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     GOOD AFTERNOON,
EVERYONE,

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WELCOME TO THE MONTHLY DNA

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REPAIR INTEREST GROUP

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CONFERENCE, WE ARE HERE TO

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DISCUSS THE RECENT ANNOUNCEMENTS

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IN DNA REPAIR, MUTAGENESIS, CELL

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CYCLE AND RELATEDDED HUMAN

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DISEASE. DR. (INAUDIBLE) AND I

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WOULD LIKE TO TAKE THIS

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OPPORTUNITY TO APPRECIATE

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PARTICIPATION AND INSIGHT FOR

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NOMINATIONS FROM ADVISORY

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COMMITTEE. BEFORE WE BEGIN,

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PLEASE MARK A CALENDAR FOR OUR

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ANNUAL YOUNG INVESTIGATOR

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WEBINAR ON THE NOVEMBER 14. WE

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WILL FEATURE TALKS ABOUT DR.

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MALIKA (INAUDIBLE),

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(INDISCERNIBLE) AND CLAUDIA

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NICOLAE. EMAIL ANNOUNCEMENT WILL

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FOLLOW SOON. TODAY WE ARE VERY

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PLEASED TO HAVE DR. WEI YANG

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WITH US, A DISTINGUISHED

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INVESTIGATOR AT THE U.S.

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NATIONAL INSTITUTES OF HEALTH. 

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WHERE SHE HAS BEEN SINCE 1995. 

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SHE BEGAN HER UNDERGRADUATE

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STUDIES IN BIOCHEMISTRY, IN

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(INAUDIBLE) UNIVERSITY SHANGHAI 

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LATER DR. YANG TRANSFERRED TO

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STONY BROOK FROM WHICH SHE

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RECEIVED A BACHELOR'S DEGREE.

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AND SHE RECEIVED DEPh.D.

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DEGREE FROM COLUMBIA UNIVERSITY

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WHERE SHE WORKED WITH WAYNE

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HENDRICKSON AND RECEIVED

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POST-DOCTORAL TRAINING AT YALE

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UNIVERSITY IN THE LAB OF TOM

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GATES.  SHE USED STRUCTURAL AND

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BIOCHEMICAL TO IN BUY LOGICAL

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CHEMISTRY SPONSORED BY CSB AND

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B. SHE'S ALSO AN ELECTED MEMBER

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FOR THE NATIONAL ACADEMY OF

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SCIENCES AND AMERICAN ACADEMY OF

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ART AND SCIENCES. NOW WE WILL

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TRANSITION TO DR. YANG'S TALK

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FOR ATTENDEES PLEASE PUT YOUR

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QUESTIONS IN THE CHAT BOX AND WE

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WILL DISCUSS AFTER TALK. THANK

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YOU. DR. YANG. THANK YOU FOR

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TAKING OVER THE DNA REPAIR SORT

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OF LIKE OUR SOCIETY MEETING AND

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THANKFUL TO CARRY OUT WITH NEWER

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TECHNOLOGY WEBEX AND ZOOM. WE

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ARE MUCH MORE SEAMLESSLY LINKED

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TO THE WORLD. TODAY I'M GOING TO

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SHARE WITH YOU OUR RECENT RESULT

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OF DNA LESION RECOGNITION.  

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EXNUCLEOTIDE EXCISION REPAIR. IT

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IS AN OLD TOPIC AND THE REASON

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I'M WILLING TO SHARE WITH YOU

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OUR RECENT RESULT IS BECAUSE

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MANY PEOPLE HAVE KNOWN AND

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STUDIED FOR MANY DECADES AND

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KNOW MUCH MORE THAN ME AND MY

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GROUP SO I'M HOPING TO LEARN

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FROM YOU AND PLEASE SEND ME YOUR

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QUESTIONS, COMMENTS AND

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SUGGESTIONS. SO IT IS AN

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OPPORTUNITY FOR ME TO LEARN. SO

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WE WERE INTERESTED STARTED

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INTEREST DNA LESION INDUCED BY

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ULTRAVIOLET ABOUT 20 SOMETHING

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YEARS AGO AND WE STARTED TO

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STUDY LESION TOLERANCE BY

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SPECIALIZED DNA POLYMERASE.  

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LESION ARE TOLERATED DURING DNA

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REPLICATION AND POST REPLICATION

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AS WELL. BUT THE TLS MUTATIONS

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CALLED XPV IS ONLY CAUSING ABOUT

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20% OF THE PATIENT. THE MAJORITY

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OF THE UV INDUCED LESION ARE

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REPAIRED OUTSIDE OF THE S PHASE.

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THE REPAIR TOLERANCE LEADS TO A

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CONDITION CHECK TESTIFILY KNOWN

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AS -- COLLECTIVELY KNOWN AS

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SERUM PIG MENTOSUM. HERE IS A

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PICTURE, NOT TOO BAD BUT COULD

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BE WORSE. THE EXCISION REPAIR

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DEPEND ON NUMBER OF ESSENTIAL

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GENES KNOWN 20, 30 DECADES AGO,

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XPA, XPC EFG.  THOSE ARE THE TWO

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TYPE OF LESION AND ACTUALLY THE

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REPAIR AND ALSO TLS OF SIMILAR

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LESIONS BECOME A PROBLEM IN

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CHEMOTHERAPY BECAUSE ONE OF THE

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MOST OFTEN USED CHEMICAL REAGENT

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IS CISPLATIN AND IT IS

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CROSS-LINKED ADJACENT SIMILAR TO

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THE RESULT OF COVALENT LINKAGE

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OF ADJACENT TWO Ts, CPD TO

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DOUBLE BOND OF (INDISCERNIBLE)

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SO UNDERSTANDING MECHANISM NOT

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ONLY HELP US TO UNDERSTAND AND

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PERHAPS TREATMENT OF THOSE XP

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PATIENTS BUT MOST IMPORTANTLY

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CAN MAYBE HELP TO HELP TREATMENT

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OF CISPLATIN CHEMOTHERAPY MORE

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EFFECTIVELY. SO ONCE WE STARTED

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TO WORK ON TRANSLATION DNA

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POLYMERASE, WE RECEIVED THE

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INVITATION FROM EXPERT IN THE

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FIELD, CA (INAUDIBLE) WHO SAID

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MAYBE WE CAN LAB RAT, THAT WAS

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MORE THAN A DOZEN YEARS AGO. WE

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STARTED TO LOOK INTO IT. 

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NUCLEOTIDE EXCISION REPAIR CAN

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BE STARTED BY TWO DIFFERENT

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PATHWAYS FOR FINDING DNA LESION

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EITHER UV OR OTHER CROSS LINK

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SPECIES BULKY ADDUCT ON DNA. ONE

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CALLED GLOBAL GENOME NER WHICH

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IS ESSENTIAL LESION RECOGNITION

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PROTEIN IS XPC, BUT SOMETIMES

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WHEN DIFFICULT TO FIND XPE IS

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INVOLVED I WILL SHOW YOU THE

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NEXT SLIDE.  THIS FINALLY IS NOT

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GUARANTEED, EVERY LESION CAN BE

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FOUND IN THE GENOME BECAUSE OF

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BILLIONS OF BASE PAIRS NOT

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GUARANTEED TO BE FOUND,

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PARTICULARLY LIKE CPD DOES NOT

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DISTORT DNA VERY MUCH. THE

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MOTION WAY IS TRANSCRIPTS

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COUPLED REPAIR BULKY LESIONS

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WILL STALL RNA POLYMERASE AND

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WHEN RNA POLYMERASE STALL, CSB

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WILL TRY TO PUSH IT OVER BUT

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WHEN THAT FAILED THEN IT IS BOTH

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PATHWAY LEADING TO RECRUITMENT

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OF TRANSCRIPTION CALLED TF 2H.

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THEN THOSE TWO PATHWAYS

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CONVERGE. TF 2H BINDING TO THE

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DNA LEADS TO NEXT PROTEIN

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BINDING DEFINED KEY PROTEIN NER

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XPA. THE TWO TOGETHER, TF 2H AND

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XP TOGETHER ABLE TO FIND WAS THE

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LESION TRULY IS THE BULKY LESION

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AND THE NEED TO BE EXCISED BY

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NUCLEASE AND IF IT IS NOT OTHER

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PATHWAYS LIKE MISMATCH REPAIR OR

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BASIC EXCISION REPAIR WILL BE

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MORE PREFERENCE THAN TO USE THIS

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BIGGER EXCISION. SO WHEN THE

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LESION IS VERIFIED BY TF 2H AND

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XPA TWO PARTICULAR NUCLEASE

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STRUCTURE SPECIFIC NUCLEASE XPF

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AND XPG RECRUITED AND CLEAVE AT

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THE 5 PRIME AND 3 PRIME JUNCTION

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OF THE LESION AND EUKARYOTS THE

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PATHWAY AND EXERCISE THE DNA

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CONTAINING LESION CAN VARY FROM

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20 SOMETHING NUCLEOTIDE TO 35

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NUCLEOTIDE. THIS IS THE END

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RESULT. AND THEN THE GAP NEED TO

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BE FILLED BY THE DNA POLYMERASE 

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MANY OUTSTANDING SCIENTISTS LAY

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THE GROUND AND CONSTITUTE

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REACTION AND LEADING US TO

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UNDERSTAND VERY WELL. THOSE ARE

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THE PEOPLE AND REMEMBER NOT MANY

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YEARS AGO NOBEL PRIZE WAS

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AWARDED TO HIM FOR HIS

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CONTRIBUTION, PHIL

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(INDISCERNIBLE) AND SO IT IS

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JUST LIKE THIS IS THE LIST OF

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PEOPLE WHO MADE IT VERY

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IMPORTANT CONTRIBUTION, OUR WORK

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IS BASED ON THOSE STUDIES.  SO

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THE (INAUDIBLE) IS RECONSTITUTED

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BUT EACH PROTEIN ROLE IS NOT

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WELL UNDERAND THE MECHANISM IS

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STILL YET TO BE REVILED.  HERE

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IS START -- REVEALED.  HERE IS

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STARTING, I'M NOT GOING TO

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ELABORATE MUCH ON THE DNA XPE,

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WHICH IS A PROTEIN SHOWN HERE

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XPE HELPS TO RECOGNIZE DNA

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LESION WHEN DNA IS NOT MUCH

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DISTORTED AND THE FORMS

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DEPENDENT DDB 1 AND HETERODIMER

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FIND THE LESION. THIS WORK IS

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DONE BY ANY C B BY NICOLE THOMAD

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CONSTANTLY STUDIED BNAB 1 AND 2

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RECOGNIZE LEAGUES LIKE PHOTO

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PRODUCT ON NUCLEOSOME. AND IN

197
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ADDITION WE FIND DDB 1

198
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INTERACTING WITH THIS WHOLE

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PROTEIN IS UBIQUITIN E 3 PROTEIN

200
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AND WORKS WITH A AND B AND

201
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UBIQUITYNATE TRANSCRIPTION

202
00:10:20,851 --> 00:10:26,624
FACTORS AND ULTIMATELY THE DDB 1

203
00:10:26,624 --> 00:10:30,161
AND 2 XPE HAND OVER THE LESION

204
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TO XPC TO START THE GLOBAL

205
00:10:31,929 --> 00:10:37,001
GENOME NER. SO HERE THE WORK IS

206
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ALSO CONTRIBUTE BY (INAUDIBLE)

207
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LAB IN PITTSBURG AND THERE IS

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MUCH WORK ESTABLISHED 30 DAYS OF

209
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XPC IN JAPAN BY (INDISCERNIBLE)

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GROUP. SO NOW WE ARE GOING TO

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00:10:48,779 --> 00:10:51,682
THE MAIN FOCUS OF TODAY. WHICH

212
00:10:51,682 --> 00:10:58,022
IS THE XPC AND VERY FIRST

213
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CRYSTAL STRUCTURE RED 4 WAS RED

214
00:11:01,592 --> 00:11:05,129
23 WERE DETERMINED BY NICOLA'S

215
00:11:05,129 --> 00:11:07,465
LAB IN 2007 AND THEY FOUND THAT

216
00:11:07,465 --> 00:11:13,170
TO THE XPC HETERODIMER DUPLEX,

217
00:11:13,170 --> 00:11:15,740
IF WE IN THIS TALK I WILL FOCUS

218
00:11:15,740 --> 00:11:17,475
ALWAYS HAVE THE LESION

219
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CONTAINING STRAND FROM 5 PRIME

220
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TO 3 PRIME LEFT TO RIGHT, LESION

221
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CONTAINING STRAND OF THE DARKER

222
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ORANGE AND NON-LESION STRAND

223
00:11:26,751 --> 00:11:28,819
SHOWING YELLOW. HERE YOU CAN SEE

224
00:11:28,819 --> 00:11:32,323
IT IS 5 PRIME TO 3 PRIME, THEN

225
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XPC BINDING DOWNSTREAM OF THE

226
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LESION AND THE LESION ITSELF IS

227
00:11:38,262 --> 00:11:40,431
DISORDERED. AND BASED OPPOSITE

228
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THE LESION ACTUALLY FLIPPED OUT

229
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AND BOUND BY XPC.  THIS

230
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STRUCTURE HAS BEEN AROUND A LOT

231
00:11:48,939 --> 00:11:50,207
OF YEARS AND DIFFERENT LEAGUES

232
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SHOWN TO BE RECOGNIZED BY RED 4,

233
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WE NEED TF 2H TO SEPARATE WHAT

234
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IS THE LESION FOR NER REPAIR OR

235
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IT IS NOT.  IT WHATTING FOR

236
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REALLY ALMOST 15 YEARS BEFORE

237
00:12:08,159 --> 00:12:13,898
THE STRUCTURE OF HOW XPC AND TF

238
00:12:13,898 --> 00:12:17,101
2H TOGETHER. TF 2H STRUCTURE WAS

239
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FIRST REVEAL IN 2016 THROUGH

240
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WORK FROM (INDISCERNIBLE) IN THE

241
00:12:28,679 --> 00:12:31,048
REPORTED PRE-INITIATION COMPLEX

242
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WHERE TF 2H INTERACTING WITH

243
00:12:34,351 --> 00:12:37,855
DOUBLE STRAND DNA AND XPB AND

244
00:12:37,855 --> 00:12:41,325
XPB IS NON-ESSENTIAL FOR LIST OF

245
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ATPASE ACTIVITIES NOT ESSENTIAL

