PROTEINS
BY ADDING ITS PARTNERS.
AND IF WE ADD BOTH OF THOSE GUYS
TOGETHER, THAT'S WHEN WE
ACTUALLY GET THE MOST ROBUST
ACTIVITY WHEN WE ARE STARTING TO
APPROACH 40% ACTIVE.
I'M GOING TO POINT OUT THERE WAS
A PAPER IN MOLECULAR CELL BY A
GUY NAMED MICHAEL SONIA, AND
THEY SEE VERY, VERY SIMILAR
BEHAVIORS IN TERMS OF BLM AND
ITS RESPONSE TO THE ADDICTION OF
PARTNER PROTEINS.
-- ADDITION OF PARTNER PROTEINS.
WHAT WE WANTED TO KNOW IS, WHAT
IS THE STATUS OF THE SINGLE
STRANDED DNA THAT THIS THING
SHOULD BE PRODUCING?
SO AGAIN, BECAUSE WE ARE LOOKING
AT GFP-BLM, WE ARE NOT ACTUALLY
LOOKING AT THE DNA AND WE CAN'T
SEE WHAT IS GOING ON.
SO TO TRY TO DO THIS, WHAT WE IF
WAS TAKE RPA, THE SINGLE
STRANDED BINDING PROTEIN, AND
FUSED IT TO mCherry SO WE
COULD WATCH IT BINDING TO THE
DNA END AS IT WAS PRODUCED.
IN THESE REACTIONS WHAT WE TEND
TO SEE IS AT THE BEGINNING WE
DON'T GET RPA BINDING BECAUSE
THERE ISN'T MUCH SINGLE STRANDED
DNA THERE.
AND THEN OVER TIME, THAT RPA
MCherry SIGNAL STARTS TO
INCREASE AND WE CAN SHOW THAT
100% OF THE MOLECULES THAT ARE
MOVING SHOW INCREASE IN RPA
BINDING THAT SCALES
PROPORTIONALLY WITH TIME AND
DISTANCE.
NOW WHEN WE FIRST STARTED DOING
THIS, WE NOTED SOMETHING QUITE
INTERESTING.
AND THAT IS, WE THOUGHT WE WERE
GOING TO BE MAKING A 3 PRIME
OVERHANG THAT SHOULD BE
EXTENDED.
BUT WHAT WE SEE IS THE RPA KEEPS
BUILDING AND BUILDING BUT
INSTEAD OF SHOWING AN EXTENDED
END, IT SEEMS TO BE TRACKING
VERY CLOSELY IN SPACE WITH THE
GPP-BLM, THAT IS SHOWN HERE IN
THIS MERGED IMAGE WHERE YOU CAN
SEE THE WHITE COLOR FROM THE
BLOOM AND THE MAGENTA SORT OF
TRACKING VERY, VERY CLOSELY WITH
IT.
AND WE SEE THIS IN 100% OF THE
CASES THAT WE LOOKED AT.
IN FACT, WE ALSO SAW THIS WITH
THE YEAST SYSTEM AND I THINK
THAT MICHAEL SAW THIS IN HIS
MOLECULAR CELL PAPER AS WELL.
NOW THE DIFFERENCE IS, IN THIS
SYSTEM, WHAT WE ALSO SAW WAS
OCCASIONALLY THESE THINGS WOULD
GET MUCH, MUCH LONGER.
AND THAT HAPPENED IN ABOUT 30%
OF THE CASES.
SO, WHEN WE SEE SOMETHING LIKE
THIS WHERE A DNA MOLECULE GOES
FROM A SHORTENED LIFE TO
SOMETHING THAT IS MUCH, MUCH
LONGER THAN IT WAS JUST IN THE
FRAME PRIOR, THAT SAYS TO US
THAT SOMEHOW THIS DNA WAS
COMPACTED INTO A STRUCTURE THAT
THEN OPENED UP VERY, VERY
QUICKLY TO LEAD TO THIS LONGER
OVERHANG.
WE KNOW THIS IS SINGLE STRANDED
BY VIRTUE OF THE BINDING OF
SINGLE STRANDED BINDING SPECIFIC
RPA.
AND SO WHAT THIS TOLD US IS THAT
MAYBE THIS DNA IS FORMING A LOOP
THAT IS SOMEHOW TRACKING WITH
GFP BLOOM AS THE PROCESS IS
GOING ON.
NOW, WHAT WE WANTED TO ASK NEXT
WAS WHAT IS GOING ON WITH THE
ACTUAL ENDS OF THE DNA
THEMSELVES?
AND SO WHAT THEY DID IS MAKE A
SUBSTRATE WHERE WE PUT A SINGLE
565 DYE EITHER ON THE 3 PRIME
END OF OUR INITIAL SUBSTRATE OR
ON THE 5 PRIME END.
HE SHOWED THEN THAT BLM BINDS TO
THESE ENDS.
YOU CAN SEE IT HERE AND SEE IT
IN THE DISTRIBUTION HIST GRAMS
AND WE CAN SEE THE END OF THE
DNA THEMSELVES NOW RATHER THAN
RELYING UPON RPA TO SEE WHERE
THINGS ARE GOING.
WE KNOW NOW THIS SYSTEM WORKS.
WHAT WE DID IS START TRACKING
THE ENDS DURING THE PROCESSING
REACTION.
SO THIS IS A SCHEMATIC OF THE
REACTION AS WE ANTICIPATE IT
MIGHT BE TAKING PLACE.
SO, THIS IS OUR SUBSTRATE.
GFP-BLM.
OUR DNA2 MOLECULE AND RPA.
WE THOUGHT MAYBE IT WILL DO
SOMETHING LIKE THIS WHERE THE 5
PRIME END IS CLEAVED.
THIS IS WHAT WE ANTICIPATED.
THE 5 PRIME END SHOULD GET CUT
OFF.
OR IN PRINCIPLE IF IT WASN'T
GETTING CLEAVED, MAYBE BOTH ENDS
TRACK WITH THE COMPLEX AND
SOMETHING FUNNY WAS GOING ON IN
TERMS OF BOTH OF THEM FORMING A
LOOP.
I SHOULD EMPHASIZE IN THIS
DIAGRAM, THIS IS WHAT WE THINK
OF AS DNA LOOP THAT MIGHT BE
FORMING.
SO THIS IS WHAT HAPPENED IN
ABOUT 87.5% OF THE CASES.
SO THIS IS GFP-BLM MOVING.
THIS FAINT SIGNAL IS NOW OUR
5-PRIME ADD 0565.
AND RIGHT HERE, RIGHT WHEN YOU
GET READY TO START MOVING, IT
GETS CUT OFF AND JUST SIMPLY
DISAPPEARS FROM VIEW.
SO THIS IS CONSISTENT WITH WHAT
WE THOUGHT MIGHT BE TAKING
PLACE.
NOW I'M GOING TO POINT OUT THAT
IN ABOUT 12 1/2% OF THE CASES WE
SEE SOMETHING DIFFERENCE AND
THAT IS THE GFP-BLM STARTS
MOVING AND THE 5 PRIME DYE ALSO
STARTS TRACKING WITH IT.
AND SO THIS TYPE OF OBSERVATION
IS CONSISTENT WITH BOTH ENDS OF
THE MOLECULE BEING BOUND SOMEHOW
AND FORMING A LOOP AS THIS THING
IS PROGRESSING ALONG THE DNA.
NOW ONE THING THAT WE CANNOT
RULE OUT IN THIS, AND THIS IS
WHAT I THINK IS PROBABLY
HAPPENING -- I SUSPECT THAT WHEN
WE SEE THE 5 PRIME END TRACKING
WITH THE RESECTION MACHINERY, WE
ARE PROBABLY MISSING THE DNA2
ENZYME.
SO I'M ASSUMING THAT IT'S NOT
THERE AND THAT'S WHY THE DNA IS
NOT GETTING CUT.
