GOOD AFTERNOON, EVERYONE,
WELCOME TO THE MONTHLY DNA
REPAIR INTEREST GROUP
CONFERENCE, WE ARE HERE TO
DISCUSS THE RECENT ANNOUNCEMENTS
IN DNA REPAIR, MUTAGENESIS, CELL
CYCLE AND RELATEDDED HUMAN
DISEASE. DR. (INAUDIBLE) AND I
WOULD LIKE TO TAKE THIS
OPPORTUNITY TO APPRECIATE
PARTICIPATION AND INSIGHT FOR
NOMINATIONS FROM ADVISORY
COMMITTEE. BEFORE WE BEGIN,
PLEASE MARK A CALENDAR FOR OUR
ANNUAL YOUNG INVESTIGATOR
WEBINAR ON THE NOVEMBER 14. WE
WILL FEATURE TALKS ABOUT DR.
MALIKA (INAUDIBLE),
(INDISCERNIBLE) AND CLAUDIA
NICOLAE. EMAIL ANNOUNCEMENT WILL
FOLLOW SOON. TODAY WE ARE VERY
PLEASED TO HAVE DR. WEI YANG
WITH US, A DISTINGUISHED
INVESTIGATOR AT THE U.S.
NATIONAL INSTITUTES OF HEALTH.
WHERE SHE HAS BEEN SINCE 1995.
SHE BEGAN HER UNDERGRADUATE
STUDIES IN BIOCHEMISTRY, IN
(INAUDIBLE) UNIVERSITY SHANGHAI
LATER DR. YANG TRANSFERRED TO
STONY BROOK FROM WHICH SHE
RECEIVED A BACHELOR'S DEGREE.
AND SHE RECEIVED DEPh.D.
DEGREE FROM COLUMBIA UNIVERSITY
WHERE SHE WORKED WITH WAYNE
HENDRICKSON AND RECEIVED
POST-DOCTORAL TRAINING AT YALE
UNIVERSITY IN THE LAB OF TOM
GATES. SHE USED STRUCTURAL AND
BIOCHEMICAL TO IN BUY LOGICAL
CHEMISTRY SPONSORED BY CSB AND
B. SHE'S ALSO AN ELECTED MEMBER
FOR THE NATIONAL ACADEMY OF
SCIENCES AND AMERICAN ACADEMY OF
ART AND SCIENCES. NOW WE WILL
TRANSITION TO DR. YANG'S TALK
FOR ATTENDEES PLEASE PUT YOUR
QUESTIONS IN THE CHAT BOX AND WE
WILL DISCUSS AFTER TALK. THANK
YOU. DR. YANG. THANK YOU FOR
TAKING OVER THE DNA REPAIR SORT
OF LIKE OUR SOCIETY MEETING AND
THANKFUL TO CARRY OUT WITH NEWER
TECHNOLOGY WEBEX AND ZOOM. WE
ARE MUCH MORE SEAMLESSLY LINKED
TO THE WORLD. TODAY I'M GOING TO
SHARE WITH YOU OUR RECENT RESULT
OF DNA LESION RECOGNITION.
EXNUCLEOTIDE EXCISION REPAIR. IT
IS AN OLD TOPIC AND THE REASON
I'M WILLING TO SHARE WITH YOU
OUR RECENT RESULT IS BECAUSE
MANY PEOPLE HAVE KNOWN AND
STUDIED FOR MANY DECADES AND
KNOW MUCH MORE THAN ME AND MY
GROUP SO I'M HOPING TO LEARN
FROM YOU AND PLEASE SEND ME YOUR
QUESTIONS, COMMENTS AND
SUGGESTIONS. SO IT IS AN
OPPORTUNITY FOR ME TO LEARN. SO
WE WERE INTERESTED STARTED
INTEREST DNA LESION INDUCED BY
ULTRAVIOLET ABOUT 20 SOMETHING
YEARS AGO AND WE STARTED TO
STUDY LESION TOLERANCE BY
SPECIALIZED DNA POLYMERASE.
LESION ARE TOLERATED DURING DNA
REPLICATION AND POST REPLICATION
AS WELL. BUT THE TLS MUTATIONS
CALLED XPV IS ONLY CAUSING ABOUT
20% OF THE PATIENT. THE MAJORITY
OF THE UV INDUCED LESION ARE
REPAIRED OUTSIDE OF THE S PHASE.
THE REPAIR TOLERANCE LEADS TO A
CONDITION CHECK TESTIFILY KNOWN
AS -- COLLECTIVELY KNOWN AS
SERUM PIG MENTOSUM. HERE IS A
PICTURE, NOT TOO BAD BUT COULD
BE WORSE. THE EXCISION REPAIR
DEPEND ON NUMBER OF ESSENTIAL
GENES KNOWN 20, 30 DECADES AGO,
XPA, XPC EFG. THOSE ARE THE TWO
TYPE OF LESION AND ACTUALLY THE
REPAIR AND ALSO TLS OF SIMILAR
LESIONS BECOME A PROBLEM IN
CHEMOTHERAPY BECAUSE ONE OF THE
MOST OFTEN USED CHEMICAL REAGENT
IS CISPLATIN AND IT IS
CROSS-LINKED ADJACENT SIMILAR TO
THE RESULT OF COVALENT LINKAGE
OF ADJACENT TWO Ts, CPD TO
DOUBLE BOND OF (INDISCERNIBLE)
SO UNDERSTANDING MECHANISM NOT
ONLY HELP US TO UNDERSTAND AND
PERHAPS TREATMENT OF THOSE XP
PATIENTS BUT MOST IMPORTANTLY
CAN MAYBE HELP TO HELP TREATMENT
OF CISPLATIN CHEMOTHERAPY MORE
EFFECTIVELY. SO ONCE WE STARTED
TO WORK ON TRANSLATION DNA
POLYMERASE, WE RECEIVED THE
INVITATION FROM EXPERT IN THE
FIELD, CA (INAUDIBLE) WHO SAID
MAYBE WE CAN LAB RAT, THAT WAS
MORE THAN A DOZEN YEARS AGO. WE
STARTED TO LOOK INTO IT.
NUCLEOTIDE EXCISION REPAIR CAN
BE STARTED BY TWO DIFFERENT
PATHWAYS FOR FINDING DNA LESION
EITHER UV OR OTHER CROSS LINK
SPECIES BULKY ADDUCT ON DNA. ONE
CALLED GLOBAL GENOME NER WHICH
IS ESSENTIAL LESION RECOGNITION
PROTEIN IS XPC, BUT SOMETIMES
WHEN DIFFICULT TO FIND XPE IS
INVOLVED I WILL SHOW YOU THE
NEXT SLIDE. THIS FINALLY IS NOT
GUARANTEED, EVERY LESION CAN BE
FOUND IN THE GENOME BECAUSE OF
BILLIONS OF BASE PAIRS NOT
GUARANTEED TO BE FOUND,
PARTICULARLY LIKE CPD DOES NOT
DISTORT DNA VERY MUCH. THE
MOTION WAY IS TRANSCRIPTS
COUPLED REPAIR BULKY LESIONS
WILL STALL RNA POLYMERASE AND
WHEN RNA POLYMERASE STALL, CSB
WILL TRY TO PUSH IT OVER BUT
WHEN THAT FAILED THEN IT IS BOTH
PATHWAY LEADING TO RECRUITMENT
OF TRANSCRIPTION CALLED TF 2H.