246
00:12:43,060 --> 00:12:45,796
FOR TRANSCRIPTION INITIATION BUT

247
00:12:45,796 --> 00:12:49,100
XPB IS AND BIND DOUBLE STRAND P

248
00:12:49,100 --> 00:12:51,702
DNA WELL. THIS STRUCTURE HELPED

249
00:12:51,702 --> 00:12:54,872
PEOPLE TO UNDERSTAND HOW TF 2H

250
00:12:54,872 --> 00:12:58,075
BINDING DOUBLE STRAND DNA BUT

251
00:12:58,075 --> 00:12:59,009
XPD ROSE VERY IMPORTANT FOR

252
00:12:59,009 --> 00:13:01,278
SCANNING THE LESION IS NOT BY

253
00:13:01,278 --> 00:13:06,383
DNA SO IT IS KIND OF LEFT

254
00:13:06,383 --> 00:13:07,685
HANDED. WHEN THE TWO STRUCTURES

255
00:13:07,685 --> 00:13:09,720
WERE TOGETHER, TWO PROTEIN

256
00:13:09,720 --> 00:13:10,921
COMPLEX WERE TOGETHER AND

257
00:13:10,921 --> 00:13:13,958
STRUCTURE DETERMINED BY

258
00:13:13,958 --> 00:13:15,659
(INDISCERNIBLE) REPORTING BY

259
00:13:15,659 --> 00:13:17,328
(INDISCERNIBLE) GROUP. THIS

260
00:13:17,328 --> 00:13:18,863
TOGETHER SEEMS TO BE NOT MUCH

261
00:13:18,863 --> 00:13:22,967
LINKED BY PROTEIN, EXCEPT TIP OF

262
00:13:22,967 --> 00:13:26,270
XPC SOMEHOW EXACT THE P 62 OF TF

263
00:13:26,270 --> 00:13:29,140
2H. MOSTLY TOGETHER IS BECAUSE

264
00:13:29,140 --> 00:13:32,776
THE DNA UPSTREAM OF THE LESION

265
00:13:32,776 --> 00:13:36,847
DUPLEX BIND BY XPD. THIS LEADS

266
00:13:36,847 --> 00:13:39,650
TO, IT IS NICE TO HAVE IN THE

267
00:13:39,650 --> 00:13:41,552
GENERAL ORGANIZATION BUT THERE

268
00:13:41,552 --> 00:13:43,921
HAS PROBLEMS BECAUSE THE LESION

269
00:13:43,921 --> 00:13:54,298
REMAINS DISORDERED IN

270
00:14:07,811 --> 00:14:09,547
(INDISCERNIBLE) WAS THE MAIN

271
00:14:09,547 --> 00:14:13,684
DRIVER. HE STUDIED TF 2H,

272
00:14:13,684 --> 00:14:17,821
TOGETHER WITH XPA AND XPA IS

273
00:14:17,821 --> 00:14:20,357
DICTATED NER. EVEN THOUGH HE

274
00:14:20,357 --> 00:14:22,593
USED THE DNA DOES NOT CONTAIN

275
00:14:22,593 --> 00:14:24,461
ANY LESION SO IT IS A DOUBLE

276
00:14:24,461 --> 00:14:26,964
STRAND DNA FOLLOWED BY SINGLE

277
00:14:26,964 --> 00:14:29,233
STRANDED, IT IS A Y SHAPE BASE

278
00:14:29,233 --> 00:14:33,237
PAIR SINGLE STRAND DNA AND XPA A

279
00:14:33,237 --> 00:14:34,705
SMALL BUT IMPORTANT PROTEIN BIND

280
00:14:34,705 --> 00:14:36,674
AT THE DOUBLE STRAND SINGLE

281
00:14:36,674 --> 00:14:38,542
STRAND JUNCTION. THE STRUCTURE

282
00:14:38,542 --> 00:14:41,512
OF XPA ALONE BY DNA CONTAIN

283
00:14:41,512 --> 00:14:44,114
LESION NEVER SEE XPA INTERACTING

284
00:14:44,114 --> 00:14:45,082
WITH LESION THOUGH IT IS KNOWN

285
00:14:45,082 --> 00:14:47,618
FOR HELPING BINDING LESION.  IT

286
00:14:47,618 --> 00:14:51,188
IS ALWAYS BIND DNA DOUBLE STRAND

287
00:14:51,188 --> 00:14:53,123
END OR HERE AT DOUBLE STRAND

288
00:14:53,123 --> 00:14:57,561
SINGLE STRAND JUNCTION. BECAUSE

289
00:14:57,561 --> 00:14:59,597
SINGLE STRAND DNA HERE IT IS

290
00:14:59,597 --> 00:15:01,799
CLEAR SINGLE STRAND DNA GOES

291
00:15:01,799 --> 00:15:03,734
THROUGH XPD VERY NICE BUT NOT

292
00:15:03,734 --> 00:15:07,171
CLEAR WHERE THE LESION IS. WE

293
00:15:07,171 --> 00:15:10,808
CAN SEE WITH TF 2H AND XPC

294
00:15:10,808 --> 00:15:14,845
WITHOUT XPA THE COMPLEX IN THAT

295
00:15:14,845 --> 00:15:18,415
COMPLEX TF 2H SEVEN CORE SUB

296
00:15:18,415 --> 00:15:20,150
UNIT IS U SHAPED.  IN IN

297
00:15:20,150 --> 00:15:24,355
PRESENCE OF XPA THE CORE 7 OF TF

298
00:15:24,355 --> 00:15:26,090
2H BECOME SIGNAL SHAPED SO A

299
00:15:26,090 --> 00:15:30,861
HUGE CONFIRMATIONAL CHANGE.

300
00:15:30,861 --> 00:15:32,730
SINCE XPD MOVES 5 PRIME TO 3

301
00:15:32,730 --> 00:15:34,164
PRIME THE LESION HAS TO BE

302
00:15:34,164 --> 00:15:35,799
SOMEWHERE OUTSIDE OF SINGLE

303
00:15:35,799 --> 00:15:40,437
STRAND DNA BOND BY XPD SO BEFORE

304
00:15:40,437 --> 00:15:43,040
XPD CAN SCAN AND FIND IT.  AND

305
00:15:43,040 --> 00:15:44,742
DECIDE IT HAS TO BE REPAIRED BY

306
00:15:44,742 --> 00:15:47,811
NER. BUT HERE THE LESIONS ARE

307
00:15:47,811 --> 00:15:50,681
HERE. AND XPD IS NOT BINDING ANY

308
00:15:50,681 --> 00:15:53,083
DNA. IF IT BINDS DNA HERE THE

309
00:15:53,083 --> 00:15:54,518
LESION WILL BE ON TO THE LEFT

310
00:15:54,518 --> 00:15:56,320
AND SCANNING TO THE RIGHT WILL

311
00:15:56,320 --> 00:15:59,256
NOT HAVE XPD ENCOUNTERING THE

312
00:15:59,256 --> 00:16:01,358
LESION SO THAT IS THE BIG

313
00:16:01,358 --> 00:16:03,460
DILEMMA WITH EVERY STRUCTURE

314
00:16:03,460 --> 00:16:04,428
KNOWN ABOUT CHEMICAL

315
00:16:04,428 --> 00:16:06,797
CHARACTERIZATION. SO WE STARTED

316
00:16:06,797 --> 00:16:11,268
PROJECT ACTUALLY IN EARLY 2010

317
00:16:11,268 --> 00:16:13,237
BUT WE WERE JUST NOT GETTING

318
00:16:13,237 --> 00:16:15,272
ANYWHERE WHERE ALL THOSE PAPER

319
00:16:15,272 --> 00:16:17,374
PUBLISHED. HOWEVER, EVENTUALLY

320
00:16:17,374 --> 00:16:18,976
WE GOT SOMEWHERE AND TODAY I

321
00:16:18,976 --> 00:16:21,045
WILL TELL YOU WHAT WE FOUND. THE

322
00:16:21,045 --> 00:16:22,346
FIRST WE WILL ADDRESS THE THREE

323
00:16:22,346 --> 00:16:23,847
QUESTIONS, HOW IS THE DNA LESION

324
00:16:23,847 --> 00:16:28,152
LOADED ON TO TF 2H SO XPD CAN

325
00:16:28,152 --> 00:16:29,553
SCAN VERIFIED LESION. ALONG WAY

326
00:16:29,553 --> 00:16:32,589
I WILL SHOW YOU HOW HUMAN XPC

327
00:16:32,589 --> 00:16:34,024
STRUCTURE AND HOW DOES IT

328
00:16:34,024 --> 00:16:38,495
RECRUIT TF 2H.  BECAUSE BEFORE

329
00:16:38,495 --> 00:16:42,866
IT IS THE RED 4 STRUCTURE. SO

330
00:16:42,866 --> 00:16:45,536
HOW THE ROLES OF XPA AND NER SO

331
00:16:45,536 --> 00:16:46,670
CRITICAL, IT IS IS SMALLEST

332
00:16:46,670 --> 00:16:50,007
PROTEIN AND CONTAIN MOST OF THE

333
00:16:50,007 --> 00:16:57,147
MUTATION THAT CAUSES XERODERMA

334
00:16:57,147 --> 00:16:58,882
PIGMENTOSA. SO WE START WITH 11

335
00:16:58,882 --> 00:17:00,584
SUB UNIT OF THE PROTEIN AS YOU

336
00:17:00,584 --> 00:17:06,690
CAN SEE XP 3, TF 2H THE CONTACT

337
00:17:06,690 --> 00:17:08,559
MOTIF IS NOT NECESSARY ACTUALLY

338
00:17:08,559 --> 00:17:11,528
INHIBIT NER SO WE ONLY USE THE 7

339
00:17:11,528 --> 00:17:15,532
SUB UNIT OF TF 2H WITH CORE 7

340
00:17:15,532 --> 00:17:18,936
AND XPA. AND WE ALSO ESTABLISHED

341
00:17:18,936 --> 00:17:20,904
THAT THROUGH THOSE PROTEIN WE

342
00:17:20,904 --> 00:17:23,907
CAN SEE LESION MIMIC SIDE 5

343
00:17:23,907 --> 00:17:26,076
OPPOSITE 2C CAN BE RECOGNIZED BY

344
00:17:26,076 --> 00:17:31,849
XPC AND BINDING AFFINITY BY LESS

345
00:17:31,849 --> 00:17:33,917
THAN 5 NANOMOLAR SO IT IS VERY

346
00:17:33,917 --> 00:17:36,353
TIGHT BINDING. BUT ADDING XPA

347
00:17:36,353 --> 00:17:39,089
DOESN'T CHANGE MUCH, ADDING TF

348
00:17:39,089 --> 00:17:41,959
2H YOU CAN SEE A SUPER SHIFT,

349
00:17:41,959 --> 00:17:44,094
THIS IS NOT MOST CLEAR BUT IN

350
00:17:44,094 --> 00:17:46,997
BETTER GELS ADDING XPA

351
00:17:46,997 --> 00:17:50,634
STABILIZES SUPER SHIFT. SO NOW

352
00:17:50,634 --> 00:17:52,836
TURNING TO STRUCTURAL STUDY. AND

353
00:17:52,836 --> 00:17:54,104
STRUCTURAL STUDY CRYSTALLOGRAPHY

354
00:17:54,104 --> 00:17:56,573
IS NOT A CHOICE, IT IS NEVER

355
00:17:56,573 --> 00:17:57,908
GOING TO CRYSTALLIZE WITH SO

356
00:17:57,908 --> 00:18:00,477
MUCH -- SO MANY SUBUNITS AND LOT

357
00:18:00,477 --> 00:18:04,248
OF MOBILITY. SO WE INITIALIZE

358
00:18:04,248 --> 00:18:08,652
AFTER MAKING THE COMPLEX, WE DID

359
00:18:08,652 --> 00:18:10,287
THE STAINING, PHILLIP

360
00:18:10,287 --> 00:18:12,990
(INDISCERNIBLE) DID IT FIRST.

361
00:18:12,990 --> 00:18:17,628
BUT WHEN PHIL PUT THE SAMPLE, IT

362
00:18:17,628 --> 00:18:20,964
BECOME HETEROGENOUS FALL APART. 

363
00:18:20,964 --> 00:18:22,933
SO HE TRIED THE GRAPHICS WHICH

364
00:18:22,933 --> 00:18:27,704
IS A GRADIENT FIXATION SO USING

365
00:18:27,704 --> 00:18:29,773
SUCROSE OF GLYCEROL GRADIENT

366
00:18:29,773 --> 00:18:33,076
APPLIED CROSS LINKING, CROSS

367
00:18:33,076 --> 00:18:36,847
LINKERS SUCH AS ALDEHYDE LOW

368
00:18:36,847 --> 00:18:39,950
CONCENTRATION, AND ESTABLISH A

369
00:18:39,950 --> 00:18:42,186
GRADIENT OF CROSS LINKERS AND

370
00:18:42,186 --> 00:18:43,921
LAY SAMPLE ON TOP, THE SAMPLE

371
00:18:43,921 --> 00:18:48,492
WHEN GOING THROUGH THE

372
00:18:48,492 --> 00:18:49,960
CENTRIFUGATION ALDEHYDE GETS

373
00:18:49,960 --> 00:18:55,399
CROSS-LINKED.  THIS IS THE

374
00:18:55,399 --> 00:18:56,033
GRADIENT CENTRIFICATION WITHOUT

375
00:18:56,033 --> 00:18:57,768
CROSS LINKING YOU CAN SEE THE

376
00:18:57,768 --> 00:19:00,704
CROSS-LINKED FORM CONTAIN 11 SUB

377
00:19:00,704 --> 00:19:03,207
UNIT AND AFTER WITH ALDEHYDE

378
00:19:03,207 --> 00:19:04,341
MOST GETS CROSS LINK TO THE

379
00:19:04,341 --> 00:19:06,844
SHIFT IN THE BAND WITH DNA. 