I'M ASSUMING THAT BUT I DON'T
KNOW IT YET.
WHAT WE REALLY WANT TO DO IS
MAKE A GFP TAG DNA2 AND START
TRACKING THAT MOLECULE AS WELL.
SO, THE VAST MAJORITY OF THE
CASES, THIS IS WHAT WE SEE.
5 PRIME END IS CUT OFF.
3 PRIME END WE ASSUME NOW IS THE
THING MAKING THE LOOP.
NOW, THIS IS TO TEST THAT EVEN
FURTHER N THIS CASE, WE ARE
LABELING THE 3 PRIME END WITH
OUR SINGLE DYE AND THIS IS WHAT
WE THOUGHT MIGHT TAKE PLACE.
IF THE 3 PRIME END IS TRACKING,
WE SHOULD SEE IT FOLLOWING THE
RESECTION WHICH WE ARE TRACKING
BY VIRTUE OF THIS BLM AND IF THE
LOOP IS RELEASED THEN WE SHOULD
SEE THAT DYE SUDDENLY MOVE
BACKWARDS.
AND SO, I POINT OUT 100% OF OUR
OBSERVATIONS THAT 3-PRIME END
DOES TRACK WITH THE RESECTION
MACHINE.
SO THE MAGENTA DYE HERE, IT
COLOCALIZES WITH THE GFP AND THE
TWO OF THEM MOVE TOGETHER ALONG
THE DNA MOLECULE AS THEY ARE
GOING.
NOW THIS IS DIFFERENT THAN WHAT
I SHOWED YOU BEFORE.
IN THIS EXPERIMENT, 99% OF THEM
SHOWED NO EVIDENCE OF SUDDEN
RELEASE.
THIS IS THE ONE THAT DID.
AND SO WE SEE THAT THE DYE GOES
FROM THIS POSITION ALL OF A
SUDDEN DOWN TO THIS POSITION AND
THAT MOVEMENT COINCIDED WITH
BLM, THE MOTION OF BLM STOPPING.
WE DON'T KNOW IF THAT'S
COINCIDENT OR NOT.
IN THE PREVIOUS EXAMPLES I SAID
SOMETHING LIKE 30% DID THIS.
HERE WE ARE ONLY SEEING 1% OR
LESS THAN 1%, I SHOULD SAY.
AND I'M GOING TO SAY THE
DIFFERENCE BETWEEN THIS
EXPERIMENT AND WHAT I SHOWED YOU
BEFORE IS THAT THESE REACTIONS
ARE INTENTIONALLY DONE WITH
UNLABELED WILDTYPE RPA.
THAT'S WHY WE CAN SEE THE END OF
THE DNA.
IF WE TRY TO LABEL RMA, THE
SIGNAL IS TOO BRIGHT TO SEE THE
DNA ITSELF.
I SUSPECT THE RELEASE OF THE
LOOP WE ARE SEEING IN REACTIONS
WITH FLUORESCENT RPA MIGHT BE
CAUSED BY HAVING A GFP OR AN
MCherry ON THE RPA ITSELF.
SO IT'S SORT OF A FORTUITOUS
DISCOVERY THAT WAS MADE IN TERMS
OF THIS LOOPING JUST BECAUSE
THERE IS SOME PARTIALITY DEFECT
WITH HAVING A GFP TAG ON THE
RPA.
IF WE DON'T HAVE IT, MOST DON'T
GET RELEASED.
SO WE THINK IT IS LOOPING.
WE THINK OUR DITA ARE MOST
CONSISTENT WITH A MODEL, WITH A
5 PRIME END THAT GETS CUT OFF,
THE 3 PRIME END TRACKS THEM WITH
THE RESECTION MACHINERY AS IT
MOVES ALONG.
SO NOW WE WANT TO ASK, WHAT
PROTEIN COMPONENT MIGHT BE
INVOLVED IN THIS?
IS IT BLM OR MIGHT IT BE DNA2?
AND SO WHAT WE DID TO TEST THIS,
OR WHAT CHOW DID WAS PERFORM THE
SAME REACTIONS IN THE ABSENCE OF
DNA2.
AND SO THIS IS AN EXAMPLE
SHOWING NO THREE PRIME END
RELEASE.
THIS IS AN EXAMPLE SHOWING SOME
3 PRIME END RELEASE.
SO WHAT CAN YOU SEE IS THAT WITH
JUST BLM AND RPA, THE RPA
COLOCALIZES WITH THE BLM AS IT
IS MOVING ALONG PROCESSING THE
DNA END AND IN THIS CASE, YOU DO
GET AN EXAMPLE OF SUDDEN
RELEASE.
NOW, AGAIN 100% OF THE SUB
STRAITS THAT WE LOOK AT SHOW
EVIDENCE FOR THIS TYPE OF LOOP
FORMATION.
42.5% IN THIS CASE REMAIN
INTACT.
THE REMAINDER SHOW EVIDENCE OF
THIS SUDDEN RELEASE.
I SHOULD POINT OUT THESE ALSO,
WHEN WE EMIT DNA2, THE REACTIONS
ARE GOING FASTER IN THIS CASE
THAN THEY WERE BEFORE.
AGAIN, WE THINK DNA2 IS ACTUALLY
STARTING TO SLOW THINGS DOWN A
BIT AS THE REACTIONS ARE GOING.
SO WE THINK IT LOOPS.
WE THINK BLOOM IS PRIMARILY
RESPONSIBLE FOR THAT.
SO WE WANTED TO TRY TO
INVESTIGATE WHAT PART OF BLOOM
MIGHT BE INVOLVED.
SO THIS IS SORT OF MY SIMPLIFIED
DIAGRAM OF WHAT BLOOM LOOKS
LIKE.
THAT IS WE HAVE AN ATP ACE
HELICASE CORE.
WE HAVE AN EXTENDED INTERNAL
DOMAIN, A LIGGERRIZATION.
IT'S ALSO INVOLVED IN A NUMBER
OF PROTEIN-PROTEIN INTERACTIONS
INCLUDING INTERACTIONS WITH RPA
AND RAD 51 AND SO ON AND SO
FORTH.
WE ALSO HAVE A RETQC TERMINAL
DOMAIN, A NUCLEAR LOCALIZATION
SIGNAL AND A HELICASE RNA-LIKE
DOMAIN AT THE C TERMINIS.
SO WE INITIALLY FOCUSED ON THE
END TERMINAL DOMAIN AND HE MADE
A BUNCH OF TRUNCATIONS WHERE HE
CUT THE WHOLE THING OFF OR CUT
OFF MARCHING SEGMENTS GOING IT
FROM ONE END TO THE OTHER.
WHAT WE ARE GOING TO ASK IS, HOW
DO THESE PROTEINS BEHAVE IN
TERMS OF DNA END RECOGNITION?
HOW DO THEY BEHAVE IN TERMS OF
MOVEMENT?
DISTANCE?
VELOCITY?
AND LOOP FORMATION?
AND I'M JUST GOING TO TELL YOU
THAT ALL OF THESE GUYS WERE
VERY, VERY SIMILAR TO
FULL-LENGTH BLM EXCEPT FOR ONE
EXCEPTION AND YOU'LL SEE WHAT
THAT IS IN JUST A MOMENT.
SO HERE IS OUR DIAGRAM AGAIN
JUST SO WE ARE ALL ORIENTED.
AS WE ARE MAKING THESE
TRUNCATIONS, EVERY SINGLE
PROTEIN WITH AN END TERMINAL
TRUNCATION WAS STILL VERY
EFFICIENTLY TARGETED TO THE DNA
ENDS AND OUR ASSAYS.
SO THERE DOESN'T SEEM TO BE ANY
DEFECT IN DNA BINDING OR DNA
RECOGNITION.