THEN THOSE TWO PATHWAYS
CONVERGE. TF 2H BINDING TO THE
DNA LEADS TO NEXT PROTEIN
BINDING DEFINED KEY PROTEIN NER
XPA. THE TWO TOGETHER, TF 2H AND
XP TOGETHER ABLE TO FIND WAS THE
LESION TRULY IS THE BULKY LESION
AND THE NEED TO BE EXCISED BY
NUCLEASE AND IF IT IS NOT OTHER
PATHWAYS LIKE MISMATCH REPAIR OR
BASIC EXCISION REPAIR WILL BE
MORE PREFERENCE THAN TO USE THIS
BIGGER EXCISION. SO WHEN THE
LESION IS VERIFIED BY TF 2H AND
XPA TWO PARTICULAR NUCLEASE
STRUCTURE SPECIFIC NUCLEASE XPF
AND XPG RECRUITED AND CLEAVE AT
THE 5 PRIME AND 3 PRIME JUNCTION
OF THE LESION AND EUKARYOTS THE
PATHWAY AND EXERCISE THE DNA
CONTAINING LESION CAN VARY FROM
20 SOMETHING NUCLEOTIDE TO 35
NUCLEOTIDE. THIS IS THE END
RESULT. AND THEN THE GAP NEED TO
BE FILLED BY THE DNA POLYMERASE
MANY OUTSTANDING SCIENTISTS LAY
THE GROUND AND CONSTITUTE
REACTION AND LEADING US TO
UNDERSTAND VERY WELL. THOSE ARE
THE PEOPLE AND REMEMBER NOT MANY
YEARS AGO NOBEL PRIZE WAS
AWARDED TO HIM FOR HIS
CONTRIBUTION, PHIL
(INDISCERNIBLE) AND SO IT IS
JUST LIKE THIS IS THE LIST OF
PEOPLE WHO MADE IT VERY
IMPORTANT CONTRIBUTION, OUR WORK
IS BASED ON THOSE STUDIES. SO
THE (INAUDIBLE) IS RECONSTITUTED
BUT EACH PROTEIN ROLE IS NOT
WELL UNDERAND THE MECHANISM IS
STILL YET TO BE REVILED. HERE
IS START -- REVEALED. HERE IS
STARTING, I'M NOT GOING TO
ELABORATE MUCH ON THE DNA XPE,
WHICH IS A PROTEIN SHOWN HERE
XPE HELPS TO RECOGNIZE DNA
LESION WHEN DNA IS NOT MUCH
DISTORTED AND THE FORMS
DEPENDENT DDB 1 AND HETERODIMER
FIND THE LESION. THIS WORK IS
DONE BY ANY C B BY NICOLE THOMAD
CONSTANTLY STUDIED BNAB 1 AND 2
RECOGNIZE LEAGUES LIKE PHOTO
PRODUCT ON NUCLEOSOME. AND IN
ADDITION WE FIND DDB 1
INTERACTING WITH THIS WHOLE
PROTEIN IS UBIQUITIN E 3 PROTEIN
AND WORKS WITH A AND B AND
UBIQUITYNATE TRANSCRIPTION
FACTORS AND ULTIMATELY THE DDB 1
AND 2 XPE HAND OVER THE LESION
TO XPC TO START THE GLOBAL
GENOME NER. SO HERE THE WORK IS
ALSO CONTRIBUTE BY (INAUDIBLE)
LAB IN PITTSBURG AND THERE IS
MUCH WORK ESTABLISHED 30 DAYS OF
XPC IN JAPAN BY (INDISCERNIBLE)
GROUP. SO NOW WE ARE GOING TO
THE MAIN FOCUS OF TODAY. WHICH
IS THE XPC AND VERY FIRST
CRYSTAL STRUCTURE RED 4 WAS RED
23 WERE DETERMINED BY NICOLA'S
LAB IN 2007 AND THEY FOUND THAT
TO THE XPC HETERODIMER DUPLEX,
IF WE IN THIS TALK I WILL FOCUS
ALWAYS HAVE THE LESION
CONTAINING STRAND FROM 5 PRIME
TO 3 PRIME LEFT TO RIGHT, LESION
CONTAINING STRAND OF THE DARKER
ORANGE AND NON-LESION STRAND
SHOWING YELLOW. HERE YOU CAN SEE
IT IS 5 PRIME TO 3 PRIME, THEN
XPC BINDING DOWNSTREAM OF THE
LESION AND THE LESION ITSELF IS
DISORDERED. AND BASED OPPOSITE
THE LESION ACTUALLY FLIPPED OUT
AND BOUND BY XPC. THIS
STRUCTURE HAS BEEN AROUND A LOT
OF YEARS AND DIFFERENT LEAGUES
SHOWN TO BE RECOGNIZED BY RED 4,
WE NEED TF 2H TO SEPARATE WHAT
IS THE LESION FOR NER REPAIR OR
IT IS NOT. IT WHATTING FOR
REALLY ALMOST 15 YEARS BEFORE
THE STRUCTURE OF HOW XPC AND TF
2H TOGETHER. TF 2H STRUCTURE WAS
FIRST REVEAL IN 2016 THROUGH
WORK FROM (INDISCERNIBLE) IN THE
REPORTED PRE-INITIATION COMPLEX
WHERE TF 2H INTERACTING WITH
DOUBLE STRAND DNA AND XPB AND
XPB IS NON-ESSENTIAL FOR LIST OF
ATPASE ACTIVITIES NOT ESSENTIAL
FOR TRANSCRIPTION INITIATION BUT
XPB IS AND BIND DOUBLE STRAND P
DNA WELL. THIS STRUCTURE HELPED
PEOPLE TO UNDERSTAND HOW TF 2H
BINDING DOUBLE STRAND DNA BUT
XPD ROSE VERY IMPORTANT FOR
SCANNING THE LESION IS NOT BY
DNA SO IT IS KIND OF LEFT
HANDED. WHEN THE TWO STRUCTURES
WERE TOGETHER, TWO PROTEIN
COMPLEX WERE TOGETHER AND
STRUCTURE DETERMINED BY
(INDISCERNIBLE) REPORTING BY
(INDISCERNIBLE) GROUP. THIS
TOGETHER SEEMS TO BE NOT MUCH
LINKED BY PROTEIN, EXCEPT TIP OF
XPC SOMEHOW EXACT THE P 62 OF TF
2H. MOSTLY TOGETHER IS BECAUSE
THE DNA UPSTREAM OF THE LESION
DUPLEX BIND BY XPD. THIS LEADS
TO, IT IS NICE TO HAVE IN THE
GENERAL ORGANIZATION BUT THERE
HAS PROBLEMS BECAUSE THE LESION
REMAINS DISORDERED IN
(INDISCERNIBLE) WAS THE MAIN
DRIVER. HE STUDIED TF 2H,
TOGETHER WITH XPA AND XPA IS
DICTATED NER. EVEN THOUGH HE
USED THE DNA DOES NOT CONTAIN
ANY LESION SO IT IS A DOUBLE
STRAND DNA FOLLOWED BY SINGLE
STRANDED, IT IS A Y SHAPE BASE
PAIR SINGLE STRAND DNA AND XPA A
SMALL BUT IMPORTANT PROTEIN BIND
AT THE DOUBLE STRAND SINGLE
STRAND JUNCTION. THE STRUCTURE
OF XPA ALONE BY DNA CONTAIN
LESION NEVER SEE XPA INTERACTING
WITH LESION THOUGH IT IS KNOWN
FOR HELPING BINDING LESION. IT
IS ALWAYS BIND DNA DOUBLE STRAND
END OR HERE AT DOUBLE STRAND
SINGLE STRAND JUNCTION. BECAUSE
SINGLE STRAND DNA HERE IT IS
CLEAR SINGLE STRAND DNA GOES
THROUGH XPD VERY NICE BUT NOT
CLEAR WHERE THE LESION IS. WE
CAN SEE WITH TF 2H AND XPC
WITHOUT XPA THE COMPLEX IN THAT
COMPLEX TF 2H SEVEN CORE SUB
UNIT IS U SHAPED. IN IN
PRESENCE OF XPA THE CORE 7 OF TF
2H BECOME SIGNAL SHAPED SO A
HUGE CONFIRMATIONAL CHANGE.
