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>>WELCOME TO THE DNA REPAIR

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INTEREST GROUP VIDEO CONFERENCE

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SERIES.

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THOSE OF YOU WHO GOT OUR MESSAGE

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CAN JOIN THE LISTSERV BY SENDING

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E-MAIL TO KRAEMERK@NIH.GOV,

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AND THE OTHERS WILL KNOW THE

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UPCOMING MEETINGS.

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DR. BOHR, MY CO-HOST WILL BE

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SPEAKING ABOUT MID O CHONT RIA

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AND NEURODEGENERATION AND IN

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MAY, DR. DEIN, ARDO FROM ITALY

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WILL TALK ABOUT MULTIFACETED

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ROLES OF N. E. R. PROTEINS AND

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THEIR IMPLICATIONS IN HUMAN

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PATHOLOGIES.

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AND IN JUNE

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 TOM KONKLE FROM NIEHS WILL

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SPEAK ABOUT EXTRINSIC PROOF

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READING OF THE DNA REPLICATION

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PROCESS.

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TODAY WE'RE HAPPY

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TO HAVE DR. RACHEL FLYNN HERE

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FROM BOSTON UNIVERSITY.

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SHE GREW UP IN NEW HAMPSHIRE AND

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GOT A LIBERAL ARTS DEGREE IN

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ST. MICHAELS COLLEGE IN VERMONT

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WHICH SHE TELLS US IS NEAR

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BURLINGTON AND SHE WENT TO THE

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UNIVERSITY OF--WHERE SHE'S STILL

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LIVING AND SHE WENT AND DID A

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POST DOCTORAL FELLOWSHIP AT THE

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MASS GENERAL AND AT HARVARD AND

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THEN, JOINED THE BOSTON

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UNIVERSITY IN THE DEPARTMENT OF

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PHARMACOLOGY AND MEDICINE AND

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SHE'S BEEN THERE EVER SINCE.

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SHE'S IN ADDITION A CO-DIRECTOR

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OF THE GENOME SCIENCE DEPARTMENT

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AT BOSTON UNIVERSITY AND NOW IS

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AN ASSOCIATE PROFESSOR IN

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PHARMACOLOGY AND MEDICINE IN

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THAT DEPARTMENT.

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SHE'S DONE SOME VERY NICE WORK

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ON TELOMERE BIOLOGY AND TODAY,

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SHE WILL SPEAK ABOUT DEFINING

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THE DNA DAMAGE RESPONSE IN ALT

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TELOMERES.

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>> TAKE IT AWAY, RACHEL.

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THANK YOU VERY MUCH

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INTERNATIONAL CLASSIFICATION ON

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ROUGH ATOM DUKS AND THE

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INVITATION TO COME AND TALK AND

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TELL YOU ABOUT THE WORK I'VE

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BEEN DOING IN MY LAB.

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SO I REALLY HAD A LONG STANDING

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INTEREST IN UNDERSTANDING HOW

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CELLS ACQUIRE DAMAGE, HOW THEY

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RESPOND TO DNA DAMAGE AND

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ULTIMATELY HOW THEY REPAIR THAT

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DNA DAMAGE.

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EARLY ON IN MY CAREER, I WAS

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REALLY FOCUSED ON THIS, THE

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WHOLE GENOME LEVEL AND I THINK

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OVER THE YEARS I'VE SORT OF

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EVOLVED THIS INTO UNDERSTANDING

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THE DYNAMICS OF THE DNA DAMAGE

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RESPONSE SPECIFICALLY AT TELEO

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MERRIC DNA AND SO TODAY I DON'T

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HAVE AN OUTLINE HERE BUT I

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WANTED TO GIVE YOU A FEW

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VIGNETTES FROM THE LAB IN OUR

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EFFORT TO COMBINE DEFINING THE

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GENETIC ALTERATIONS IN CERTAIN

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TYPES OF CANCERS AND HOW THAT

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AFFECTS THE DNA DAMAGE REPAIR

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PATHWAYS.

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AND SO DNA DAMAGE AT THE

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TELOMERE IS NOT UNLIKE ANY OTHER

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DNA DAMAGE, REPLICATION BREAKS,

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SINGLE STRANDED DNA, BREAKS AND

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ALL THESE INVOKE A DNA DAMAGE

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RESPONSE IN TELOMERES SO REALLY

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UNDERSTANDING THIS MECHANISM OF

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THE RESPONSE AND THE REPAIR IS

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SOMETHING THAT I BUILT MY LAB

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UPON.

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SO THE MOST OBVIOUS SOURCES OF

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DNA DAMAGE AT TELOMERES REALLY

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ARISE IN THE TELOMERE REPEAT

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THEMSELVES SO BEING THESE

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HEXAMORE TTA, GGO, G REPEATS,

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THEE ARE PRONE TO REPLICATION

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SLIPPAGE, THEY FORM SECONDARY

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STRUCTURES LIKE G-DUPLEXS AND

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THEY HAVE THIS SINGLE STRANDED

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DNA TAIL AT THE END AND THAT

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COMBINED WITHLET LINEAR NATURE

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OF THE CHROMOSOME AND THE

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TERMINAL POSITION OF THE

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TELOMERE ITSELF, REALLY RENDERS

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THIS REGION SUSCEPTIBLE AS

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RECOGNITION TO A DNA DOUBLE

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STRAND BREAK.

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SO AT TELOMERES THEY FORM A

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SECONDARY STRUCTURE REALLY

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FOLDING THAT SINGLE STRANDED DNA

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OVERHANG BACK INTO ITSELF,

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CREATING A LOCALIZED D-LOOP OR

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THESE ARE T-LOOPS AND THIS IS TO

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PROTECT THE END OF THAT

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CHROMOSOME FROM BEING RECOGNIZED

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AS A DNA DOUBLE STRABD BREAK.

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THIS DOESN'T HAPPEN ON ITS OWN.

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THE FORMATION OF THAT T-LOOP IS

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FACILITATED BY THE PROTEIN

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COMPLEX REFERRED TO AS

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SHELTERIN, IT'S A MULTISUBUNIT

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COMPLEX, MANY COMPONENTS

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INCLUDING DOUBT STRANDED DNA

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BINDINGS INCLUDING TRF 1 AND 2

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EXPW PROTEIN POT 1.

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I WOULD SAY ABOUT 15 YEARS AGO,

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THIS COMPLEX REALLY EXPAND TO

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INCLUDE THIS NONCODING RNA

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TRANSCRIBING FROM ENDS REFERRED

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TO AS TELEMERRIC REPEAT

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CONTAINING RNA, AND MY LAB AND

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OTHER VS GONE ON TO SHOW THAT

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THIS LONG NONCODING RNA

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CONTRIBUTES TO THE DNA RESPONSE

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AT TELOMERES AND NOT

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SURPRISINGLY, WE NOTICED THAT

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THERE'S AN INCREASE IN THIS LONG

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NONCODING RNA TRANSCRIBED IN A

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SUBSET'VE CANCERS AND SO WHILE

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THIS HAS BECOME A FOCUS OF MY

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LAB, I WON'T BE TALKING ABOUT

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TARA TODAY, WHAT I'M MORE

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FOCUSED ON IS AS I MENTIONED

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THIS DNA DAMAGE RESPONSE TO

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TELOMERES AND SO, COLLECTIVE

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EFFORTS OF CHILDREN AND TARA,

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BEING TO PROTECT THE TELOMERES

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FROM MACHINE DAMAGE, AND

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MAINTENANCE AND PROMOTES THE

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ELONGATION OF TELOMERES VIA A

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NUMBER OF NECKANISMS AND SO THE

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MOST COMMONLY KNOWN MECHANISM

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RIGHT, BEING TELEMERACE AND

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THOSE ARE COMMON IN THE MAJORITY

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OF CANCERS AND PROMOTES VERMEN

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INFECTED VERSE TRANSCRIPTION.

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AND I WOULD SAY THAT THE--SORRY

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THE SLIDES WERE SLOW TO CHANGE

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THERE.

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SO IN ADDITION TO TELEMERACE,

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THIS ALTERNATIVE MECHANISM OF

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TELOMERE LENGTHENS THAT WE REFER

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TO AS  ALT.

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THIS IS A PATHWAY FOR TELOMERE

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ELONGATION, SO WHILE THE VAST

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MAJORITY OF CANCERS AS A

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MENTIONED RELY ON THE

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TELEMERACE, SO ABOUT 85-95%--AND

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ACHIEVE REPLICATIVE IMMORTALITY.

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ABOUT 5-10% RELY ON THIS OLD

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MECHANISM OR ALTERNATIVE

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LENGTHENING OF TELOMERES, IT'S

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NOT DISTRIBUTED ACROSS ALL

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CANCER, SO BREAST CANCER DOESN'T

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HAVE 10%, LIVER DOESN'T HAVE

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10%, THIS IS DISTRIBUTED QUITE

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UNEACHLY AND THE VAST MAJORITY

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OF CANCERS THAT RELY ON THEM ARE

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MEZENCAMALL, AND ENDOTHELIAL AND

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THESE PICTURES AS DEPICTED HERE

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ARE OFTIO SARCOMAS, GLUE MARIOUS

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O BLASTOMAS, AND ENDOCRINE

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TUMORS

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THESE CANCERS HAVE POOR 5 YEAR

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SURVIVAL RATES AND THE

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THERAPEUTIC MODALITIES FOR THEZ

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CANCERS HAVEN'T CHANGED IN 40

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YEARS.

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SO I THINK IT WAS CLEAR EARLIER

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THAT HAVING A NEW REGULATED

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TELOMERE PATHWAY WOULD PROVIDE

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INSIGHT NOT ONLY INTO THE

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MECHANISM BUT ALSO IN THE

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OPPORTUNITY FOR THERAPEUTIC

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DEVELOPMENT AND SO ABOUT A 11

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YEARS AGO, I THINK THE FIRST

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MUTATIONS WERE IDENTIFY INDEED

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THE CHROMATIN REMODELING GENES

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AND ATRX AND DAXX, THESE

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DISCOVERIES WERE MADE BY CHRIS

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[INDISCERNIBLE] IN THE LAB, HE'S

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DONE AN AMAZING JOB OF THESE

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COMPLEXES.

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DAXX, BINDS THE EFTHIMIOS OPEN

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VARIANT SPECIFICALLY H3 PBT 3

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AND ATRX FACILITATES THE

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DEFINITION OF THESE REGIONS,

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INCLUDING TELOMERES, AND IN THE

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ABSENCE OR LOSS OF FUNCTION OF

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THESE PROTEINS, YOU GET LOSS OF

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H3.3 DEPOSITION, YOU GET

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CHROMATIN DECOMPACTION AND THIS

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IS BELIEVED TO PROMOTE MORE

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PERMISSIVE ENVIRONMENT FOR

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RECOMBINATION AT THE TELOMERES.

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SO WHEN I STARTED MY LAB, I WAS

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REALLY INTERESTED IN

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UNDERSTANDING THE BASIC

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MECHANISMS OF THE ALT PATHWAY,

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AND SO, AT THE TIME I STARTED MY

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LAB, THERE WERE VERY FEW

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RESOURCES AVAILABLE IN THE WORLD

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OF ALT.

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THERE WERE A FEW CELL LINES THAT

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WERE NOTE INDEED THE LITERATURE

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AND OF COURSE A FEW TUMORS BUT

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WE DIDN'T HAVE ANY OF OUR OWN

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AND SO I STARTED COLLECTING A

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NUMBER OF THESE CELL LINES AND

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TUMORS BECAUSE I WAS VERY

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CURIOUS NOT JUST ABOUT THE

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MECHANISM BUT ABOUT THE GENETICS

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UNDERLYING ALT.

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AND SO 1 OF THE THINGS THAT

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STRUCK ME WHEN I STARTED TO

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ACQUIRE THESE TUMORS IS I HAD NO

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IDEA WHETHER THEY WERE ALT OR

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TELEMERACE, AND SO I HAVEN'T

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LISTED ALL OF THEM HERE.

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THESE ARE THE 1S THAT ENDED UP

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BEING ALT POSITIVE.

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SO IN AN EFFORT TO CHARACTERIZE

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THESE TUMORS AND CELL LINES WE

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USE A NUMBER OF DIFFERENT

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ASSAYS, SO FIRST STARTING OFF WE

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CAN ANALYZE FOR TELE MERAISE ACE

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ACTIVITY AND WE CAN RULE OUT

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THAT THAT DO OR DO NOT HAVE THE

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ACTIVITY, WE LIKE TO CONFIRM ALT

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ACTIVITY AND WE HAVE TO DO THIS

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USING A DIFFERENT NUMBER OF

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ASSAYS AND THESE WILL BE

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RELEVANT THROUGHOUT THE TALK SO

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I HAVE JOUST OUTLINES THEM HERE.

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SO 1 OF THEM IS THE FORMATION OF

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1 OF THESE WHAT WE CALL APB AND

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THESE ARE PML BODIES AND SO THAT

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IS WHEN THE TELEOR MERRIC

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PROTEINS CO LOCALIZE WITH THE

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LEUKEMIA PROTEIN, AND YOU CAN

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SEE, THEY GENERATE THESE

259
00:09:41,600 --> 00:09:43,680
COLOCALIZATION EVENTS AND THESE

260
00:09:43,680 --> 00:09:46,520
OCCUR IN ALT CELLS AND ALMOST

261
00:09:46,520 --> 00:09:47,720
NEVER IN TELEORDER OF MICRONSER

262
00:09:47,720 --> 00:09:50,480
ACE CELLS SO THIS IS A STRONG

263
00:09:50,480 --> 00:09:51,800
INDICATION OF ALT POSITIVITY IN

264
00:09:51,800 --> 00:09:54,720
A CELL OR TUMOR AND IN ADDITION

265
00:09:54,720 --> 00:09:56,200
TO THE APBs, I WOULD SAY THE

266
00:09:56,200 --> 00:09:57,080
GOLD STANDARD IN THE FIELD MAY

267
00:09:57,080 --> 00:10:00,240
BE IS TO ANALYZE THE COMA TELEO

268
00:10:00,240 --> 00:10:01,800
MERRIC REPEATS AND THESE ARE

269
00:10:01,800 --> 00:10:03,480
CIRCULAR SPECIES OF DNA, THESE

270
00:10:03,480 --> 00:10:04,760
1S IN PARTICULAR ARE C-RICH AND

271
00:10:04,760 --> 00:10:07,960
THEY HAVE A PARTIAL DIEWP LEX

272
00:10:07,960 --> 00:10:10,160
REGION AND THIS DUPLEX REGION

273
00:10:10,160 --> 00:10:11,720
SERVES AS A PRIMER IF YOU USE

274
00:10:11,720 --> 00:10:15,320
THIS SUBSTRATE IN A ROLLING

275
00:10:15,320 --> 00:10:16,360
CIRCLE AMLIAISONIFICATION

276
00:10:16,360 --> 00:10:17,720
REACTION, CAN YOUASMLIFY THESE

277
00:10:17,720 --> 00:10:19,440
C-CIRCLE PRODUCTS AND IF YOU RUN

278
00:10:19,440 --> 00:10:23,120
THEM OUT IN A DOT BLOT AND

279
00:10:23,120 --> 00:10:24,560
PRESCRIBE, YOU CAN SEE THAT THE

280
00:10:24,560 --> 00:10:25,880
ALT CELLS HAVE A DRAMATIC

281
00:10:25,880 --> 00:10:27,680
INCREASE IN THE PREVALENCE OF

282
00:10:27,680 --> 00:10:29,680
THESE CIRCLES AND THIS IS IMAGED

283
00:10:29,680 --> 00:10:31,920
AND SORT OF A GOLD STANDARD OF

284
00:10:31,920 --> 00:10:34,760
ALT ACTIVITY IN CELLS AND

285
00:10:34,760 --> 00:10:35,000
TUMORS.