WE ALSO LOOKED AT VELOCITY AND
I'M GOING TO POINT OUT, THESE
ARE VELOCILITY MEASURED IN
REACTIONS WITH ONLY BMLM AND
BOUND WHEREVER IT HAPPENED.
I THINK THE WAY THE REACTION WAS
DONE IN THIS CASE, WE DIDN'T
HAVE FREE DNA.
WE HAD BOTH ENDS TETHERED.
BUT THAT IS NEITHER HERE NOR
THERE.
THE MOST IMPORTANT PART IS THEY
ARE ALL GOING ROUGHLY THE SAME
SPEED EXEMPT THIS END TERMINAL
DELETION WHERE WE CUT THE ENTIRE
THING OFF.
AND I HAVE THIS AS 614.
IT SHOULD BE 641.
BUT IT'S THE ENTIRE END TERMINAL
DELETION.
IT'S GOING FASTER THAN THE
WILDTYPE FULL-LENGTH BLOOM.
AND IT'S GOING AHEC OF A LOT
FURTHER THAN THE FULL-LENGTH
BLOOM AS WELL.
WHEREAS THE OTHERS ALL SEEM TO
GO ROUGHLY THE SAME DISTANCE.
SO SOMETHING ABOUT CHOPPING THAT
WHOLE END TERMINAL DOMAIN OFF IS
MAKING US GO A LITTLE FASTER AND
A LITTLE FURTHER AS WE ARE
MARCHING ALONG THE DNA MOLECULE.
SO NOW, THIS IS SOMETHING ELSE
THAT,NOTICED RIGHT AWAY.
AND THAT IS IF YOU CAN PICTURE
IN YOUR MIND WHAT THOSE GFP FOCI
I SHOWED IN THE EARLY SLIDES
LOOKED LIKE, WE HAVE NO PROBLEM
DETECTING THAT SIGNAL.
IT'S BRIGHT.
IT'S MUCH BRIGHTER THAN ONE
WOULD EXPECT IF WE WERE SIMPLY
LOOKING AT A SINGLE GFP JUST
BASED ON OUR OWN EXPERIENCES.
BUT NOW WHEN HE IS LOOKING AT
THIS END TERMINAL DELETION
CONSTRUCT, ALL OF A SUDDEN IT'S
MUCH DIMMER THAN ALL OF THE
OTHER GUYS THAT WE LOOKED AT,
INCLUDING FULL LENGTH BLOOM AND
ANY OF THE OTHER TRUNCATIONS.
SO THERE IS A SORT OF LARGE
DECREASE, ABOUT 80% DECREASE, IN
TERMS OF INTEGRATED SIGNAL
INTENSITY.
WHEN WE HAVE THAT MUTANT.
SO EVEN THOUGH IT'S GOING FASTER
AND FURTHER, IT LOOKS LIKE THERE
IS LESS AT THE END.
AND HE DID A LITTLE BIT OF A
FANCIER MEASUREMENT WHERE HE
BOMBARDED THESE END-BOUND
PROTEINS WITH LASER LIGHT AND
ASKED, OKAY, HOW MANY
PHOTOBLEACHING STEPS DO WE SEE?
THIS IS AN EXAMPLE WITH
FULL-LENGTH BLM, GFP-TAGGED BLM.
WE ARE SEEING FOUR STEPS.
AND THAT INDICATES TO US THAT
THERE SHOULD BE FOUR MOLECULES
OF GFP BOUND.
AND IF WE LOOK AT ONE EXAMPLE,
IF WE LOOK AT A POPULATION
DISTRIBUTION, THAT IS MORE OR
LESS WHAT WE ARE SEEING ON THE
ORDER OF 3.6 PLUS OR MINUS .5.
WE ALSO SEE WHAT WE THINK ARE
PROBABLY AGGREGATES WHERE WE
CAN'T ACTUALLY MEASURE THE
STEPS.
IT'S JUST A LOT OF PROTEIN.
WE ARE JUST CATEGORIZING THESE
THAT ARE GREATER THAN 6.
NOW WHEN WE LOOK AT OUR END
TERMINAL DELETION, AND THAT'S A
TYPO.
IT SHOULD BE 641.
PRIMARILY, WHAT WE ARE SEEING
ARE SINGLE-STEP PHOTOBLEACHING
EVENTS.
THE PROTEIN IS THERE AND THEN IT
ALL OF A SUDDEN DISAPPEARS.
THAT IS MOST CONSISTENT WITH THE
IDEA THAT WE HAVE A SINGLE GFP
BOUND THERE, MEANING A SINGLE
COMPLEX OF GFP-BLM PROTEIN AND
THEN THERE ARE EXAMPLES WHERE WE
HAVE A FEW MORE BUT NOT VERY
MANY.
SO WE THINK SOMEHOW CHOPPING OFF
THE END TERMINAL DOMAIN MADE US
GO FASTER AND FURTHER AND
REDISED OUR LIGMERRIC STATE.
AND HERE IS ONE OF THE THINGS WE
FOUND WAS INTERESTING.
AND THIS IS SHOWING BLM WHERE
THE DNA GETS LOOPED AND LIE
LOOPED, I MEAN THE 3 PRIME END
IN THIS CASE IS TRACKING WITH
THE RESECTION MACHINERY, WHICH
WE DEMARKED WITH RPA-GFP IN THIS
CASE.
YOU'LL SEE WHY IN JUST A MINUTE.
THIS IS WHEN WE CUT OFF THE END
TERMINAL DOMAIN.
INSTEAD OF SEEING THIS
COLOCALIZATION THROUGH TIME,
WHAT WE SEE IS THAT 3 PRIME END
STAYS PUT OVER TIME.
IT STARTS TO GET A LITTLE BIT
LONGER OUT HERE AND YOU START TO
GENERATE THIS LONG TRACK OF RPA.
SO WHAT WE THINK IS HAPPENING IS
THAT AT THE LEADING EDGE OF THIS
RPA, THAT IS WHERE OUR GFP END
TERMINAL DELETION WILL BE
SITTING.
B WILL.
M WILL BE SITTING, MOVING,
TRACKING DNA2 WITH IT, CHOPPING
UP ONE OF THE DNA STRANDS.
BUT THE 3 PRIME END IS
UNAFFECTED.
IT'S NOT GENERATING A LOOP SO WE
GET THIS LONG EXTENDED STRUCTURE
OF SINGLE STRANDED DNA LIT UP BY
GFP-RPA.
I'LL POINT OUT THAT MOST OF OUR
TRUNCATIONS LOOK COMPARABLE TO
FULL-LENGTH BLM IN TERMS OF THE
PERCENT THAT UNDERGO LOOPING AND
IT'S THIS SINGLE, THIS ONE
MUTANT WHERE WE CHOPPED OFF THE
ENTIRE END TERMINAL DOMAIN THAT
SEEMS TO GO FROM A LOOPING
PROFICIENT ENZYME NOW TO
SOMETHING THAT IS NO LONGER
GENERATING DNA LOOPS.
SO, WE FOUND THAT EXCITING.
NO YOU WE WANTED TO ASK WHAT
ABOUT THE END TERMINAL OR THE C
TERMINAL DOMAIN?
AND SO HE WENT ABOUT GENERATING
A NUMBER OF DIFFERENT CONSTRUCTS
HERE WHERE HE EITHER CHOPPED OFF
THE ENTIRE C TERMINAL DOMAIN UP
TO THE ROCQ LIKE INCLUDING THE
HRDC AND THE DARK BLUE AND
YELLOW REGIONS.
WE KNOW THEY ARE INVOLVED IN
PROTEIN-PROTEIN INTERACTIONS.
HE ALSO CUT OFF THE C TERMINAL
DEMAIN BUT INCLUDED HRDC.