SINCE XPD MOVES 5 PRIME TO 3
PRIME THE LESION HAS TO BE
SOMEWHERE OUTSIDE OF SINGLE
STRAND DNA BOND BY XPD SO BEFORE
XPD CAN SCAN AND FIND IT. AND
DECIDE IT HAS TO BE REPAIRED BY
NER. BUT HERE THE LESIONS ARE
HERE. AND XPD IS NOT BINDING ANY
DNA. IF IT BINDS DNA HERE THE
LESION WILL BE ON TO THE LEFT
AND SCANNING TO THE RIGHT WILL
NOT HAVE XPD ENCOUNTERING THE
LESION SO THAT IS THE BIG
DILEMMA WITH EVERY STRUCTURE
KNOWN ABOUT CHEMICAL
CHARACTERIZATION. SO WE STARTED
PROJECT ACTUALLY IN EARLY 2010
BUT WE WERE JUST NOT GETTING
ANYWHERE WHERE ALL THOSE PAPER
PUBLISHED. HOWEVER, EVENTUALLY
WE GOT SOMEWHERE AND TODAY I
WILL TELL YOU WHAT WE FOUND. THE
FIRST WE WILL ADDRESS THE THREE
QUESTIONS, HOW IS THE DNA LESION
LOADED ON TO TF 2H SO XPD CAN
SCAN VERIFIED LESION. ALONG WAY
I WILL SHOW YOU HOW HUMAN XPC
STRUCTURE AND HOW DOES IT
RECRUIT TF 2H. BECAUSE BEFORE
IT IS THE RED 4 STRUCTURE. SO
HOW THE ROLES OF XPA AND NER SO
CRITICAL, IT IS IS SMALLEST
PROTEIN AND CONTAIN MOST OF THE
MUTATION THAT CAUSES XERODERMA
PIGMENTOSA. SO WE START WITH 11
SUB UNIT OF THE PROTEIN AS YOU
CAN SEE XP 3, TF 2H THE CONTACT
MOTIF IS NOT NECESSARY ACTUALLY
INHIBIT NER SO WE ONLY USE THE 7
SUB UNIT OF TF 2H WITH CORE 7
AND XPA. AND WE ALSO ESTABLISHED
THAT THROUGH THOSE PROTEIN WE
CAN SEE LESION MIMIC SIDE 5
OPPOSITE 2C CAN BE RECOGNIZED BY
XPC AND BINDING AFFINITY BY LESS
THAN 5 NANOMOLAR SO IT IS VERY
TIGHT BINDING. BUT ADDING XPA
DOESN'T CHANGE MUCH, ADDING TF
2H YOU CAN SEE A SUPER SHIFT,
THIS IS NOT MOST CLEAR BUT IN
BETTER GELS ADDING XPA
STABILIZES SUPER SHIFT. SO NOW
TURNING TO STRUCTURAL STUDY. AND
STRUCTURAL STUDY CRYSTALLOGRAPHY
IS NOT A CHOICE, IT IS NEVER
GOING TO CRYSTALLIZE WITH SO
MUCH -- SO MANY SUBUNITS AND LOT
OF MOBILITY. SO WE INITIALIZE
AFTER MAKING THE COMPLEX, WE DID
THE STAINING, PHILLIP
(INDISCERNIBLE) DID IT FIRST.
BUT WHEN PHIL PUT THE SAMPLE, IT
BECOME HETEROGENOUS FALL APART.
SO HE TRIED THE GRAPHICS WHICH
IS A GRADIENT FIXATION SO USING
SUCROSE OF GLYCEROL GRADIENT
APPLIED CROSS LINKING, CROSS
LINKERS SUCH AS ALDEHYDE LOW
CONCENTRATION, AND ESTABLISH A
GRADIENT OF CROSS LINKERS AND
LAY SAMPLE ON TOP, THE SAMPLE
WHEN GOING THROUGH THE
CENTRIFUGATION ALDEHYDE GETS
CROSS-LINKED. THIS IS THE
GRADIENT CENTRIFICATION WITHOUT
CROSS LINKING YOU CAN SEE THE
CROSS-LINKED FORM CONTAIN 11 SUB
UNIT AND AFTER WITH ALDEHYDE
MOST GETS CROSS LINK TO THE
SHIFT IN THE BAND WITH DNA.
INITIALLY WHEN WE PUT ON THE EM
GRID, THE COMPLEX STILL FALL
APART AFTER CROSS LINKING. AND
THEN WE USE SPECIAL GRID WITH
FILM SO SOMEWHAT HAVE ONE SIDE
SUPPORTING OF THOSE PARTICLES
THAT YOU CAN SEE THOSE COMPLEX
LOOK VERY UNIFORM AND HOMOGENOUS
IN TERM OF SIZ SIZE. STRUCTURE E
THEY ARE STILL DIFFERENT. LONG
STORY SHORT, WE WERE ABLE TO
SOLVE THE STRUCTURE AND ACTUALLY
THE WORK BY (INDISCERNIBLE) AND
ERIC LEE. THEY WERE ON THE SAME
CRYOEM GRID. THEY FOUND THREE
TYPE OF COMPLEX WAS THE MIX OF
11 SUB UNIT AND DNA LESION
CONTAINING SIDE 5. SO THE FIRST
TYPE OF COMPLEX IS XPC WITH TF
2H AND DNA BUT YOU CAN SEE NOW
YOU SEE MORE XPC. THIS IS
SIMILAR TO (INDISCERNIBLE) WORK,
SO WE SEE MORE DETAIL AND AT
MUCH HIGHER RESOLUTION. THE
SECOND COMPLEX WE OBSERVE IS TF
2H WITH XPA. XPC STILL THERE
BECAUSE WE KNOW THE C TERMINAL
OF XPC STUCK ON TO THE TF 2H
HERE BUT THE BULK OF THE DOUBLE
STRAND DNA INCLUDING LESION AND
XPC WAS NOT THERE BECAUSE IT IS
-- WE CAN SEE A BLOB MOVING. SO
THE REMAINING STRUCTURE BECAUSE
THE PRESENCE OF XPA, THIS TF 2H
DNA XPA IS VERY SIMILAR TO
PATRICK KRAMER AND
(INDISCERNIBLE) STRUCTURE SO IN
SENSE OF XPA THE CORE 7 IS U
SHAPE AND IN THE PRESENCE OF XPA
THERE IS A BIG CONFIRMATIONAL
CHANGE. AND LESIONS OUTSIDE OF
THE CORE 7. THE MOST REWARDING
COMPLEX IS OBSERVE XPCC. WITH XA
AND TF 2H ON DNA WE CAN SEE
WHERE THE LESION IS AND THE
RELATIVE TO THE TF 2H. AND YOU
CAN SEE THIS LOWER PORTION OF
THE TF 2H IS SIMILAR TO THE
STRUCTURE WITHOUT XPC OR WITH
XPC MOBILE. HERE WE SEE BOTH XPC
AND XPA PRESENT. SO IN THE
FOLLOWING SLIDE I WILL SHOW YOU
A FEW DETAIL, HOW XPC RECOGNIZE
DNA LESION. AND RECRUIT TF 2H.