380
00:19:06,844 --> 00:19:11,582
INITIALLY WHEN WE PUT ON THE EM

381
00:19:11,582 --> 00:19:13,517
GRID, THE COMPLEX STILL FALL

382
00:19:13,517 --> 00:19:16,119
APART AFTER CROSS LINKING. AND

383
00:19:16,119 --> 00:19:18,922
THEN WE USE SPECIAL GRID WITH

384
00:19:18,922 --> 00:19:21,225
FILM SO SOMEWHAT HAVE ONE SIDE

385
00:19:21,225 --> 00:19:22,459
SUPPORTING OF THOSE PARTICLES

386
00:19:22,459 --> 00:19:24,428
THAT YOU CAN SEE THOSE COMPLEX

387
00:19:24,428 --> 00:19:27,865
LOOK VERY UNIFORM AND HOMOGENOUS

388
00:19:27,865 --> 00:19:30,367
IN TERM OF SIZ SIZE. STRUCTURE E

389
00:19:30,367 --> 00:19:32,302
THEY ARE STILL DIFFERENT. LONG

390
00:19:32,302 --> 00:19:34,471
STORY SHORT, WE WERE ABLE TO

391
00:19:34,471 --> 00:19:36,306
SOLVE THE STRUCTURE AND ACTUALLY

392
00:19:36,306 --> 00:19:40,477
THE WORK BY (INDISCERNIBLE) AND

393
00:19:40,477 --> 00:19:43,080
ERIC LEE. THEY WERE ON THE SAME

394
00:19:43,080 --> 00:19:46,450
CRYOEM GRID. THEY FOUND THREE

395
00:19:46,450 --> 00:19:48,919
TYPE OF COMPLEX WAS THE MIX OF

396
00:19:48,919 --> 00:19:53,790
11 SUB UNIT AND DNA LESION

397
00:19:53,790 --> 00:19:55,526
CONTAINING SIDE 5. SO THE FIRST

398
00:19:55,526 --> 00:19:58,595
TYPE OF COMPLEX IS XPC WITH TF

399
00:19:58,595 --> 00:20:01,265
2H AND DNA BUT YOU CAN SEE NOW

400
00:20:01,265 --> 00:20:06,303
YOU SEE MORE XPC. THIS IS

401
00:20:06,303 --> 00:20:08,038
SIMILAR TO (INDISCERNIBLE) WORK,

402
00:20:08,038 --> 00:20:09,673
SO WE SEE MORE DETAIL AND AT

403
00:20:09,673 --> 00:20:12,976
MUCH HIGHER RESOLUTION.  THE

404
00:20:12,976 --> 00:20:15,445
SECOND COMPLEX WE OBSERVE IS TF

405
00:20:15,445 --> 00:20:18,282
2H WITH XPA. XPC STILL THERE

406
00:20:18,282 --> 00:20:20,250
BECAUSE WE KNOW THE C TERMINAL

407
00:20:20,250 --> 00:20:24,154
OF XPC STUCK ON TO THE TF 2H

408
00:20:24,154 --> 00:20:26,356
HERE BUT THE BULK OF THE DOUBLE

409
00:20:26,356 --> 00:20:28,592
STRAND DNA INCLUDING LESION AND

410
00:20:28,592 --> 00:20:30,193
XPC WAS NOT THERE BECAUSE IT IS

411
00:20:30,193 --> 00:20:33,096
-- WE CAN SEE A BLOB MOVING. SO

412
00:20:33,096 --> 00:20:34,464
THE REMAINING STRUCTURE BECAUSE

413
00:20:34,464 --> 00:20:38,602
THE PRESENCE OF XPA, THIS TF 2H

414
00:20:38,602 --> 00:20:41,738
DNA XPA IS VERY SIMILAR TO

415
00:20:41,738 --> 00:20:46,577
PATRICK KRAMER AND

416
00:20:46,577 --> 00:20:55,719
(INDISCERNIBLE) STRUCTURE SO IN

417
00:20:55,719 --> 00:20:58,622
SENSE OF XPA THE CORE 7 IS U

418
00:20:58,622 --> 00:21:00,490
SHAPE AND IN THE PRESENCE OF XPA

419
00:21:00,490 --> 00:21:03,293
THERE IS A BIG CONFIRMATIONAL

420
00:21:03,293 --> 00:21:07,097
CHANGE. AND LESIONS OUTSIDE OF

421
00:21:07,097 --> 00:21:09,766
THE CORE 7. THE MOST REWARDING

422
00:21:09,766 --> 00:21:14,237
COMPLEX IS OBSERVE XPCC. WITH XA

423
00:21:14,237 --> 00:21:18,141
AND TF 2H ON DNA WE CAN SEE

424
00:21:18,141 --> 00:21:19,610
WHERE THE LESION IS AND THE

425
00:21:19,610 --> 00:21:23,013
RELATIVE TO THE TF 2H. AND YOU

426
00:21:23,013 --> 00:21:24,581
CAN SEE THIS LOWER PORTION OF

427
00:21:24,581 --> 00:21:29,119
THE TF 2H IS SIMILAR TO THE

428
00:21:29,119 --> 00:21:32,723
STRUCTURE WITHOUT XPC OR WITH

429
00:21:32,723 --> 00:21:35,792
XPC MOBILE. HERE WE SEE BOTH XPC

430
00:21:35,792 --> 00:21:37,661
AND XPA PRESENT. SO IN THE

431
00:21:37,661 --> 00:21:38,629
FOLLOWING SLIDE I WILL SHOW YOU

432
00:21:38,629 --> 00:21:42,599
A FEW DETAIL, HOW XPC RECOGNIZE

433
00:21:42,599 --> 00:21:46,637
DNA LESION. AND RECRUIT TF 2H.

434
00:21:46,637 --> 00:21:51,842
SO THIS IS A HUMAN XPC TRIMER

435
00:21:51,842 --> 00:21:58,415
AND CONTAIN MOSTLY XPC WHICH HAE

436
00:21:58,415 --> 00:22:02,185
STABILIZE THE XPC THOUGH RED 23

437
00:22:02,185 --> 00:22:03,553
DOES NOT INTERACT WITH DNA

438
00:22:03,553 --> 00:22:04,855
DIRECTLY BUT STABILIZE THE

439
00:22:04,855 --> 00:22:07,924
STRUCTURE OF XPC TO BIND DOUBLE

440
00:22:07,924 --> 00:22:09,126
STRAND DNA DOWNSTREAM OF THE

441
00:22:09,126 --> 00:22:13,363
LESION. AND WHAT WE FOUND ALSO

442
00:22:13,363 --> 00:22:14,998
IS THE CENTERING 2 WHICH IS

443
00:22:14,998 --> 00:22:18,969
KNOWN TO STABILIZE XPC ON DNA

444
00:22:18,969 --> 00:22:22,572
BEFORE WITH YEAST STRUCTURE AND

445
00:22:22,572 --> 00:22:24,307
THE NEW PORTION OF THE STRUCTURE

446
00:22:24,307 --> 00:22:28,311
THAT IS NOT OBSERVED HERE JUST

447
00:22:28,311 --> 00:22:31,148
SHOWS THE RED 23 DIFFERENT VIEW

448
00:22:31,148 --> 00:22:35,018
FROM MERE RED 3 AND CENTERING 2

449
00:22:35,018 --> 00:22:36,353
NEITHER TOUCH DNA BUT STABILIZE

450
00:22:36,353 --> 00:22:38,855
XPC. AND THE NEW STRUCTURE WE

451
00:22:38,855 --> 00:22:41,892
FOUND WHICH IS NOT OBSERVE

452
00:22:41,892 --> 00:22:45,829
PREVIOUS WITH YEAST PROTEIN IS

453
00:22:45,829 --> 00:22:47,764
HERE END TERMINAL VERY LONG

454
00:22:47,764 --> 00:22:50,801
HELIX EXTENDING ACROSS DNA IN

455
00:22:50,801 --> 00:22:52,736
THE EXTENDING FOR NON-HELIX END

456
00:22:52,736 --> 00:22:55,806
TERMINAL SO OHM. THERE IS ALSO A

457
00:22:55,806 --> 00:23:00,444
LONG HELIX AT C TERMINAL SO LONG

458
00:23:00,444 --> 00:23:02,012
HELIC C. THE SAME THING I

459
00:23:02,012 --> 00:23:04,281
MENTIONED WAS NOT KNOWN BEFORE. 

460
00:23:04,281 --> 00:23:07,017
AND COMPARED TO THE PREVIOUS

461
00:23:07,017 --> 00:23:09,586
STRUCTURES OBSERVED BY

462
00:23:09,586 --> 00:23:11,288
(INDISCERNIBLE) LAB OF THE YEAST

463
00:23:11,288 --> 00:23:12,989
WITH DNA, THERE'S SOME

464
00:23:12,989 --> 00:23:14,958
DIFFERENCE NOT ONLY PROTEIN WAS

465
00:23:14,958 --> 00:23:16,460
NOW WE OBSERVE IN THE PRESENCE

466
00:23:16,460 --> 00:23:22,099
OF TF 2H, THE DNA DOWNSTREAM

467
00:23:22,099 --> 00:23:23,834
BINDING BY RAD 4 XPC IS SIMILAR

468
00:23:23,834 --> 00:23:27,003
BUT AFTER THE LESION UPSTREAM

469
00:23:27,003 --> 00:23:28,872
DNA HAVE VERY DIFFERENT BENDING

470
00:23:28,872 --> 00:23:31,508
ANGLE AND IN THE PRESENCE OF TH

471
00:23:31,508 --> 00:23:33,343
TF 2H DNA IS MORE STRAIGHT AND

472
00:23:33,343 --> 00:23:37,981
HAVE MORE INTERACTION WITH BETA

473
00:23:37,981 --> 00:23:39,382
DOMAIN 3 WHICH INTERACTIONS WITH

474
00:23:39,382 --> 00:23:42,552
THE LESION AND UPSTREAM DNA. 

475
00:23:42,552 --> 00:23:46,990
OKAY. SO WHY WE HAVE THE LONG

476
00:23:46,990 --> 00:23:48,959
END TERMINAL HELIX AND LONG C

477
00:23:48,959 --> 00:23:51,661
TERMINAL HELIX. SO WE CAN SEE

478
00:23:51,661 --> 00:23:55,065
THAT IS WHERE EXTENSION OF THE

479
00:23:55,065 --> 00:24:00,570
XPC RECRUIT THE TF 2H. AND OHN

480
00:24:00,570 --> 00:24:02,806
INTERACTING WITH THE -- WE CALL

481
00:24:02,806 --> 00:24:08,178
THE XPD ARM OF THE TF 2H, OHC

482
00:24:08,178 --> 00:24:11,715
INTERACTING WITH THE XPB ARM OF

483
00:24:11,715 --> 00:24:15,585
THE TF 2H. HERE IS THE ZOOM IN

484
00:24:15,585 --> 00:24:19,756
OF THE C TERMINAL OF OHC XPC

485
00:24:19,756 --> 00:24:22,626
INTERACTING WITH XPB, P 8 AND P

486
00:24:22,626 --> 00:24:25,095
52 SUB UNIT AND LITTLE BIT OF P

487
00:24:25,095 --> 00:24:29,432
32 SUB UNIT OF CORE 7. END

488
00:24:29,432 --> 00:24:32,736
TERMINAL HELIX INTERACTING WITH

489
00:24:32,736 --> 00:24:35,438
P 62 AND ALSO XPD. THIS

490
00:24:35,438 --> 00:24:36,706
INTERFACE IS VERY SMALL AS YOU

491
00:24:36,706 --> 00:24:39,876
CAN SEE HELIX AND EXTENSION TINY

492
00:24:39,876 --> 00:24:43,013
BIT BUT WE DIDN'T START FROM THE

493
00:24:43,013 --> 00:24:46,249
RESIDUE 1 OF XPC, BEFORE RESIDUE

494
00:24:46,249 --> 00:24:48,218
167 XPC IS DISORDERED. EVEN

495
00:24:48,218 --> 00:24:50,287
THOUGH IT IS PRESENT. BUT

496
00:24:50,287 --> 00:24:52,088
PREVIOUS STUDY OF ISOLATE

497
00:24:52,088 --> 00:24:55,525
PEPTIDE OF XPC BEFORE RESIDUE

498
00:24:55,525 --> 00:24:57,093
167 HAS BEEN SHOWN TO

499
00:24:57,093 --> 00:24:59,629
INTERACTING WITH P 62 SUB UNIT

500
00:24:59,629 --> 00:25:03,733
WHICH IS LP -- XPD ARM. SO WE

501
00:25:03,733 --> 00:25:06,069
DIDN'T OBSERVE THIS BECAUSE THIS

502
00:25:06,069 --> 00:25:08,205
MODEL PROBABLY IS FLEXIBLE

503
00:25:08,205 --> 00:25:10,640
RELATIVE TO REST OF IT SO WE

504
00:25:10,640 --> 00:25:12,576
COULDN'T SEE BUT SUSPECT I MUST

505
00:25:12,576 --> 00:25:14,578
EXIST BECAUSE EVEN THIS ISOLATED

506
00:25:14,578 --> 00:25:17,314
PEPTIDE OF XPC CAN INTERACT WITH

507
00:25:17,314 --> 00:25:21,418
THE DOMAIN PH DOMAIN OF P 62. 

508
00:25:21,418 --> 00:25:23,687
THIS KIND OF ANCHORING NOT ONLY

509
00:25:23,687 --> 00:25:26,623
ORIENT HOW TF 2H APPROACH DNA

510
00:25:26,623 --> 00:25:29,459
WITH XPD BINDING DOUBLE STRAND

511
00:25:29,459 --> 00:25:31,394
DNA UPSTREAM OF THE LESION. 

512
00:25:31,394 --> 00:25:33,230
MOREOVER, THIS ANCHORING ALLOWS

513
00:25:33,230 --> 00:25:35,565
THE FLEXIBILITY OF XPC WHICH YOU

514
00:25:35,565 --> 00:25:37,834
CAN SEE IN THE CRYOEM STRUCTURE

515
00:25:37,834 --> 00:25:40,337
EVEN WHEN BINDING TO SITE 5 WE

516
00:25:40,337 --> 00:25:43,406
STILL SEE XPC HAVE AT LEAST TWO

517
00:25:43,406 --> 00:25:46,476
MAJOR CONFIRMATION AND MANY IN

518
00:25:46,476 --> 00:25:49,512
BETWEEN. THIS FLEXIBILITY

519
00:25:49,512 --> 00:25:53,116
PROBABLY ENABLES XPC TO BIND ALL

520
00:25:53,116 --> 00:25:54,918
KIND OF LESION HAS THAT

521
00:25:54,918 --> 00:25:56,553
ADAPTABILITY AND IT IS NOT ONLY

522
00:25:56,553 --> 00:26:00,223
THIS MOVEMENT RELATIVE TO TF 2H

523
00:26:00,223 --> 00:26:03,660
BUT BETWEEN EVERY DOMAIN TDB TO

524
00:26:03,660 --> 00:26:05,695
BETA DOMAIN 123, EVERY DOMAIN

525
00:26:05,695 --> 00:26:09,466
CAN HAVE RELATIVE MOVEMENT.