286
00:10:35,000 --> 00:10:36,880
AND THEN FINALLY, WE HAVE A

287
00:10:36,880 --> 00:10:39,320
TENDENCY TO ANALYZE THE ACTUAL

288
00:10:39,320 --> 00:10:43,600
EXCHANGE EVENTS WHICH HAPPEN AT

289
00:10:43,600 --> 00:10:44,920
TELOMERE EVENTS AND WE CALL

290
00:10:44,920 --> 00:10:47,360
THESE EXSCHAINCHS AND WE DO THIS

291
00:10:47,360 --> 00:10:49,120
BY PREPARING ENDS AND WE CAN

292
00:10:49,120 --> 00:10:53,120
ANALYZE THE ENDS USING PROBES,

293
00:10:53,120 --> 00:10:54,720
SO RED OR GREEN PROBES THAT

294
00:10:54,720 --> 00:10:55,480
RECOGNIZE THE ARM OF THE

295
00:10:55,480 --> 00:10:57,120
CHROMOSOME AND YOU CAN SEE WHERE

296
00:10:57,120 --> 00:10:58,760
THERE'S INDEPENDENT RED AND

297
00:10:58,760 --> 00:11:01,120
GREEN SIGNAL BUT WHEN THERE'S A

298
00:11:01,120 --> 00:11:02,240
REON COMBINATION, YOU GET AN

299
00:11:02,240 --> 00:11:04,360
OVERLAP OF THE PROBES AND YOU

300
00:11:04,360 --> 00:11:05,600
CAN SEE THESE ARE VISUALIZED AND

301
00:11:05,600 --> 00:11:07,520
THEY ARE OVERLAP NOTHING THAT

302
00:11:07,520 --> 00:11:08,720
COLOR SIGNAL SO ONCE I

303
00:11:08,720 --> 00:11:10,400
ESTABLISHED A PANEL OF ALT

304
00:11:10,400 --> 00:11:11,600
TUMORS AND CELL LINES IN THE

305
00:11:11,600 --> 00:11:14,000
LAB, I WAS CURIOUS ABOUT THE

306
00:11:14,000 --> 00:11:15,640
GENETICS SOY INITIATED A

307
00:11:15,640 --> 00:11:16,920
SIGNIFYQUENCING CAMPAIGN OF

308
00:11:16,920 --> 00:11:18,520
THESE ALT POSITIVE SAMPLES TO

309
00:11:18,520 --> 00:11:20,000
SEE IF WE COULD DEFINE ANY

310
00:11:20,000 --> 00:11:27,200
ADDITIONAL MUTATIONS THAT MAY BE

311
00:11:27,200 --> 00:11:28,120
CONTRIBUTING TO THE--PERCENT OF

312
00:11:28,120 --> 00:11:30,480
THESE TUMORS AND CELL LINES HAD

313
00:11:30,480 --> 00:11:31,520
THE MUTATIONS IN ATX.

314
00:11:31,520 --> 00:11:32,960
SO THERE'S A VARIETY OF

315
00:11:32,960 --> 00:11:35,160
DIFFERENT MUTATIONS AND BECAUSE

316
00:11:35,160 --> 00:11:37,440
OFTIO SARCOMAS ARE HIGHLY

317
00:11:37,440 --> 00:11:39,080
REARRANGED, SOME OF THEM WERE

318
00:11:39,080 --> 00:11:40,600
DEEP DELETIONS, INVERSION

319
00:11:40,600 --> 00:11:41,680
MUTATIONS BUT AGAIN 50% RIGHT

320
00:11:41,680 --> 00:12:09,280
OFF THE TOP HAD MUTATIONS ATRX.

321
00:12:09,280 --> 00:12:11,400
--PUBLISHED IN THE LITERATURE TO

322
00:12:11,400 --> 00:12:14,320
BE WILD TYPE FOR ATRX AND DAXX.

323
00:12:14,320 --> 00:12:16,120
BUT WHEN WE DID OUR INITIAL

324
00:12:16,120 --> 00:12:18,080
ANALYSIS, WHAT WE NOTICE SIDE

325
00:12:18,080 --> 00:12:20,640
LOOKING AT THOSE STRUCTURAL

326
00:12:20,640 --> 00:12:23,200
REARANKMENTS, WE COULD SEE A

327
00:12:23,200 --> 00:12:26,120
STRONG OR SIGNIFICANT

328
00:12:26,120 --> 00:12:29,000
TRANSLOCATION BETWEEN DAXX ON

329
00:12:29,000 --> 00:12:32,280
FIFTH YEAR AND KIPS-ON 16, SO WE

330
00:12:32,280 --> 00:12:34,880
LOOK TO CONFIRM THIS BY USING

331
00:12:34,880 --> 00:12:37,680
CARIO TYPING AND I THINK THIS IS

332
00:12:37,680 --> 00:12:39,480
IMEAN A BEAUTIFUL VISUAL

333
00:12:39,480 --> 00:12:41,080
REPRESENTATION NOT ONLY THE

334
00:12:41,080 --> 00:12:43,560
CHANGES IN EMPLOYEDY BUT THE

335
00:12:43,560 --> 00:12:44,920
INCREASES IN STRUCTURAL

336
00:12:44,920 --> 00:12:46,360
REARRANGEMENTS THAT ARE

337
00:12:46,360 --> 00:12:47,480
PREVALENT THROUGHOUT THESE OFTIO

338
00:12:47,480 --> 00:12:50,480
SARCOMA TUMORS AND WE CAN

339
00:12:50,480 --> 00:12:52,240
HIGHLIGHT HERE THE TRANSLOCATION

340
00:12:52,240 --> 00:12:53,800
WE IECIALIZED IN OUR SLOTS WHERE

341
00:12:53,800 --> 00:12:56,040
WE SEE THIS TRANSLOCATION

342
00:12:56,040 --> 00:12:57,640
BETWEEN CHROMOSOME 6 AND THE

343
00:12:57,640 --> 00:12:58,680
PINK AND CHROMOSOME 16 IN THE

344
00:12:58,680 --> 00:12:58,920
BLUE.

345
00:12:58,920 --> 00:13:04,360
SO WHEN WE TOOK A REAL BIT OF A

346
00:13:04,360 --> 00:13:06,360
DEEPER DIVE INTO THAT STRUCTURAL

347
00:13:06,360 --> 00:13:07,560
REARRANGEMENT, WE COULD LOOK AT

348
00:13:07,560 --> 00:13:13,360
THE DNA LEVEL AND WE COULD SEE

349
00:13:13,360 --> 00:13:14,440
THAT THIS DAXXKIFC3, HAD A BREAK

350
00:13:14,440 --> 00:13:16,520
POINT THAT WAS IN THE PRIME

351
00:13:16,520 --> 00:13:18,960
POINT OF DAXX, AND WHEN I

352
00:13:18,960 --> 00:13:19,800
PUBLISHED BEFORE THESE WERE WILD

353
00:13:19,800 --> 00:13:21,280
TYPE IN THE PREVIOUS CELLS, A

354
00:13:21,280 --> 00:13:24,480
LOT OF THESE WERE USING WHOLE

355
00:13:24,480 --> 00:13:25,520
EXOME SEQUENCING TO ANALYZE

356
00:13:25,520 --> 00:13:27,160
THESE TUMORS AND THIS WAS A

357
00:13:27,160 --> 00:13:31,440
LESSON IN WHY THIS IS DIFFICULT

358
00:13:31,440 --> 00:13:33,400
IN OFTIO SARCOMA, SOMETHING

359
00:13:33,400 --> 00:13:34,240
THAT'S HIGHLY REARRANGED BECAUSE

360
00:13:34,240 --> 00:13:36,800
IT WAS FULLY INTACT AND WHILED

361
00:13:36,800 --> 00:13:39,080
TYPE DAX JEANS AND THERE WAS NO

362
00:13:39,080 --> 00:13:39,880
EFFECT TO THEAXONS THEMSELVES

363
00:13:39,880 --> 00:13:41,920
BUT WHEN WE WENT TO ANALYZE THE

364
00:13:41,920 --> 00:13:45,960
RNA WE FOUND THAT IN FACT, THIS

365
00:13:45,960 --> 00:13:49,480
FUSION RESULTED IN A TRANSCRIPT

366
00:13:49,480 --> 00:13:50,640
THAT WAS ALTERNATIVELY SPLICED

367
00:13:50,640 --> 00:13:53,200
AND AS A CONSEQUENCE SPLICED OUT

368
00:13:53,200 --> 00:13:54,480
AXON 8.

369
00:13:54,480 --> 00:14:00,200
SO FOLLOWING AXON 7, DAXX WAS

370
00:14:00,200 --> 00:14:02,400
FUSED TOAXON 10 OF KIFC3.

371
00:14:02,400 --> 00:14:04,600
SO THIS TOLD US WE HAD A KHIHIGH

372
00:14:04,600 --> 00:14:05,800
MERRIC RIGHT GENE PRODUCT BUT

373
00:14:05,800 --> 00:14:07,080
LIKELY IF THERE WERE TRANSLATED

374
00:14:07,080 --> 00:14:09,280
WE MIGHT ALSO HAVE A KHIHIGH

375
00:14:09,280 --> 00:14:10,080
MERRIC PROTEIN AND THAT'S WHAT

376
00:14:10,080 --> 00:14:11,840
WE SAW SO IF WE LOOK AT THESE

377
00:14:11,840 --> 00:14:14,360
CONTROL CELLS WE CAN SEE WILD

378
00:14:14,360 --> 00:14:16,560
TYPE DAX PROTEIN HERE WE CAN OF

379
00:14:16,560 --> 00:14:21,120
COURSE KNOCK DOWN WITH SIRNA TO

380
00:14:21,120 --> 00:14:25,760
DAXX, AND YOU CAN ALSO SEE THIS

381
00:14:25,760 --> 00:14:31,400
HIGHER DAKS AND IF YOU USE DAKS

382
00:14:31,400 --> 00:14:33,360
WE CAN ALSO KNOCK DOWN THIS

383
00:14:33,360 --> 00:14:34,280
FIEWGDZ PRODUCT SO THIS

384
00:14:34,280 --> 00:14:35,480
SUGGESTED TO US THAT NOT ONLY

385
00:14:35,480 --> 00:14:38,800
WAS THERE A TRANSWELL ONICATION

386
00:14:38,800 --> 00:14:40,240
BUT IT MAINTAINS EXPRESSION OF A

387
00:14:40,240 --> 00:14:42,560
PROTEIN PRODUCT SO OF COURSE THE

388
00:14:42,560 --> 00:14:43,280
QUESTION BECOMES WELL, WHAT'S

389
00:14:43,280 --> 00:14:45,080
THE FUNCTION OF THIS PROTEIN OR

390
00:14:45,080 --> 00:14:48,120
IS IT A LOSS OF FUNCTION FOR

391
00:14:48,120 --> 00:14:49,240
DAXX.

392
00:14:49,240 --> 00:14:50,680
AND THAT DAXX, C-TERMINUS IS

393
00:14:50,680 --> 00:14:53,800
CRITICAL FOR A NUMBER OF

394
00:14:53,800 --> 00:14:55,200
DAXX FUNCTIONS, SO SPECIFICALLY

395
00:14:55,200 --> 00:14:58,640
REGIONS IN THAT AXON 8 WHICH IS

396
00:14:58,640 --> 00:15:00,320
ELIMINATED FROM THE TRANSCRIPT

397
00:15:00,320 --> 00:15:01,480
AND NOT PRODUCED AS PART OF THE

398
00:15:01,480 --> 00:15:01,720
PROTEIN.

399
00:15:01,720 --> 00:15:03,200
SO 1 THING WE LOOKED AT RIGHT

400
00:15:03,200 --> 00:15:08,480
WAIRKS WAS THE IDEA OF

401
00:15:08,480 --> 00:15:10,280
DAXX INTERACTIONS SO IT'S KNOWN

402
00:15:10,280 --> 00:15:13,360
TO INTERACT WITH THE PML.

403
00:15:13,360 --> 00:15:17,080
AND AS I MENTIONED IN THE

404
00:15:17,080 --> 00:15:19,360
PREVALENCE OF CAPSERS OR IN THE

405
00:15:19,360 --> 00:15:20,040
COLOCALIZATION OF TELEO PLEERS.

406
00:15:20,040 --> 00:15:22,520
SO WHEN WE LOOK AT THESE G2 FINE

407
00:15:22,520 --> 00:15:25,360
2 CELLS, AND WE STAIN WITH A

408
00:15:25,360 --> 00:15:26,440
DAXX ANTIBODY WITH THE

409
00:15:26,440 --> 00:15:27,680
ENDOGENOUS PROTEIN, WE SEE

410
00:15:27,680 --> 00:15:30,240
LITTLE SIGNAL AND THE ANTIBODY

411
00:15:30,240 --> 00:15:31,280
DOES IN FACT RECOGNIZE A PART OF

412
00:15:31,280 --> 00:15:33,080
THE PROTEIN IT EXPRESSED, IT

413
00:15:33,080 --> 00:15:34,160
DOESN'T RECOGNIZE THE

414
00:15:34,160 --> 00:15:37,520
C-TERMINUS, SO WE SEE ALMOST NO

415
00:15:37,520 --> 00:15:41,280
SIGNAL FROM DAXX, BUT WE DO SEE

416
00:15:41,280 --> 00:15:42,400
COLOCALIZATION AS WE WOULD

417
00:15:42,400 --> 00:15:44,160
EXPECT TO INDICATE THE PRESENCE

418
00:15:44,160 --> 00:15:46,880
OF APB, SO WE DECIDED, CAN WE

419
00:15:46,880 --> 00:15:48,640
RESCUE THIS ALT STATUS OF THIS

420
00:15:48,640 --> 00:15:51,000
CELL AND REPRESS APB BY

421
00:15:51,000 --> 00:15:52,600
EXPRESSING WILD TYPE DAXX, SO WE

422
00:15:52,600 --> 00:15:54,280
DID THAT AGAIN BY EXPRESSING

423
00:15:54,280 --> 00:15:56,640
THIS FLAG TAGGED WILD TYPE

424
00:15:56,640 --> 00:15:58,280
DAXXFOR EITHER 2 OR SEX DAYS AND

425
00:15:58,280 --> 00:15:59,880
WHAT WE COULD SEE IS NOT ONLY DO

426
00:15:59,880 --> 00:16:01,840
WE START TO ACCUMULATE THE

427
00:16:01,840 --> 00:16:05,680
EXPRESSION OF THIS PROTEIN AND

428
00:16:05,680 --> 00:16:07,760
THESE, BUT YOU CAN SEE THEY

429
00:16:07,760 --> 00:16:10,600
ACCUMULATE WITH PML AS WOULD BE

430
00:16:10,600 --> 00:16:12,400
PREDICTED FOR NORMAL DAXXSPAY

431
00:16:12,400 --> 00:16:14,360
FUNCTION AND THESE FOCI NO

432
00:16:14,360 --> 00:16:17,280
LONGER CO LOCALIZE WITH THESE

433
00:16:17,280 --> 00:16:18,800
TELOMERES SUGGESTS THAT APB AND

434
00:16:18,800 --> 00:16:19,560
THESE WERE QUANTIFIED ON THE

435
00:16:19,560 --> 00:16:20,960
RIGHT AND THIS SUGGESTED THAT IN

436
00:16:20,960 --> 00:16:28,680
THE FACT IN THE CELL LINES, THIS

437
00:16:28,680 --> 00:16:35,160
DAXX KIFC3 FUNCTIONALLY

438
00:16:35,160 --> 00:16:35,760
ENACTIVATES DAXX.

439
00:16:35,760 --> 00:16:37,480
AND UNDERSTANDING THE MUTATIONAL

440
00:16:37,480 --> 00:16:38,960
PROFILE, BUT IF WE WANT TO DIG

441
00:16:38,960 --> 00:16:40,040
FURTHER AND CHARACTERIZE SOME OF

442
00:16:40,040 --> 00:16:41,280
THESE OTHER UNKNOWNS THAT WERE

443
00:16:41,280 --> 00:16:42,760
SORT OF GLARING AT THE TIME TO

444
00:16:42,760 --> 00:16:45,600
US, WE NEEDED TO REALLY DIG INTO

445
00:16:45,600 --> 00:16:48,080
THE MECHANISTIC DETAILS OF THE

446
00:16:48,080 --> 00:16:48,320
PATHWAY.