SO THIS IS A HUMAN XPC TRIMER
AND CONTAIN MOSTLY XPC WHICH HAE
STABILIZE THE XPC THOUGH RED 23
DOES NOT INTERACT WITH DNA
DIRECTLY BUT STABILIZE THE
STRUCTURE OF XPC TO BIND DOUBLE
STRAND DNA DOWNSTREAM OF THE
LESION. AND WHAT WE FOUND ALSO
IS THE CENTERING 2 WHICH IS
KNOWN TO STABILIZE XPC ON DNA
BEFORE WITH YEAST STRUCTURE AND
THE NEW PORTION OF THE STRUCTURE
THAT IS NOT OBSERVED HERE JUST
SHOWS THE RED 23 DIFFERENT VIEW
FROM MERE RED 3 AND CENTERING 2
NEITHER TOUCH DNA BUT STABILIZE
XPC. AND THE NEW STRUCTURE WE
FOUND WHICH IS NOT OBSERVE
PREVIOUS WITH YEAST PROTEIN IS
HERE END TERMINAL VERY LONG
HELIX EXTENDING ACROSS DNA IN
THE EXTENDING FOR NON-HELIX END
TERMINAL SO OHM. THERE IS ALSO A
LONG HELIX AT C TERMINAL SO LONG
HELIC C. THE SAME THING I
MENTIONED WAS NOT KNOWN BEFORE.
AND COMPARED TO THE PREVIOUS
STRUCTURES OBSERVED BY
(INDISCERNIBLE) LAB OF THE YEAST
WITH DNA, THERE'S SOME
DIFFERENCE NOT ONLY PROTEIN WAS
NOW WE OBSERVE IN THE PRESENCE
OF TF 2H, THE DNA DOWNSTREAM
BINDING BY RAD 4 XPC IS SIMILAR
BUT AFTER THE LESION UPSTREAM
DNA HAVE VERY DIFFERENT BENDING
ANGLE AND IN THE PRESENCE OF TH
TF 2H DNA IS MORE STRAIGHT AND
HAVE MORE INTERACTION WITH BETA
DOMAIN 3 WHICH INTERACTIONS WITH
THE LESION AND UPSTREAM DNA.
OKAY. SO WHY WE HAVE THE LONG
END TERMINAL HELIX AND LONG C
TERMINAL HELIX. SO WE CAN SEE
THAT IS WHERE EXTENSION OF THE
XPC RECRUIT THE TF 2H. AND OHN
INTERACTING WITH THE -- WE CALL
THE XPD ARM OF THE TF 2H, OHC
INTERACTING WITH THE XPB ARM OF
THE TF 2H. HERE IS THE ZOOM IN
OF THE C TERMINAL OF OHC XPC
INTERACTING WITH XPB, P 8 AND P
52 SUB UNIT AND LITTLE BIT OF P
32 SUB UNIT OF CORE 7. END
TERMINAL HELIX INTERACTING WITH
P 62 AND ALSO XPD. THIS
INTERFACE IS VERY SMALL AS YOU
CAN SEE HELIX AND EXTENSION TINY
BIT BUT WE DIDN'T START FROM THE
RESIDUE 1 OF XPC, BEFORE RESIDUE
167 XPC IS DISORDERED. EVEN
THOUGH IT IS PRESENT. BUT
PREVIOUS STUDY OF ISOLATE
PEPTIDE OF XPC BEFORE RESIDUE
167 HAS BEEN SHOWN TO
INTERACTING WITH P 62 SUB UNIT
WHICH IS LP -- XPD ARM. SO WE
DIDN'T OBSERVE THIS BECAUSE THIS
MODEL PROBABLY IS FLEXIBLE
RELATIVE TO REST OF IT SO WE
COULDN'T SEE BUT SUSPECT I MUST
EXIST BECAUSE EVEN THIS ISOLATED
PEPTIDE OF XPC CAN INTERACT WITH
THE DOMAIN PH DOMAIN OF P 62.
THIS KIND OF ANCHORING NOT ONLY
ORIENT HOW TF 2H APPROACH DNA
WITH XPD BINDING DOUBLE STRAND
DNA UPSTREAM OF THE LESION.
MOREOVER, THIS ANCHORING ALLOWS
THE FLEXIBILITY OF XPC WHICH YOU
CAN SEE IN THE CRYOEM STRUCTURE
EVEN WHEN BINDING TO SITE 5 WE
STILL SEE XPC HAVE AT LEAST TWO
MAJOR CONFIRMATION AND MANY IN
BETWEEN. THIS FLEXIBILITY
PROBABLY ENABLES XPC TO BIND ALL
KIND OF LESION HAS THAT
ADAPTABILITY AND IT IS NOT ONLY
THIS MOVEMENT RELATIVE TO TF 2H
BUT BETWEEN EVERY DOMAIN TDB TO
BETA DOMAIN 123, EVERY DOMAIN
CAN HAVE RELATIVE MOVEMENT.