HE MADE ANOTHER CONSTRUCT WHERE
HE DELIGHTED THE DARK BLUE AND
THE YELLOW BUT LEFT THE NUCLEAR
LOCALIZATION SIGNAL INTACT.
HE MADE ANOTHER GUY THAT SORT OF
THE TWO DARK AND A LIGHT BLUE
AND THEN ANOTHER LITTLE GUY THAT
IS 40 AMINO ACIDS OF THIS BLUE
REGION.
HE IS ASKING, HOW DO THESE GUYS
BEHAVE?
AND SO, THE FIRST THING THAT
STRUCK US WHEN HE STARTED
LOOKING AT THESE CONSTRUCTS IS
THAT THEY USE THE CAPACITY TO
RECOGNIZE DNA ENDS.
SO THIS IS GFP-BLM.
NICELY LINED UP IN OUR DNA ENDS.
THING THIS IS THE SAME SLIDE I
SHOWED AT THE BEGINNING.
THIS IS OUR C TERMINAL DELETION
CONSTRUCT.
WHAT IS HAPPENING IS IT IS GOING
EVERYWHERE.
SO IT'S NOT SIMPLY GOING TO
ENDS.
IT'S GOING ALL OVER THE PLACE.
AND THIS IS TRUE FOR EVERY
SINGLE C TERMINAL DOMAIN
TRUNCATION CONSTRUCT HE LOOKED
AT, EVEN THE SMALLEST GUY, DELTA
1291 TO 1330T GOES ALL OVER THE
DNA.
WE ARE NOT LOSING THE CAPACITY
TO BIND, WE ARE LOSING TARGET
SPECIFICILITY.
NOW HE ALSO TESTED THESE GUYS IN
TERMS OF BULK, ATPI DRAW
ACTIVITY.
I'M GOING TO POINT OUT WHERE WE
CHOP OFF THE WHOLE C TERMINAL
DOMAIN.
YOU END UP WITH A BETTER ATP
ACE.
THESE ARE ALL THE OTHER
CONSTRUCTS COMPARED TO
FULL-LENGTH BLOOM.
THIS IS ATP HIDEALIZED PER TIME.
AND HE MEASURED THE VELOCITIES
ON DNA.
THE FUN THING HERE IS THIS
PROTEIN, EVEN THOUGH IT'S GOING
MUCH, MUCH FASTER IN TERMS OF
TURNING OVER ATP, IT'S GOING
MUCH, MUCH SLOWER IN TERMS OF
ITS MOVEMENT ON THE DNA.
SO WHAT WE ARE DOING HERE WITH
THIS MUTATION OR THIS TRUNCATION
IS DECOUPLING ATP HYDROLYSIS
FROM THE PROTEIN'S MOTOR
ACTIVITY.
AND THEN IN THIS CASE, IT'S
ABOUT 64% SLOWER.
I'M GOING POINT OUT THIS PROTEIN
ACTUALLY WAS CHARACTERIZED IN
BOTH CHEMICAL ASSAYS A NUMBER OF
YEARS AGO BY TWO DIFFERENT LABS.
AND BOTH OF THEM SHOWED THAT IT
HAD DECREASED HELICASE ACTIVITY
BUT INCREASE ATP ACE ACTIVITY.
WE THINK OUR RESULT IS FULLY
CONSISTENT WITH THE LITERATURE
AND WHAT WE ARE ADDING TO THIS
IS SHOWING THAT IT IS ACTUALLY
GOING SLOWER.
SO YOU'RE DECOUPLING MOTOR FROM
ATP HYDROLYSIS.
NOW, SORRY, NEEDED ANGDRINK.
EVERYTHING I SHOWED YOU UPON UP
TO THIS POINT HAS BEEN WORK BY
CHOW YOU.
I'M GOING TO START TALKING ABOUT
EXPERIMENTS DONE IN PATRICK'S
LAB.
FIRST OF WHICH IS DONE BY HIS
LONG TIME ASSOCIATE WHO HAS BEEN
AMAZING TO WORK WITH OVER THE
YEARS.
HE DID SIMPLE BULK BIOCHEMICAL
ASSAYS WITH TWO MUTANTS.
WE SETTLED ON THESE TWO GUYS AS
THE MOST INTERESTING.
OUR END TERMINAL DELETION
CONSTRUCT WHICH NO LONGER
GENERATES DNA LOOPS AND OUR C
TERMINAL PROTEIN WHERE WE
DELETED THIS SHORT TRACK OF
AMINO ACIDS, 40 ROUGHLY.
AND IT NO LONGER RECOGNIZES DNA
ENDS VERY WELL.
THIS IS A SIMPLE MEASUREMENT OF
DNA UNWINDING TAKING A 2 1/2 KB
FRAGMENT AND ASKING HOW WELL DO
YOU UNWIND?
WE SEE END TERMINAL GUY UNWINDS
IT BETTER CONSISTENT WITH THE
MORE RAPID VELOCITY.
AND THIS GUY ALSO UNWINDS.
IT'S COMPARABLE TO WILDTYPE.
IT'S NOT SO BAD.
AND WE THINK EVEN THOUGH IT'S
NOT RECOGNIZING ENDS, WHEN YOU
HAVE EYE SHORT SUBSTRATE IN A
TEST TUBE, THAT DOESN'T REALLY
MATTER IT'S ENOUGH TO GET THE
JOB DONE.
AND SO IT'S STILL CAN UNWINE
THIS SUBSTRATE.
PUTTING ENOUGH ENZYME ON THE DNA
IT'S NOT A PROBLEM.
WE THEN ASK, WHAT ABOUT
PROCESSING?
WHEN WE ADD DNA2 TO THESE SAME
REACTIONS.
WHAT WE SEE HERE IS INTERESTING
IN THAT OUR END TERMINAL
DELETION, A BETTER HELICASE,
ACTUALLY SHOWS PROCESSING LEVELS
THAT ARE COMPARABLE TO WILDTYPE
AND WE THINK WHAT THIS IS
TELLING US IS SOMETHING ABOUT
DNA2.
IT'S LIKELY THAT DNA2,
DEGRADATION, THE NUCLEASE,
REMEMBER.
IT'S LIKELY RATE LIMITING
REACTIONS.
BUT NOW OUR END TERMINAL --
SORRY, OUR C TERMINAL DELETION
CONSTRUCT IS NOT DOING SUCH A
GOOD JOB.
AGAIN WE THINK THAT IS BECAUSE
IT'S NOT GETTING TO THE ENDS
VERY WELL.
AND THESE PROPERTIES IN GENERAL
ARE SCALING WITH WHAT WE ARE
SEEING IN OUR SINGLE MOLECULE
REACTIONS.
AND SO WHAT WE NOW WANTED TO DO
IS FUN EXPERIMENTS AND ASK, WHAT
HAPPENS WHEN WE PUT THESE
MUTANTS INTO CELLS?
AGAIN I'M TALKING ABOUT THE END
TERMINAL AND C TERMINAL
TRUNCATION.
AND SO, THIS IS ALL WORK DONE BY
SA MERE WHERE HE IS TAKING THESE
U20S CELLS AND KNOCKING DOWN
EITHER BLOOM OR XO1 OR IN EVERY
EXPERIMENT WE ARE KNOCKING DOWN
BOTH THAT I WILL SHOW YOU.
THE REASON WE ARE KNOCKING XO1
DOWN IT OFFERS ALTERNATIVE
PATHWAY TO END PROCESSING IN THE
EVENT THAT BLOOM IS NOT THERE.
SO WE WANT TO GET RID OF BOTH OF
THESE WHEN LOOKING AT OUR
MUTANTS.
SO WE ARE KNOCKING THEM BOTH
DOWN AND THEN PUTTING IN
FULL-LENGTH BLOOM, A VECTOR
CONTROL OR OUR, IN THIS CASE, C
AND END TERMINAL CONSTRUCTS.