526
00:26:09,466 --> 00:26:10,400
FLEXIBILITY MAY HAVE HIGHEST

527
00:26:10,400 --> 00:26:12,402
BINDING AFFINITY. NOW WE SWITCH

528
00:26:12,402 --> 00:26:14,004
OUR ATTENTION TO WHAT IS THE

529
00:26:14,004 --> 00:26:18,074
ROLE OF XPA. SOME YEARS AGO IN

530
00:26:18,074 --> 00:26:20,143
2015 BEFORE ANY STRUCTURE OF

531
00:26:20,143 --> 00:26:23,346
EVEN TF 2H WE LOOK AT HELOCASE

532
00:26:23,346 --> 00:26:28,084
ACTIVITY OF TF 2H. WE DESIGN DNA

533
00:26:28,084 --> 00:26:30,520
SUBSTRATE WITH 5 PRIME OVERHANG

534
00:26:30,520 --> 00:26:32,889
BECAUSE XPD MOVES 5 PRIME TO 3

535
00:26:32,889 --> 00:26:34,424
PRIME. THE MOVEMENT IF THERE IS

536
00:26:34,424 --> 00:26:37,494
A LESION, THE HELOCASE SUBSTRATE

537
00:26:37,494 --> 00:26:40,297
BECOME PRODUCT AND THE LESION

538
00:26:40,297 --> 00:26:41,965
WILL STALL LEE LA CASE SO LESS

539
00:26:41,965 --> 00:26:45,735
PRODUCT IS FORMED. HELOCASE, SO

540
00:26:45,735 --> 00:26:47,237
LESS PRODUCT IS FORMD BY THE

541
00:26:47,237 --> 00:26:49,039
LESION. THE DIFFERENCE IS NOW

542
00:26:49,039 --> 00:26:50,273
HUGE, THESE ARE VERY LIMITED

543
00:26:50,273 --> 00:26:52,742
ASSAY BUT IF WE ADD XPA IN

544
00:26:52,742 --> 00:26:56,046
ADDITION TO TF 2H NOW WE SEE THE

545
00:26:56,046 --> 00:26:58,248
HELOCASE ACTIVITY ARE NORMAL DNA

546
00:26:58,248 --> 00:27:02,218
IS MUCH BETTER AND ON LESION DNA

547
00:27:02,218 --> 00:27:05,121
IS (INAUDIBLE) SO CLEARLY XPA IS

548
00:27:05,121 --> 00:27:06,756
DOING SOMETHING TO HELP HELOCASE

549
00:27:06,756 --> 00:27:10,727
IN GENERAL, SPECIFICALLY

550
00:27:10,727 --> 00:27:14,764
RECOGNIZE LESION STALLED XPD. SO

551
00:27:14,764 --> 00:27:16,333
HERE IS THE STRUCTURE I SHOW

552
00:27:16,333 --> 00:27:21,638
BEFORE WITH TF 2H HERE, AND XPD

553
00:27:21,638 --> 00:27:25,542
-- XPC ON TOP, THIS IS THE

554
00:27:25,542 --> 00:27:27,644
COMPLETION JUST BETWEEN XPC AND

555
00:27:27,644 --> 00:27:30,647
TF 2H. WHEN XP IS PRESENT, WHAT

556
00:27:30,647 --> 00:27:33,616
YOU SEE IS DRAMATIC

557
00:27:33,616 --> 00:27:36,052
CONFIRMATIONAL CHANGE OF THE

558
00:27:36,052 --> 00:27:39,322
CORE 7 FROM U2 SIGMA, MOREOVER

559
00:27:39,322 --> 00:27:42,192
IN THIS COMPLEX XPD DOES NOT

560
00:27:42,192 --> 00:27:44,127
INTERACT WITH DNA AT ALL, VERY

561
00:27:44,127 --> 00:27:47,063
MUCH LIKE PRE-INITIATION

562
00:27:47,063 --> 00:27:49,332
COMPLETION IN TRANSCRIPTION. 

563
00:27:49,332 --> 00:27:52,268
HERE IN THE PRESENCE OF XPA XPD

564
00:27:52,268 --> 00:27:52,969
INTERACTS WITH DOUBLE STRAND

565
00:27:52,969 --> 00:27:57,707
DNA. AND MOREOVER INSTEAD OF

566
00:27:57,707 --> 00:28:00,276
LESION BETWEEN XPD AND XPD, NOW

567
00:28:00,276 --> 00:28:04,114
LESION IS SHIFTED TO OUTSIDE AND

568
00:28:04,114 --> 00:28:07,384
DOWNSTREAM OF XPD, IF XPD GETS

569
00:28:07,384 --> 00:28:11,888
THE LESION CONTAINING STRAND OF

570
00:28:11,888 --> 00:28:14,791
ORANGE COLOR IT WILL BE ABLE TO

571
00:28:14,791 --> 00:28:15,959
SCAN FINDS THE LESION AND STALL

572
00:28:15,959 --> 00:28:17,961
IT. HOW DOES XPA ACHIEVE IS

573
00:28:17,961 --> 00:28:23,066
THIS? XPA IS FUNNY, IT IS

574
00:28:23,066 --> 00:28:25,802
INSERTED IN TO BETWEEN XPC AND

575
00:28:25,802 --> 00:28:30,040
DNA AND C TERMINAL OF XPA IS

576
00:28:30,040 --> 00:28:34,744
ANCHORED.  PH P 52 AND XPD ARM

577
00:28:34,744 --> 00:28:37,781
AND IT IS 35-ANGSTROM AWAY FROM

578
00:28:37,781 --> 00:28:39,315
XPC ANCHORING SO THIS PORTION OF

579
00:28:39,315 --> 00:28:42,752
THE TH 2H YOU CAN SEE IS REALLY

580
00:28:42,752 --> 00:28:45,989
IMPORTANT FOR HOLDING XPC DOING

581
00:28:45,989 --> 00:28:49,125
INTERESTING CHOREOGRAPHY OF

582
00:28:49,125 --> 00:28:50,126
MOVEMENT PROTEIN CHAIN

583
00:28:50,126 --> 00:28:52,062
CONFIRMATION AND SHIFTING DNA.

584
00:28:52,062 --> 00:28:56,232
HERE XPC RESULT IN XPA IN THE

585
00:28:56,232 --> 00:28:59,102
PRESENCE OF XPA XPC DIDN'T LET

586
00:28:59,102 --> 00:29:05,475
GO OF THE LESION AND THE HD 3

587
00:29:05,475 --> 00:29:06,109
DOMAIN TRAIN TAPING HOLD OF THE

588
00:29:06,109 --> 00:29:06,843
NON-LESION STRAND AND FLIPPING

589
00:29:06,843 --> 00:29:10,880
OF THE LESION. SO POSITION OF

590
00:29:10,880 --> 00:29:13,183
LESION IS SHIFTED AND IS

591
00:29:13,183 --> 00:29:14,617
WONDERFUL NOW I WILL JUST GIVE

592
00:29:14,617 --> 00:29:19,255
YOU A LITTLE MORE DETAIL HOW TF

593
00:29:19,255 --> 00:29:20,457
2A STRUCTURE CAN CHANGE THIS

594
00:29:20,457 --> 00:29:21,925
MUCH WHERE ARE THE FLEXIBLE

595
00:29:21,925 --> 00:29:25,462
PORTION. HERE IS A REVIEW OF TF

596
00:29:25,462 --> 00:29:28,598
2H WITH XPC AGAIN AND COLORING

597
00:29:28,598 --> 00:29:34,070
IS XPB XPD ARE HIGHLIGHTED.  WE

598
00:29:34,070 --> 00:29:37,707
FOCUS ON CORE 7 OF TF 2H ONLY.

599
00:29:37,707 --> 00:29:42,412
SO THIS LOOK LIKE XPB AND XPD ON

600
00:29:42,412 --> 00:29:47,417
THE TIP OF THE U ARM XPD AND XPD

601
00:29:47,417 --> 00:29:52,055
ARM, U SHAPED STRUCTURE. P 44 IS

602
00:29:52,055 --> 00:29:57,360
ON THE XPD ARM AND MIDDLE IS P

603
00:29:57,360 --> 00:30:01,397
34 AND P 52. HOLD TOGETHER IS P

604
00:30:01,397 --> 00:30:04,601
62 IS VERY IMPORTANT TO TIE THE

605
00:30:04,601 --> 00:30:09,672
P 62 OF PXPD, P 44, P 34 AND P

606
00:30:09,672 --> 00:30:13,810
52 TOGETHER. SO IT IS THE XPB

607
00:30:13,810 --> 00:30:19,582
ARM AND XPD AROUND. XPD ARM

608
00:30:19,582 --> 00:30:22,218
INVOLVED THE MOVEMENT OF P 44

609
00:30:22,218 --> 00:30:24,320
WITHIN THE P 44 THERE ARE

610
00:30:24,320 --> 00:30:25,622
MULTIPLE DOMAIN THEY CAN SEE THE

611
00:30:25,622 --> 00:30:28,691
BIGGEST MOVEMENT IS TWO DOMAIN,

612
00:30:28,691 --> 00:30:30,994
BIG ROTATION WITHIN P 44 AND

613
00:30:30,994 --> 00:30:34,197
THAT MOVES THE XPD RELATIVE TO

614
00:30:34,197 --> 00:30:38,735
THE XPD ARM. P 62 IS VERY

615
00:30:38,735 --> 00:30:40,970
IMPORTANT TO HOLD ARM TOGETHER. 

616
00:30:40,970 --> 00:30:43,406
NOW WE FOCUS ON THE XPB AROUND.

617
00:30:43,406 --> 00:30:45,675
THE MOVEMENT ACTUALLY OCCUR

618
00:30:45,675 --> 00:30:49,379
WITHIN P 52, P 52 HAVE FOUR

619
00:30:49,379 --> 00:30:51,214
DOMAINS.  THERE IS MOVEMENT

620
00:30:51,214 --> 00:30:52,582
BETWEEN FIRST DOMAIN AND LATER

621
00:30:52,582 --> 00:30:57,820
TWO DOMAIN. AND AT THE MIDDLE OF

622
00:30:57,820 --> 00:31:01,124
THE U SHAPE P 34 AND P 52, THE

623
00:31:01,124 --> 00:31:03,660
INTERFACE IS STABLE. AND NOW YOU

624
00:31:03,660 --> 00:31:06,229
CAN SEE THE INTERFACE BETWEEN

625
00:31:06,229 --> 00:31:10,200
XPD AND P 44 STABLE, XPB BETWEEN

626
00:31:10,200 --> 00:31:12,936
PA AND P 52 ARE STABLE SO THE

627
00:31:12,936 --> 00:31:15,672
MOVEMENT OCCURS REALLY WITHIN P

628
00:31:15,672 --> 00:31:18,942
44 AND P 52 AND LEADING TO THIS

629
00:31:18,942 --> 00:31:24,981
BIG CONFIRMATIONAL CHANGE. AND

630
00:31:24,981 --> 00:31:28,218
WHAT P -- XPA DOES IS NOW WE

631
00:31:28,218 --> 00:31:32,422
ZOOM INTO THE XPA XPC ARM XPV

632
00:31:32,422 --> 00:31:35,658
AND XPD STRUCTURE. XPA IS A VERY

633
00:31:35,658 --> 00:31:38,428
SMALL PROTEIN, TOTAL 273 RESIDUE

634
00:31:38,428 --> 00:31:46,336
SO WE MISSED THE FIRST 100

635
00:31:46,336 --> 00:31:49,072
RESIDUE OF XPA REMAIN DISORDERD

636
00:31:49,072 --> 00:31:50,406
IN STRUCTURE AND IT IS IMPORTANT

637
00:31:50,406 --> 00:31:54,277
FOR INTERACTING WITH RPA AND

638
00:31:54,277 --> 00:31:55,845
XPF, ET CETERA. HERE WHAT WE SAW

639
00:31:55,845 --> 00:31:58,081
IS THE MIDDLE CENTER CORE

640
00:31:58,081 --> 00:32:00,650
STRUCTURE WE CALL BE-- ALPHA

641
00:32:00,650 --> 00:32:03,186
BETA DOMAIN OF FPA, ENTEROR VERY

642
00:32:03,186 --> 00:32:05,521
INTERESTING.  IT IS THE FIRST

643
00:32:05,521 --> 00:32:08,157
TIME WE SEE INTERACTING WITH

644
00:32:08,157 --> 00:32:10,560
DOUBLE STRAND DNA AND PREVE --

645
00:32:10,560 --> 00:32:11,794
UNLIKE PREVIOUS STRUCTURES. THIS

646
00:32:11,794 --> 00:32:14,998
ALPHA BETA DOMAIN OF XPA MIMIC

647
00:32:14,998 --> 00:32:19,636
LIKE THE BETA DOMAIN OF THE XPC.

648
00:32:19,636 --> 00:32:24,240
SO THIS BHD 3 OF XPC SIN SETTED

649
00:32:24,240 --> 00:32:26,075
BETWEEN THE TWO STRAND FLIP OUT

650
00:32:26,075 --> 00:32:29,045
THE LESION AND BINDS THE

651
00:32:29,045 --> 00:32:31,214
NON-LESION BASE, RECOGNIZE

652
00:32:31,214 --> 00:32:33,483
SPECIFICALLY NOW LESION, MOST XP

653
00:32:33,483 --> 00:32:36,953
ALPHA BETA DOMAIN ALSO BINDS THE

654
00:32:36,953 --> 00:32:39,122
DNA AND THE DNA CLEARLY BANNING

655
00:32:39,122 --> 00:32:43,092
50 DEGREES AND HAS IN THE

656
00:32:43,092 --> 00:32:44,961
TRYPTOPHAN AND INTER COLLATED

657
00:32:44,961 --> 00:32:48,831
DNA, SO CAUSES BINDING IN THE

658
00:32:48,831 --> 00:32:52,201
BAND SO XPA BINDS 7 BASE PAIR

659
00:32:52,201 --> 00:32:54,203
UPSTREAM OF THE XPC WITHOUT

660
00:32:54,203 --> 00:32:56,406
INTERFERING THE DNA BINDING BY

661
00:32:56,406 --> 00:33:00,543
XPC BUT IT IS INSERTED BETWEEN

662
00:33:00,543 --> 00:33:05,048
OHC AND DNA UPSTREAM HERE. AND

663
00:33:05,048 --> 00:33:10,186
THE RESULT IS THAT BY XPA

664
00:33:10,186 --> 00:33:12,322
SHIFTING THE DNA AND BENDING DNA

665
00:33:12,322 --> 00:33:16,292
NOW THE WHOLE PART OF CORE 7 TF

666
00:33:16,292 --> 00:33:19,929
2H RELATIVE TO XPC WHICH DOESN'T

667
00:33:19,929 --> 00:33:22,932
CHANGE ITS POSITION IS SHIFTED.

668
00:33:22,932 --> 00:33:25,468
SO WITHOUT XPA THE DNA IS

669
00:33:25,468 --> 00:33:28,371
SHOWING GRAY AND IT IS MORE OR

670
00:33:28,371 --> 00:33:30,306
LESS STRAIGHT. IN THE PRESENCE

671
00:33:30,306 --> 00:33:34,344
OF XPA THE DNA SHIFTED NEARLY

672
00:33:34,344 --> 00:33:37,280
TEN BASE PAIR WHOLE HELICAL TURN

673
00:33:37,280 --> 00:33:38,948
OR XPC BIND WHICH IS HERE IN THE

674
00:33:38,948 --> 00:33:42,652
ABSENCE OF XPA, IN THE PRESENCE

675
00:33:42,652 --> 00:33:46,222
OF XPA NOW SHIFTED THERE.  SO IT

676
00:33:46,222 --> 00:33:48,391
IS COMPLETELY CHANGED THE

677
00:33:48,391 --> 00:33:50,526
REGISTER OF XPC LESION RELATIVE

678
00:33:50,526 --> 00:33:55,631
TO THE TF 2H OR 7. AS I

679
00:33:55,631 --> 00:33:58,801
MENTIONED BEFORE, THIS

680
00:33:58,801 --> 00:34:00,770
MANIPULATION OF PROTEIN IN DNA

681
00:34:00,770 --> 00:34:02,872
DEPEND ON XPA C TERMINAL DOCKING

682
00:34:02,872 --> 00:34:07,276
TO THE XPB ARM OF THE TF 2H. SO

683
00:34:07,276 --> 00:34:10,413
THIS IS THE LINKER OF THE XPA

684
00:34:10,413 --> 00:34:15,017
SHOWN IN RED FROM TRYPTOPHAN 235

685
00:34:15,017 --> 00:34:17,587
TO 273, VERY C TERMINAL FOR BETA

686
00:34:17,587 --> 00:34:19,622
DOMAIN INTERACTING WITH P 8 AND

687
00:34:19,622 --> 00:34:23,559
P 52.  HERE IS XPV. THIS

688
00:34:23,559 --> 00:34:26,095
STRUCTURE EXPLAIN VERY WELL MANY

689
00:34:26,095 --> 00:34:29,732
OF THE XPA MUTATION ARE DUE TO C

690
00:34:29,732 --> 00:34:31,934
TERMINAL TRUNCATION, WITHOUT

691
00:34:31,934 --> 00:34:34,237
THIS ANCHORING XPA COULDN'T

692
00:34:34,237 --> 00:34:38,107
ACHIEVE THE REPOSITION OF TF 2H

693
00:34:38,107 --> 00:34:41,844
RELATIVE TO PC AND PARTICULARLY

694
00:34:41,844 --> 00:34:45,214
THIS HISTIDINE 244 TO ARGININE

695
00:34:45,214 --> 00:34:50,586
IS ONE MYCATION THAT CAUSE

696
00:34:50,586 --> 00:34:51,854
XERODERMA PIG MEN TOE SUM. SO

697
00:34:51,854 --> 00:34:58,394
HERE IS HOW XPA -- PIGMENTOSA. 