FLEXIBILITY MAY HAVE HIGHEST
BINDING AFFINITY. NOW WE SWITCH
OUR ATTENTION TO WHAT IS THE
ROLE OF XPA. SOME YEARS AGO IN
2015 BEFORE ANY STRUCTURE OF
EVEN TF 2H WE LOOK AT HELOCASE
ACTIVITY OF TF 2H. WE DESIGN DNA
SUBSTRATE WITH 5 PRIME OVERHANG
BECAUSE XPD MOVES 5 PRIME TO 3
PRIME. THE MOVEMENT IF THERE IS
A LESION, THE HELOCASE SUBSTRATE
BECOME PRODUCT AND THE LESION
WILL STALL LEE LA CASE SO LESS
PRODUCT IS FORMED. HELOCASE, SO
LESS PRODUCT IS FORMD BY THE
LESION. THE DIFFERENCE IS NOW
HUGE, THESE ARE VERY LIMITED
ASSAY BUT IF WE ADD XPA IN
ADDITION TO TF 2H NOW WE SEE THE
HELOCASE ACTIVITY ARE NORMAL DNA
IS MUCH BETTER AND ON LESION DNA
IS (INAUDIBLE) SO CLEARLY XPA IS
DOING SOMETHING TO HELP HELOCASE
IN GENERAL, SPECIFICALLY
RECOGNIZE LESION STALLED XPD. SO
HERE IS THE STRUCTURE I SHOW
BEFORE WITH TF 2H HERE, AND XPD
-- XPC ON TOP, THIS IS THE
COMPLETION JUST BETWEEN XPC AND
TF 2H. WHEN XP IS PRESENT, WHAT
YOU SEE IS DRAMATIC
CONFIRMATIONAL CHANGE OF THE
CORE 7 FROM U2 SIGMA, MOREOVER
IN THIS COMPLEX XPD DOES NOT
INTERACT WITH DNA AT ALL, VERY
MUCH LIKE PRE-INITIATION
COMPLETION IN TRANSCRIPTION.
HERE IN THE PRESENCE OF XPA XPD
INTERACTS WITH DOUBLE STRAND
DNA. AND MOREOVER INSTEAD OF
LESION BETWEEN XPD AND XPD, NOW
LESION IS SHIFTED TO OUTSIDE AND
DOWNSTREAM OF XPD, IF XPD GETS
THE LESION CONTAINING STRAND OF
ORANGE COLOR IT WILL BE ABLE TO
SCAN FINDS THE LESION AND STALL
IT. HOW DOES XPA ACHIEVE IS
THIS? XPA IS FUNNY, IT IS
INSERTED IN TO BETWEEN XPC AND
DNA AND C TERMINAL OF XPA IS
ANCHORED. PH P 52 AND XPD ARM
AND IT IS 35-ANGSTROM AWAY FROM
XPC ANCHORING SO THIS PORTION OF
THE TH 2H YOU CAN SEE IS REALLY
IMPORTANT FOR HOLDING XPC DOING
INTERESTING CHOREOGRAPHY OF
MOVEMENT PROTEIN CHAIN
CONFIRMATION AND SHIFTING DNA.
HERE XPC RESULT IN XPA IN THE
PRESENCE OF XPA XPC DIDN'T LET
GO OF THE LESION AND THE HD 3
DOMAIN TRAIN TAPING HOLD OF THE
NON-LESION STRAND AND FLIPPING
OF THE LESION. SO POSITION OF
LESION IS SHIFTED AND IS
WONDERFUL NOW I WILL JUST GIVE
YOU A LITTLE MORE DETAIL HOW TF
2A STRUCTURE CAN CHANGE THIS
MUCH WHERE ARE THE FLEXIBLE
PORTION. HERE IS A REVIEW OF TF
2H WITH XPC AGAIN AND COLORING
IS XPB XPD ARE HIGHLIGHTED. WE
FOCUS ON CORE 7 OF TF 2H ONLY.
SO THIS LOOK LIKE XPB AND XPD ON
THE TIP OF THE U ARM XPD AND XPD
ARM, U SHAPED STRUCTURE. P 44 IS
ON THE XPD ARM AND MIDDLE IS P
34 AND P 52. HOLD TOGETHER IS P
62 IS VERY IMPORTANT TO TIE THE
P 62 OF PXPD, P 44, P 34 AND P
52 TOGETHER. SO IT IS THE XPB
ARM AND XPD AROUND. XPD ARM
INVOLVED THE MOVEMENT OF P 44
WITHIN THE P 44 THERE ARE
MULTIPLE DOMAIN THEY CAN SEE THE
BIGGEST MOVEMENT IS TWO DOMAIN,
BIG ROTATION WITHIN P 44 AND
THAT MOVES THE XPD RELATIVE TO
THE XPD ARM. P 62 IS VERY
IMPORTANT TO HOLD ARM TOGETHER.
NOW WE FOCUS ON THE XPB AROUND.
THE MOVEMENT ACTUALLY OCCUR
WITHIN P 52, P 52 HAVE FOUR
DOMAINS. THERE IS MOVEMENT
BETWEEN FIRST DOMAIN AND LATER
TWO DOMAIN. AND AT THE MIDDLE OF
THE U SHAPE P 34 AND P 52, THE
INTERFACE IS STABLE. AND NOW YOU
CAN SEE THE INTERFACE BETWEEN
XPD AND P 44 STABLE, XPB BETWEEN
PA AND P 52 ARE STABLE SO THE
MOVEMENT OCCURS REALLY WITHIN P
44 AND P 52 AND LEADING TO THIS
BIG CONFIRMATIONAL CHANGE. AND
WHAT P -- XPA DOES IS NOW WE
ZOOM INTO THE XPA XPC ARM XPV
AND XPD STRUCTURE. XPA IS A VERY
SMALL PROTEIN, TOTAL 273 RESIDUE
SO WE MISSED THE FIRST 100
RESIDUE OF XPA REMAIN DISORDERD
IN STRUCTURE AND IT IS IMPORTANT
FOR INTERACTING WITH RPA AND
XPF, ET CETERA. HERE WHAT WE SAW
IS THE MIDDLE CENTER CORE
STRUCTURE WE CALL BE-- ALPHA
BETA DOMAIN OF FPA, ENTEROR VERY
INTERESTING. IT IS THE FIRST
TIME WE SEE INTERACTING WITH
DOUBLE STRAND DNA AND PREVE --
UNLIKE PREVIOUS STRUCTURES. THIS
ALPHA BETA DOMAIN OF XPA MIMIC
LIKE THE BETA DOMAIN OF THE XPC.
SO THIS BHD 3 OF XPC SIN SETTED
BETWEEN THE TWO STRAND FLIP OUT
THE LESION AND BINDS THE
NON-LESION BASE, RECOGNIZE
SPECIFICALLY NOW LESION, MOST XP
ALPHA BETA DOMAIN ALSO BINDS THE
DNA AND THE DNA CLEARLY BANNING
50 DEGREES AND HAS IN THE
TRYPTOPHAN AND INTER COLLATED
DNA, SO CAUSES BINDING IN THE
BAND SO XPA BINDS 7 BASE PAIR
UPSTREAM OF THE XPC WITHOUT
INTERFERING THE DNA BINDING BY
XPC BUT IT IS INSERTED BETWEEN
OHC AND DNA UPSTREAM HERE. AND
THE RESULT IS THAT BY XPA
SHIFTING THE DNA AND BENDING DNA
NOW THE WHOLE PART OF CORE 7 TF
2H RELATIVE TO XPC WHICH DOESN'T
CHANGE ITS POSITION IS SHIFTED.