447
00:16:48,320 --> 00:16:49,680
AND SO I MENTIONED TO YOU

448
00:16:49,680 --> 00:16:52,920
EARLIER THAT THE ALT PATHWAY

449
00:16:52,920 --> 00:16:53,640
RELYS ON RECOMBINATION, THIS

450
00:16:53,640 --> 00:16:56,640
EVENT IS NOT UNLIKE OTHER TYPES

451
00:16:56,640 --> 00:16:58,480
OF RECOMBINATION FOR EXAMPLE IN

452
00:16:58,480 --> 00:16:59,760
A DOUBLE STRAND BREAK, WHERE YOU

453
00:16:59,760 --> 00:17:01,680
HAVE PROCESS THAGOREAN CREATES

454
00:17:01,680 --> 00:17:03,920
THIS 3 PRIME SINGLE STRANDED

455
00:17:03,920 --> 00:17:06,240
OVERHANG AND THIS 3 PRIME

456
00:17:06,240 --> 00:17:08,680
OVERHANG CAN INITIATE STRAND

457
00:17:08,680 --> 00:17:10,560
INVASION AND HOMOLOGYY SEARCH

458
00:17:10,560 --> 00:17:12,680
AND CAPTURE, IT DOESN'T HAVE TO

459
00:17:12,680 --> 00:17:16,400
BE A HOMOLOGOUS CHROMOSOME, ANY

460
00:17:16,400 --> 00:17:17,640
CHROMOSOME THAT HAS TELEOR

461
00:17:17,640 --> 00:17:18,680
MERRIC SEQUENCE, AND SOME OF

462
00:17:18,680 --> 00:17:21,240
THOSE PREETS I MENTIONED

463
00:17:21,240 --> 00:17:21,480
EARLIER.

464
00:17:21,480 --> 00:17:24,880
SO IN THESE INSTANCES YOU CAN

465
00:17:24,880 --> 00:17:27,120
ENVISION, YOU GET THE SMALLER

466
00:17:27,120 --> 00:17:29,120
CAPTURE AND EXTENSION AND

467
00:17:29,120 --> 00:17:30,880
TELOMERE SYNTHESIS, BUT WHAT I

468
00:17:30,880 --> 00:17:33,400
DIDN'T MENTION IS THAT IN

469
00:17:33,400 --> 00:17:35,920
ADDITION TO THE CANNONICLE GG8,

470
00:17:35,920 --> 00:17:37,920
THAT ARE PREULENT AT THE

471
00:17:37,920 --> 00:17:38,960
TELOMERE ENDS, WE'VE COME TO

472
00:17:38,960 --> 00:17:40,760
LEARN THERE IS THE INCORPORATION

473
00:17:40,760 --> 00:17:42,640
OF DEGENERATE OR VARIANT REPEATS

474
00:17:42,640 --> 00:17:45,120
AND SO INSTEAD OF TTA, GGG,

475
00:17:45,120 --> 00:17:47,600
THESE MAY CONSIST OF TTA, GGG,

476
00:17:47,600 --> 00:17:49,600
SO A SLIGHT VARIATION IN THE

477
00:17:49,600 --> 00:17:50,680
NUCLEOTIDE SEQUENCE, AND SO YOU

478
00:17:50,680 --> 00:17:53,360
CAN ENVISION NOW, IF YOU HAVE

479
00:17:53,360 --> 00:17:54,480
INITIATION OF THIS HOMOLOGY

480
00:17:54,480 --> 00:17:57,240
SEARCH AND CAPTURE AND A REGION

481
00:17:57,240 --> 00:17:59,600
OF HOMOLOGY THAT HAS DEGENERATE

482
00:17:59,600 --> 00:18:02,280
OR REPEATS DOWN STREAM, YOU CAN

483
00:18:02,280 --> 00:18:04,360
SEE HOW EXTENSION DNA SYNTHESIS

484
00:18:04,360 --> 00:18:06,480
AND MIGHT THROUGH THIS REGION

485
00:18:06,480 --> 00:18:07,200
MIGHT INCORPORATE VARIANT

486
00:18:07,200 --> 00:18:11,240
REPEATS THROUGHOUT THAT

487
00:18:11,240 --> 00:18:11,480
SEQUENCE.

488
00:18:11,480 --> 00:18:13,000
AND SO WHY DOES THAT MATTER?

489
00:18:13,000 --> 00:18:15,280
IT MATTERS BECAUSE AS I TOLD YOU

490
00:18:15,280 --> 00:18:17,960
BEFORE, THE SHELTER AND PROTEIN

491
00:18:17,960 --> 00:18:20,720
COMPLEX BINDS TO THESE WITH

492
00:18:20,720 --> 00:18:22,240
THESE SPECIFICITIES AND THESE

493
00:18:22,240 --> 00:18:23,520
BINDINGS PROTEINS, AND THESE

494
00:18:23,520 --> 00:18:25,400
VARIANT REPEATS LEAD TO DECREASE

495
00:18:25,400 --> 00:18:27,320
IN THE EFFICIENCY OF CHILDREN

496
00:18:27,320 --> 00:18:29,480
BINDING IT AT THESE ALT TELEOR

497
00:18:29,480 --> 00:18:32,240
MERES AND IF THIS IS THE CASE,

498
00:18:32,240 --> 00:18:34,720
YOU WOULD IMAGINE GIVEN THE

499
00:18:34,720 --> 00:18:35,800
ABNORMALITIES SENSZ OF

500
00:18:35,800 --> 00:18:37,080
SHELTERIN, THAT THIS WOULD

501
00:18:37,080 --> 00:18:38,880
INDUCE SOME LEVEL OF REPLICATION

502
00:18:38,880 --> 00:18:41,720
STRESS AND ULTIMATELY A DNA

503
00:18:41,720 --> 00:18:42,080
DAMAGE RESPONSE.

504
00:18:42,080 --> 00:18:43,560
AND SO THIS LED TO OUR FIRST

505
00:18:43,560 --> 00:18:46,720
QUESTION WHICH WAS DOES CHRONIC

506
00:18:46,720 --> 00:18:47,840
REPLICATION STRESS PERPETUATE

507
00:18:47,840 --> 00:18:48,360
ALT ACTIVITY.

508
00:18:48,360 --> 00:18:53,200
AND SO, WE SORT OF RELIED ON

509
00:18:53,200 --> 00:18:56,280
SOME PUBLISHED DATA FROM DAVID

510
00:18:56,280 --> 00:18:57,640
CORTEZ'S GROUP WHERE E IDENTIFY

511
00:18:57,640 --> 00:19:00,280
THAD THIS WAS 1 OF THE MOST

512
00:19:00,280 --> 00:19:03,600
ABUNDANT PROTEINS BOUND OF SITES

513
00:19:03,600 --> 00:19:07,280
STALLED BY REPLICATION OR

514
00:19:07,280 --> 00:19:09,560
REPPLICATION STRESS.

515
00:19:09,560 --> 00:19:12,200
THIS IS A KNEELING HELIX CASE

516
00:19:12,200 --> 00:19:17,880
AND WE WANTED TO KNOW WHETHER

517
00:19:17,880 --> 00:19:18,520
SMARCAL LOCALIZED TO TELOMERES.

518
00:19:18,520 --> 00:19:20,080
SO ON OUR INITIAL PASS AND

519
00:19:20,080 --> 00:19:21,640
THROUGHOUT THE TALK I WILL USE A

520
00:19:21,640 --> 00:19:23,320
NUMBER OF THESE ALT CELL LINES

521
00:19:23,320 --> 00:19:24,600
THAT WE'VE GATHERED OVER THE

522
00:19:24,600 --> 00:19:26,440
YEARS AND THIS PARTICULAR

523
00:19:26,440 --> 00:19:27,720
INSTANCE, THERE ARE OFTENNAN

524
00:19:27,720 --> 00:19:29,080
OITATED AS SO BEING ALT.

525
00:19:29,080 --> 00:19:31,520
AND O IN THESE 2 ALT CELL LINES

526
00:19:31,520 --> 00:19:34,080
WE CAN SEE THESE REALLY

527
00:19:34,080 --> 00:19:35,600
BEAUTIFUL FOCI THAT CO LOCALIZE

528
00:19:35,600 --> 00:19:37,640
IN TELOMERES IN THE ALT POSITIVE

529
00:19:37,640 --> 00:19:43,880
CELL LINES AND WE SEE MINIMAL

530
00:19:43,880 --> 00:19:45,400
COLOCALIZATION BETWEEN SMARCAL,

531
00:19:45,400 --> 00:19:47,720
AND TELEOR MERES THESE ARE

532
00:19:47,720 --> 00:19:48,280
REGULATED BY TELEORDER OF

533
00:19:48,280 --> 00:19:48,760
MICRONS RACE.

534
00:19:48,760 --> 00:19:53,640
SO WE CAN FURTHER SUPPORT THIS

535
00:19:53,640 --> 00:19:56,280
DATA USING CHP, USING SMARCALL,

536
00:19:56,280 --> 00:19:59,160
IN THIS INSTANCE AND WE IPsING

537
00:19:59,160 --> 00:20:02,840
IT HERE OR AS A POSITIVE CONTROL

538
00:20:02,840 --> 00:20:05,160
TRF2, AND YOU CAN SEE IT

539
00:20:05,160 --> 00:20:08,280
ASSOCIATES BOTH WITH TELOMERES

540
00:20:08,280 --> 00:20:10,400
AND ALT-POSITIVE TELOMERES, THIS

541
00:20:10,400 --> 00:20:13,200
IS SIGNIFICANTLY RICHED IN THE

542
00:20:13,200 --> 00:20:15,720
ALT TELEO MERRIC SAMPLES,

543
00:20:15,720 --> 00:20:19,000
SUGGESTING OR HIGHLIGHTING THE

544
00:20:19,000 --> 00:20:20,360
PREVALENCE ALT 1 TELOMERES.

545
00:20:20,360 --> 00:20:22,080
SO MORE RECENTLY WE'VE BEEN PUT

546
00:20:22,080 --> 00:20:23,120
NOTHING A CONCERTED EXPOSURE TO

547
00:20:23,120 --> 00:20:23,920
RADIATION TO CHARACTERIZE THE DO

548
00:20:23,920 --> 00:20:25,320
PAIN IN AMERICAS THAT ARE

549
00:20:25,320 --> 00:20:28,680
RESPONSIBLE FOR THE

550
00:20:28,680 --> 00:20:31,240
RECRUELTIMENT OF SMARCALL, 2

551
00:20:31,240 --> 00:20:32,600
SITES OF TELEMERRIC STRESS AND

552
00:20:32,600 --> 00:20:36,040
THOSE HAVE BEEN IDENTIFIED IN

553
00:20:36,040 --> 00:20:36,400
THE LITERATURE.

554
00:20:36,400 --> 00:20:38,920
SO STRUCTURALLY THIS IS THE

555
00:20:38,920 --> 00:20:41,040
SMARCALL 1 PROTEIN WHICH

556
00:20:41,040 --> 00:20:43,640
CONTAINS A BINDING DOMAIN THE

557
00:20:43,640 --> 00:20:45,080
EPPED TERMINUS, CONTAINS 2

558
00:20:45,080 --> 00:20:46,160
SEQUENTIAL HEART DOMAINS THAT

559
00:20:46,160 --> 00:20:47,400
WERE THOUGHT TO MEDIATE ACTION

560
00:20:47,400 --> 00:20:51,680
WITH DNA AND AS I MENTIONED IT'S

561
00:20:51,680 --> 00:20:55,080
A SWITCH TO CHROMATIN REMODELING

562
00:20:55,080 --> 00:21:00,520
DESIGN AND CONTAINS A SNF 2.

563
00:21:00,520 --> 00:21:02,600
DELETION OF THE RNA BINDING

564
00:21:02,600 --> 00:21:04,360
DOMAIN MAIN, OR WE CREATED POINT

565
00:21:04,360 --> 00:21:06,720
MUTATIONS WITHIN THE INITIAL

566
00:21:06,720 --> 00:21:07,720
HARP DOMAIN, THE SECOND HARP

567
00:21:07,720 --> 00:21:11,560
DOMAIN AND OF COURSE THE

568
00:21:11,560 --> 00:21:13,600
COMBINED HARP DOMAINS AND

569
00:21:13,600 --> 00:21:15,680
FINALLY, THIS ENACTIVATES THE

570
00:21:15,680 --> 00:21:18,040
ATP ACE OF SMACALL AND WE CAN

571
00:21:18,040 --> 00:21:24,880
SEE BY THE IMAGE --IMAGES, WE

572
00:21:24,880 --> 00:21:25,280
EXPRESS TELOMERES.

573
00:21:25,280 --> 00:21:26,720
AND THE SAME COULD BE SAID FOR

574
00:21:26,720 --> 00:21:30,080
THE DELETION OF THE RNA BINDING

575
00:21:30,080 --> 00:21:34,000
DOMAIN AS TO SOME EXTENT IN THE

576
00:21:34,000 --> 00:21:36,200
MUTANTS AND THE RDQ.

577
00:21:36,200 --> 00:21:40,720
AND THE ONLY 1 THAT'S--WAS THIS

578
00:21:40,720 --> 00:21:42,680
DUAL HARP DOMAIN MUTANT.

579
00:21:42,680 --> 00:21:44,480
AND SO WE CAN QUANTIFY THIS HERE

580
00:21:44,480 --> 00:21:53,040
AGAIN WHERE WE SEE THAT EVEN THE

581
00:21:53,040 --> 00:21:54,640
[INDISCERNIBLE] INTERACTING WITH

582
00:21:54,640 --> 00:21:57,480
THE TELOMERES, IT'S THESE DUAL

583
00:21:57,480 --> 00:22:00,680
HARP DOMAIN MUTANTS THAT SEEM TO

584
00:22:00,680 --> 00:22:03,280
INCREASE SMARCALL 1 BINDING OF

585
00:22:03,280 --> 00:22:03,520
TELOMERES.

586
00:22:03,520 --> 00:22:05,080
THIS SAN INTERESTING FINDING,

587
00:22:05,080 --> 00:22:08,080
SPECIFICALLY BECAUSE IN THE

588
00:22:08,080 --> 00:22:09,360
LITERATURE SMARCALL IS MENTIONED

589
00:22:09,360 --> 00:22:12,360
TO BIND WITH RFA, AND THIS RPA

590
00:22:12,360 --> 00:22:14,600
BINDING DOMAIN IS REQUIRED FOR

591
00:22:14,600 --> 00:22:16,480
THE LOCAL SITES OF DNA DAMAGE

592
00:22:16,480 --> 00:22:18,680
WIDE AND IT SUGGESTS THAT

593
00:22:18,680 --> 00:22:20,880
PERHAPS THESE HARP DOMAINS ARE

594
00:22:20,880 --> 00:22:22,400
REQUIRED FOR TELEO MERRIC DNA

595
00:22:22,400 --> 00:22:23,360
AND THIS IS ONGOING IN THE LAB

596
00:22:23,360 --> 00:22:26,120
AND WE HOPE TO BE FOLLOWING UP

597
00:22:26,120 --> 00:22:26,480
ON THIS SOON.

598
00:22:26,480 --> 00:22:29,840
BUT I THINK IT SUGGESTED TO US

599
00:22:29,840 --> 00:22:31,520
IN GENERAL, WAS THAT IF OR AT

600
00:22:31,520 --> 00:22:34,440
LEAST IT LED TOLET HYPOTHESIS

601
00:22:34,440 --> 00:22:37,480
THAT IF IN GENERAL SMARCALL WAS

602
00:22:37,480 --> 00:22:39,520
REQUIRED TO MEDIATE REPLICATION

603
00:22:39,520 --> 00:22:41,160
STRESS THROUGH THESE CHALLENGING

604
00:22:41,160 --> 00:22:42,880
REGIONS IT MIGHT ALSO CONTRIBUTE

605
00:22:42,880 --> 00:22:45,960
TO THE PROMOTION OF TELOMERE

606
00:22:45,960 --> 00:22:46,240
STABILITY.

607
00:22:46,240 --> 00:22:50,360
SO WE ASKED IF WE ELIMINATE

608
00:22:50,360 --> 00:22:51,520
SMARCALL, AND INCREASED

609
00:22:51,520 --> 00:22:53,280
REPLICATION FORKS WOULD THEY BE

610
00:22:53,280 --> 00:22:54,760
SUSCEPTIBLE TO CLEAVAGE THAT

611
00:22:54,760 --> 00:22:56,360
PROMOTE DOUBLE STRAND BREAKS BY

612
00:22:56,360 --> 00:22:56,800
THESE ENZYMES?