698
00:34:58,394 --> 00:35:01,164
HOW IT CHANGE TO XPC. YOU CAN

699
00:35:01,164 --> 00:35:03,232
SEE THE DNA BANDING CHANGE OF

700
00:35:03,232 --> 00:35:09,772
POSITION IN THE STRUCTURE OF

701
00:35:09,772 --> 00:35:12,642
TFIIH, YOU CAN SEE THE DNA

702
00:35:12,642 --> 00:35:14,143
BANNING AS WELL. THE CHANGE. NOW

703
00:35:14,143 --> 00:35:15,945
I SHOW YOU THE ADDITIONAL VIEW,

704
00:35:15,945 --> 00:35:18,114
YOU CAN SEE THE CHANGE OF THE

705
00:35:18,114 --> 00:35:20,283
STRUCTURE AND THE DNA BANNING

706
00:35:20,283 --> 00:35:23,085
AND ALSO THE LIFTING OF LONG

707
00:35:23,085 --> 00:35:25,421
HELIX AT THE C TERMINAL OF XPC

708
00:35:25,421 --> 00:35:32,395
TO ACCOMMODATE XPA. PULLING

709
00:35:32,395 --> 00:35:34,564
TOGETHER THE SHIFT OF THIS

710
00:35:34,564 --> 00:35:38,367
CHANGE OF THE CONFIRMATION SHIFT

711
00:35:38,367 --> 00:35:43,105
OF TFIIH ALLOW THE LESION TO

712
00:35:43,105 --> 00:35:46,642
FALL OUTSIDE OF THE TF 2H. THIS

713
00:35:46,642 --> 00:35:50,279
BANDING PLUS THE XPC DISTORTION

714
00:35:50,279 --> 00:35:53,616
OF DNA EVENTUALLY WILL ENABLE

715
00:35:53,616 --> 00:35:55,251
UNWINDING OF THIS PORTION OF

716
00:35:55,251 --> 00:35:56,786
DUPLEX AND DELIVER THE LESION

717
00:35:56,786 --> 00:36:00,323
STRAND TO PPD 4 BINDING. BECAUSE

718
00:36:00,323 --> 00:36:02,225
IN THE STRUCTURE STUDY WE DIDN'T

719
00:36:02,225 --> 00:36:06,496
ADD ATP AT ALL, SO HELOCASE NOT

720
00:36:06,496 --> 00:36:17,006
MOBILE, ALL THE STRUCTURE JUST

721
00:36:17,840 --> 00:36:21,744
EQUALIBRIUM OF THE ADOPTED. HERE

722
00:36:21,744 --> 00:36:25,081
IS XPC RECRUITING LONG TERMINAL

723
00:36:25,081 --> 00:36:28,417
TO RECRUIT TFIIH AND PRESENCE OF

724
00:36:28,417 --> 00:36:29,886
XPA CHANGE CONFIRMATION AND

725
00:36:29,886 --> 00:36:32,255
SHIFT THE TFIIH TO BE UPSTREAM

726
00:36:32,255 --> 00:36:34,590
OF THE LESION. AND WE HAVEN'T

727
00:36:34,590 --> 00:36:37,760
SEEN THE TOTAL DISSOCIATION SOFT

728
00:36:37,760 --> 00:36:40,363
XPC BECAUSE IT IS C TERMINAL

729
00:36:40,363 --> 00:36:42,064
STILL ANCHORED AROUND THE XPB

730
00:36:42,064 --> 00:36:45,034
ARM OF THE TFIIH. SO WE ARE

731
00:36:45,034 --> 00:36:47,303
WAITING FOR FUTURE STUDY. I WILL

732
00:36:47,303 --> 00:36:51,774
USE THE REMAINING TIME TO

733
00:36:51,774 --> 00:36:52,642
ADDRESS TWO BURNING QUESTIONS

734
00:36:52,642 --> 00:36:53,876
FOR US.  FOR US WE ARE

735
00:36:53,876 --> 00:36:57,079
INTERESTED IN THE MECHANISM FOR

736
00:36:57,079 --> 00:37:01,851
HOW XPB AND XPD ARE BOTH ATPASE

737
00:37:01,851 --> 00:37:04,020
AVOID AND THEY HAVE OPPOSITE

738
00:37:04,020 --> 00:37:06,222
POLARITY AND HOW THEY CAN AVOID

739
00:37:06,222 --> 00:37:09,859
TUG OF WAR AND COOPERATE TO

740
00:37:09,859 --> 00:37:12,094
FACILITATE DNA LESION REMOVAL.

741
00:37:12,094 --> 00:37:16,065
SO HYPOTHESIS IS TWO HE LA CASE

742
00:37:16,065 --> 00:37:18,534
WORK TOGETHER TO GENERATE DNA

743
00:37:18,534 --> 00:37:20,136
BUBBLE OF THE 20 SOMETHING

744
00:37:20,136 --> 00:37:22,371
NUCLEOTIDE TO 30 NUCLEOTIDE LONG

745
00:37:22,371 --> 00:37:26,108
SO XPF AND XPG CAN CLEAVE AT

746
00:37:26,108 --> 00:37:28,110
BORDER OF THE BUBBLE AND THE

747
00:37:28,110 --> 00:37:32,248
COMPLEX DNA. -- DUPLEX DNA. WE

748
00:37:32,248 --> 00:37:35,117
WILL TOUCH HOW GLOBAL GENOME NER

749
00:37:35,117 --> 00:37:37,420
AND TRANSCRIPTION COUPLED NER

750
00:37:37,420 --> 00:37:40,723
CAN CONVERGE ON TFIIH AND FOR

751
00:37:40,723 --> 00:37:47,330
NER.  SO AS I MENTIONED, BEFORE

752
00:37:47,330 --> 00:37:49,732
WE STARTED STRUCTURAL STUDYING

753
00:37:49,732 --> 00:37:51,834
OF BIOCHEMICAL STUDY WAS ALREADY

754
00:37:51,834 --> 00:37:55,371
ESTABLISHED THAT XPB IS A 3

755
00:37:55,371 --> 00:37:57,540
PRIME TO 5 PRIME DNA TRANSLOW

756
00:37:57,540 --> 00:38:00,076
CASE AND XPB IS A 5 PRIME TO 3

757
00:38:00,076 --> 00:38:03,079
PRIME HELOCASE.  IN 2015 OR

758
00:38:03,079 --> 00:38:06,816
BEFORE WE HAVE NO IDEA HOW THE

759
00:38:06,816 --> 00:38:09,752
TFIIH BIND DNA SO WE WERE

760
00:38:09,752 --> 00:38:13,623
PUZZLED WHETHER TWO HELOCASE

761
00:38:13,623 --> 00:38:14,857
BOOST AND TRANSLOCATE ON THE

762
00:38:14,857 --> 00:38:16,993
LESION STRAND. IT IS A BIT OF

763
00:38:16,993 --> 00:38:20,363
TROUBLESOME BECAUSE WE MOVE

764
00:38:20,363 --> 00:38:22,598
OPPOSITE DIRECTION THOUGH XPD IS

765
00:38:22,598 --> 00:38:25,935
A STRONG STRONG MOTOR AND XPB IS

766
00:38:25,935 --> 00:38:27,837
A WEAK MOTOR SO IT CREATES A TUG

767
00:38:27,837 --> 00:38:31,574
OF WAR. THE POSSIBILITY ALSO IS

768
00:38:31,574 --> 00:38:34,110
XPB XPD MOVE ON THE LESION

769
00:38:34,110 --> 00:38:35,544
STRAND WHICH WE KNOW ANY LESION

770
00:38:35,544 --> 00:38:40,016
DOWNSTREAM OF XPD WILL BE FOUND

771
00:38:40,016 --> 00:38:42,952
AND CAN DETERMINE WHETHER WHOLE

772
00:38:42,952 --> 00:38:44,186
HELOCASE CAN BE STALLED OR KEEP

773
00:38:44,186 --> 00:38:45,955
MOVING. WE HYPOTHESIS, IT IS

774
00:38:45,955 --> 00:38:49,458
ALSO POSSIBLE FOR XPB MOVING ON

775
00:38:49,458 --> 00:38:51,127
THE OTHER STRAND, NOT ONLY

776
00:38:51,127 --> 00:38:52,828
CONTAINING STRAND AND THE RESULT

777
00:38:52,828 --> 00:38:54,830
IS SUCH WE WOULD BE MOVING IN

778
00:38:54,830 --> 00:38:57,099
PARALLEL AND HELPING EACH OTHER

779
00:38:57,099 --> 00:39:01,871
SO THIS BECOMES SORT OF IN MY

780
00:39:01,871 --> 00:39:03,839
MIND MAKES SENSE SO WE DID MORE

781
00:39:03,839 --> 00:39:05,241
EXPERIMENT, FIRST WE USE THE

782
00:39:05,241 --> 00:39:07,677
SUBSTRATE WITH 5 PRIME OVERHEAD

783
00:39:07,677 --> 00:39:12,114
SO XPD CAN BIND DNA. WE DID

784
00:39:12,114 --> 00:39:15,051
BINDING ACTIVITY ASSAY USING MZA

785
00:39:15,051 --> 00:39:17,653
YOU CAN SEE THE SHIFT IS

786
00:39:17,653 --> 00:39:19,255
MINIMAL. YET THE HELOCASE IS

787
00:39:19,255 --> 00:39:23,059
VERY GOOD. YOU CAN SEE UNWIND

788
00:39:23,059 --> 00:39:26,395
THE DNA. WHEN WE TRY A DIFFERENT

789
00:39:26,395 --> 00:39:28,764
SUBSTRATE WITH 3 PRIME OVERHANG

790
00:39:28,764 --> 00:39:30,766
SUPPOSEDLY XPB CAN UNWIND. WE

791
00:39:30,766 --> 00:39:33,869
FOUND THE BINDING IS EXTREMELY

792
00:39:33,869 --> 00:39:36,005
GOOD, THE HELOCASE ACTIVITY IS

793
00:39:36,005 --> 00:39:42,478
NEXT TO NOTHING.  SO NOW WE USE

794
00:39:42,478 --> 00:39:44,480
THE BEST SUBSTRATE AND TRIED

795
00:39:44,480 --> 00:39:47,817
FIGURE OUT WHICH DOES WHAT. SO

796
00:39:47,817 --> 00:39:51,020
THERE'S TWO POSSIBILITIES. WE

797
00:39:51,020 --> 00:39:54,490
KNOCK OUT XPB HELOCASE ACTIVITY,

798
00:39:54,490 --> 00:39:58,627
TAKE THE ATPASE ACTIVE SITE AND

799
00:39:58,627 --> 00:40:02,498
THE LEFT XPD TO WORK ALONE. THE

800
00:40:02,498 --> 00:40:03,632
SUBSTRATE GETS UNWOUND AND IF

801
00:40:03,632 --> 00:40:05,101
THERE IS A LESION IN THE

802
00:40:05,101 --> 00:40:07,002
SUBSTRATE NOW THE UNWINDING

803
00:40:07,002 --> 00:40:09,271
ACTIVITY IS VERY MUCH

804
00:40:09,271 --> 00:40:12,441
DIMINISHED. SO THAT MAKES SENSE.