SO WITHOUT XPA THE DNA IS
SHOWING GRAY AND IT IS MORE OR
LESS STRAIGHT. IN THE PRESENCE
OF XPA THE DNA SHIFTED NEARLY
TEN BASE PAIR WHOLE HELICAL TURN
OR XPC BIND WHICH IS HERE IN THE
ABSENCE OF XPA, IN THE PRESENCE
OF XPA NOW SHIFTED THERE. SO IT
IS COMPLETELY CHANGED THE
REGISTER OF XPC LESION RELATIVE
TO THE TF 2H OR 7. AS I
MENTIONED BEFORE, THIS
MANIPULATION OF PROTEIN IN DNA
DEPEND ON XPA C TERMINAL DOCKING
TO THE XPB ARM OF THE TF 2H. SO
THIS IS THE LINKER OF THE XPA
SHOWN IN RED FROM TRYPTOPHAN 235
TO 273, VERY C TERMINAL FOR BETA
DOMAIN INTERACTING WITH P 8 AND
P 52. HERE IS XPV. THIS
STRUCTURE EXPLAIN VERY WELL MANY
OF THE XPA MUTATION ARE DUE TO C
TERMINAL TRUNCATION, WITHOUT
THIS ANCHORING XPA COULDN'T
ACHIEVE THE REPOSITION OF TF 2H
RELATIVE TO PC AND PARTICULARLY
THIS HISTIDINE 244 TO ARGININE
IS ONE MYCATION THAT CAUSE
XERODERMA PIG MEN TOE SUM. SO
HERE IS HOW XPA -- PIGMENTOSA.
HOW IT CHANGE TO XPC. YOU CAN
SEE THE DNA BANDING CHANGE OF
POSITION IN THE STRUCTURE OF
TFIIH, YOU CAN SEE THE DNA
BANNING AS WELL. THE CHANGE. NOW
I SHOW YOU THE ADDITIONAL VIEW,
YOU CAN SEE THE CHANGE OF THE
STRUCTURE AND THE DNA BANNING
AND ALSO THE LIFTING OF LONG
HELIX AT THE C TERMINAL OF XPC
TO ACCOMMODATE XPA. PULLING
TOGETHER THE SHIFT OF THIS
CHANGE OF THE CONFIRMATION SHIFT
OF TFIIH ALLOW THE LESION TO
FALL OUTSIDE OF THE TF 2H. THIS
BANDING PLUS THE XPC DISTORTION
OF DNA EVENTUALLY WILL ENABLE
UNWINDING OF THIS PORTION OF
DUPLEX AND DELIVER THE LESION
STRAND TO PPD 4 BINDING. BECAUSE
IN THE STRUCTURE STUDY WE DIDN'T
ADD ATP AT ALL, SO HELOCASE NOT
MOBILE, ALL THE STRUCTURE JUST
EQUALIBRIUM OF THE ADOPTED. HERE
IS XPC RECRUITING LONG TERMINAL
TO RECRUIT TFIIH AND PRESENCE OF
XPA CHANGE CONFIRMATION AND
SHIFT THE TFIIH TO BE UPSTREAM
OF THE LESION. AND WE HAVEN'T
SEEN THE TOTAL DISSOCIATION SOFT
XPC BECAUSE IT IS C TERMINAL
STILL ANCHORED AROUND THE XPB
ARM OF THE TFIIH. SO WE ARE
WAITING FOR FUTURE STUDY. I WILL
USE THE REMAINING TIME TO
ADDRESS TWO BURNING QUESTIONS
FOR US. FOR US WE ARE
INTERESTED IN THE MECHANISM FOR
HOW XPB AND XPD ARE BOTH ATPASE
AVOID AND THEY HAVE OPPOSITE
POLARITY AND HOW THEY CAN AVOID
TUG OF WAR AND COOPERATE TO
FACILITATE DNA LESION REMOVAL.
SO HYPOTHESIS IS TWO HE LA CASE
WORK TOGETHER TO GENERATE DNA
BUBBLE OF THE 20 SOMETHING
NUCLEOTIDE TO 30 NUCLEOTIDE LONG
SO XPF AND XPG CAN CLEAVE AT
BORDER OF THE BUBBLE AND THE
COMPLEX DNA. -- DUPLEX DNA. WE
WILL TOUCH HOW GLOBAL GENOME NER
AND TRANSCRIPTION COUPLED NER
CAN CONVERGE ON TFIIH AND FOR
NER. SO AS I MENTIONED, BEFORE
WE STARTED STRUCTURAL STUDYING
OF BIOCHEMICAL STUDY WAS ALREADY
ESTABLISHED THAT XPB IS A 3
PRIME TO 5 PRIME DNA TRANSLOW
CASE AND XPB IS A 5 PRIME TO 3
PRIME HELOCASE. IN 2015 OR
BEFORE WE HAVE NO IDEA HOW THE
TFIIH BIND DNA SO WE WERE
PUZZLED WHETHER TWO HELOCASE
BOOST AND TRANSLOCATE ON THE
LESION STRAND. IT IS A BIT OF
TROUBLESOME BECAUSE WE MOVE
OPPOSITE DIRECTION THOUGH XPD IS
A STRONG STRONG MOTOR AND XPB IS
A WEAK MOTOR SO IT CREATES A TUG
OF WAR. THE POSSIBILITY ALSO IS
XPB XPD MOVE ON THE LESION
STRAND WHICH WE KNOW ANY LESION
DOWNSTREAM OF XPD WILL BE FOUND
AND CAN DETERMINE WHETHER WHOLE
HELOCASE CAN BE STALLED OR KEEP
MOVING. WE HYPOTHESIS, IT IS
ALSO POSSIBLE FOR XPB MOVING ON
THE OTHER STRAND, NOT ONLY
CONTAINING STRAND AND THE RESULT
IS SUCH WE WOULD BE MOVING IN
PARALLEL AND HELPING EACH OTHER
SO THIS BECOMES SORT OF IN MY
MIND MAKES SENSE SO WE DID MORE
EXPERIMENT, FIRST WE USE THE
SUBSTRATE WITH 5 PRIME OVERHEAD
SO XPD CAN BIND DNA. WE DID
BINDING ACTIVITY ASSAY USING MZA
YOU CAN SEE THE SHIFT IS
MINIMAL. YET THE HELOCASE IS
VERY GOOD. YOU CAN SEE UNWIND
THE DNA. WHEN WE TRY A DIFFERENT
SUBSTRATE WITH 3 PRIME OVERHANG
SUPPOSEDLY XPB CAN UNWIND. WE
FOUND THE BINDING IS EXTREMELY
GOOD, THE HELOCASE ACTIVITY IS
NEXT TO NOTHING. SO NOW WE USE
THE BEST SUBSTRATE AND TRIED
FIGURE OUT WHICH DOES WHAT. SO
THERE'S TWO POSSIBILITIES. WE
KNOCK OUT XPB HELOCASE ACTIVITY,
TAKE THE ATPASE ACTIVE SITE AND
THE LEFT XPD TO WORK ALONE. THE
SUBSTRATE GETS UNWOUND AND IF
THERE IS A LESION IN THE
SUBSTRATE NOW THE UNWINDING
ACTIVITY IS VERY MUCH
DIMINISHED. SO THAT MAKES SENSE.