613
00:22:56,800 --> 00:22:59,120
AND THESE COULD BEING RESECTED

614
00:22:59,120 --> 00:23:01,160
TO PROMOTE THE RECOMBINATION

615
00:23:01,160 --> 00:23:02,600
INDICATIVE OF DNA DAMAGE REPAIR.

616
00:23:02,600 --> 00:23:03,520
AND SO, THE FIRST QUESTION WAS

617
00:23:03,520 --> 00:23:06,520
IF WE GET RID OF SMARCALL, DO WE

618
00:23:06,520 --> 00:23:08,160
SEE AN INCREASE IN DOUBLE STRAND

619
00:23:08,160 --> 00:23:09,960
BREAKS AND INITIALLY WE LOOKEDDA

620
00:23:09,960 --> 00:23:12,520
THE IN THE CONTEXT OF GAMAH 2 X

621
00:23:12,520 --> 00:23:16,120
AND WE COULD SEE THAT IN THE

622
00:23:16,120 --> 00:23:18,280
ABSENCE OF SMARCALL, WE SEE THE

623
00:23:18,280 --> 00:23:21,200
FOCI, BUT THEY CO-LEGALIZE WITH

624
00:23:21,200 --> 00:23:22,600
THE TELOMERES AND THIS IS

625
00:23:22,600 --> 00:23:24,040
INCREASED WITH THE CONTROL

626
00:23:24,040 --> 00:23:25,200
CELLS, THAT LACKS AND LIKEWISE

627
00:23:25,200 --> 00:23:28,040
WE SEE A SIMILAR THING IN THE

628
00:23:28,040 --> 00:23:29,120
ABSENCE OF SMARCALL, AND WE CAN

629
00:23:29,120 --> 00:23:31,000
SEE THAT WE ALSO GET AN INCREASE

630
00:23:31,000 --> 00:23:33,120
IN THE RECRUITMENT OF RAD 51 TO

631
00:23:33,120 --> 00:23:33,560
THESE SITES.

632
00:23:33,560 --> 00:23:37,320
AND I HOPE IT'S NOT LOST ON ALL

633
00:23:37,320 --> 00:23:39,120
OF YOU THAT IN LOOKING AT THESE

634
00:23:39,120 --> 00:23:41,760
IMAGES YOU CAN GET A SENSE THAT

635
00:23:41,760 --> 00:23:45,120
THESE TELEOR MERE FOCI IN THE

636
00:23:45,120 --> 00:23:46,240
ABSENCE OF SMARCALL, ARE GIANT.

637
00:23:46,240 --> 00:23:50,280
AND IN THE LAB WE WOULD REFER TO

638
00:23:50,280 --> 00:23:53,000
THEM AS THE GIANT TELOMERE FOCI,

639
00:23:53,000 --> 00:23:54,080
BECAUSE WE DIDN'T KNOW WHY THEY

640
00:23:54,080 --> 00:23:55,960
WERE SO LARGE AND WE WANTED TO

641
00:23:55,960 --> 00:23:57,160
QUANTIFY THEM.

642
00:23:57,160 --> 00:23:59,600
SO WE FOUND THESE TELEMERRIC

643
00:23:59,600 --> 00:24:01,800
FOCI, IN THE ABNORMALITIES SENSZ

644
00:24:01,800 --> 00:24:03,480
OF SMARCALL WERE GREATER IF FOLD

645
00:24:03,480 --> 00:24:04,960
AND SIZE THAN THE OTHER

646
00:24:04,960 --> 00:24:05,920
TELOMERES THROUGHOUT THE CELL OR

647
00:24:05,920 --> 00:24:08,080
IN THE CONTROL CELLS.

648
00:24:08,080 --> 00:24:10,120
SO THERE WAS REALLY THE

649
00:24:10,120 --> 00:24:12,080
SIGNIFICANT INCREASE IN THE

650
00:24:12,080 --> 00:24:13,800
TELOMERE FOCI, AND SO ROGER

651
00:24:13,800 --> 00:24:14,880
GREEN BERG'S LAB HAD

652
00:24:14,880 --> 00:24:18,600
DEMONSTRATED THAT IN FACT,

653
00:24:18,600 --> 00:24:20,160
THIS--THIS TELOMERE CLUSTERING

654
00:24:20,160 --> 00:24:21,680
WAS REALLY PREVALENT IN ALT

655
00:24:21,680 --> 00:24:24,240
CELLS AND THAT IN FACT THIS WAS

656
00:24:24,240 --> 00:24:26,040
DRIVEN BY RAD 51 SO YOU CAN

657
00:24:26,040 --> 00:24:26,760
ENVISION THAT IN THE PRESCRIBING

658
00:24:26,760 --> 00:24:28,320
ESTIMATE THAD RESEARCH A DOUBLE

659
00:24:28,320 --> 00:24:30,480
STRAND BREAK AND YOU SEE THE

660
00:24:30,480 --> 00:24:32,200
FORMATION, YOU COULD PROMOTE THE

661
00:24:32,200 --> 00:24:33,880
CAPTURE, SEARCH AND CAPTURE OF

662
00:24:33,880 --> 00:24:36,600
THE HOMOLOGOUS SEQUENCE TO

663
00:24:36,600 --> 00:24:38,320
PROMOTE DNA DOUBLE STRAND REPAIR

664
00:24:38,320 --> 00:24:40,080
AND THIS CONTEXT THE TELOMERE

665
00:24:40,080 --> 00:24:42,120
AND WE REFER TO THESE AS

666
00:24:42,120 --> 00:24:43,320
CLUSTERING EVENT WHERE IS

667
00:24:43,320 --> 00:24:45,200
THERE'S AN EVENT WHERE 1 IS

668
00:24:45,200 --> 00:24:47,560
INVADING ANOTHER AND THERE'S

669
00:24:47,560 --> 00:24:49,480
MULTIPLE TELEOR MERES COALESCING

670
00:24:49,480 --> 00:24:51,280
IN 1 REGION AND SO THIS IS NOT

671
00:24:51,280 --> 00:24:53,840
UNIQUE TO THOSE TYPE 2 CELLS, IN

672
00:24:53,840 --> 00:24:55,800
FACT WHEN WE KNOCK DOWN THE

673
00:24:55,800 --> 00:24:58,280
GOALS AND THE CELL LINES WE SEE

674
00:24:58,280 --> 00:25:01,240
THESE GIANT FOCI AND THE AVERAGE

675
00:25:01,240 --> 00:25:03,520
ABUNDANCE OF THESE IS BETWEEN 20

676
00:25:03,520 --> 00:25:07,240
AND 25% OF CELLS CONTAIN THESE

677
00:25:07,240 --> 00:25:10,880
REALLY GIANT TELOMERE FOCI.

678
00:25:10,880 --> 00:25:13,000
AND SO IN THIS FACT THAT'S A

679
00:25:13,000 --> 00:25:15,080
PRODUCT OF THE MECHANISM, WHERE

680
00:25:15,080 --> 00:25:17,080
IT'S MEDIATING THIS SEARCH AND

681
00:25:17,080 --> 00:25:19,240
CAPTURE TO PROMOTE RECOMBINATION

682
00:25:19,240 --> 00:25:22,200
OF TELOMERE ENDS AND IF YOU GET

683
00:25:22,200 --> 00:25:23,960
RID OF RAD 51 YOU COULD

684
00:25:23,960 --> 00:25:25,200
ELIMINATE THESE CLUSTERING

685
00:25:25,200 --> 00:25:27,840
EVENTS AND THESE GIANT F OCI AND

686
00:25:27,840 --> 00:25:29,120
WE DID THAT AND IMKED IT DOWN

687
00:25:29,120 --> 00:25:32,080
AND IN THE ABSENCE OF SMARCALL,

688
00:25:32,080 --> 00:25:35,200
YOU GET A PERCENTAGE OF INCREASE

689
00:25:35,200 --> 00:25:36,960
WITH THESE FOCI, BUT WHEN YOU

690
00:25:36,960 --> 00:25:39,720
KNOCK DOWN RAD 51 YOU CAN RESCUE

691
00:25:39,720 --> 00:25:41,360
THE PHENOTYPE.

692
00:25:41,360 --> 00:25:44,160
AND SO LIKEWISE IF WE TAKE A

693
00:25:44,160 --> 00:25:45,240
STEP BACKWARDS WE CAN ENVISION

694
00:25:45,240 --> 00:25:49,280
THAT IF YOU NEVER INDUCED THE

695
00:25:49,280 --> 00:25:50,320
NUCLEOLYTIC CLEAVEERAGE THAT

696
00:25:50,320 --> 00:25:51,160
PROMOTES THE DOUBLE STRAND

697
00:25:51,160 --> 00:25:52,600
BREAK, YOU WOULD NEVER ACHIEVE

698
00:25:52,600 --> 00:25:53,880
THE CLUSTERING EVENTS THAT WE'RE

699
00:25:53,880 --> 00:25:54,880
OBSERVING IN THESE CELLS.

700
00:25:54,880 --> 00:25:59,440
SO WE ASKED IF YOU ELIMINATE

701
00:25:59,440 --> 00:26:00,880
NUCLEOLYTIC CLEAVAGE DO YOU

702
00:26:00,880 --> 00:26:01,560
ELIMINATE THESE CLUSTERING

703
00:26:01,560 --> 00:26:03,600
EVENTS AS WELL, SO SIMILAR TO

704
00:26:03,600 --> 00:26:07,040
WHAT WE DESCRIBED BEFORE, IN THE

705
00:26:07,040 --> 00:26:09,960
ABSENCE OF SMARCALL, YOU SEE THE

706
00:26:09,960 --> 00:26:12,000
INCREASE FOR THESE GIANT TELEO

707
00:26:12,000 --> 00:26:14,840
MERRIC FOCI AND IF YOU COMBINE

708
00:26:14,840 --> 00:26:18,280
THIS WITH DELETION OR LOSS OF

709
00:26:18,280 --> 00:26:20,240
MOSS 81 AND [INDISCERNIBLE] AND

710
00:26:20,240 --> 00:26:21,640
THOSE PROMOTE THE STALLED

711
00:26:21,640 --> 00:26:25,760
REPLICATION FORKS YOU CAN RESCUE

712
00:26:25,760 --> 00:26:26,280
THE PHENOTYPE.

713
00:26:26,280 --> 00:26:28,080
AND SO TO FURTHER SUPPORT THE

714
00:26:28,080 --> 00:26:33,280
IDEA THAT IF FACT IN THE ABSENCE

715
00:26:33,280 --> 00:26:35,680
OF SMARCALL, YOU ARE PROMOTING

716
00:26:35,680 --> 00:26:36,000
RECOMBINATION.

717
00:26:36,000 --> 00:26:37,760
WE STARTED TO LOOK AT SPREADS

718
00:26:37,760 --> 00:26:38,840
FOR KNOCK DOWN, AND WE CAN SEE

719
00:26:38,840 --> 00:26:45,080
IN THE CONTROL CELLS WE HAVE

720
00:26:45,080 --> 00:26:46,000
THESE REALLY DISCREET--CAN

721
00:26:46,000 --> 00:26:46,960
APPRECIATE THIS ON THE SCREEN.

722
00:26:46,960 --> 00:26:49,080
A NUMBER OF THESE REALLY UNIQUE

723
00:26:49,080 --> 00:26:51,040
FUSION EVENTS AND IF WE CAN BLOW

724
00:26:51,040 --> 00:26:52,000
THESE UP A BIT ON THE RIGHT

725
00:26:52,000 --> 00:26:54,840
HERE, YOU CAN SEE THAT THESE

726
00:26:54,840 --> 00:26:57,560
FUSION EVENTS ARE ACTUALLY

727
00:26:57,560 --> 00:27:01,280
WITHIN THE TELOMERES AND THEIR

728
00:27:01,280 --> 00:27:01,760
TELOMERE BRIDGING.

729
00:27:01,760 --> 00:27:03,400
AND SO AS I MENTIONED BEFORE, 1

730
00:27:03,400 --> 00:27:05,280
OF THE GOLD STANDARDS IN THE

731
00:27:05,280 --> 00:27:08,000
FIELD IN LOOKING AT TELOMERE

732
00:27:08,000 --> 00:27:09,880
RECOMBINATION IS THE ANALYSIS OF

733
00:27:09,880 --> 00:27:10,600
C-CIRCLES AND THESE ARE

734
00:27:10,600 --> 00:27:13,600
GENERALLY THOUGHT TO BE A

735
00:27:13,600 --> 00:27:14,440
BYPRODUCT OF THOSE RECOMBINATION

736
00:27:14,440 --> 00:27:16,360
EVENTS AND SO IF YOU HAVE

737
00:27:16,360 --> 00:27:17,080
HEIGHTENED RECOMBINATION IN

738
00:27:17,080 --> 00:27:18,720
THESE CELLS YOU CAN ENVISION YOU

739
00:27:18,720 --> 00:27:20,240
WOULD HAVE AN INCREASE IN THESE

740
00:27:20,240 --> 00:27:24,600
CIRCLES AND IN FACT WHEN WE

741
00:27:24,600 --> 00:27:26,280
KNOCK DOWN SMARCALL, WE SEE AN

742
00:27:26,280 --> 00:27:28,080
INCREASE IN THE C-CIRCLE

743
00:27:28,080 --> 00:27:30,960
ABUNDANCE IN THESE CELLS AGAIN

744
00:27:30,960 --> 00:27:32,280
SUGGESTING THAT WE'RE REALLY

745
00:27:32,280 --> 00:27:32,880
PUSHING THE RECOMBINATION

746
00:27:32,880 --> 00:27:35,880
EFFECTS IN THE ABSENCE OF

747
00:27:35,880 --> 00:27:36,400
SMARCALL.

748
00:27:36,400 --> 00:27:39,680
SO WHAT ARE THE CONSEQUENCES OF

749
00:27:39,680 --> 00:27:40,600
THESE RECOMBINATION EEIVETS?

750
00:27:40,600 --> 00:27:42,400
I THINK WE NOTICED VERY EARLY ON

751
00:27:42,400 --> 00:27:44,120
THAT IN THESE CELLS SPECIFICALLY

752
00:27:44,120 --> 00:27:45,520
WE WOULD START TO SEE A LOT OF

753
00:27:45,520 --> 00:27:48,520
CELL DEATH IN THE ABSENCE OF

754
00:27:48,520 --> 00:27:50,280
SMARCALL AND WE WERE CURIOUS

755
00:27:50,280 --> 00:27:51,440
WHETHER THEY WERE CONSISTENT

756
00:27:51,440 --> 00:27:53,280
ACROSS ALL OF OUR CELL LINES AND

757
00:27:53,280 --> 00:27:55,080
USING A PANEL OF THESE CELL

758
00:27:55,080 --> 00:27:58,440
LINES WOO WE'VE KNOCKED DOWN

759
00:27:58,440 --> 00:28:27,800
SMARCALL USING THE COMBINATION

760
00:28:27,800 --> 00:28:31,320
OF THESE TOOLS--ALSO FUNCTION TO

761
00:28:31,320 --> 00:28:32,160
REPRESS TELOMERE COMBINATION.

762
00:28:32,160 --> 00:28:36,320
SO THE QUESTION CAME IF SMARCALL

763
00:28:36,320 --> 00:28:38,320
WAS LIKE ATRX, WAS IT POSSIBLE

764
00:28:38,320 --> 00:28:40,360
THAT IT WAS LOST IN THE ALT

765
00:28:40,360 --> 00:28:41,600
POSITIVE CANCERS AND SO WHAT WE

766
00:28:41,600 --> 00:28:45,360
FOUND IS IN FACT, YES, THAT IN A

767
00:28:45,360 --> 00:28:47,120
NUMBER OF THESE CELL LINES WE

768
00:28:47,120 --> 00:28:49,080
HAD DAMAGING MUTATIONS IN

769
00:28:49,080 --> 00:28:54,960
SMARCALL, THAT LED TO A ALMOST

770
00:28:54,960 --> 00:28:55,960
COMPLETE LOSS IN THE CELLS.