NOW WE ARE THEN ASKING IF WE
TAKE THESE CELLS AND SIMPLY
TREAT THEM WITH AGENTS TO CAUSE
PROBLEMS, DNA DAMAGE, EITHER A
HOW DO THE CELLS DO?
SO THIS IS OUR VECTOR-ONLY
CONTROL AT THE BEGINNING.
CELLS DON'T SURVIVE VERY WELL.
IF WE PUT IN FULL-LEAPT BLOOM,
WE GET AN UPTICK IN SURVIVAL IN
THE CASE OF BOTH.
WHEREAS END TERMINAL AND C
TERMINAL DELETION CONSTRUCTS
SHOW POOR SURVIVAL.
SO WE NOW WANT TO ASK WHY IS
THAT?
WHY ARE THESE GUYS NOT SURVIVING
VERY WELL?
AND AGAIN, I REMIND YOU THIS IS
THE ONE THAT DOESN'T DO LOOPING.
THIS IS THE ONE THAT IS NOT
TARGETED TO ENDS.
IT'S PROBABLY PRETTY EASY TO
IMAGINE WHY THIS ONE IS NOT
DOING SO WELL.
SO NOW WHAT SA MERE DID WAS TO
ASK, WHAT HAPPENS WITH THESE
GUYS WHEN WE TREAT THESE CELLS
IONIZING RADIATION?
CAN WE SEE FOCI FORMING?
PROTEIN FOCI?
BOTH FOR BLM, RPA AND ALSO FOR
Rad51.
SO WE ARE USING Rad51 AS AN
INDICATORS HOMOLOGOUS
RECOMBINATION CAN TAKE PLACE
BECAUSE WE ARE MAKING A
PROCESSED END WHERE WE CAN
ASSEMBLE OUR RECOMBINASE
FILAMENT.
AND WHAT I'M GOING TO SHOW YOU
AS WE CAN SEE BLM FOCI IN ALL
THESE GUYS, THESE FOCI IN TERMS
OF THE INTERNAL DELETION LOOK A
LITTLE BIT BIG.
IT'S NOT UNUSUAL.
WE ARE OVEREXPRESSIONING A GFP
TAG PROTEIN.
IN ALL CASES WE ARE SEEING RPA,
TO SOME EXTENT.
AND THEN Rad51 WE ARE SEEING
IT AND IT VARIES QUITE A BIT.
I'LL SHOW YOU QUANTITATION OF
THIS DATA NOW.
SO FIRST THING WE LOOKED AT WAS
53BP1 WHICH WE ARE TAKING AS
EARLY INDICATOR OF DNA DAMAGE.
AND ALL THEE THERE ARE SOME
DIFFERENCES FROM CELL LINE TO
CELL LINE BACKGROUND TO
BACKGROUND, PRETTY MUCH THEY ARE
ALL MAKING DNA DOUBLE STRAND
BREAKS.
WE KNOW THAT WE ARE INDUCING DNA
DAMAGE WHEN WE EXPOSE THESE
CELLS TO IONIZING RADIATION.
NOW ALL CELLS ALSO SHOW EVIDENCE
OF GFP-BLM FOCI AND THERE ARE
DIFFERENCES FROM ONE CONSTRUCT
TO THE NEXT.
BUT THEY ARE STILL FORMING FOCI.
IT'S INTERESTING TO THINK ABOUT
THIS GUY, WHICH IS NOT TARGETED
TO DNA ENDS BUT IT IS STILL
FORMING FOCI CONSISTENT WITH IT
GOING TO WHERE THE DNA DAMAGE
IS.
I'LL BRING THAT UP AGAIN LATER,
AT THE END.
NOW, WHAT WE WANTED TO DO THEN
WAS LOOK AT RPA FOCI.
SO RPA FOCI ARE AN INDICATION
THAT END PROCESSING SELL
ACTUALLY TAKING PLACE.
NOW THIS IS OUR VECTOR-ONLY
CONTROL.
THIS IS WHEN WE PUT IN
FULL-LENGTH BLM.
SO GOING FROM HERE TO HERE,
STARTING TO SEE MORE FOCI SAYING
WE ARE GENERATING PROCESSED
BREAKS.
WE SEE A SIMILAR INCREASE WHEN
WE DELETE THE END TERMINIS OF
BLM.
WE STILL SEIZE RPA FOCI FORMING.
THAT'S TELLING US THAT THE
PROTEIN IS PROCESSING THE DNA
ENDS.
BUT IF WE TAKE OUR C TERMINAL
CONSTRUCT, NOW WE DON'T SEE THAT
INCREASE IN RPA FOCI.
SO WE THINK THIS GUY IS NO
LONGER MAKING RPA FOCI BECAUSE
IT'S NO LONGER SUPPORTING RPA
FOCI FORMATION BECAUSE IT'S NOT
PROCESSING THE DNA ENDS.
AND I'M JUST GOING TO POINT OUT
THAT WE ALSO HAVE PHYSICAL
ASSAYS WHERE WE ARE DOING PCR
NEAR THE BREAK TO DETECT THE
PRODUCTION OF SINGLE STRANDED
DNA AND IT ALSO SAYS THAT THIS
GUY IS NOT GENERATING SINGLE
STRANDED DNA WHEREAS THESE GUYS
ARE.
SO NOW IF WE LOOK AT Rad51
FOCI, WHAT WE SEE IS THAT WITH
BLM, WE GET AN UPTICK COMPARED
TO VECTOR-ONLY CONTROL.
BUT NOW NEITHER OF OUR DELETION
CONSTRUCTS ARE SHOWING EVIDENCE
OF STRONG UPTICKS IN Rad51
FOCI FORMATION.
SO, WE THINK THAT THE C TERMINAL
GUY IS NOT MAKING Rad51 FOCI
BECAUSE THE ENDS AREN'T GETTING
RESECTED SO NO RPA OR Rad51
LOADING.
WHEREAS THIS ONE, THE END
TERMINAL CONSTRUCT, WE THINK IT
IS RESECTING THE ENDS.
WE DO SEE RPA FOCI, BUT IT SEEMS
TO BE SPECIFICALLY DEFECTIVE IN
Rad51 FILAMENT FORMATION OR
FOCI FORMATION.
AND SO WE THINK THERE IS SOME
KIND OF DEFECT ASSOCIATED WITH
THIS GUY THAT DOESN'T LET
RAD51 NOW BIND TO THE DNA.
NOW I'M GOING TO GO THROUGH TWO
ASSAYS RELATIVELY QUICK AND GET
TO THE CONCLUSIONS.
IN THESE TWO ASSAYS, WHAT WE ARE
DOING ARE TAKING GFP CONSTRUCTS
WHERE WE HAVE A GFP GENE THAT IS
INACTIVE AND ONE THAT IS ACTIVE
AND THEN WE ARE USING DNA
BREAK-IN DUDES BY RESTRICTION IN
THE NUCLEASE, ICE1, TO INDUCE
REPAIR THAT RESULTS IN REPAIR OF
THE GFP GENE SO IT GOES FROM NOT
FLUORESCENT TO NOW FLUORESCENT.
AND WE CAN TUNE THESE OR I
SHOULD SAY, THE ASSAYS WE HAVE
ADAPTED, CAN ATTUNE THESE TO
DETECT HOMOLOGOUS RECOMBINATION
OR SYNTHESIS DEPENDENT STRAND
KNEELING.
SSA.
I SHOULD SAY THAT.
OKAY, SO LET ME WALK YOU THROUGH
THIS DATA REALLY QUICK.
THESE ARE CONTROLLED CELL WHERE
WE DO KNOWING.
THIS IS A CELL WHERE WE BREAK
THE DNA AND HAVE AN siRNA NOT
TARGETED AGAINST ANYTHING AND WE
SEE HOMOLOGOUS RECOMBINATION
TAKING PLACE.