THEN WE KNOCK OUT PPD ACTIVITY
AND LET PPD WORK ALONE WITH THIS
SUBSTRATE WHAT YOU SEE IS EVEN
WITHOUT XPD PPD WHICH IS
SUPPOSEDLY MOVING THIS
DIRECTION, HERE WE MOVE OPPOSITE
DIRECTION, WE STILL SEE THE DNA
STRAND BEING SEPARATED AND
LESION STILL HAVE EFFECT ON
STALLING THE TFIIH. SO AT THAT
TIME WITH LIMITED SUBSTRATE
STUDY AND RESULT, WE SORT OF
FAVOR THIS MODEL. THIS DATA
LOOKS CONVINCING BUT WITH
STRUCTURE OF TFIIH REPORTED A
YEAR LATER AND WITH OUR MORE
RECENT STUDY, ACTUALLY HOW IT
WORKS. IT IS THIS WAY. I WILL
TELL YOU WHY. IT IS XPB AND XPD
ON THE SAME STRAND. POOR BINDING
AND POOR UNWINDING THIS
FAVORABLE SUBSTRATE IS BECAUSE
XPB HAVE HIGH AFFINITY FOR
DOUBLE STRAND DNA AND XPD
DOESN'T HAVE STRONG AFFINITY. SO
THIS IS A POOR BINDING SUBSTRATE
I SHOW IN THE PREVIOUS SLIDE BUT
XPB STILL HAVE HELOCASE ACTIVITY
SOMEWHAT. THEN XPD THOUGH
WITHOUT ATPASE STILL RECOGNIZE
LESION. AND STALL LESION PREVENT
PPB FURTHER MOVING. THOUGH XPD
ALONE HAVE WEAK BINDING AFFINITY
FOR SINGLE STRAND DNA RELATIVE
TO XPB FOR DOUBLE STRAND DNA XPB
IS PA GOOD HELOCASE AND HAVE
GOOD UNWINDING. THE OTHER
SUBSTRATE WE TRIED XPB AND XPD
HAS GOOD BINDING BUT DOMINANT
MOTOR OF XPB MOVES OPPOSITE
DIRECTION TO THE DOUBLE STRAND
DNA SO NO UNWINDING, OR VERY
LITTLE. AND THE PROBLEM WHEN WE
HAVE THIS MODEL PUT TWO HELOCASE
ANTI-PARALLEL TWO STRAND INSTEAD
OF ONE STRAND WE DIDN'T USE THE
PROPER SUBSTRATE WHICH IS THE
BUBBLE DNA. WITH LESION
DOWNSTREAM OF XPD. SO THIS IS
THE KILLER AND I WANT TO REMIND
MYSELF AND EVERYONE OUT THERE,
WHEN WE DO ANY EXPERIMENT IF THE
-- WE HAVE GROPING THE ELEPHANT
BY USING OUR TWO HANDS SO WE
REALLY DON'T SEE THE WHOLE IMAGE
UNTIL THERE'S WHOLE IMAGE. AND
WE SHOULD BE CAREFUL IN TERMS --
INTERPRETING OUR RESULT. BUT
MODEL IS WRONG AND IT IS GOOD
STARTING PLACE AND WE CORRECT
THE MODEL SO HERE IS THE WHOLE
STRUCTURE OF TFIIH WITH XPC AND
XPA ON DNA. HERE WHEN PXC IS
MOBILE AND THE LESION IS ALMOST
INVISIBLE BECAUSE IT IS MOVE XPC
AND ALL STRUCTURE AVERAGED OUT.
IN OUR STRUCTURE WHAT WE CAN SEE
IS DOUBLE STRAND BIND TO XPB AND
THERE'S TWO NUCLEOTIDE, ALMOST
SINGLE STRAND BIND TO XPD BUT
NOT THE LESION ITSELF CANNOT BE
OBSERVED. BORROWING THE
STRUCTURE FROM PATRICK KRAMERS
LAB THOUGH THE DNA HAVE NO
LESIONS CLEAR THIS HAS TO BE THE
LESION STRAND. AND THE LESION
HAS TO BE DOWNSTREAM OF THIS
SUPPOSEDLY CLUSTER AND RECOGNIZE
THE LESION AND POSSIBLY WE FIND
HERE IS ANOTHER GATE WHERE
LESION MAYBE STALLED. SO WE KNOW
THE LESION STRANGLES THROUGH
THIS. THAT IS VERY NICE, WE CAN
TRY TO UNDERSTAND WHY THE TWO
MOTOR WORK TOGETHER TO SEPARATE
THE DNA. SO IN THIS FULL
SUBSTRATE XPB BIND DOUBLE STRAND
DNA. BUT THE TWO MOTOR DOMAIN
HELOCASE DOMAIN 1 AND 2 GRABS
ONLY ONE STRAND, THAT'S THE
ORANGE STRAND, LESION CONTAINING
STRAND. AND THIS IS A BASED ON
MANY STUDIES SO WE KNOW AND ALSO
THE CLOSE INTERACTION OF HD 1
AND 2 IS THE LESION STRAND. AND
WE KNOW FROM PREVIOUS STUDY XPB
MOVES 3 PRIME TO 5 PRIME. AND IF
THE DNA IS MOBILE BUT IF --
IMMOBILE. IF DNA IS MOBILE
PROTEIN IS MOBILE THE DOUBLE
STRAND DNA WILL BE MOVING
TOWARDS THE RIGHT. XPA IS THE
KEY PROTEIN TO MAKE XPB A
HELOCASE. WITHOUT XPA, XPB IS A
SINGLE TRANSLOW CASE, MOVING THE
DOUBLE STRAND TO THE LEFT. AND
THAT IS HOW IT WORKS IN
TRANSCRIPTION JUST MOVING DOUBLE
STRAND TOWARDS RNA POLYMERASE.
AND THE RNA POLYMERASE IS ONE
TRAY SEPARATE THE DOUBLE STRAND
DNA. HERE IN NUCLEOTIDE EXCISION
REPAIR PPB WORK WITH XPA WHICH
IS A WEDGE STRAITING HERE IS
TIPTOE FAN I TOLD YOU BEFORE --
TRYPTOPHAN I TOLD YOU BEFORE IN
DNA MAKE A BAN BAND. WHEN THE 2
STRAND IS SEPARATED IT BECOME A
WEDGE TO SEPARATE TWO STRANDS.
SO THE TWO STRAND MOVING HERE
ONE IN EXACT BA ALPHA BETA
DOMAIN AND TRYPTOPHAN 175 TWO
STRAND SEPARATE AND XPB CAN BE A
HELOCASE. XPA IS A HELOCASE WITH
A GOOD MOTOR AND CAN MOVE ITSELF
DOWN THIS WAY OR AL ATTORNEY
TESTIFILY IF PROTEIN NOW MOVING
IT CAN PULL IN THE SINGLE STRAND
DNA THIS WAY SO THE LESION WILL
ENTER THE PROTEIN AND BE SCANNED
AND WORK AS PAST THE XPD OR
STALL THE HELOCASE. WE FIND THE
TO BE POSSIBILITY LOCATION WHERE
LESION CAN BE STALLED, EITHER AT
THE IRON SULFUR CLUSTER ANOTHER
THE GATEWAY WHERE THE SINGLE
STRAND INTO EXIT XPD AND EXIT IS
VERY SMALL, IF IT IS A BULKY
LESION IT CANNOT GET OUT. SO
PUT TOGETHER I SHOW YOU HERE,
HOW THE WE THINK HOW THE TFIIH
WITH HELP WITH XPA BECOME A
MOTOR AND GENERATE A ENLARGE THE
BUBBLE OF OVER 20 SOMETHING
NUCLEOTIDE FOR XPF AND XPD TO
CLEAVE. SO ONCE LOADED THE DNA
LOADING BY XPC OR BY RNA
POLYMERASE, XPB WANT TO MOVE TO
THE RIGHT, XPB WANT TO MOVE
LEFT, THIS IS A DOMINANT MOTOR,
IT SEES THE SINGLE STRAND DNA,
IT GOES. THE LESION IS A SMALL
LESION. SUCH AS MISMATCH DNA
WHICH IS A VERY WELL RECOGNIZE
XPC OR BASIC SITE. XPD GOES
THROUGH. WITHOUT SEEING IT AND
NO NUCLEOTIDE EXCISION REPAIR.