771
00:28:55,960 --> 00:28:57,640
SO CAN YOU GET THE PICTURE THAT

772
00:28:57,640 --> 00:29:00,160
WE'RE FILLING IN THE GAPS WE

773
00:29:00,160 --> 00:29:02,040
HAVE IN THE SAMPLES WE HAVE

774
00:29:02,040 --> 00:29:03,560
UNDERSTANDING THE MUTATION

775
00:29:03,560 --> 00:29:05,440
PROFILE OF THESE ALT POSITIVE

776
00:29:05,440 --> 00:29:07,760
TUMORS AND SINCE SOME OF THESE

777
00:29:07,760 --> 00:29:10,320
DISCOVERIES, WE'VE ACTUALLY BEEN

778
00:29:10,320 --> 00:29:12,920
ABLE TO SEE THESE COME OUT IN

779
00:29:12,920 --> 00:29:17,280
THE LITERATURE, SO, ATRX, DAX

780
00:29:17,280 --> 00:29:19,080
AND SMARCALL HAVE BEEN

781
00:29:19,080 --> 00:29:22,520
IDENTIFIED NOT JUST IN AFTIO

782
00:29:22,520 --> 00:29:25,280
SARCOMAS, SUBSETS OF LIVER

783
00:29:25,280 --> 00:29:26,840
CANCERS, AND THESE ARE IN THE

784
00:29:26,840 --> 00:29:28,280
CONTEXT OF ALT AND THESE WERE

785
00:29:28,280 --> 00:29:29,120
SOME OF THE FIRST INDKAIGDZS

786
00:29:29,120 --> 00:29:30,280
THAT THESE WERE THE CRITICAL

787
00:29:30,280 --> 00:29:32,800
PLAYERS IN IN PATHWAY.

788
00:29:32,800 --> 00:29:34,200
AND SO AS SATISFYING AS IT WAS

789
00:29:34,200 --> 00:29:36,720
TO SEE THESE MUTATIONS, IT'S

790
00:29:36,720 --> 00:29:39,880
CLEAR, ESPECIALLY IN THE TUMOR

791
00:29:39,880 --> 00:29:41,080
SAMPLES THAT WE WEREN'T THERE IN

792
00:29:41,080 --> 00:29:43,080
THE BEDDING OF THE GENETICS OF

793
00:29:43,080 --> 00:29:46,760
ALT, AND WE WANTED TO DIG DEEPER

794
00:29:46,760 --> 00:29:47,200
INTO THE NECKANISM.

795
00:29:47,200 --> 00:29:49,920
SO 1 OF THE THINGS THAT WE

796
00:29:49,920 --> 00:29:53,240
EALZED AT THE TIME, THERE HAD

797
00:29:53,240 --> 00:29:54,720
BEEN SEMINOLE PAPERS IN THE

798
00:29:54,720 --> 00:29:57,440
FIELD FROM ROGER GREEN BERG AND

799
00:29:57,440 --> 00:29:58,640
HILDA PICKET'S LAB WHICH LAID

800
00:29:58,640 --> 00:30:00,920
THE FOUNDATION FOR UNDERSTANDING

801
00:30:00,920 --> 00:30:02,040
OF TELOMERE ELONGATION AND ALT

802
00:30:02,040 --> 00:30:04,800
AND THAT THIS IS A PROCESS

803
00:30:04,800 --> 00:30:07,120
MEDIATED BY BREAK INDUCED

804
00:30:07,120 --> 00:30:08,600
REPLICATION, AND SO FOLLOWING

805
00:30:08,600 --> 00:30:11,880
FORK COLLAPSE OR JUST AT

806
00:30:11,880 --> 00:30:13,880
CRITICALLY SHORT TELOMERES THESE

807
00:30:13,880 --> 00:30:14,520
GROUPS COLLECTIVELY DEMONSTRATED

808
00:30:14,520 --> 00:30:17,520
THAT THAT THE BTR COMPLEX OR

809
00:30:17,520 --> 00:30:22,920
BLOOM TAUPE 3 AND RMI 1 AND 2,

810
00:30:22,920 --> 00:30:24,680
ACTUALLY REGULATE DNA SYNTHESIS

811
00:30:24,680 --> 00:30:26,640
ASK TELEOR MERE ELONGATION AND

812
00:30:26,640 --> 00:30:26,920
DISSOLUTION.

813
00:30:26,920 --> 00:30:31,120
AND IN AN OPPOSING AND PARALLEL

814
00:30:31,120 --> 00:30:35,040
PATHWAY THE SMX OR SLX 1 AND 4

815
00:30:35,040 --> 00:30:36,880
AND 81 COMPLEX REGULATES THESE

816
00:30:36,880 --> 00:30:38,480
CROSS OVER EVENTS WITHOUT

817
00:30:38,480 --> 00:30:41,480
ELONGATION WHICH WE REFER TO AS

818
00:30:41,480 --> 00:30:42,960
THE TELOMERE ASHES CYSTED

819
00:30:42,960 --> 00:30:43,200
EVENTS.

820
00:30:43,200 --> 00:30:44,920
SO I THINK WHAT WAS LEFT

821
00:30:44,920 --> 00:30:47,280
LINGERING WHEN THESE PAPERS CAME

822
00:30:47,280 --> 00:30:50,680
OUT WAS HAQUE REGULATES THESE

823
00:30:50,680 --> 00:30:52,240
PATHWAYS HOW ARE THEY BALANCED?

824
00:30:52,240 --> 00:30:56,320
SO AT THIS TIME WE WERE LOOKING

825
00:30:56,320 --> 00:30:58,000
INTO THESE TUMORS WE HAD THAT WE

826
00:30:58,000 --> 00:30:59,640
KNEW WERE ALT AND WE NOTICED

827
00:30:59,640 --> 00:31:02,200
THESE INTERESTING MUTATIONS IN A

828
00:31:02,200 --> 00:31:05,280
PROTEIN CALLED SLICKS 4 IP AND

829
00:31:05,280 --> 00:31:06,840
SO WHEN WE CROSS CHECKED OUR

830
00:31:06,840 --> 00:31:09,200
GENETIC DATA WE COULD SEE THERE

831
00:31:09,200 --> 00:31:11,680
WERE THESE SIGNIFICANT DAMAGE OF

832
00:31:11,680 --> 00:31:12,880
MUTATIONS, FOR SLICKSS 4 IP AND

833
00:31:12,880 --> 00:31:16,480
SO AT THE TIME IT WAS KNOWN THAT

834
00:31:16,480 --> 00:31:19,320
SLICKs 4 IP INTERACTED WITH

835
00:31:19,320 --> 00:31:22,360
SLICKS 4 BUT IT WASN'T CLEAR

836
00:31:22,360 --> 00:31:23,640
THAT CONTRIBUTED TO THE DNA

837
00:31:23,640 --> 00:31:25,640
RESPONSE AND WHAT WAS IT DOING

838
00:31:25,640 --> 00:31:28,400
AT TELOMERES AND SO AROUND THE

839
00:31:28,400 --> 00:31:30,280
TIME OF THIS DISCOVERY FOR US WE

840
00:31:30,280 --> 00:31:33,800
WERE CHATTING WITH SIMON

841
00:31:33,800 --> 00:31:39,280
FULTON'S LAB AT FRANCIS CREEK

842
00:31:39,280 --> 00:31:41,120
AND THEY STARTED TO HOW IP

843
00:31:41,120 --> 00:31:42,760
TRIBUTES TO THE MECHANISM OF ALT

844
00:31:42,760 --> 00:31:52,320
SO IN A COLLABORATION WITH THEIR

845
00:31:52,320 --> 00:31:54,280
GROUP SIMON WAS AND 1 OF THE

846
00:31:54,280 --> 00:31:55,360
CRITICAL REGULATORS OF THE

847
00:31:55,360 --> 00:31:57,280
BALANCE OF THESE 2 ARMS OF THIS

848
00:31:57,280 --> 00:32:03,280
BREAK INDUCED REPLICATION

849
00:32:03,280 --> 00:32:05,080
PATHWAY.

850
00:32:05,080 --> 00:32:08,640
WHERE SLX4 AND BLM TO TOLERATE

851
00:32:08,640 --> 00:32:10,280
AND BALANCE THIS REPLICATION

852
00:32:10,280 --> 00:32:13,480
THROUGH BLOOM AND DNA SYNTHESIS

853
00:32:13,480 --> 00:32:15,680
AND TELOMERE ELONGATION AND

854
00:32:15,680 --> 00:32:17,080
DISSOLUTION, BALANCING THAT WITH

855
00:32:17,080 --> 00:32:19,560
THE CLEAVAGE AND CROSS OVER

856
00:32:19,560 --> 00:32:22,160
EVENTS THAT ARE IN THE EXCHANGES

857
00:32:22,160 --> 00:32:24,080
AND SO SLX 4 IP BECAME A

858
00:32:24,080 --> 00:32:25,040
CRITICAL REGULATOR OF THE

859
00:32:25,040 --> 00:32:27,520
BALANCE OF THIS PATHWAY.

860
00:32:27,520 --> 00:32:28,960
IT WAS AT THIS TIME THAT WE

861
00:32:28,960 --> 00:32:34,080
REALLY BECAME VERY INTERESTED IN

862
00:32:34,080 --> 00:32:34,920
UNDERSTANDING THE MECHANISM THAT

863
00:32:34,920 --> 00:32:37,560
IS REQUIRED TO PROMOTE THESE DNA

864
00:32:37,560 --> 00:32:38,880
SYNTHESIS EEIVETS SPECIFICALLY

865
00:32:38,880 --> 00:32:41,720
THROUGH BLM, AND PART OF THIS

866
00:32:41,720 --> 00:32:45,440
CURIOSITY CAME FROM SOME OF THE

867
00:32:45,440 --> 00:32:46,440
MECHANISTIC UNDERSTANDING WE HAD

868
00:32:46,440 --> 00:32:48,040
FROM PREVIOUS STUDIES THAT

869
00:32:48,040 --> 00:32:49,360
DEMONSTRATED THAT THESE VARIANT

870
00:32:49,360 --> 00:32:51,880
REPEATS THAT I MENTIONED BEFORE

871
00:32:51,880 --> 00:32:53,480
ARE REALLY PREVALENT THROUGHOUT

872
00:32:53,480 --> 00:32:54,560
THE TELOMERE AND WHAT'S

873
00:32:54,560 --> 00:32:55,840
INTERESTING ABOUT THIS IS THAT

874
00:32:55,840 --> 00:32:59,080
AS I HAD MENTIONED WHEN YOU HAVE

875
00:32:59,080 --> 00:33:01,400
STRAND INVASION AND HOMOLOGY

876
00:33:01,400 --> 00:33:02,840
SEARCH FOR THIS HOMOLOGOUS

877
00:33:02,840 --> 00:33:05,160
SEQUENCE IN A NEIGHBORING

878
00:33:05,160 --> 00:33:13,440
CHROMOSOME, YOU CAN ENVISION A

879
00:33:13,440 --> 00:33:16,760
SCENARIO THAT ARE BY BUT NOT

880
00:33:16,760 --> 00:33:23,920
ASENTIOUS FICIENT FOR

881
00:33:23,920 --> 00:33:24,440
HETEROGENEOUSERROLOGIOUS

882
00:33:24,440 --> 00:33:26,000
EXTEBDERS SO HOW WERE THESE

883
00:33:26,000 --> 00:33:28,080
BLOOMING BEHIND THESE MIGRATION

884
00:33:28,080 --> 00:33:30,960
EVENTS GIVEN THAT BLM IS NOT AN

885
00:33:30,960 --> 00:33:33,880
EFFICIENTLE HELIOS POSITIVE

886
00:33:33,880 --> 00:33:35,280
EXCITATORY CASE OVER THIS I

887
00:33:35,280 --> 00:33:36,880
HAVEROLOGY, SO WE KNEW THAT RAD

888
00:33:36,880 --> 00:33:41,600
4 WAS THE MOST WELL

889
00:33:41,600 --> 00:33:43,680
CHARACTERIZED THAT REGULATES

890
00:33:43,680 --> 00:33:44,080
MIGRATION THROUGH

891
00:33:44,080 --> 00:33:44,480
HETEROGENEOUSROLOGY.

892
00:33:44,480 --> 00:33:47,680
SO WE ASKED DOES RAD54

893
00:33:47,680 --> 00:33:49,480
FACILITATE BREAK INDUCED

894
00:33:49,480 --> 00:33:52,920
REPLICATION AT ALT TELOMERES?

895
00:33:52,920 --> 00:33:54,640
AND SO IN QUEST QUEE STARTED OFF

896
00:33:54,640 --> 00:33:56,360
OF COURSE LOOKING ACROSS OUR

897
00:33:56,360 --> 00:33:58,240
PANEL OF ALT TELEOR MERES AND WE

898
00:33:58,240 --> 00:34:07,320
CAN SEE THAT RAD 54 BUT THESE CO

899
00:34:07,320 --> 00:34:09,400
LOCALIZE WITH THE TELOMERE CELL

900
00:34:09,400 --> 00:34:11,320
LINES AND THESE ARE OFTEN

901
00:34:11,320 --> 00:34:13,600
ENRICHED AS COMPARED TO NONALT

902
00:34:13,600 --> 00:34:16,000
TELOMERES AND WE CAN EXACERBATE

903
00:34:16,000 --> 00:34:17,240
THIS PHENOTYPE BY TAKING

904
00:34:17,240 --> 00:34:19,040
ADVANTAGE OF A CELL LINE THAT

905
00:34:19,040 --> 00:34:21,520
HAD BEEN PREVIOUSLY PUBLISHED IN

906
00:34:21,520 --> 00:34:27,840
THE LITERATURE, A UTO AN S CELL

907
00:34:27,840 --> 00:34:32,880
LINE THAT HAS A FUSION PROBLEM

908
00:34:32,880 --> 00:34:35,480
AND IT HAS THE ENZYME THAT CAN

909
00:34:35,480 --> 00:34:37,080
PROMOTE CLEAVE LAGE OF DOUBLE

910
00:34:37,080 --> 00:34:40,280
STRANDED DNA AND THIS TRF 1 AND

911
00:34:40,280 --> 00:34:45,840
FOK 1 IS DRIVEN BY TAMOXIFEN AND

912
00:34:45,840 --> 00:34:46,920
FOLLOWING EXPRESSION--FOLLOWING

913
00:34:46,920 --> 00:34:48,160
INDUCTION WITH TAMOXIFEN, YOU

914
00:34:48,160 --> 00:34:49,320
CAN SEE EXPRESSION OF NOT JUST

915
00:34:49,320 --> 00:34:53,560
THE WILD TYPE BUT ALSO AN

916
00:34:53,560 --> 00:34:57,480
ENZYMEATICALLY INACTIVE D450 A,

917
00:34:57,480 --> 00:35:00,800
TRF FAK 1, AND WE CAN SEE WHEN

918
00:35:00,800 --> 00:35:02,520
WE INDUCE WITH TAMOXIFEN, YOU

919
00:35:02,520 --> 00:35:05,880
GET THE INCREASE IN RAD 54 FOCI

920
00:35:05,880 --> 00:35:08,880
FORMATION AND THESE FOCI CO

921
00:35:08,880 --> 00:35:09,960
LOCALIZE WITH TELOMERES ONLY

922
00:35:09,960 --> 00:35:12,360
WHEN YOU HAVE WILD TYPE FAK 1

923
00:35:12,360 --> 00:35:14,960
AND NOT WITH THE D450 A SO WHEN

924
00:35:14,960 --> 00:35:16,280
WE QUANTIFY THIS, WE CAN SEE

925
00:35:16,280 --> 00:35:22,440
THIS AS AN INCREASE IN THE

926
00:35:22,440 --> 00:35:23,880
PERCENTAGE OF RAD 54

927
00:35:23,880 --> 00:35:24,880
COLOCALIZATIONS PER SE, AGAIN

928
00:35:24,880 --> 00:35:28,880
ONLY IN THE WILD TYPE FAK WILD

929
00:35:28,880 --> 00:35:29,920
TYPE SPRIPPING CELLS AND NOT

930
00:35:29,920 --> 00:35:32,440
ONLY THAT BUT WE NOTICED WE HAD

931
00:35:32,440 --> 00:35:33,280
A SIGNIFICANT INCREASE IN THE

932
00:35:33,280 --> 00:35:37,080
PERCENTAGE OF CELLS THAT HAD FAR

933
00:35:37,080 --> 00:35:39,640
MORE RAD 54 TELEMERRIC FOCI THAN

934
00:35:39,640 --> 00:35:41,400
WE HAD SEEN IN THE UNTREATED

935
00:35:41,400 --> 00:35:41,600
CELLS.