THIS IS WHERE WE CUT THE DNA BUT
INHIBIT BRCA2 AND SO WE SEE A
DOWNTICK IN HR, MOM OL GUS
RECOMBINATION.
THIS IS WHERE WE KNOCKOUT B R&D
O1.
NOT VERY GOOD RECOMBINATION.
THIS IS OUR VECTOR-ONLY CONTROL.
THIS IS WHERE WE PUT THAT
FULL-LENGTH BLM.
SO WE DO GET A PARTIAL REGULARS
CUE OF HOMOLOGOUS RECOMBINATION
AS LONG AS WHEAT HAVE
FULL-LENGTH.
NOW NEITHER OF THESE GUYS ARE
SUPPORTING HOMOLOGOUS
RECOMBINATION IN THESE CELLS
COMPARED TO OUR VECTOR-ONLY
CONTROL.
AND SO THAT IS TELLING US THAT
CONSISTENT WITH OUR PRIOR
RESULTS, THERE IS SOME SORT OF
DEFECT ASSOCIATED WITH REPAIR.
NOW IF WE LOOK AT SINGLE STRAND
KNEELING WHICH IS NOT RadPOP
DEPENDENT -- Rad51 DEPENDENT,
WHAT WE SEE IS WHEN WE KNOCKOUT
BRCA2, WE GET INCREASE IN SINGLE
STRANDED KNEELING BECAUSE WE ARE
TAKING THINGS THAT NORMALLY
WOULD BE REPAIRED BY HR AND
CHANNELING THEM INTO THIS NEW
PATHWAY THAT IS Rad51
INDEPENDENT.
NOW IF WE LOOK AT OUR
VECTOR-ONLY CONTROL THIS IS WHAT
WE GET.
ADDING THE BLOOM DOESN'T REALLY
HAVE MUCH OF AN EFFECT.
OUR C TERMINAL DELETION ALSO
DOESN'T HAVE AN EFFECT.
BUT ADDING THIS END TERMINAL
DELETION NOW WE GET A
SUBSTANTIAL INCREASE IN SINGLE
STRANDED KNEELING.
AND WE THINK THAT IS BECAUSE
THIS GUY IS MAKING SINGLE
STRANDED ENDS THAT ARE LOADED BY
RPA BUT THEY ARE NOT LOADED
WITH Rad51.
SO Rad51 ACTUALLY IN HIBITS
PATHWAY.
AND SO WE THINK THAT THAT IS
FULLY CONSISTENT WITH THIS IDEA
THAT SOMEHOW THIS END TERMINAL
DELETION CONSTRUCT IS DEFICIENT
FOR Rad51 FILAMENT FORMATION.
SO NOW I'M GOING TO GO TO MY
SUMMARY SLIDE.
AND I HOPE THAT I HAVE CONVINCED
YOU THAT END RESECONDS IN
EUKARYOTES IS SOMEHOW COUPLED TO
THE FORMATION OF THIS LARGE
SINGLE STRANDED LOOP AND THAT
THE END TERMINAL DOMAIN OF BLM
HELICASE IS SOMEHOW CONTRIBUTING
TO THIS FORMATION.
AND CELLS EXPRESSING THIS MUTANT
ARE DEFICIENT FOR DNA REPAIR IN
THE PRESENCE OF DNA DAMAGING
DRUGS.
AND IT SEEMS AS THOUGH THIS
MUTANT CAN SUPPORT END RESECTION
IN-VIVO BUT Rad51 IS FAILING
TO FORM ON THIS.
AND SO OUR HYPOTHESES GOING
FORWARD IS THERE MAY BE SOME
KIND OF LINK BETWEEN LOOP
FORMATION DURING END RESECTION
AND THE ASSEMBLY OF THE Rad51
PRESYNAPTIC COMPLEX.
I COLORED THIS BLUE AND ADDED A
QUESTION MARK BECAUSE THIS IS
SPECULATIVE AT THIS POINT.
AND WE ARE CURIOUS WHAT IS
DRIVING THESE LOOPING
INTERACTIONS?
IS IT BLM INTERACTIONS WITH
SINGLE STRANDED DNA OR PERHAPS
BLM INTERACTIONS WITH RP!
WE DON'T KNOW.
AND WE ARE ALSO CURIOUS WHETHER
BLM ITSELF MAY BE SOMEHOW LOADED
OR INVOLVED IN LOADING Rad51
ON TO THE DNA ENDS DURING
RESECTION.
OF COURSE WE ALWAYS HAVE THE
QUESTION OF CAN WE SEPARATE OR
GENERATE MUTANTS THAT SEPARATE
LOOPING FROM Rad51 LOADING?
WE DON'T KNOW THE ANSWER TO THAT
YET.
I ALSO WANT TO MAKE A POINT
HERE.
THE KEY BACTERIAL SYSTEM, SO FOR
EXAMPLE, E.COLI, IT ALSO MAKES
DNA LOOPS DURING END RESECTION.
BUT WE DON'T KNOW WHY.
SO IT SEEMS AS THOUGH, AND I
WANT TO MAKE THIS CLEAR,
PROCESSING DNA ENDS IN E.COLI IS
COMPLETELY DIFFERENT FROM BLM,
DNA2, RPA, WHICH IS INVOLVED IN
PROCESSING IN EUKARYOTES.
BUT BOTH SYSTEMS ARE FORMING
LOOPS.
IS IT A COINCIDENCE OR SOMETHING
TO LOOP FORMATION THAT IS
IMPORTANT FOR RECOMBINATION?
WE DON'T KNOW THE ANSWER TO
THAT.
NOW IN TERMS OF OUR C TERMINAL
DOMAIN, WE KNOW THAT HDRC MOTIF
HELPS COUPLE ATP HYDROLYSIS TO
TRANSLOCATION.
WE HAVE NO IDEA HOW THAT TAKES
PLACE IT'S INTERESTING BECAUSE
WITHIN THE CRYSTAL STRUCTURE,
THIS DOMAIN SEEMS TO BE WELL
AWAY FROM ANY DNA BINDING MOTIF.
ALTHOUGH YOU CAN ALWAYS THINK
ABOUT NEW STRUCTURES THAT MIGHT
CHANGE THAT.
WE HAVE IDENTIFIED A NEW PEPTIDE
WITHIN BLM.
IT'S FRANKING HDRC BUT NOT
WITHIN HDRC ITSELF THAT SEEM TO
BE NECESSARY FOR END RESECTION.
EVEN THOUGH IT IS NOT MESSING UP
ANY OF THE OTHER PROPERTIES D-NA
BINDING OR HELICASE ACTIVITIES.
SPECIFICALLY AFFECTING
RECOGNITION.
AND WE CAN SHOW THAT THIS MULE
ANNUAL IS RECRUITED TO SITES OF
OF DNA REPAIR BUT IT'S NOT
TARGETED TO D-NA AND ENDS IT
SEEMS TO BE SERIOUSLY
COMPROMISED FOR END PROCESSING
BOTH, FOR FORMATION OF RPA AND
RAD 51 FOCI AND IT DOESN'T DO
HR.
AND SO ONE THING THAT WE ARE
THINKING THIS IS TELLING US, IS
THAT BECAUSE IT'S GOING TO FOCI,
ACTUAL RECRUITMENT TO THE REPAIR
FOCI MAY BE A SEPARATE PROCESS
FROM DIRECTORY CRUELTY TO THE
ACTUAL DNA ENDS.
AND AGAIN, THAT IS SORT OF A
SPECULATIVE THINKING WE HAVE TO
SORT OF WORK OUT IN THE FUTURE.
SO I'M HAPPY TO END THERE AND
ADDRESS ANY QUESTIONS THAT
ANYONE MIGHT HAVE.
NOT ENTIRELY SURE HOW THIS WORKS
BUT I THINK SUSAN WILL READ YOUR
QUESTIONS TO ME.