805
00:40:12,441 --> 00:40:15,911
THEN WE KNOCK OUT PPD ACTIVITY

806
00:40:15,911 --> 00:40:18,581
AND LET PPD WORK ALONE WITH THIS

807
00:40:18,581 --> 00:40:20,382
SUBSTRATE WHAT YOU SEE IS EVEN

808
00:40:20,382 --> 00:40:25,988
WITHOUT XPD PPD WHICH IS

809
00:40:25,988 --> 00:40:27,757
SUPPOSEDLY MOVING THIS

810
00:40:27,757 --> 00:40:29,291
DIRECTION, HERE WE MOVE OPPOSITE

811
00:40:29,291 --> 00:40:30,893
DIRECTION, WE STILL SEE THE DNA

812
00:40:30,893 --> 00:40:33,295
STRAND BEING SEPARATED AND

813
00:40:33,295 --> 00:40:35,231
LESION STILL HAVE EFFECT ON

814
00:40:35,231 --> 00:40:38,367
STALLING THE TFIIH. SO AT THAT

815
00:40:38,367 --> 00:40:40,970
TIME WITH LIMITED SUBSTRATE

816
00:40:40,970 --> 00:40:43,672
STUDY AND RESULT, WE SORT OF

817
00:40:43,672 --> 00:40:49,578
FAVOR THIS MODEL.  THIS DATA

818
00:40:49,578 --> 00:40:51,247
LOOKS CONVINCING BUT WITH

819
00:40:51,247 --> 00:40:52,815
STRUCTURE OF TFIIH REPORTED A

820
00:40:52,815 --> 00:40:55,017
YEAR LATER AND WITH OUR MORE

821
00:40:55,017 --> 00:40:56,519
RECENT STUDY, ACTUALLY HOW IT

822
00:40:56,519 --> 00:41:00,289
WORKS.  IT IS THIS WAY. I WILL

823
00:41:00,289 --> 00:41:04,994
TELL YOU WHY. IT IS XPB AND XPD

824
00:41:04,994 --> 00:41:07,296
ON THE SAME STRAND. POOR BINDING

825
00:41:07,296 --> 00:41:09,064
AND POOR UNWINDING THIS

826
00:41:09,064 --> 00:41:11,333
FAVORABLE SUBSTRATE IS BECAUSE

827
00:41:11,333 --> 00:41:13,302
XPB HAVE HIGH AFFINITY FOR

828
00:41:13,302 --> 00:41:15,638
DOUBLE STRAND DNA AND XPD

829
00:41:15,638 --> 00:41:17,006
DOESN'T HAVE STRONG AFFINITY. SO

830
00:41:17,006 --> 00:41:19,508
THIS IS A POOR BINDING SUBSTRATE

831
00:41:19,508 --> 00:41:22,812
I SHOW IN THE PREVIOUS SLIDE BUT

832
00:41:22,812 --> 00:41:25,648
XPB STILL HAVE HELOCASE ACTIVITY

833
00:41:25,648 --> 00:41:28,017
SOMEWHAT. THEN XPD THOUGH

834
00:41:28,017 --> 00:41:31,821
WITHOUT ATPASE STILL RECOGNIZE

835
00:41:31,821 --> 00:41:36,759
LESION. AND STALL LESION PREVENT

836
00:41:36,759 --> 00:41:39,995
PPB FURTHER MOVING. THOUGH XPD

837
00:41:39,995 --> 00:41:41,297
ALONE HAVE WEAK BINDING AFFINITY

838
00:41:41,297 --> 00:41:42,932
FOR SINGLE STRAND DNA RELATIVE

839
00:41:42,932 --> 00:41:47,770
TO XPB FOR DOUBLE STRAND DNA XPB

840
00:41:47,770 --> 00:41:49,305
IS PA GOOD HELOCASE AND HAVE

841
00:41:49,305 --> 00:41:55,010
GOOD UNWINDING. THE OTHER

842
00:41:55,010 --> 00:41:58,314
SUBSTRATE WE TRIED XPB AND XPD

843
00:41:58,314 --> 00:42:00,716
HAS GOOD BINDING BUT DOMINANT

844
00:42:00,716 --> 00:42:02,351
MOTOR OF XPB MOVES OPPOSITE

845
00:42:02,351 --> 00:42:03,052
DIRECTION TO THE DOUBLE STRAND

846
00:42:03,052 --> 00:42:05,955
DNA SO NO UNWINDING, OR VERY

847
00:42:05,955 --> 00:42:09,225
LITTLE. AND THE PROBLEM WHEN WE

848
00:42:09,225 --> 00:42:13,162
HAVE THIS MODEL PUT TWO HELOCASE

849
00:42:13,162 --> 00:42:14,997
ANTI-PARALLEL TWO STRAND INSTEAD

850
00:42:14,997 --> 00:42:17,066
OF ONE STRAND WE DIDN'T USE THE

851
00:42:17,066 --> 00:42:19,668
PROPER SUBSTRATE WHICH IS THE

852
00:42:19,668 --> 00:42:21,370
BUBBLE DNA. WITH LESION

853
00:42:21,370 --> 00:42:25,975
DOWNSTREAM OF XPD. SO THIS IS

854
00:42:25,975 --> 00:42:27,977
THE KILLER AND I WANT TO REMIND

855
00:42:27,977 --> 00:42:29,178
MYSELF AND EVERYONE OUT THERE,

856
00:42:29,178 --> 00:42:33,282
WHEN WE DO ANY EXPERIMENT IF THE

857
00:42:33,282 --> 00:42:35,217
-- WE HAVE GROPING THE ELEPHANT

858
00:42:35,217 --> 00:42:38,153
BY USING OUR TWO HANDS SO WE

859
00:42:38,153 --> 00:42:40,256
REALLY DON'T SEE THE WHOLE IMAGE

860
00:42:40,256 --> 00:42:42,391
UNTIL THERE'S WHOLE IMAGE. AND

861
00:42:42,391 --> 00:42:45,327
WE SHOULD BE CAREFUL IN TERMS --

862
00:42:45,327 --> 00:42:47,196
INTERPRETING OUR RESULT. BUT

863
00:42:47,196 --> 00:42:49,531
MODEL IS WRONG AND IT IS GOOD

864
00:42:49,531 --> 00:42:51,300
STARTING PLACE AND WE CORRECT

865
00:42:51,300 --> 00:42:53,602
THE MODEL SO HERE IS THE WHOLE

866
00:42:53,602 --> 00:42:59,041
STRUCTURE OF TFIIH WITH XPC AND

867
00:42:59,041 --> 00:43:02,444
XPA ON DNA. HERE WHEN PXC IS

868
00:43:02,444 --> 00:43:05,114
MOBILE AND THE LESION IS ALMOST

869
00:43:05,114 --> 00:43:07,549
INVISIBLE BECAUSE IT IS MOVE XPC

870
00:43:07,549 --> 00:43:09,685
AND ALL STRUCTURE AVERAGED OUT.

871
00:43:09,685 --> 00:43:11,153
IN OUR STRUCTURE WHAT WE CAN SEE

872
00:43:11,153 --> 00:43:15,090
IS DOUBLE STRAND BIND TO XPB AND

873
00:43:15,090 --> 00:43:17,927
THERE'S TWO NUCLEOTIDE, ALMOST

874
00:43:17,927 --> 00:43:20,462
SINGLE STRAND BIND TO XPD BUT

875
00:43:20,462 --> 00:43:22,698
NOT THE LESION ITSELF CANNOT BE

876
00:43:22,698 --> 00:43:24,333
OBSERVED. BORROWING THE

877
00:43:24,333 --> 00:43:26,201
STRUCTURE FROM PATRICK KRAMERS

878
00:43:26,201 --> 00:43:28,671
LAB THOUGH THE DNA HAVE NO

879
00:43:28,671 --> 00:43:31,273
LESIONS CLEAR THIS HAS TO BE THE

880
00:43:31,273 --> 00:43:33,342
LESION STRAND. AND THE LESION

881
00:43:33,342 --> 00:43:38,414
HAS TO BE DOWNSTREAM OF THIS

882
00:43:38,414 --> 00:43:40,049
SUPPOSEDLY CLUSTER AND RECOGNIZE

883
00:43:40,049 --> 00:43:41,684
THE LESION AND POSSIBLY WE FIND

884
00:43:41,684 --> 00:43:44,620
HERE IS ANOTHER GATE WHERE

885
00:43:44,620 --> 00:43:47,323
LESION MAYBE STALLED. SO WE KNOW

886
00:43:47,323 --> 00:43:48,290
THE LESION STRANGLES THROUGH

887
00:43:48,290 --> 00:43:50,960
THIS. THAT IS VERY NICE, WE CAN

888
00:43:50,960 --> 00:43:54,496
TRY TO UNDERSTAND WHY THE TWO

889
00:43:54,496 --> 00:43:55,597
MOTOR WORK TOGETHER TO SEPARATE

890
00:43:55,597 --> 00:44:00,302
THE DNA. SO IN THIS FULL

891
00:44:00,302 --> 00:44:01,503
SUBSTRATE XPB BIND DOUBLE STRAND

892
00:44:01,503 --> 00:44:04,606
DNA. BUT THE TWO MOTOR DOMAIN

893
00:44:04,606 --> 00:44:07,076
HELOCASE DOMAIN 1 AND 2 GRABS

894
00:44:07,076 --> 00:44:09,111
ONLY ONE STRAND, THAT'S THE

895
00:44:09,111 --> 00:44:11,013
ORANGE STRAND, LESION CONTAINING

896
00:44:11,013 --> 00:44:12,581
STRAND. AND THIS IS A BASED ON

897
00:44:12,581 --> 00:44:14,616
MANY STUDIES SO WE KNOW AND ALSO

898
00:44:14,616 --> 00:44:17,853
THE CLOSE INTERACTION OF HD 1

899
00:44:17,853 --> 00:44:20,689
AND 2 IS THE LESION STRAND. AND

900
00:44:20,689 --> 00:44:25,661
WE KNOW FROM PREVIOUS STUDY XPB

901
00:44:25,661 --> 00:44:28,364
MOVES 3 PRIME TO 5 PRIME. AND IF

902
00:44:28,364 --> 00:44:32,234
THE DNA IS MOBILE BUT IF --

903
00:44:32,234 --> 00:44:36,905
IMMOBILE. IF DNA IS MOBILE

904
00:44:36,905 --> 00:44:38,374
PROTEIN IS MOBILE THE DOUBLE

905
00:44:38,374 --> 00:44:39,208
STRAND DNA WILL BE MOVING

906
00:44:39,208 --> 00:44:42,244
TOWARDS THE RIGHT. XPA IS THE

907
00:44:42,244 --> 00:44:45,914
KEY PROTEIN TO MAKE XPB A

908
00:44:45,914 --> 00:44:48,751
HELOCASE. WITHOUT XPA, XPB IS A

909
00:44:48,751 --> 00:44:50,719
SINGLE TRANSLOW CASE, MOVING THE

910
00:44:50,719 --> 00:44:52,221
DOUBLE STRAND TO THE LEFT.  AND

911
00:44:52,221 --> 00:44:55,124
THAT IS HOW IT WORKS IN

912
00:44:55,124 --> 00:44:58,193
TRANSCRIPTION JUST MOVING DOUBLE

913
00:44:58,193 --> 00:45:00,329
STRAND TOWARDS RNA POLYMERASE.

914
00:45:00,329 --> 00:45:03,165
AND THE RNA POLYMERASE IS ONE

915
00:45:03,165 --> 00:45:03,832
TRAY SEPARATE THE DOUBLE STRAND

916
00:45:03,832 --> 00:45:09,104
DNA. HERE IN NUCLEOTIDE EXCISION

917
00:45:09,104 --> 00:45:11,173
REPAIR PPB WORK WITH XPA WHICH

918
00:45:11,173 --> 00:45:12,708
IS A WEDGE STRAITING HERE IS

919
00:45:12,708 --> 00:45:15,511
TIPTOE FAN I TOLD YOU BEFORE --

920
00:45:15,511 --> 00:45:17,846
TRYPTOPHAN I TOLD YOU BEFORE IN

921
00:45:17,846 --> 00:45:20,182
DNA MAKE A BAN BAND. WHEN THE 2

922
00:45:20,182 --> 00:45:21,483
STRAND IS SEPARATED IT BECOME A

923
00:45:21,483 --> 00:45:23,719
WEDGE TO SEPARATE TWO STRANDS.

924
00:45:23,719 --> 00:45:26,388
SO THE TWO STRAND MOVING HERE

925
00:45:26,388 --> 00:45:29,525
ONE IN EXACT BA ALPHA BETA

926
00:45:29,525 --> 00:45:31,794
DOMAIN AND TRYPTOPHAN 175 TWO

927
00:45:31,794 --> 00:45:36,565
STRAND SEPARATE AND XPB CAN BE A

928
00:45:36,565 --> 00:45:39,201
HELOCASE. XPA IS A HELOCASE WITH

929
00:45:39,201 --> 00:45:42,271
A GOOD MOTOR AND CAN MOVE ITSELF

930
00:45:42,271 --> 00:45:43,839
DOWN THIS WAY OR AL ATTORNEY

931
00:45:43,839 --> 00:45:45,707
TESTIFILY IF PROTEIN NOW MOVING

932
00:45:45,707 --> 00:45:48,043
IT CAN PULL IN THE SINGLE STRAND

933
00:45:48,043 --> 00:45:49,478
DNA THIS WAY SO THE LESION WILL

934
00:45:49,478 --> 00:45:54,583
ENTER THE PROTEIN AND BE SCANNED

935
00:45:54,583 --> 00:45:57,753
AND WORK AS PAST THE XPD OR

936
00:45:57,753 --> 00:46:00,656
STALL THE HELOCASE. WE FIND THE

937
00:46:00,656 --> 00:46:01,723
TO BE POSSIBILITY LOCATION WHERE

938
00:46:01,723 --> 00:46:04,493
LESION CAN BE STALLED, EITHER AT

939
00:46:04,493 --> 00:46:06,929
THE IRON SULFUR CLUSTER ANOTHER

940
00:46:06,929 --> 00:46:09,098
THE GATEWAY WHERE THE SINGLE

941
00:46:09,098 --> 00:46:11,500
STRAND INTO EXIT XPD AND EXIT IS

942
00:46:11,500 --> 00:46:13,202
VERY SMALL, IF IT IS A BULKY

943
00:46:13,202 --> 00:46:16,505
LESION IT CANNOT GET OUT.  SO

944
00:46:16,505 --> 00:46:19,374
PUT TOGETHER I SHOW YOU HERE,

945
00:46:19,374 --> 00:46:23,779
HOW THE WE THINK HOW THE TFIIH

946
00:46:23,779 --> 00:46:26,115
WITH HELP WITH XPA BECOME A

947
00:46:26,115 --> 00:46:29,785
MOTOR AND GENERATE A ENLARGE THE

948
00:46:29,785 --> 00:46:31,753
BUBBLE OF OVER 20 SOMETHING

949
00:46:31,753 --> 00:46:36,358
NUCLEOTIDE FOR XPF AND XPD TO

950
00:46:36,358 --> 00:46:38,360
CLEAVE. SO ONCE LOADED THE DNA

951
00:46:38,360 --> 00:46:42,764
LOADING BY XPC OR BY RNA

952
00:46:42,764 --> 00:46:44,399
POLYMERASE, XPB WANT TO MOVE TO

953
00:46:44,399 --> 00:46:46,768
THE RIGHT, XPB WANT TO MOVE

954
00:46:46,768 --> 00:46:49,071
LEFT, THIS IS A DOMINANT MOTOR,

955
00:46:49,071 --> 00:46:50,939
IT SEES THE SINGLE STRAND DNA,

956
00:46:50,939 --> 00:46:54,176
IT GOES.  THE LESION IS A SMALL

957
00:46:54,176 --> 00:46:55,911
LESION. SUCH AS MISMATCH DNA

958
00:46:55,911 --> 00:46:58,780
WHICH IS A VERY WELL RECOGNIZE

959
00:46:58,780 --> 00:47:01,817
XPC OR BASIC SITE. XPD GOES

960
00:47:01,817 --> 00:47:05,554
THROUGH. WITHOUT SEEING IT AND

961
00:47:05,554 --> 00:47:07,789
NO NUCLEOTIDE EXCISION REPAIR.

962
00:47:07,789 --> 00:47:12,127
BUT IF THE LESION IS BULKY, XPA

963
00:47:12,127 --> 00:47:16,398
HAPPILY CHUCKING ALONG BUT THE

964
00:47:16,398 --> 00:47:18,200
LESION IS STALL AND MOTOR CAN NO

965
00:47:18,200 --> 00:47:21,069
LONGER WORK. NOW XPB IS THE ONLY

966
00:47:21,069 --> 00:47:25,340
MOTOR. AND IT WANTS TO GO TO THE

967
00:47:25,340 --> 00:47:27,976
LEFT BECAUSE THE WHOLE TFIIH

968
00:47:27,976 --> 00:47:29,444
WITH XPA IS STUCK ON THE DNA

969
00:47:29,444 --> 00:47:31,813
BECAUSE THE LESION. SO THE WHOLE

970
00:47:31,813 --> 00:47:34,383
CONQUEST CANNOT MOVE AND PROTEIN

971
00:47:34,383 --> 00:47:36,818
BECOME IMMOBILE. AND

972
00:47:36,818 --> 00:47:39,188
ALTERNATIVELY, IS THE DNA IS

973
00:47:39,188 --> 00:47:41,123
BEING SHUFFLED TO THE RIGHT

974
00:47:41,123 --> 00:47:43,659
BECAUSE THE XPB TRANSLOW CASE

975
00:47:43,659 --> 00:47:47,796
ACTIVITY. XPA IS THE WEDGE,

976
00:47:47,796 --> 00:47:49,331
SEPARATED DUPLEX. THE TWO

977
00:47:49,331 --> 00:47:51,600
TOGETHER MAKING BIGGER BUBBLE.