936
00:35:41,600 --> 00:35:43,200
SO IT WAS NOT ONLY THE

937
00:35:43,200 --> 00:35:45,960
PERCENTAGE OF CELLS WITH THIS

938
00:35:45,960 --> 00:35:48,960
HIGH FREQUENCY OF

939
00:35:48,960 --> 00:35:49,400
COLOCALIZATION.

940
00:35:49,400 --> 00:35:53,000
AND SO, THERE HAD BEEN A LOT OF

941
00:35:53,000 --> 00:35:59,280
RESEARCH IN THE FIELD ON RAD 54

942
00:35:59,280 --> 00:36:01,560
GIVING BOTH PRESYNAPTIC AND POST

943
00:36:01,560 --> 00:36:03,080
SYNAPTIC FUNCTION OR ASSIGNING

944
00:36:03,080 --> 00:36:04,440
BOTH OF THESE FUNCTIONS TO RAD

945
00:36:04,440 --> 00:36:06,840
54 AND THERE WAS A LOT OF DATA

946
00:36:06,840 --> 00:36:08,800
SWIRLING AROUND THE REGULATION

947
00:36:08,800 --> 00:36:10,360
OF RAD51 AND MEDIATED STRAND

948
00:36:10,360 --> 00:36:13,200
INVASION AND THAT IT MIGHT BE

949
00:36:13,200 --> 00:36:14,080
REQUIRED FOR THIS FUNCTION AND

950
00:36:14,080 --> 00:36:16,800
SO WE STARTED OFF BY ASKING

951
00:36:16,800 --> 00:36:20,320
WHETHER RAD54 WAS IN FACT

952
00:36:20,320 --> 00:36:23,160
REQUIRED FOR RAD51 FUNCTION AND

953
00:36:23,160 --> 00:36:25,280
SO, WHILE OUR INITIAL ANALYSIS

954
00:36:25,280 --> 00:36:26,800
REALLY STARTED WITH LOOKING AT

955
00:36:26,800 --> 00:36:28,680
RAD 51 AND SO I DON'T HAVE ALL

956
00:36:28,680 --> 00:36:30,800
OF THE DATA, I'M NOT GOING TO

957
00:36:30,800 --> 00:36:31,520
PRESENT THIS HERE TODAY, BUT

958
00:36:31,520 --> 00:36:33,400
WHAT WE CAN SEER IN THESE CELLS

959
00:36:33,400 --> 00:36:35,280
IS THAT IN BOTH OF THESE ALT

960
00:36:35,280 --> 00:36:38,880
CELL LINES YOU CAN SEE RAD51

961
00:36:38,880 --> 00:36:40,880
FOCI THAT CO LOCALIZE IN

962
00:36:40,880 --> 00:36:42,720
TELOMERES WE SEE NO

963
00:36:42,720 --> 00:36:44,080
STATISTICALLY SIGNIFICANT CHANGE

964
00:36:44,080 --> 00:36:48,040
IN THE NUMBER OF PREVALENCE OF

965
00:36:48,040 --> 00:36:50,520
THESE RAD51 FOCI AT THAT CO

966
00:36:50,520 --> 00:36:51,640
LOCALIZE AT THE TELEO MERENDS

967
00:36:51,640 --> 00:36:59,480
AND THIS IS NOT ACTUALLY SO

968
00:36:59,480 --> 00:37:01,680
WHILE IT DOESN'T XCTLY GET THE

969
00:37:01,680 --> 00:37:03,880
AT IDEA WHETHER RAD IS REQUIRED

970
00:37:03,880 --> 00:37:04,720
FOR FILAMENT FORMATION, WE

971
00:37:04,720 --> 00:37:06,840
THOUGHT WE WOULD FOLLOW THIS UP

972
00:37:06,840 --> 00:37:10,880
BY SAYING THAT IF WE ANALYZE

973
00:37:10,880 --> 00:37:11,760
THESE SYNAPTIC STRUCTURES, YOU

974
00:37:11,760 --> 00:37:14,080
WOULD IMAGINE IF THE

975
00:37:14,080 --> 00:37:14,720
NUCLEOFILAMENT WAS DISRUPTED,

976
00:37:14,720 --> 00:37:16,920
YOU MIGHT ALSO SEE A DECREASE IN

977
00:37:16,920 --> 00:37:18,000
THE RECRUITMENT OF SUBSEQUENT

978
00:37:18,000 --> 00:37:19,440
ENZYMES THAT ARE REQUIRED FOR

979
00:37:19,440 --> 00:37:21,920
THIS BREAK INDUCED REPLICATION

980
00:37:21,920 --> 00:37:22,160
PROCESS.

981
00:37:22,160 --> 00:37:25,160
AND SOME OF THOSE WOULD BE PC NA

982
00:37:25,160 --> 00:37:27,520
AND THE POLYMERASES THEMSELVES

983
00:37:27,520 --> 00:37:28,880
RESPONSIBLE FOR BREAK INDUCED

984
00:37:28,880 --> 00:37:30,840
REPLICATION SO I DON'T HAVE THE

985
00:37:30,840 --> 00:37:32,640
PC NA DATA HERE BUT WE NOTICED

986
00:37:32,640 --> 00:37:38,800
THAT PC NA LOOKS SIMILAR TO

987
00:37:38,800 --> 00:37:43,840
ANALYSIS OF POLA AND POLB, ARE

988
00:37:43,840 --> 00:37:48,200
THE 2 THAT HAVE BEEN IMPLICATED

989
00:37:48,200 --> 00:37:50,720
IN BREAK REPLICATION, IN THE

990
00:37:50,720 --> 00:37:52,040
TELOMERES SO USING A THESE WE

991
00:37:52,040 --> 00:37:55,560
CAN SEE THE FOCI REGULATION AND

992
00:37:55,560 --> 00:37:57,520
THESE CO LOCALIZE WITH

993
00:37:57,520 --> 00:37:57,880
TELOMERES.

994
00:37:57,880 --> 00:38:01,120
WHEN YOU KNOCK DOWN RAD54 WE

995
00:38:01,120 --> 00:38:05,760
DIDN'T SEE ANY CHANGE OF POL 8A,

996
00:38:05,760 --> 00:38:07,360
FROM THIS ANALIS COMBINED WITH

997
00:38:07,360 --> 00:38:10,240
THE RAD 51 THAT IT DOESN'T SEEM

998
00:38:10,240 --> 00:38:12,600
LIKE RAD54 IS CONTRIBUTING TO

999
00:38:12,600 --> 00:38:15,080
ANY PRESYNAPTIC FUNCTIONS OF THE

1000
00:38:15,080 --> 00:38:18,320
BREAK INDUCED REPLICATION

1001
00:38:18,320 --> 00:38:19,120
PROCESS.

1002
00:38:19,120 --> 00:38:22,680
AND SO WHAT ABOUT MATURATION OF

1003
00:38:22,680 --> 00:38:23,920
THESE D-LOOP STRUCTURES, AND HOW

1004
00:38:23,920 --> 00:38:26,400
DO WE ANALYZE THOSE?

1005
00:38:26,400 --> 00:38:29,280
AND SO, MATURATION OF THESE

1006
00:38:29,280 --> 00:38:31,120
D-LOOPS REALLY INVOLVES NOT JUST

1007
00:38:31,120 --> 00:38:32,920
RECRUITMENT OF THESE ENZYMES BUT

1008
00:38:32,920 --> 00:38:34,200
ALSO PRODUCTIVITY OF THESE

1009
00:38:34,200 --> 00:38:36,520
ENZYMES, SO FOR EXAMPLE, CAN WE

1010
00:38:36,520 --> 00:38:38,080
MONITOR EDITORROR DNA SYNTHESIS

1011
00:38:38,080 --> 00:38:39,480
FROM THESE D-LOOP STRUCTURES.

1012
00:38:39,480 --> 00:38:41,480
AND SO WE CAN DO THIS BY

1013
00:38:41,480 --> 00:38:44,640
TREATING CELLS WITH THE THYMA

1014
00:38:44,640 --> 00:38:46,520
DINE ANALOGUE EDU AND USING

1015
00:38:46,520 --> 00:38:48,280
CLICK CHEMIST RADIOY TO DETECT

1016
00:38:48,280 --> 00:38:50,800
EDU OR THE FISH PROBES TO DETECT

1017
00:38:50,800 --> 00:38:54,080
TELOMERES AND WE CAN MONITOR EDU

1018
00:38:54,080 --> 00:38:55,640
CORPORATION IN TELOMERES IN

1019
00:38:55,640 --> 00:38:56,600
NONS-PHASE CELLS AND YOU CAN

1020
00:38:56,600 --> 00:38:58,800
APPRECIATE THAT WE GET THESE

1021
00:38:58,800 --> 00:39:02,080
FOCI EDU POSITIVE CELLS THAT CO

1022
00:39:02,080 --> 00:39:03,960
LOCALIZE WITH TELOMERES

1023
00:39:03,960 --> 00:39:05,480
INDICATIVE OF ALT TERTELOMERES

1024
00:39:05,480 --> 00:39:06,840
AND WE SAW IN BOTH OF THESE CELL

1025
00:39:06,840 --> 00:39:08,480
LINES WHEN YOU KNOCK DOWN RAD

1026
00:39:08,480 --> 00:39:09,680
51, NOT ONLY DO YOU SEE A

1027
00:39:09,680 --> 00:39:12,680
DECREASE IN THE NUMBER OF EDU

1028
00:39:12,680 --> 00:39:13,760
POSITIVE TELOMERES BUT WE

1029
00:39:13,760 --> 00:39:16,680
ACTUALLY SEE A DECREASE IN THE

1030
00:39:16,680 --> 00:39:17,720
FLUORESCENTS INTENSITY AND THE

1031
00:39:17,720 --> 00:39:19,360
SIGNAL FROM THIS EDU, SUGGESTING

1032
00:39:19,360 --> 00:39:21,640
IN FACT, THAT THERE IS A DEFECT

1033
00:39:21,640 --> 00:39:27,960
IN THE ACCUMULATION OF EDU AT

1034
00:39:27,960 --> 00:39:29,200
ALT TELOMERES.

1035
00:39:29,200 --> 00:39:32,480
AND SO RAD 54 IS A MONITOR

1036
00:39:32,480 --> 00:39:33,440
MERSOLUTION AND OLIGMERRIZES IN

1037
00:39:33,440 --> 00:39:35,560
THE PREPS ENSEL OF DNA AND SO WE

1038
00:39:35,560 --> 00:39:38,400
THOUGHT WE MAY BE BETTER ABLE TO

1039
00:39:38,400 --> 00:39:40,920
CHARACTERIZE RAD 54 FUNCTION AT

1040
00:39:40,920 --> 00:39:42,880
ALT TELOMERES BY LOOKINGA THE

1041
00:39:42,880 --> 00:39:43,960
THESE PREVIOUSLY CHARACTERIZED

1042
00:39:43,960 --> 00:39:45,800
MUTANTS IN THE FIELD.

1043
00:39:45,800 --> 00:39:48,520
SO THIS OLIGMERRIZATION IS

1044
00:39:48,520 --> 00:39:49,800
ACTUALLY ANTAGONIZED BY

1045
00:39:49,800 --> 00:39:50,640
PHOSPHORYLATION AT SERIES

1046
00:39:50,640 --> 00:39:54,280
POINTSINE 49, AND SO IN THE

1047
00:39:54,280 --> 00:39:59,280
PRESENCE OF PHOSPHORYLATION,

1048
00:39:59,280 --> 00:40:01,680
THESE MONOMERS OLIGMERRIZE AND

1049
00:40:01,680 --> 00:40:04,680
NO LONGER MAINTAIN BRANCH 

1050
00:40:04,680 --> 00:40:05,360
MIGRATION ACTIVITIES.

1051
00:40:05,360 --> 00:40:08,160
SO WE ASKED, CAN WE RESCUE THIS

1052
00:40:08,160 --> 00:40:11,720
DEFECT IN DNA SYNTHESIS AT ALT

1053
00:40:11,720 --> 00:40:15,720
TELOMERES WITH WILD TYPE RAD 54

1054
00:40:15,720 --> 00:40:18,800
OR A K-R TRANSLO CASE CASE

1055
00:40:18,800 --> 00:40:21,760
MUSEUM TABT OR A VERSION OF RAD

1056
00:40:21,760 --> 00:40:25,240
54 WHERE WE MAKE SERIES

1057
00:40:25,240 --> 00:40:27,360
POINTSINE 49 CONSTITTATIVELY

1058
00:40:27,360 --> 00:40:29,240
PHOSPHORALATED USING AN SDE

1059
00:40:29,240 --> 00:40:29,480
MUTANT.

1060
00:40:29,480 --> 00:40:30,760
SO WHAT WE FOWBD FROM THIS

1061
00:40:30,760 --> 00:40:31,960
ANALIS WAS THAT IN FACT, AGAIN

1062
00:40:31,960 --> 00:40:34,080
AS WE SAW BEFORE, WHEN WHEN YOU

1063
00:40:34,080 --> 00:40:36,760
GET RID OF RAD 54, YOU SEE AIAN

1064
00:40:36,760 --> 00:40:39,680
DECREASE IN THE TELOMERES WHEN

1065
00:40:39,680 --> 00:40:41,400
RECONSITUTE WITH RAD 54 YOU CAN

1066
00:40:41,400 --> 00:40:44,680
RESCUE THAT FEIGNEE TYPE BUT

1067
00:40:44,680 --> 00:40:46,440
NEITHER THE TRANSLOCATION LOCATE

1068
00:40:46,440 --> 00:40:57,480
A 9 MUTANT NOR THE--BUT IN FACT,

1069
00:40:57,480 --> 00:40:58,960
THE BRANCH MIGRATION ACTIVITY OF

1070
00:40:58,960 --> 00:41:04,080
RAD 54 IS CRITICAL FOR THIS

1071
00:41:04,080 --> 00:41:04,320
FUNCTION.

1072
00:41:04,320 --> 00:41:07,800
AND SO, IF IN FACT, ALT RELIES

1073
00:41:07,800 --> 00:41:11,120
ON BREAK INDUCED REPLICATION,

1074
00:41:11,120 --> 00:41:13,280
AND RAD54 IS MEDIATING THIS

1075
00:41:13,280 --> 00:41:18,880
PATHWAY, POTENTIALLY IN

1076
00:41:18,880 --> 00:41:21,000
CONJUNCTION WITH TD BTR INDUCED

1077
00:41:21,000 --> 00:41:22,520
REPLICATION, YOU CAN IMAGINE IF

1078
00:41:22,520 --> 00:41:24,360
YOU ELIMINATE YOU WOULD SHIFT

1079
00:41:24,360 --> 00:41:26,080
THE BALANCE FAVORING CLEAVAGE

1080
00:41:26,080 --> 00:41:28,440
AND CROSS OVER PRODUCT FORMATION

1081
00:41:28,440 --> 00:41:33,560
VIA THIS SMX PATHWAY.

1082
00:41:33,560 --> 00:41:35,480
SOY THE FIRST QUESTION QUEE ASK

1083
00:41:35,480 --> 00:41:38,880
IS IF YOU GET RID OF RAD 54 IN

1084
00:41:38,880 --> 00:41:40,880
CELLS, AND IN THIS PARTICULAR

1085
00:41:40,880 --> 00:41:43,640
INSTANCE WE USE MUS81 AS A

1086
00:41:43,640 --> 00:41:45,480
SURROGATE FOR THE SMX COMPLEX

1087
00:41:45,480 --> 00:41:47,040
AND YOU CAN APPRECIATE IN THESE

1088
00:41:47,040 --> 00:41:49,800
IMAGES THAT IN THE ABSENCE OF

1089
00:41:49,800 --> 00:41:52,280
RAD54 WE GET A SIGNIFICANT

1090
00:41:52,280 --> 00:41:54,560
INCREASE AND THEY CO LOCALIZE

1091
00:41:54,560 --> 00:41:55,200
WITH TELOMERE ENDS SUGGESTING

1092
00:41:55,200 --> 00:42:03,680
THAT WE ARE FORCING RESOLUTION

1093
00:42:03,680 --> 00:42:06,800
WITH A LOOK AT THESE TELOMERE

1094
00:42:06,800 --> 00:42:08,320
EXCHANGES I DESCRIBED EARLIER.