SO WE'LL SEE HOW THAT GOES.
AND I'LL JUST THANK EVERYBODY
FOR LISTENING TO ME.
>> SUSAN: THANK YOU, ERIC.
I AM SURE IF WE COULD SEE
PEOPLE, THERE WOULD BE A LOT OF
HANDS CLAPPING.
I'M NOT SURE HOW THIS WORKS
EITHER BUT I THINK WE'LL WORK IT
OUT.
IT WAS A BEAUTIFUL TALK AND
THERE ARE SOME QUESTIONS IN THE
CHAT.
PLEASE CONTINUE EVERYBODY TO ADD
YOUR QUESTIONS AND I WILL GO
THROUGH AS MANY AS WE CAN.
SO THE FIRST ONE IS FROM RACHEL
JOHNSTON AND IT'S MORE OF A
TECHNICAL SET OF QUESTIONS ABOUT
CONTROLS FOR YOUR CURTAIN ASSAY.
SHE ASKS, YOU NOTED THAT THE BLM
LOCALIZED TO THE DNA ENDS USING
GFP LABELING.
DID YOU HAVE A CONTROL FOR
LOCALIZATION FOR A PROTEIN?
WHICH WOULD LOCALIZE TO ANOTHER
LOCATION ON THE DNA?
SO RATHER THAN IT BEING BUFFER
FLOAT ISSUE, YOU KNOW IT IS TRUE
LOCALIZATION.
>> DR. GREENE: YES.
SO OVER THE YEARS WE PUBLISHED
100 -- NORTH OF 100 PAPERS NOW
AND IN SEVERAL OF THOSE WE HAVE
PUBLISHED PROTEINS THAT BIND TO
SPECIFIC SITES WITHIN THE DNA.
I THINK ONE OF THE MORE FUN ONES
WE DID WAS WITH CAS9 ACTUALLY.
AND WE COULD TARGET IT BASICALLY
WHEREVER WE WANTED WITHIN THE
DNA JUST BY CHANGING THE GUIDE
RNA.
WE HAVE ALSO DID STUDIES -- I
SHOULD HAVE MENTIONED THIS.
THIS IS LAMBDA PHAGE DNA WE ARE
USING.
IT'S NOTHING FANCY.
NICE THING ABOUT IT HAS RNA
POLYMERASE PROMOTORS AND IT GOES
TO ALL THE RIGHT PROMOTORS.
WE CAN PUT IN ALSO -- LET ME
THINK ABOUT THIS -- A NUCLEASE
DEFICIENT VERSION OF FOR1.
IT GOES TRITE THAT SITE.
AND WE CAN PUT IN A MISMATCH IN
THAT DNA AND WE CAN TARGET THE
MISMATCH RECOGNITION PROTEINS.
MSH26 RIGHT TO THE MISMATCH.
WE DON'T THINK FLOW IS CAUSING
IT TO GO TO THE END OF THE DNA.
I SHOULD ALSO NOTE WE ARE OFTEN
ASKED, HOW DO YOU KNOW FORCE IS
NOT INFLUENCING THIS?
AND I MEAN, I CAN'T SAY WITH
CERTAINTY THAT IT'S NOT, BUT
WHAT I CAN SAY IS THAT WE ARE
NOT EXERTING A TREMENDOUS AMOUNT
OF FORCE ON THESE MOLECULES.
THIS IS ONE OF THE REASONS THEY
SORT OF LOOK LIKE THEY ARE
FLIPPING BACK-AND-FORTH A LITTLE
BIT.
WE ARE SORT OF DOWN IN THE
SUBSINGLE PEEKA NEWTON DIGITS
JUST ENOUGH TO EXTEND THE DNA
ABOUT 70% OF ITS FULL CONTOUR
LENGTH.
SO WE DON'T THINK WE ARE DOING
THAT.
NOW I WILL NOTE WE HAVE SEEN
EXAMPLES OF ENZYMES WHERE WE
PUSH IT OFF THE D IN.
A, AT LEAST WE THINK WE DO, BUT
I DON'T THINK IT IS TAKING
PLAGUES IN THIS EXAMPLE.
>> GREAT.
THANK YOU.
SO TOM SNYDER HAS A COUPLE OF
QUESTIONLESS I'LL ASK ON HIS
BEHALF.
THE FIRST IS, DID YOU GET THE
NUMBER OF ATP HYDROL SIZED PER
STEP IN WILDTYPE BLM?
>> DR. GREENE:
[ LAUGHS ]
TOM, I LOVE THAT QUESTION.
I THOUGHT TOO.
[ LAUGHS ]
I HATE IT TOO.
THIS IS OF COURSE -- HE MUST BE
A MOTOR PROTEIN AFFICIONADO.
THIS IS A QUESTION THAT OFTEN
COMES UP.
THAT IS HOW MUCH ATP YOU
HYDROLYZE PER STEP?
I ALWAYS MAKE THE SIMPLEST
ASSUMPTION AND I THINK THIS IS
CONSISTENT WITH STRUCTURAL DATA
FOR ALL HELICASES I KNOW OF OR
MOST OF THEM ANYWAY, THAT YOU'RE
TAKING A SINGLE NUCLEOTIDE STEP.
SO IT'S OF COURSE A GREAT
INTEREST TO KNOW, IS ONE STEP
CORRESPOND TO 1ATP?
AND YOU KNOW, TOM, I WOULD LOVE
TO BE ABLE TO ANSWER THAT.
I CAN'T.
WE HAVEN'T NADE MEASUREMENT YET.
IT IS NOT SUCH AN EASY
MEASUREMENT TO MAKE CORRECTLY.
AND REALLY THE ONLY WAY THAT WE
CAN DO THIS IS TO DO BULK
CHEMICAL MEASUREMENTS MEASURING
ATP HYDROLYSIS PER UNIT TIME AND
THEN COMPARE TO THE VELOCITY.
THE CHALLENGE WITH THAT IN THIS
PARTICULAR SYSTEM IS THAT WE
KNOW THAT BLM BINDS TO ENDS.
WE ALSO KNOW THAT IT BINDS
INTERNAL POSITIONS.
AND THE VELOCITY IT MOVES IN
THOSE TWO DIFFERENT
CONFIGURATIONS IS VERY
DIFFERENT.
IT MOVES MUCH FASTER WHEN IT'S
BOUND TO AN INTERNAL SITE.
SO ANY BULK BIOCHEMICAL ATP
HYDROLYSIS DATA IS GOING TO BE A
MIXTURE OF THINGS THAT ARE BOUND
TO ENDS AND THINGS THAT ARE
BOUND TO INTERNAL SITES THAT WE
JUST DON'T HAVE A GOOD WAY OF
KNOWING.
I DO APPRECIATE THE QUESTION,
TOM AND I WOULD LOVE TO KNOW THE
ANSWER MYSELF.
>> THAT'S A GOOD POINT.
BIOCHEMISTRY IS NOT REALLY GOING
TO ANSWER THAT WELL, I DON'T
THINK, EITHER.
HE ALSO ASKS, HAVE YOU LOOKED AT
THE LOOP USING CRYO-EM?
MAYBE IT'S A COIL HELICAL
STRUCTURE?
>> WE HAVE NOT DONE THAT YET.
YES, WE ARE REALLY -- I'LL
JUSTSADE A LOT OF THE PROJECTS
WE ARE WORKING ON, WE HAVE KIND
OF COME INTO THESE BARRIERS
WHERE IT'S LIKE, MAN I WOULD
REALLY LIKE TO HAVE A STRUCTURE
OF THAT.
THIS IS NOT JUST THIS PROJECT
BUT OF COURSE IT APPLIES HERE AS
WELL.
AND SO, WE REALLY NEED TO START
DOING SOME CRYO-EM ON SOME OF
THE THINGS WE ARE LOOKING AT.