978
00:47:51,600 --> 00:47:53,302
EVENTUALLY IT IS BIG ENOUGH WITH

979
00:47:53,302 --> 00:47:56,738
THE RPA SINGLE STRAND BINDING

980
00:47:56,738 --> 00:47:59,041
PROTEIN HELP XPD WILL CLEAVE

981
00:47:59,041 --> 00:48:02,077
HERE AND XPF WILL CLEAVE ON THE

982
00:48:02,077 --> 00:48:04,479
5 PRIME END OF THE LESION.  HERE

983
00:48:04,479 --> 00:48:07,449
SHOWS TO US XPB IS REALLY

984
00:48:07,449 --> 00:48:10,285
IMPORTANT TO GENERATE THIS

985
00:48:10,285 --> 00:48:15,357
BUBBLE XPD ONCE XPD IS STUCK.

986
00:48:15,357 --> 00:48:18,927
MANY YEARS AGO WHEN THE DUAL

987
00:48:18,927 --> 00:48:23,398
INCISION NER DUAL EXCISION WAS

988
00:48:23,398 --> 00:48:25,934
ASSAYED USING CIRCULAR PLASMID

989
00:48:25,934 --> 00:48:28,904
DNA SUPER COILED, THERE WAS AN

990
00:48:28,904 --> 00:48:31,773
INTERNAL LABEL OF THE DNA, SO

991
00:48:31,773 --> 00:48:35,377
THE LAB CARRY OUT THIS STUDY AND

992
00:48:35,377 --> 00:48:38,447
THEY FOUND IS OF COURSE WITH

993
00:48:38,447 --> 00:48:42,351
EITHER TFIIH TEN SUBRAHMANYAN OR

994
00:48:42,351 --> 00:48:44,219
CORE 7 THE ACTIVITY DOING

995
00:48:44,219 --> 00:48:46,021
EXCISION PRODUCT IS SIMILAR. BUT

996
00:48:46,021 --> 00:48:53,262
IF WE KNOCK OUT XPB ATPASE

997
00:48:53,262 --> 00:48:54,363
ACTIVITY YOU SEE NO DUAL

998
00:48:54,363 --> 00:48:58,033
INCISION AT ALL. WHEN WE KNOCK

999
00:48:58,033 --> 00:49:01,937
OUT XPB ATPASE YOU CAN LOOK

1000
00:49:01,937 --> 00:49:03,772
CLOSE AND STILL SEE SOME PRODUCT

1001
00:49:03,772 --> 00:49:06,842
SO XPB IS VERY IMPORTANT,

1002
00:49:06,842 --> 00:49:10,712
ESSENTIAL AND XPD WITHOUT ATPASE

1003
00:49:10,712 --> 00:49:11,947
CAN STALL LESION AND MAY STILL

1004
00:49:11,947 --> 00:49:15,350
HAVE NER ACTIVITY.  SO I THINK I

1005
00:49:15,350 --> 00:49:18,754
WOULD LIKE TO PUT THE --

1006
00:49:18,754 --> 00:49:21,890
TOGETHER IN SPECULATION IN A WAY

1007
00:49:21,890 --> 00:49:26,228
HOW NER AND TC NER CONVERGE ONCE

1008
00:49:26,228 --> 00:49:29,898
XPA IS RECRUITED AND TFIIH AND

1009
00:49:29,898 --> 00:49:32,034
XPA VERIFY LESION. SO YOU CAN

1010
00:49:32,034 --> 00:49:34,102
ALREADY SEE XPC MAINLY BIND

1011
00:49:34,102 --> 00:49:36,538
DOWNSTREAM DOUBLE STRAND DNA. 

1012
00:49:36,538 --> 00:49:38,573
AND ALSO RECOGNIZE THE

1013
00:49:38,573 --> 00:49:40,275
NON-LESION STRAND OPPOSITE THE

1014
00:49:40,275 --> 00:49:42,110
LESION. AND THE END TERMINAL AND

1015
00:49:42,110 --> 00:49:45,280
C TERMINAL LONG HELIX OF XPC

1016
00:49:45,280 --> 00:49:49,217
ORIENT THE LOW TFIIH XPB AND

1017
00:49:49,217 --> 00:49:51,953
XPD. XP ENABLED TRANSFORMATION

1018
00:49:51,953 --> 00:49:56,191
OF TFIIH AND REPOSITION OF TFI,H

1019
00:49:56,191 --> 00:50:00,829
RELATIVE TO LESION XPC. IN TC

1020
00:50:00,829 --> 00:50:03,131
NER I'M BORROWING THE STRUCTURE

1021
00:50:03,131 --> 00:50:05,200
OF PRE-INITIATION COMPLEX. THE

1022
00:50:05,200 --> 00:50:09,905
RNA POLYMERASE IS ALSO LOCATED

1023
00:50:09,905 --> 00:50:13,942
DOWNSTREAM OF THE XPB AND XPD.

1024
00:50:13,942 --> 00:50:15,811
IN THIS COMPLEX HOWEVER RNAP

1025
00:50:15,811 --> 00:50:18,280
REALLY READING OUT THE LESION

1026
00:50:18,280 --> 00:50:20,682
STRAND RATHER THAN

1027
00:50:20,682 --> 00:50:22,250
NON-TRANSCRIBED LESION,

1028
00:50:22,250 --> 00:50:26,088
NON-LESION STRAND. XPD, THE SAME

1029
00:50:26,088 --> 00:50:27,522
HERE, IS NOT TOUCHING DNA AT

1030
00:50:27,522 --> 00:50:29,624
ALL. XPB IS INTERACTING WITH

1031
00:50:29,624 --> 00:50:30,992
DOUBLE STRAND DNA AS I POINTED

1032
00:50:30,992 --> 00:50:36,231
OUT.  WITHOUT XPA AS A WEDGE

1033
00:50:36,231 --> 00:50:38,300
SEPARATING THE TWO STRANDS. XPB

1034
00:50:38,300 --> 00:50:41,603
SERVING AS A TRANSLOW CASE. SO

1035
00:50:41,603 --> 00:50:44,339
IT PUSH THE DNA TOWARDS RNAP SO

1036
00:50:44,339 --> 00:50:48,143
ALLOW THE TWO STRAND TO

1037
00:50:48,143 --> 00:50:49,044
SEPARATE. ONCE THE INITIATION

1038
00:50:49,044 --> 00:50:53,115
BUBBLE IS FORMED ACTUALLY

1039
00:50:53,115 --> 00:50:57,486
PHOSPHORYLATION, ET CETERA, PXB

1040
00:50:57,486 --> 00:50:59,955
XPD IS RELEASED AN TRANSCRIPTION

1041
00:50:59,955 --> 00:51:02,691
CAN START. BUT IN NUCLEOTIDE

1042
00:51:02,691 --> 00:51:05,894
EXCISION REPAIR IN THE PRESENCE

1043
00:51:05,894 --> 00:51:07,963
OF LESION AND VARIOUS PROTEIN

1044
00:51:07,963 --> 00:51:10,999
INCLUDING CSB WHICH MAKES SURE

1045
00:51:10,999 --> 00:51:14,136
XP -- RNAP IS REALLY STUCK, WE

1046
00:51:14,136 --> 00:51:18,173
DON'T KNOW EXACTLY HOW THE TFIIH

1047
00:51:18,173 --> 00:51:22,377
IS RECRUITED BACK. CSB CSA AND

1048
00:51:22,377 --> 00:51:25,113
VARIOUS MODIFICATION

1049
00:51:25,113 --> 00:51:27,282
UBIQUITINATION INVOLVED.  BUT

1050
00:51:27,282 --> 00:51:30,352
THE RESULT OF THIS COMPLEX

1051
00:51:30,352 --> 00:51:32,788
FORMATION IS RNAP IS LEADING

1052
00:51:32,788 --> 00:51:36,458
RATHER THAN TFIIH LEADING. WITH

1053
00:51:36,458 --> 00:51:39,294
HELP OF XPA, THEN THIS IS THE

1054
00:51:39,294 --> 00:51:42,731
COMPLEX WE OBSERVED THAT REALLY

1055
00:51:42,731 --> 00:51:44,566
READS OUT WHETHER THERE IS A

1056
00:51:44,566 --> 00:51:46,067
LESION OR NO LESION WITH NER

1057
00:51:46,067 --> 00:51:49,438
SHOULD BE INVOLVED. I THINK I

1058
00:51:49,438 --> 00:51:52,340
WILL JUST USE LAST COUPLE OF

1059
00:51:52,340 --> 00:51:53,809
MINUTES TO LET YOU KNOW WHAT WE

1060
00:51:53,809 --> 00:51:57,479
ARE WORKING ON NOW. SO WE

1061
00:51:57,479 --> 00:52:03,618
ACTUALLY PURIFIED RPA XPD AND

1062
00:52:03,618 --> 00:52:05,887
XPF AS WELL SO WE COME TO

1063
00:52:05,887 --> 00:52:08,790
RECONSTITUTE WHOLE NER AND WE

1064
00:52:08,790 --> 00:52:11,126
USE DIFFERENT SUBSTRATE OF BULKY

1065
00:52:11,126 --> 00:52:12,427
LESION AND NOT A BULKY LESION

1066
00:52:12,427 --> 00:52:14,729
BOTH IN BINDING AND EXCISION

1067
00:52:14,729 --> 00:52:17,165
REPAIR ASSAY AND THE BULKY

1068
00:52:17,165 --> 00:52:21,102
LESION TURN OUT DOES NOT BIND BY

1069
00:52:21,102 --> 00:52:26,908
XPC TFIIH AND SPA AS WELL AS THE

1070
00:52:26,908 --> 00:52:28,210
NON-LESION, NON-BULKY LESION,

1071
00:52:28,210 --> 00:52:31,379
TWO BASIC SITE HAVE DISSOCIATION

1072
00:52:31,379 --> 00:52:35,617
LIKE LESS THAN 2.5 NANOMOLAR OF

1073
00:52:35,617 --> 00:52:38,186
THIS IS CY 5 AND CY 5 IS NOT A

1074
00:52:38,186 --> 00:52:42,257
NATURAL SUBSTRATE ANYWAY. BUT

1075
00:52:42,257 --> 00:52:43,191
INTERESTINGLY, EVEN THOUGH IT IS

1076
00:52:43,191 --> 00:52:47,963
A WEAKER BINDING, XPA AND XPC

1077
00:52:47,963 --> 00:52:52,033
AND TFIIH, IN REPAIR THERE IS A

1078
00:52:52,033 --> 00:52:53,201
DIFFERENCE, WE DEVELOPED ASSAY

1079
00:52:53,201 --> 00:52:56,938
USING OLIGODNA, THE REPAIR

1080
00:52:56,938 --> 00:53:01,009
EFFICIENCY WAS VERY GOOD, WE CAN

1081
00:53:01,009 --> 00:53:05,814
OBSERVE EVEN NEARLY 80% OF

1082
00:53:05,814 --> 00:53:08,316
SUBSTRATE BECOME DUAL EXCISION

1083
00:53:08,316 --> 00:53:10,719
PRODUCT SHOW HERE SUBSTRATE 94

1084
00:53:10,719 --> 00:53:12,954
BASE PAIR PAIR AND DUAL EXCISION

1085
00:53:12,954 --> 00:53:15,790
RESULT SPREAD FROM 27 TO ABOUT

1086
00:53:15,790 --> 00:53:19,461
34. AND WITH CY 5 WE SEE GOOD

1087
00:53:19,461 --> 00:53:21,663
PRODUCT FORMATION, DOING

1088
00:53:21,663 --> 00:53:24,599
EXCISION WITH TWO BASIC SITE IS

1089
00:53:24,599 --> 00:53:28,169
MUCH WEAKERER. SO SOMEWHERE

1090
00:53:28,169 --> 00:53:32,774
SOMEHOW AFTER TFIIH XPA BINDING,

1091
00:53:32,774 --> 00:53:35,911
XPG AND RPA FURTHER DETERMINES

1092
00:53:35,911 --> 00:53:38,446
IN THE PRESENCE OF ATP THERE IS

1093
00:53:38,446 --> 00:53:39,648
A DIFFERENCE IN THE EXCISION

1094
00:53:39,648 --> 00:53:43,251
EFFICIENCY. SO OUR GOAL IS TO

1095
00:53:43,251 --> 00:53:48,356
REALLY FIGURE OUT HOW BULKY

1096
00:53:48,356 --> 00:53:52,127
LESION STALLS TFIIH AND HOW DNA

1097
00:53:52,127 --> 00:53:53,562
EXCISION REALLY -- SO WITH THIS

1098
00:53:53,562 --> 00:53:57,032
I WANT TO THANK THE REAL HEROES

1099
00:53:57,032 --> 00:53:58,833
OF THIS PROJECT. ERIC STARTED

1100
00:53:58,833 --> 00:54:00,735
THIS PROJECT MORE THAN TEN YEARS

1101
00:54:00,735 --> 00:54:04,105
AGO AND PHILLIP DID A LOT OF IN

1102
00:54:04,105 --> 00:54:07,342
VITRO HELOCASE ACTIVITY ASSAY

1103
00:54:07,342 --> 00:54:12,013
AND INITIAL EM CHARACTERIZATION,

1104
00:54:12,013 --> 00:54:13,715
JINSEOK CARRIED OUT FROM PHILLIP

1105
00:54:13,715 --> 00:54:16,985
TO COMPLETION AND ERIC IMAGING

1106
00:54:16,985 --> 00:54:18,119
WORKING TOGETHER OBJECT

1107
00:54:18,119 --> 00:54:20,989
FOLLOWING STRUCTURE WE HOPE TO

1108
00:54:20,989 --> 00:54:25,527
GET. AND (INDISCERNIBLE) WORKED

1109
00:54:25,527 --> 00:54:29,564
ON THE PROJECT BEFORE ERIC.

1110
00:54:29,564 --> 00:54:33,702
NADINE CLONED XPA XPA PURIFIED

1111
00:54:33,702 --> 00:54:35,470
THE PROTEIN RPA AND REALLY LAID

1112
00:54:35,470 --> 00:54:37,872
GROUND. WITH FELLOWSHIP OF THREE

1113
00:54:37,872 --> 00:54:42,310
YEARS, JUST NOT POSSIBLE TO

1114
00:54:42,310 --> 00:54:44,145
COMPLETE. AND TO WORK ON

1115
00:54:44,145 --> 00:54:46,181
SOMETHING WITH BARING RESULT

1116
00:54:46,181 --> 00:54:50,885
ENABLE TO MOVE ON AND NOW IS A

1117
00:54:50,885 --> 00:54:53,655
TENURE TRACK FACULTY AT NIH.

1118
00:54:53,655 --> 00:54:57,258
CRYOEM WORK CAN BE ACCOMPLISHED

1119
00:54:57,258 --> 00:54:58,093
WITHOUT (INAUDIBLE) HELP AND

1120
00:54:58,093 --> 00:55:00,795
WITH FACILITY AT NIDDK,

1121
00:55:00,795 --> 00:55:03,598
SUPERVISED BY (INDISCERNIBLE).