1095
00:42:08,320 --> 00:42:10,960
IF IN FACT, YOU ARE FORCING

1096
00:42:10,960 --> 00:42:12,360
CLEAVAGE AND CROSS OVER EVENTS

1097
00:42:12,360 --> 00:42:14,480
YOU MENTD ENVISION YOU HAVE A

1098
00:42:14,480 --> 00:42:15,960
INCREASE IN TSCE IN THE CELLS

1099
00:42:15,960 --> 00:42:18,080
AND IN FACT, AGAIN IF WE TAKE

1100
00:42:18,080 --> 00:42:21,200
THESE SAME ALT POSITIVE CELLS

1101
00:42:21,200 --> 00:42:22,960
AND WE KNOCKED DOWN RAD54, CAN

1102
00:42:22,960 --> 00:42:24,680
YOU SEE IN THE CONTROL SAMPLES

1103
00:42:24,680 --> 00:42:27,000
YOU HAVE THIS NICE STAINING OF

1104
00:42:27,000 --> 00:42:28,400
TELOMERE ENDS INDEPENDENTLY OF 1

1105
00:42:28,400 --> 00:42:28,640
ANOTHER.

1106
00:42:28,640 --> 00:42:32,440
AND IN THE ABSENCE OF RAD54, YOU

1107
00:42:32,440 --> 00:42:35,600
SEE AN INCREASE OF THESE

1108
00:42:35,600 --> 00:42:36,680
RECOMBINATION EVENTS AND

1109
00:42:36,680 --> 00:42:37,720
OVERLAPPING SIGNAL AT THESE

1110
00:42:37,720 --> 00:42:40,680
ENDS, SO AGAIN, THIS WAS WAS

1111
00:42:40,680 --> 00:42:42,840
INDICATIVE TO US THAT IN THE

1112
00:42:42,840 --> 00:42:44,680
ABSENCE OF RAD54, YOU ARE

1113
00:42:44,680 --> 00:42:46,120
PROMOTING THESE CROSS OVER

1114
00:42:46,120 --> 00:42:47,240
EVENTS AT TELOMERE ENDS.

1115
00:42:47,240 --> 00:42:51,400
AND SO IF YOU GET RID OF THE BTR

1116
00:42:51,400 --> 00:42:53,040
COMPLEX AND THE SMS COMPLEX, CAN

1117
00:42:53,040 --> 00:42:54,880
YOU IMAGINE THAT IN THE END YOU

1118
00:42:54,880 --> 00:42:57,680
TAKE OUT BOGHT PATHWAYS YOU

1119
00:42:57,680 --> 00:42:58,480
COULD PROMOTE THESE

1120
00:42:58,480 --> 00:42:59,440
RECOMBINATION ISHT MEDIATES AND

1121
00:42:59,440 --> 00:43:00,880
THIS IS ACTUALLY VISUALIZE AS

1122
00:43:00,880 --> 00:43:04,280
WHAT WE REFER TO AS BRIDGES AND

1123
00:43:04,280 --> 00:43:06,480
SO WE ASKED IF IN THE ABSENCE OF

1124
00:43:06,480 --> 00:43:11,160
RAD 54 AND THE IN ABSENCE OF THE

1125
00:43:11,160 --> 00:43:14,160
SMX PATHWAY DO WE SEE INCREASES

1126
00:43:14,160 --> 00:43:15,040
IN ULTRA FIND ANAPHASE BRIDGES

1127
00:43:15,040 --> 00:43:17,040
AND THE WAY WE ANALYZE THIS IS

1128
00:43:17,040 --> 00:43:19,880
USING THE PROTEIN PITCH WHICH

1129
00:43:19,880 --> 00:43:20,920
SPECIFICALLY BINDS THE ULTRA

1130
00:43:20,920 --> 00:43:24,840
FINE BRIDGES AND THESE ARE

1131
00:43:24,840 --> 00:43:25,440
ANAPHRASE PREPARATIONS WHERE

1132
00:43:25,440 --> 00:43:26,760
WE'VE STAINED FOR PITCH AND YOU

1133
00:43:26,760 --> 00:43:28,080
CAN SEE THAT IN THE CONTROL

1134
00:43:28,080 --> 00:43:31,560
SAMPLES IF YOU GET RID OF BLOO

1135
00:43:31,560 --> 00:43:35,120
BLM AND SLX 4, YOU GET THE

1136
00:43:35,120 --> 00:43:35,800
BEAUTIFUL ANAPHASE BRIDGES CAN

1137
00:43:35,800 --> 00:43:38,680
YOU VISUALIZE WITH PITCH.

1138
00:43:38,680 --> 00:43:42,320
AND LIKEWISE, IF YOU GET RID OF

1139
00:43:42,320 --> 00:43:46,120
SLX 4 AND RAD54, YOU CAN SEE AN

1140
00:43:46,120 --> 00:43:47,480
INCREASE IN THE THESE BRIDGES SO

1141
00:43:47,480 --> 00:43:48,760
DATA I'M NOT SHOWING HERE IS

1142
00:43:48,760 --> 00:43:51,280
THAT IN ADDITION IT SEEING AN

1143
00:43:51,280 --> 00:43:53,360
INCREASE IN CELLS, THAT HAVE

1144
00:43:53,360 --> 00:43:54,760
THESE ANAPHASE BRIDGES, I THINK

1145
00:43:54,760 --> 00:43:55,880
YOU CAN APPRECIATE, WE SEE AN

1146
00:43:55,880 --> 00:43:57,480
INCREASE IN THE NUMBER OF

1147
00:43:57,480 --> 00:44:00,800
ANAPHASE BRIDGES BETWEEN THESE

1148
00:44:00,800 --> 00:44:01,160
CELLS.

1149
00:44:01,160 --> 00:44:02,560
SO 1 MORE POINT ABOUT THIS

1150
00:44:02,560 --> 00:44:07,160
ANALYSIS WAS THAT IF WE ACTUALLY

1151
00:44:07,160 --> 00:44:09,280
COMBINED BLM, SLX 4 AND RAD54

1152
00:44:09,280 --> 00:44:10,800
KNOCK DOWN, WE DON'T SEE A

1153
00:44:10,800 --> 00:44:11,600
FURTHER INCREASE WHEN WE

1154
00:44:11,600 --> 00:44:13,040
QUANTIFY HERE ON THE RIGHT.

1155
00:44:13,040 --> 00:44:16,840
WE DON'T SEE A FURTHER INCREASE

1156
00:44:16,840 --> 00:44:19,720
SUGGESTING THAT BLM AND RAD 54

1157
00:44:19,720 --> 00:44:22,640
ARE IN FACT EPISTATIC.

1158
00:44:22,640 --> 00:44:23,840
AND SO, THAT WAS INTERESTING

1159
00:44:23,840 --> 00:44:26,800
DATA TO US BUT IN FACT, RIGHT,

1160
00:44:26,800 --> 00:44:29,480
PITCH IS A GENERIC PROTEIN THAT

1161
00:44:29,480 --> 00:44:30,960
MIGHT MARK ANY ANAPHASE BRIDGE

1162
00:44:30,960 --> 00:44:33,600
AND WE WANTED TO NOTE WHETHER

1163
00:44:33,600 --> 00:44:35,240
THESE BRIDGES WERE TELOMERES AND

1164
00:44:35,240 --> 00:44:37,080
A PRODUCT OF TELOMERE

1165
00:44:37,080 --> 00:44:37,400
DYSFUNCTION.

1166
00:44:37,400 --> 00:44:39,000
SO INSTEAD OF USING PITCH, WE

1167
00:44:39,000 --> 00:44:41,000
DECIDED WE WERE GOING TO ANALYZE

1168
00:44:41,000 --> 00:44:43,800
THESE BRIDGES USING THE TELOMERE

1169
00:44:43,800 --> 00:44:45,000
BINDING PROTEIN TRF 2, WHICH HAS

1170
00:44:45,000 --> 00:44:46,840
BEEN ANOTHER PROTEIN THAT'S BEEN

1171
00:44:46,840 --> 00:44:48,200
ESTABLISHED TO STAIN THESE

1172
00:44:48,200 --> 00:44:50,560
BRIDGES AND SPECIFICALLY FOR

1173
00:44:50,560 --> 00:44:52,480
TELOMERIC SEQUENCES AND SO,

1174
00:44:52,480 --> 00:44:54,840
WE'RE IN CONTROL CELLS OR SINGLE

1175
00:44:54,840 --> 00:44:56,360
DEPLETION ALONE, WE DON'T REALLY

1176
00:44:56,360 --> 00:44:58,320
SEE ANY CHANGES IN THE FORMATION

1177
00:44:58,320 --> 00:44:59,880
OF THESE UMENT RAFINE BRIDGES

1178
00:44:59,880 --> 00:45:01,840
AND SPECIFICALLY 1S THAT CONTAIN

1179
00:45:01,840 --> 00:45:04,920
TELOMERES WHEN WE KNOCK DOWN RAD

1180
00:45:04,920 --> 00:45:06,560
54 AND SLX 4 TOGETHER, AGAIN WE

1181
00:45:06,560 --> 00:45:08,280
SEE THIS INCREASE IN THESE ULTRA

1182
00:45:08,280 --> 00:45:10,080
FINE BRIDGES THAT ARE STAINED

1183
00:45:10,080 --> 00:45:11,520
WITH THE TELOMERE BINDING

1184
00:45:11,520 --> 00:45:12,880
PROTEIN SUGGESTING THAT IN FACT,

1185
00:45:12,880 --> 00:45:19,880
THESE ULTRA FINE BRIDGES ARE

1186
00:45:19,880 --> 00:45:20,440
ACTUALLY TELOMERE BRIDGES.

1187
00:45:20,440 --> 00:45:23,880
AND SO IF THESE ULTRA FINE

1188
00:45:23,880 --> 00:45:27,160
BRIDGES ACTUALLY PERSIST THROUGH

1189
00:45:27,160 --> 00:45:28,920
TELEO PHASE, THEY WOULD CAUSE

1190
00:45:28,920 --> 00:45:30,800
FRAGMENTATION AND YOU WOULD GET

1191
00:45:30,800 --> 00:45:33,440
THE MICRONUCLEI AND THESE MAY

1192
00:45:33,440 --> 00:45:34,960
CONTAIN TELEO MERRIC SEQUENCE IF

1193
00:45:34,960 --> 00:45:37,680
THE BRIDGES ARISE FROM TELOMERE

1194
00:45:37,680 --> 00:45:40,280
DYSFUNCTION AND SO, HERE WHAT WE

1195
00:45:40,280 --> 00:45:42,800
ASK WAS, DO WE SEE AN INCREASE

1196
00:45:42,800 --> 00:45:45,680
IN THE PERCENTAGE OF MICRONUCLEI

1197
00:45:45,680 --> 00:45:46,280
THAT CONTAIN TELEO MERRIC

1198
00:45:46,280 --> 00:45:49,400
SEQUENCE IN THE ABSENCE OF

1199
00:45:49,400 --> 00:45:53,040
COMBINED DEPLETION OF RAD 54 AND

1200
00:45:53,040 --> 00:45:53,240
SLX 4.

1201
00:45:53,240 --> 00:45:54,080
SO POTENTIALLY DIFFICULT TO SEE

1202
00:45:54,080 --> 00:45:55,840
ON THE SCREEN BUT HERE'S AN

1203
00:45:55,840 --> 00:45:58,200
EXAMPLE OF THE MICRO NUCLEI

1204
00:45:58,200 --> 00:46:00,480
WE'RE COUNTING AND THE SIGNAL

1205
00:46:00,480 --> 00:46:02,000
INSIDE OF THAT OF TELOMERES AND

1206
00:46:02,000 --> 00:46:04,320
WHEN WE DO THAT IN THE COMBINED

1207
00:46:04,320 --> 00:46:07,880
DEPLETION DO WE SEE A COMBINED

1208
00:46:07,880 --> 00:46:10,480
INCREASE OF CELLS MICRONUCLEI

1209
00:46:10,480 --> 00:46:13,760
SUGGESTING THAT IN FACT, LOSS OF

1210
00:46:13,760 --> 00:46:15,480
RAD54 AND SLX 4 PATHWAYS IS

1211
00:46:15,480 --> 00:46:17,720
LEADING TO FRAGMENTATION OF THE

1212
00:46:17,720 --> 00:46:23,920
DNA OR THE TELEO MERRIC DNA, AND

1213
00:46:23,920 --> 00:46:24,480
CREATING THESE MICRODNA.

1214
00:46:24,480 --> 00:46:25,880
SO I HOPE FROM THIS DATA I'VE

1215
00:46:25,880 --> 00:46:29,880
GIVEN YOU A SENSE THAT RAD54

1216
00:46:29,880 --> 00:46:31,640
ACTUALLY FUNCTIONS TO PROMOTE

1217
00:46:31,640 --> 00:46:33,480
BREAK INDUCED REPLICATION BY

1218
00:46:33,480 --> 00:46:35,680
PROCESSING OF THESE D-LOOP

1219
00:46:35,680 --> 00:46:38,760
STRUCTURES AND WORKS

1220
00:46:38,760 --> 00:46:40,600
COORDINATELY WITH BLM AND

1221
00:46:40,600 --> 00:46:42,600
TOP1311 TWIN STUDIES 2 TO

1222
00:46:42,600 --> 00:46:44,280
PROMOTE BRANCH MIGRATION, AND

1223
00:46:44,280 --> 00:46:46,800
DNA SYNTHESIS AND ULTIMATELY

1224
00:46:46,800 --> 00:46:48,760
TELOMERE ELONGATION AND THAT IN

1225
00:46:48,760 --> 00:46:50,320
THE ABSENCE OF RAD 54, THIS

1226
00:46:50,320 --> 00:46:52,400
PATHWAY IS FORCED TO RELY ON

1227
00:46:52,400 --> 00:46:54,880
RESOLUTION OF THESE

1228
00:46:54,880 --> 00:46:56,400
STRUBLGHTURES THROUGH CLEAVAGE

1229
00:46:56,400 --> 00:47:03,200
AND CHROMA TID EXCHANGES.

1230
00:47:03,200 --> 00:47:04,560
SO WHILE I QUISH I COULD SAY

1231
00:47:04,560 --> 00:47:06,400
THAT THE FINAL SAMPLE THAT

1232
00:47:06,400 --> 00:47:07,880
LINGERS IN OUR MIND IN OUR LAB

1233
00:47:07,880 --> 00:47:13,040
WAS IN FACT MUTATED--LAB LOOKING

1234
00:47:13,040 --> 00:47:16,400
TO DEFINE THE GENETICS OF THESE

1235
00:47:16,400 --> 00:47:17,400
ALT TUMORS, --I LIKE TO SAY I

1236
00:47:17,400 --> 00:47:19,400
THINK WE HAVE FIGURED OUT WITH

1237
00:47:19,400 --> 00:47:21,160
THIS MUTATION, WHAT IT IS, BUT

1238
00:47:21,160 --> 00:47:23,040
WE'RE IN THE EARLY STAGES.