AND TRY TO GET SOME STRUCTURAL
INSIGHT.
ANOTHER GREAT QUESTION, WHICH I
UNFORTUNATELY CAN'T ANSWER VERY
WELL RIGHT NOW.
SORRY, TOM.
>> YOU HAVE A LOT OF COLLEAGUES
AT COLUMBIA THAT COULD HELP WITH
THAT.
>> THEY ARE ALL TRYING TO SOLVE
A VIRUS STRUCTURE, ANTIBODY
STRUCTURE NOW.
>> NEXT QUESTION.
>> WE ARE GOING TO DO IT
OURSELVES.
>> GOOD FOR YOU!
YOU CAN.
ALSO INTERESTING TO NOTE THAT A
RECK A LOADING FUNCTION HAS BEEN
SUGGESTED FOR RECBCD.
>> SO IS THAT MICHAEL?
I WISH I COULD SEE YOU AND HEAR
YOU.
THIS IS ONE OF THE THINGS I
DON'T LIKE SO MUCH ABOUT ZOOM.
THAT'S A GREAT POINT, MICHAEL.
IT'S VERY CLEAR THAT RECB IS
SOMEHOW INVOLVED IN LOADING RECA
AND WE ALSO KNOW THAT BLM
INTERACTS WITH Rad51.
SO WE WOULD LOVE TO BE ABLE TO
SEE THIS, TO SEE COUPLED Rad51
LOADING IN OUR SYSTEM.
WE ARE NOT THERE.
WE ARE GOING TO MOVE IN THAT
DIRECTION.
THE CHALLENGE MICHAEL, IS THAT
AND YOU'RE PROBABLY WELL AWARE
OF THIS IN A TEST TUBE, BECAUSE
IT'S AN ARTIFICIAL SYSTEM, AN
IMPORTANT CAVEAT YOU HAVE TO
CONTEND WITH IS THAT Rad51
DOESN'T DISTINGUISH VERY WELL
BETWEEN DOUBLE AND SINGLE
STRANDED DNA.
SO I KNOW THAT IF WE JUST THROW
Rad51 IN THERE WILLY NILLY, IT
WILL GO EVERYWHERE.
IT WILL GET IN FRONT OF THIS
THING, BEHIND IT, IT WILL CAUSE
ALL KINDS OF CHAOS.
I SUSPECT WE CAN OVERCOME THAT
BY PERHAPS ONE OR TWO WAYS.
THE FIRST OF WHICH IS TO PERFORM
THE REACTIONS UNDER CONDITIONS
IN WHICH Rad51 LOADING ITSELF
ON ITS OWN IS COMPROMISED.
MEANING A LOWER CONCENTRATION
OF Rad51 THAT IS NECESSARY TO
GET ON WITH DNA ITSELF UNDER THE
ASSUMPTION THAT BLM MIGHT HELP
IT GET ON THERE.
SO WE ARE DEFINITELY THINKING
ABOUT THAT.
AND THEN THE OTHER WAY, AS YOU
KNOW, THERE IS A NUMBER OF
DIFFERENT PROTEINS THAT ARE
INVOLVED IN HELPING TO GET
RAD51 ON DNA.
SO MEDIATOR COMPLEXES LIKE
BRCA2, FOR EXAMPLE.
OR Rad51 PARALOGS POSSIBLY.
WE WOULD LOVE TO BE ABLE TO SEE
COUPLED REACTIONS AND WHERE WE
ARE DOING THIS PROCESSING.
PUTTH IN THE MEDIATORS AND
PUTTING IN Rad51 AND TRYING TO
SEE WHERE DOES EVERYONE FALL IN
PLACE.
WE ARE NOT THERE YET.
SORRY, MICHAEL.
>> WE ARE GOING TO TAKE ONE
FINAL QUESTION BUT BEFORE THAT,
LET ME REPEAT THE CME CODE FOR
TODAY FOR THOSE WHO NEED IT IT'S
37930.
SO I THOUGHT THE LAST QUESTION
SHOULD COME FROM A FELLOW LMB
PERSON.
ONE OF OUR NEWER INVESTIGATORS
AT LMB AND HE ASKS, FANTASTIC
WHAT HE SAYS.
FANTASTIC TALK, ERIC.
DO YOU THINK THE SINGLE STRANDED
DNA FORMS A GIANT SINGLE LOOM OR
MANY SMALLER LOOPS?
COULD IT BE FORMING NUMEROUS DNA
HAIR PINS AS ALONG SINGLE STRAND
RNA LOOP DOES?
>> YES, SO THAT'S A GREAT
QUESTION.
I DON'T KNOW IF WE MET.
I HOPE I'M NOT FORGETTING YOU.
IF NOT, I'M SORRY I COULDN'T
COME THERE AND MEET YOU.
SO THOSE ARE TWO DIFFERENT
THINGS, KIND OF.
SO EXACTLY AS YOU SAY, YOU COULD
BE FORMING MULTIPLE SORT OF HAIR
PINS THAT ARE NOT INVOLVED IN A
INTERACTION BUT ARE INVOLVED IN
A DNA-DNA PAIRING INTERACTION.
OR YOU COULD FORM MULTIPLE
CONTACTS PERHAPS WITH THE
RESECTION MACHINERY.
I DON'T THINK THAT EITHER OF
THOSE IS TAKING PLACE.
AND THE REASON I DON'T THINK SO
IS WHEN WE SEE, THERE IS A
COUPLE OF THINGS.
FIRST OF ALL YOU, WHEN WE SEE
THE LOOPS BREAK AND THAT DNA
GETS LONG AGAIN, IT TENDS TO
HAPPEN ALL IN ONE STEP.
SO THAT TELLS ME THAT WE HAVE
RUPTURED A SINGLE CONTACT.
OF COURSE I CAN'T SAY THAT MAYBE
THERE WERE MULTIPLE CONTACTS AND
IT'S THE LAST ONE YOU SEE.
THAT'S ALSO A POSSIBILITY.
I DON'T THINK HAIR PINS ARE
FORMING AT LEAST NOT TO AN
EXCESSIVE AMOUNT AND THE REASON
FOR THAT IS THAT WE KNOW RPA,
ONE OF THE PRIMARY ROLLS IN
THESE REACTIONS LIKE THIS IS TO
PREVENT HAIR PIN FORMATION.
SO I'M SURE IF WE DIDN'T HAVE
RPA THERE, THAT WHOLE THING
WOULD BE KNOTTED UP LIKE CRAZY
LIKE YOU WOULD ENVISION IN THE
RNA MOLECULE.
BUT WHEN WE HAVE RPA THERE, I
THINK IT'S PREVENTING THAT FROM
TAKING PLACE.
SO HOPEFULLY I ANSWERED YOUR
QUESTION AND HOPEFULLY I CAN
MEET YOU SOME DAY TOO.
>> WE ALL HAVE THAT.
THAT WOULD BE REALLY NICE.
SO I THINK IT'S PROBABLY UP TO
ME TO THANK ERIC AGAIN FOR A
LOVELY TALK.
I HAVE QUESTIONS TOO BUT WE
COULDN'T GET TO EVERYTHING AND
WE HAVE A FULL TRAINEE SCHEDULE.
WHICH WAS SUPPOSED TO GIVE ERIC
A LITTLE BIT OF A BREAK BUT
THERE IS NOT MUCH OF A BREAK
LEFT.
THIS IS A GORGEOUS CITIZEN.
IT'S SO ELEGANT.
SO MUCH YOU CAN DO WITH IT.
-- SYSTEM.
IT'S GOING TO BE VERY EXCITING
WHERE THIS GOES FROM HERE.
SO ON BEHALF OF AL ALL OF US AT
NIH, WE MISS YOU AND THANK YOU
VERY MUCH FOR GIVING THIS TALK
TODAY.
>> THANK YOU, SUSAN.