1122
00:55:03,598 --> 00:55:07,102
LAST BUT NOT LEAST,

1123
00:55:07,102 --> 00:55:09,738
(INDISCERNIBLE) APPROACH ME IN

1124
00:55:09,738 --> 00:55:10,705
EARLY 2010 AND STARTED THIRD

1125
00:55:10,705 --> 00:55:12,641
DEGREE PROJECT. IT TAKES A LONG

1126
00:55:12,641 --> 00:55:14,509
TIME BUT ACTUALLY IT IS

1127
00:55:14,509 --> 00:55:21,483
REWARDING. THANK YOU. 

1128
00:55:21,483 --> 00:55:23,451
>> THANK YOU, DR. YANG FOR
THE

1129
00:55:23,451 --> 00:55:24,386
WONDERFUL TALK. I BELIEVE

1130
00:55:24,386 --> 00:55:26,121
EVERYBODY AGREE THAT THE RESULT

1131
00:55:26,121 --> 00:55:28,590
IS VERY STRIKING AND VERY

1132
00:55:28,590 --> 00:55:32,527
IMPRESSIVE. CAN SEE THE

1133
00:55:32,527 --> 00:55:33,662
DEDICATED MOVEMENT OF THE

1134
00:55:33,662 --> 00:55:35,096
COMPRESSION TO HELP US

1135
00:55:35,096 --> 00:55:38,400
UNDERSTAND HOW IT WORKS. SO I

1136
00:55:38,400 --> 00:55:40,502
CAN TELL THERE ARE SOME

1137
00:55:40,502 --> 00:55:41,703
QUESTIONS TYPED IN THE CHAT BOX.

1138
00:55:41,703 --> 00:55:43,872
SO THE FIRST ONE IS BY CHERYL

1139
00:55:43,872 --> 00:55:47,942
WHICH IS MOSTLY ABOUT GENERAL

1140
00:55:47,942 --> 00:55:50,512
DNA REPAIR PATHWAY. IS ATR AND

1141
00:55:50,512 --> 00:55:52,647
ATM MEDIATED DNA DAMAGE RESPONSE

1142
00:55:52,647 --> 00:55:56,351
SOMEHOW PLAY A ROLE WITH TFIIH

1143
00:55:56,351 --> 00:55:59,954
RECRUITMENT? LIKEWISE HOW TFIIH

1144
00:55:59,954 --> 00:56:02,323
RESPOND TO PARP ACTIVATION?

1145
00:56:02,323 --> 00:56:07,962
>> SO AT THIS POINT, I
CANNOT

1146
00:56:07,962 --> 00:56:12,200
SPEAK OF HOW ATR AND OTHER

1147
00:56:12,200 --> 00:56:16,604
PROTEIN GENERAL GENOMIC

1148
00:56:16,604 --> 00:56:18,406
INSTABILITY AFFECT TFI,H.

1149
00:56:18,406 --> 00:56:19,741
PROBABLY SHARING THOSE MORE THAN

1150
00:56:19,741 --> 00:56:23,311
I DO AND I'M ALL EARS.  AND

1151
00:56:23,311 --> 00:56:26,915
RECRUITMENT OF TFIIH AS FAR AS I

1152
00:56:26,915 --> 00:56:37,258
CAN TELL WE CAN TEL

1153
00:56:49,204 --> 00:56:53,274
>> WE DO HAVE ONE COMMENT
FROM

1154
00:56:53,274 --> 00:56:54,542
WILLIAM (INDISCERNIBLE) SAYING

1155
00:56:54,542 --> 00:56:55,844
BEAUTIFUL VISUALS, REALLY

1156
00:56:55,844 --> 00:56:56,611
ENJOYING THE PRESENTATION. I

1157
00:56:56,611 --> 00:57:01,116
THINK HE SPEAKS FOR ALL OF US.  

1158
00:57:01,116 --> 00:57:02,183
THERE'S ALSO A QUESTION -- DO

1159
00:57:02,183 --> 00:57:03,885
YOU WANT TO GO AHEAD?

1160
00:57:03,885 --> 00:57:06,988
>> SURE. THERE IS A
QUESTION

1161
00:57:06,988 --> 00:57:10,024
FROM DMITRI. WHAT WOULD HAPPEN

1162
00:57:10,024 --> 00:57:12,560
WITH BULKY LESION CLOSE TO A

1163
00:57:12,560 --> 00:57:15,096
DSBN? DO WE EXPECT HELOCASE

1164
00:57:15,096 --> 00:57:18,466
ACTIVITY CREATING A LONG SSDI

1165
00:57:18,466 --> 00:57:18,700
REGION?

1166
00:57:18,700 --> 00:57:23,805
>> IT IS POSSIBLE BECAUSE
IT IS

1167
00:57:23,805 --> 00:57:26,975
(INDISCERNIBLE) WORK IN 2009, SO

1168
00:57:26,975 --> 00:57:29,444
BASICALLY ONCE TFI,H IS

1169
00:57:29,444 --> 00:57:32,747
UNLOADED, EVEN LOADED SAY BY A

1170
00:57:32,747 --> 00:57:35,517
MISMATCH BASE PAIR, XPD IS SUCH

1171
00:57:35,517 --> 00:57:37,952
A STRONG HELOCASE, ONCE LOADED

1172
00:57:37,952 --> 00:57:40,121
ON DOUBLE STRAND DNA IT CAN MOVE

1173
00:57:40,121 --> 00:57:44,559
ALONG THE DNA. DR.

1174
00:57:44,559 --> 00:57:45,426
(INDISCERNIBLE) PLAYS A REAL

1175
00:57:45,426 --> 00:57:47,595
BULKY LESION, 100 BASE PAIR AWAY

1176
00:57:47,595 --> 00:57:50,398
AND XPD STILL CAN FIND IT AS

1177
00:57:50,398 --> 00:57:52,367
LONG AS IT IS LOADED ON THE

1178
00:57:52,367 --> 00:57:55,970
RIGHT STRAND. IF IT IS LOADED ON

1179
00:57:55,970 --> 00:57:58,173
THE OTHER -- MISMATCH YOU CANNOT

1180
00:57:58,173 --> 00:58:00,141
TELL WHICH STRAND IT IS BUT IF

1181
00:58:00,141 --> 00:58:03,344
YOU HAVE AN A ASIC SITE, A BASIC

1182
00:58:03,344 --> 00:58:04,379
IS THE LESION AND IF YOU

1183
00:58:04,379 --> 00:58:05,747
DOWNSTREAM THERE IS A BULKY

1184
00:58:05,747 --> 00:58:07,348
LESION, IT WILL BE RECOGNIZED SO

1185
00:58:07,348 --> 00:58:08,983
IT CAN TRAVEL AND I DON'T KNOW

1186
00:58:08,983 --> 00:58:13,021
HOW LONG IT CAN TRAVEL. AND ALSO

1187
00:58:13,021 --> 00:58:15,089
I HAVE TO GIVEN THAT THERE IS A

1188
00:58:15,089 --> 00:58:19,294
NUCLEOSOME ON THE DNA, SO THERE

1189
00:58:19,294 --> 00:58:21,830
IS A -- PROBABLY REMOVAL OF

1190
00:58:21,830 --> 00:58:25,200
NUCLEOSOME LOCALLY THROUGH THE

1191
00:58:25,200 --> 00:58:30,805
DDBE 1, DDB 1, DDB 23 BECAUSE

1192
00:58:30,805 --> 00:58:35,243
THEY ACTIVATE THE

1193
00:58:35,243 --> 00:58:36,377
(INDISCERNIBLE) DISSOCIATE THE

1194
00:58:36,377 --> 00:58:37,745
NUCLEOSOME BUT PROBABLY NOT LONG

1195
00:58:37,745 --> 00:58:39,480
DISTANCE BECAUSE IT DOESN'T DO

1196
00:58:39,480 --> 00:58:44,953
GOOD FOR THE GENOME.

1197
00:58:44,953 --> 00:58:46,487
>> A QUESTION FROM BOB,
HAVE YOU

1198
00:58:46,487 --> 00:58:47,789
BEEN ABLE TO EXCLUDE THE

1199
00:58:47,789 --> 00:58:49,891
POSSIBILITY THAT XPD STALLED BY

1200
00:58:49,891 --> 00:58:51,426
THE LESION AT A STRAND SWITCHES

1201
00:58:51,426 --> 00:58:53,361
TO THE OTHER SIDE OF THE NEW

1202
00:58:53,361 --> 00:58:55,630
BUBBLE? OR ANOTHER XPD MOLECULE

1203
00:58:55,630 --> 00:58:56,631
LOADS TO THE NON-LESION STRAND

1204
00:58:56,631 --> 00:58:58,733
TO EXPAND THE BUBBLE?

1205
00:58:58,733 --> 00:59:00,869
>> YEAH, IT IS POSSIBLE
BUT I

1206
00:59:00,869 --> 00:59:03,705
FEEL LIKE THAT IS VERY MUCH A

1207
00:59:03,705 --> 00:59:09,344
SETUP IN VITRO. WE CAN SEE IT.  

1208
00:59:09,344 --> 00:59:15,250
AND THE REASON IS I GUESS I CAN

1209
00:59:15,250 --> 00:59:19,287
USE THIS SLIDE. IN THE PRESENCE

1210
00:59:19,287 --> 00:59:21,723
OF XPC THE LOADING IS REALLY

1211
00:59:21,723 --> 00:59:23,925
DICTATED BY XPC IN THE LESION.

1212
00:59:23,925 --> 00:59:31,566
AND THE SAME WITH RNAP AND TFIIH

1213
00:59:31,566 --> 00:59:33,334
RECRUITMENT. WITHOUT THOSE

1214
00:59:33,334 --> 00:59:34,636
RECRUITERS, IF YOU JUST HAVE A

1215
00:59:34,636 --> 00:59:37,372
BUBBLE, TFI,H CAN LOAD ON EITHER

1216
00:59:37,372 --> 00:59:40,642
SIDE, EITHER XPD ON THE LESION

1217
00:59:40,642 --> 00:59:41,876
STRAND OR NON-LESION STRAND IF

1218
00:59:41,876 --> 00:59:47,081
YOU GIVE A BUBBLE. RIGHT? BUT IF

1219
00:59:47,081 --> 00:59:50,285
YOU HAVE THE XPC OR RNAP TO

1220
00:59:50,285 --> 00:59:51,853
REPORT WHERE THE PROBLEM STRAND

1221
00:59:51,853 --> 00:59:53,788
IS, THEN THE LOADING IS

1222
00:59:53,788 --> 01:00:01,829
ABSOLUTELY ONLY ONE WAY. SO THAT

1223
01:00:01,829 --> 01:00:06,401
IS WHAT I LEARNED, WE KNOW AS A

1224
01:00:06,401 --> 01:00:08,803
BIOLOGIST WE DIVIDE AND CONQUER,

1225
01:00:08,803 --> 01:00:10,605
EVEN TODAY WE STILL DIVIDE AND

1226
01:00:10,605 --> 01:00:12,807
CONQUER, WE DON'T STUDY WHOLE

1227
01:00:12,807 --> 01:00:15,109
ANIMAL, HAVE TO GET TO TISSUE

1228
01:00:15,109 --> 01:00:17,045
AND ANIMAL GETS YOU CERTAIN AM

1229
01:00:17,045 --> 01:00:19,447
OF READING.  AT READING OF

1230
01:00:19,447 --> 01:00:20,748
MECHANISM WE GET SMALLER PIECE

1231
01:00:20,748 --> 01:00:23,685
AND CRYOEM IS A SAVIOR FOR OUR

1232
01:00:23,685 --> 01:00:24,786
STRUCTURAL BIOLOGISTS BECAUSE

1233
01:00:24,786 --> 01:00:26,020
ALL THOSE STRUCTURES WE WOULD

1234
01:00:26,020 --> 01:00:28,089
NEVER BE ABLE TO CRYSTALLIZE IF

1235
01:00:28,089 --> 01:00:30,558
WE HAVE THE BIG -- THERE'S

1236
01:00:30,558 --> 01:00:32,293
CONFIRMATIONAL CHANGE SO THIS IS

1237
01:00:32,293 --> 01:00:33,728
THE INTACT ONE AND I'M NOT

1238
01:00:33,728 --> 01:00:36,731
SHOWING THE XPC AND RNAP. THIS

1239
01:00:36,731 --> 01:00:40,401
IS THE WHOLE ENZYME, WHEN YOU

1240
01:00:40,401 --> 01:00:42,971
HAVE RNAP OR XPC TFI,H IS LOADED

1241
01:00:42,971 --> 01:00:46,541
CORRECTLY. WE ALREADY HAVE SOME

1242
01:00:46,541 --> 01:00:49,911
DATA TO INDICATE THE

1243
01:00:49,911 --> 01:00:50,545
CONFIRMATIONAL CHANGE I SHOW YOU

1244
01:00:50,545 --> 01:00:52,013
THE BIG MOVIE, SURE IT IS KIND

1245
01:00:52,013 --> 01:00:54,015
OF HARD TO GET YOUR HEAD AROUND.

1246
01:00:54,015 --> 01:00:57,452
IT IS NOT THE END. WITH ATP

1247
01:00:57,452 --> 01:00:59,954
PRESENCE, WITH RPA BINDING

1248
01:00:59,954 --> 01:01:10,665
SINGLE STRAND DNA AND TWOWE ALWO

1249
01:01:25,680 --> 01:01:28,683
KEEP IN MIND WHAT WE SEE IS

1250
01:01:28,683 --> 01:01:33,688
MAYBE JUST THE LEG OR EAR OF

1251
01:01:33,688 --> 01:01:34,088
ELEPHANT.

1252
01:01:34,088 --> 01:01:37,558
VERY TRUE. 

1253
01:01:37,558 --> 01:01:38,926
WELL, THANK YOU, I
THINK

1254
01:01:38,926 --> 01:01:40,862
THAT'S FOR ALL THE QUESTIONS.

1255
01:01:40,862 --> 01:01:43,598
AND FOR ALL THE AUDIENCE, PLEASE

1256
01:01:43,598 --> 01:01:45,500
JOIN ME TO THANK AGAIN DR.

1257
01:01:45,500 --> 01:01:46,868
YANG'S WONDERFUL TALK. THANK

1258
01:01:46,868 --> 01:01:48,503
YOU, EVERYBODY'S PARTICIPATION.

1259
01:01:48,503 --> 01:01:51,005
DR. YANG, THANK YOU AGAIN.

1260
01:01:51,005 --> 01:01:53,041
REALLY GREAT TO HAVE YOU HERE.

1261
01:01:53,041 --> 01:01:54,842
WE WILL KEEP IN TOUCH.

1262
01:01:54,842 --> 01:01:57,712
>> THANK YOU VERY MUCH.

1263
01:01:57,712 --> 01:01:59,280
LOVELY STORY, THANK YOU
SO

1264
01:01:59,280 --> 01:01:59,514
MUCH.

1265
01:01:59,514 --> 01:02:02,050
THANK YOU. GOODBYE.

1266
01:02:02,050 --> 01:02:12,460
THANK YOU, BYE-BYE.