1239
00:47:23,040 --> 00:47:24,920
SO AS AN EARLY TEASER, I HOPE I

1240
00:47:24,920 --> 00:47:25,920
CAN SHARE THAT DATA SOON, WITH

1241
00:47:25,920 --> 00:47:27,000
THAT I WOULD LIKE TO THANK THE

1242
00:47:27,000 --> 00:47:28,360
PEOPLE WHO DID THE WORK IN THE

1243
00:47:28,360 --> 00:47:31,440
LAB, THIS IS REALLY A

1244
00:47:31,440 --> 00:47:32,880
KIEWMRATIVE EFFORT OF CURRENT

1245
00:47:32,880 --> 00:47:39,880
AND FORMER STUDENTS INCLUDING

1246
00:47:39,880 --> 00:47:41,320
KELLEY, MASON-OSANN WHO DID A

1247
00:47:41,320 --> 00:47:42,880
QUITE A BIT OF WORK, SUPPORTED

1248
00:47:42,880 --> 00:47:48,000
BY A NUMBER OF PEOPLE AT THE

1249
00:47:48,000 --> 00:47:52,040
LAB, INCLUDING HIMABINDU, GA,

1250
00:47:52,040 --> 00:47:55,320
IR, AND ADAM LABADORF AND ÁNGEL

1251
00:47:55,320 --> 00:47:58,560
DAI, AND SOME OF THE SMARCALL

1252
00:47:58,560 --> 00:48:01,800
WORK WAS DONE AT THE UNIVERSITY

1253
00:48:01,800 --> 00:48:04,320
OF SHERBROOK, AND THE SLX

1254
00:48:04,320 --> 00:48:06,760
COLLABORATION WAS IN

1255
00:48:06,760 --> 00:48:08,120
COLLABORATION WITH COLLEAGUES AT

1256
00:48:08,120 --> 00:48:09,080
THE FRANCIS CRICK INSTITUTE.

1257
00:48:09,080 --> 00:48:10,600
WITH THAT I WILL BE HAPPY TO

1258
00:48:10,600 --> 00:48:14,520
TAKE ANY QUESTIONS.

1259
00:48:14,520 --> 00:48:19,880
>> THANK YOU VERY MUCH FOR THIS

1260
00:48:19,880 --> 00:48:21,080
EXCELLENT TALK.

1261
00:48:21,080 --> 00:48:28,680
DR. BOHR AND I WILL TAKE TURNS

1262
00:48:28,680 --> 00:48:29,240
ASKING THE QUESTIONS.

1263
00:48:29,240 --> 00:48:32,120
THERE YOU ARE, DO YOU WANT TO GO

1264
00:48:32,120 --> 00:48:32,320
FIRST?

1265
00:48:32,320 --> 00:48:32,880
>> YES, YEAH.

1266
00:48:32,880 --> 00:48:36,480
THANK YOU FOR THAT VERY

1267
00:48:36,480 --> 00:48:38,360
INTERESTING TALK AND I HAVE

1268
00:48:38,360 --> 00:48:40,840
ANOTHER QUESTION, BUT FIRST OF

1269
00:48:40,840 --> 00:48:44,920
ALL, READ THE 1 HERE FROM GORDON

1270
00:48:44,920 --> 00:48:48,400
AND BASED ON YEAST STUDIES LONG

1271
00:48:48,400 --> 00:48:51,880
STRETCHES OF B-RICH BIR INVADING

1272
00:48:51,880 --> 00:48:53,880
STRAND CAN STAY SINGLE STRANDED

1273
00:48:53,880 --> 00:48:56,000
FOR A SIGNIFICANT TIME.

1274
00:48:56,000 --> 00:48:59,640
THIS WILL MAKE IT PRONE TO

1275
00:48:59,640 --> 00:49:01,120
FORMING G-GAWR AT THE TIMES

1276
00:49:01,120 --> 00:49:04,000
WHICH MAY CAUSE A BIR BREAKAGE

1277
00:49:04,000 --> 00:49:05,760
IN THE SUBSEQUENT REPLICATIONS.

1278
00:49:05,760 --> 00:49:07,920
SHOULD THIS REALLY BE HAPPENING?

1279
00:49:07,920 --> 00:49:12,120
WOULD IT BE VISIBLE IN YOUR

1280
00:49:12,120 --> 00:49:12,680
ASSAY?

1281
00:49:12,680 --> 00:49:12,920
>> OOH.

1282
00:49:12,920 --> 00:49:14,360
THAT'S A GOOD QUESTION.

1283
00:49:14,360 --> 00:49:18,520
WHEN WE THINK ABOUT THE

1284
00:49:18,520 --> 00:49:20,480
CONTRIBUTIONS FOR G-QUADRUP LEX

1285
00:49:20,480 --> 00:49:21,560
FORMATION ON RUPLICATION STRESS,

1286
00:49:21,560 --> 00:49:23,520
IT'S NOT SOMETHING WE HAVE

1287
00:49:23,520 --> 00:49:24,800
CEREBELLUMS PETTER EASE

1288
00:49:24,800 --> 00:49:26,520
VISUALIZING, IT'S NOT SOMETHING

1289
00:49:26,520 --> 00:49:27,480
WE LOOK AT.

1290
00:49:27,480 --> 00:49:29,080
WOULD WE BE ABLE TO VISUALIZE IN

1291
00:49:29,080 --> 00:49:31,160
THE ASSAY, I WOULD SAY PROBABLY

1292
00:49:31,160 --> 00:49:33,480
NOT. 

1293
00:49:33,480 --> 00:49:35,720
>> OKAY, THANK YOU.

1294
00:49:35,720 --> 00:49:38,680
MIKE AT LICHTON, ASKED THIS

1295
00:49:38,680 --> 00:49:39,480
QUESTION, SHARED FUNCTION

1296
00:49:39,480 --> 00:49:41,160
BETWEEN BLM AND RAD54 AND THE

1297
00:49:41,160 --> 00:49:48,080
KNOCK DOWN FOR EITHER IS

1298
00:49:48,080 --> 00:49:50,360
SYNERGISTIC WITH XLX4 KNOCK DOWN

1299
00:49:50,360 --> 00:49:51,200
FOR UPF FORMATION.

1300
00:49:51,200 --> 00:49:55,520
HAVE YOU EXAMINED A PHENOTYPE OF

1301
00:49:55,520 --> 00:50:01,080
A BLM RAD54 DOUBLE KNOCK DOWN?

1302
00:50:01,080 --> 00:50:02,680
>> JUST IN THE ULTRA FINE BRIDGE

1303
00:50:02,680 --> 00:50:05,040
ANALYSIS WHERE WE SEE THAT

1304
00:50:05,040 --> 00:50:10,080
COMBINED DEPLETION OF SLX 4 AND

1305
00:50:10,080 --> 00:50:11,680
RAD54 BLM ARE IN THAT FORMATION,

1306
00:50:11,680 --> 00:50:15,680
SO OTHER THAN THAT WHEN WE KNOCK

1307
00:50:15,680 --> 00:50:18,480
DOWN JUST BLM AND RAD54 ALONE WE

1308
00:50:18,480 --> 00:50:22,600
DON'T SEE ANY ADDITIONAL

1309
00:50:22,600 --> 00:50:22,880
PHENOTYPES.

1310
00:50:22,880 --> 00:50:26,600
>> GOOD DEAL.

1311
00:50:26,600 --> 00:50:29,680
>> FROM JIM, YEAST BIR ALSO

1312
00:50:29,680 --> 00:50:34,360
DEPENDS ON RAD54'S COULD YOU

1313
00:50:34,360 --> 00:50:35,440
SAYIN RDH54, RAD54 B, WHAT ABOUT

1314
00:50:35,440 --> 00:50:37,360
THAT IN YOUR CELLS?

1315
00:50:37,360 --> 00:50:38,800
>> YEAH, GOOD QUESTION.

1316
00:50:38,800 --> 00:50:41,640
WE DIDN'T ANALYZE RAD54 B BUT

1317
00:50:41,640 --> 00:50:43,680
IT'S SOMETHING WE'RE REALLY

1318
00:50:43,680 --> 00:50:45,680
INTERESTED IN.

1319
00:50:45,680 --> 00:50:46,880
>> SO MICHAELLICATEON SAYS, HE

1320
00:50:46,880 --> 00:50:49,480
MEANT SLX 4 BUT I THINK THAT

1321
00:50:49,480 --> 00:50:52,120
WAS--KEN READ THAT. 

1322
00:50:52,120 --> 00:50:53,080
>> YEAH, I UNDERSTAND.

1323
00:50:53,080 --> 00:50:57,800
SNIMENT --I WANT TO ASK YOU
ABOUT

1324
00:50:57,800 --> 00:50:59,400
THESE CELLS AND FOLLOWING THEM

1325
00:50:59,400 --> 00:50:59,640
ALT.

1326
00:50:59,640 --> 00:51:04,160
YOU SAY YOU USE THESE DIFFERENT

1327
00:51:04,160 --> 00:51:07,200
3 APPROACHES LOOKING AT ALT

1328
00:51:07,200 --> 00:51:11,400
BODIES AND CIRCLES AND TELEO

1329
00:51:11,400 --> 00:51:13,240
MERRIC SYSTEM CHROMA TID

1330
00:51:13,240 --> 00:51:15,560
EXCHANGES TO WHAT EXTENT DO ALL

1331
00:51:15,560 --> 00:51:17,080
THESE CELLS HAVE THESE CRITERIA

1332
00:51:17,080 --> 00:51:18,320
OR SOME CELLS HAVE THEM AND SOME

1333
00:51:18,320 --> 00:51:21,920
OF THEM NOT, AND OTHERS HOW AND

1334
00:51:21,920 --> 00:51:23,320
DOES THAT AFFECT WHAT YOU CALL

1335
00:51:23,320 --> 00:51:24,960
ALL CELLS AND HOW YOU CAN

1336
00:51:24,960 --> 00:51:28,320
COMPARE THEM? 

1337
00:51:28,320 --> 00:51:28,880
>> YEAH, GOOD QUESTION.

1338
00:51:28,880 --> 00:51:31,480
I WOULD SAY IN GENERAL THERE'S

1339
00:51:31,480 --> 00:51:32,240
CERTAIN REQUIREMENTS OR

1340
00:51:32,240 --> 00:51:34,360
THRESHOLDS THAT WE USE TO DEFINE

1341
00:51:34,360 --> 00:51:36,880
ALT STATUS AND SO, FROM THE 1S

1342
00:51:36,880 --> 00:51:38,960
THAT I SHOWED TODAY, THEY ALL

1343
00:51:38,960 --> 00:51:42,560
HAVE ALL OF THOSE FEATURES, SO

1344
00:51:42,560 --> 00:51:44,480
FOR EXAMPLE, THEY ALL HAVE

1345
00:51:44,480 --> 00:51:45,760
C-CIRCLES, SOME MAY HAVE MORE

1346
00:51:45,760 --> 00:51:47,600
THAN OTHER AND IN FACT, I THINK

1347
00:51:47,600 --> 00:51:50,200
CELLS THAT ARE DEFICIENT FOR

1348
00:51:50,200 --> 00:51:51,520
ATRX AND DAXX, DEMONSTRATE

1349
00:51:51,520 --> 00:51:53,320
HIGHER LEVELS OF C-CIRCLES THAN

1350
00:51:53,320 --> 00:51:56,080
THE OTHER MUTANTS BUT THEY ALL

1351
00:51:56,080 --> 00:52:01,520
HAVE THEM. 

1352
00:52:01,520 --> 00:52:03,480
APBs AND TELOMERE SISTER

1353
00:52:03,480 --> 00:52:04,880
CHROMA TID EXCHANGE ANALYSIS,

1354
00:52:04,880 --> 00:52:06,680
BUT IT WOULD BE IMPOSSIBLE, NOT

1355
00:52:06,680 --> 00:52:07,920
IMPOSSIBLE BUT VERY DIFFICULT TO

1356
00:52:07,920 --> 00:52:10,680
DO ON A TUMOR SAMPLE SO THAT'S

1357
00:52:10,680 --> 00:52:16,760
LIMITED TO CELL LINES.

1358
00:52:16,760 --> 00:52:20,240
>> A COMMENT, I WAS PLEASED TO

1359
00:52:20,240 --> 00:52:24,760
SEE THAT THE--YOU ARE DOING LOTS

1360
00:52:24,760 --> 00:52:28,640
OF CARIO TYPES, THESE DAYS WHEN

1361
00:52:28,640 --> 00:52:31,880
WE'RE LOOKING FOR DISEASE

1362
00:52:31,880 --> 00:52:33,680
PHENOTYPES AND GENETICS, THEY

1363
00:52:33,680 --> 00:52:34,920
JUST DO EXOME SEQUENCING, AND

1364
00:52:34,920 --> 00:52:37,680
YOU CLEARLY POINTED OUT, YOU

1365
00:52:37,680 --> 00:52:38,320
WILL--YOU WILL MISS

1366
00:52:38,320 --> 00:52:39,480
TRANSLOCATIONS WHEN YOU DO THAT

1367
00:52:39,480 --> 00:52:44,040
WHICH MAY BE A VERY IMPORTANT

1368
00:52:44,040 --> 00:52:48,280
AND CARIO TYPING SEEMS TO BE

1369
00:52:48,280 --> 00:52:49,040
NEGLECTED IN GENERAL WHEN

1370
00:52:49,040 --> 00:53:05,640
THEY'RE LOOKING FOR GENETIC

1371
00:53:05,640 --> 00:53:08,920
SYSTEMS, WE WORK WITH PATIENTS

1372
00:53:08,920 --> 00:53:11,600
WITH 0 PIG MATOSA, AND THE

1373
00:53:11,600 --> 00:53:13,880
VARIANT AND IN POL8A, AND IF YOU

1374
00:53:13,880 --> 00:53:15,240
LOOKED AT AND WHAT WOULD BE THE

1375
00:53:15,240 --> 00:53:19,320
EFFECT OF DEFICIENCIES IN POL8A

1376
00:53:19,320 --> 00:53:21,120
IN YOUR ALT TELOMERES.

1377
00:53:21,120 --> 00:53:24,360
>> YEAH, I THINK THOSE STUDIES

1378
00:53:24,360 --> 00:53:25,560
HAVE BEEN DONE AND I WOULD NEED

1379
00:53:25,560 --> 00:53:27,440
TO THINK ABOUT OR RECALL WHAT

1380
00:53:27,440 --> 00:53:28,880
THE EXACT RESULT WAS BUT THE

1381
00:53:28,880 --> 00:53:31,960
PRIMING EVENTS ARE THOUGHT TO

1382
00:53:31,960 --> 00:53:34,000
BE--I THINK THE PRIMING EVENTS

1383
00:53:34,000 --> 00:53:37,960
ARE THOUGHT TO BE REGULATED BY

1384
00:53:37,960 --> 00:53:40,600
POLA BUT IT'S REGULATED BY POL

1385
00:53:40,600 --> 00:53:42,680
D, BUT WE DON'T KNOW ENOUGH OF

1386
00:53:42,680 --> 00:53:45,600
THE RECRUITMENT OF THOSE

1387
00:53:45,600 --> 00:53:46,200
POLYMERASES TELEO MERRIC, TO

1388
00:53:46,200 --> 00:53:47,440
KNOW WHAT THE CONSEQUENCE OF

1389
00:53:47,440 --> 00:53:48,560
LOSS OF 1 ON OTHER.

1390
00:53:48,560 --> 00:53:50,880
SO I THINK THOSE STUDIES HAVEN'T

1391
00:53:50,880 --> 00:53:51,840
SPECIFICALLY BEEN DONE.

1392
00:53:51,840 --> 00:53:56,840
>> YEAH, WE DON'T PARTICULARLY

1393
00:53:56,840 --> 00:53:58,120
SEE STUDY GENETIC ABNORMALITIES

1394
00:53:58,120 --> 00:53:59,680
IN THE PATIENTS, OR MAYBE WE

1395
00:53:59,680 --> 00:54:02,800
HAVEN'T LOOKED HARD ENOUGH AS

1396
00:54:02,800 --> 00:54:11,080
THE POSSIBILITY.

1397
00:54:11,080 --> 00:54:12,000
YES.

1398
00:54:12,000 --> 00:54:12,720
>> ANYMORE QUESTIONS, WILL?

1399
00:54:12,720 --> 00:54:13,720
>> I'M LOOKING AT IF THERE ARE

1400
00:54:13,720 --> 00:54:21,840
ANY MORE IN THE LIST HERE?

1401
00:54:21,840 --> 00:54:22,880
IT'S REALLY GREAT, THANK YOU.

1402
00:54:22,880 --> 00:54:23,480
>> OKAY, GREAT.

1403
00:54:23,480 --> 00:54:24,000
THANK YOU SO MUCH.

1404
00:54:24,000 --> 00:00:00,000
IT WAS A GREAT TALK.