BUT IF THE LESION IS BULKY, XPA
HAPPILY CHUCKING ALONG BUT THE
LESION IS STALL AND MOTOR CAN NO
LONGER WORK. NOW XPB IS THE ONLY
MOTOR. AND IT WANTS TO GO TO THE
LEFT BECAUSE THE WHOLE TFIIH
WITH XPA IS STUCK ON THE DNA
BECAUSE THE LESION. SO THE WHOLE
CONQUEST CANNOT MOVE AND PROTEIN
BECOME IMMOBILE. AND
ALTERNATIVELY, IS THE DNA IS
BEING SHUFFLED TO THE RIGHT
BECAUSE THE XPB TRANSLOW CASE
ACTIVITY. XPA IS THE WEDGE,
SEPARATED DUPLEX. THE TWO
TOGETHER MAKING BIGGER BUBBLE.
EVENTUALLY IT IS BIG ENOUGH WITH
THE RPA SINGLE STRAND BINDING
PROTEIN HELP XPD WILL CLEAVE
HERE AND XPF WILL CLEAVE ON THE
5 PRIME END OF THE LESION. HERE
SHOWS TO US XPB IS REALLY
IMPORTANT TO GENERATE THIS
BUBBLE XPD ONCE XPD IS STUCK.
MANY YEARS AGO WHEN THE DUAL
INCISION NER DUAL EXCISION WAS
ASSAYED USING CIRCULAR PLASMID
DNA SUPER COILED, THERE WAS AN
INTERNAL LABEL OF THE DNA, SO
THE LAB CARRY OUT THIS STUDY AND
THEY FOUND IS OF COURSE WITH
EITHER TFIIH TEN SUBRAHMANYAN OR
CORE 7 THE ACTIVITY DOING
EXCISION PRODUCT IS SIMILAR. BUT
IF WE KNOCK OUT XPB ATPASE
ACTIVITY YOU SEE NO DUAL
INCISION AT ALL. WHEN WE KNOCK
OUT XPB ATPASE YOU CAN LOOK
CLOSE AND STILL SEE SOME PRODUCT
SO XPB IS VERY IMPORTANT,
ESSENTIAL AND XPD WITHOUT ATPASE
CAN STALL LESION AND MAY STILL
HAVE NER ACTIVITY. SO I THINK I
WOULD LIKE TO PUT THE --
TOGETHER IN SPECULATION IN A WAY
HOW NER AND TC NER CONVERGE ONCE
XPA IS RECRUITED AND TFIIH AND
XPA VERIFY LESION. SO YOU CAN
ALREADY SEE XPC MAINLY BIND
DOWNSTREAM DOUBLE STRAND DNA.
AND ALSO RECOGNIZE THE
NON-LESION STRAND OPPOSITE THE
LESION. AND THE END TERMINAL AND
C TERMINAL LONG HELIX OF XPC
ORIENT THE LOW TFIIH XPB AND
XPD. XP ENABLED TRANSFORMATION
OF TFIIH AND REPOSITION OF TFI,H
RELATIVE TO LESION XPC. IN TC
NER I'M BORROWING THE STRUCTURE
OF PRE-INITIATION COMPLEX. THE
RNA POLYMERASE IS ALSO LOCATED
DOWNSTREAM OF THE XPB AND XPD.
IN THIS COMPLEX HOWEVER RNAP
REALLY READING OUT THE LESION
STRAND RATHER THAN
NON-TRANSCRIBED LESION,
NON-LESION STRAND. XPD, THE SAME
HERE, IS NOT TOUCHING DNA AT
ALL. XPB IS INTERACTING WITH
DOUBLE STRAND DNA AS I POINTED
OUT. WITHOUT XPA AS A WEDGE
SEPARATING THE TWO STRANDS. XPB
SERVING AS A TRANSLOW CASE. SO
IT PUSH THE DNA TOWARDS RNAP SO
ALLOW THE TWO STRAND TO
SEPARATE. ONCE THE INITIATION
BUBBLE IS FORMED ACTUALLY
PHOSPHORYLATION, ET CETERA, PXB
XPD IS RELEASED AN TRANSCRIPTION
CAN START. BUT IN NUCLEOTIDE
EXCISION REPAIR IN THE PRESENCE
OF LESION AND VARIOUS PROTEIN
INCLUDING CSB WHICH MAKES SURE
XP -- RNAP IS REALLY STUCK, WE
DON'T KNOW EXACTLY HOW THE TFIIH
IS RECRUITED BACK. CSB CSA AND
VARIOUS MODIFICATION
UBIQUITINATION INVOLVED. BUT
THE RESULT OF THIS COMPLEX
FORMATION IS RNAP IS LEADING
RATHER THAN TFIIH LEADING. WITH
HELP OF XPA, THEN THIS IS THE
COMPLEX WE OBSERVED THAT REALLY
READS OUT WHETHER THERE IS A
LESION OR NO LESION WITH NER
SHOULD BE INVOLVED. I THINK I
WILL JUST USE LAST COUPLE OF
MINUTES TO LET YOU KNOW WHAT WE
ARE WORKING ON NOW. SO WE
ACTUALLY PURIFIED RPA XPD AND
XPF AS WELL SO WE COME TO
RECONSTITUTE WHOLE NER AND WE
USE DIFFERENT SUBSTRATE OF BULKY
LESION AND NOT A BULKY LESION
BOTH IN BINDING AND EXCISION
REPAIR ASSAY AND THE BULKY
LESION TURN OUT DOES NOT BIND BY
XPC TFIIH AND SPA AS WELL AS THE
NON-LESION, NON-BULKY LESION,
TWO BASIC SITE HAVE DISSOCIATION
LIKE LESS THAN 2.5 NANOMOLAR OF
THIS IS CY 5 AND CY 5 IS NOT A
NATURAL SUBSTRATE ANYWAY. BUT
INTERESTINGLY, EVEN THOUGH IT IS
A WEAKER BINDING, XPA AND XPC
AND TFIIH, IN REPAIR THERE IS A
DIFFERENCE, WE DEVELOPED ASSAY
USING OLIGODNA, THE REPAIR
EFFICIENCY WAS VERY GOOD, WE CAN
OBSERVE EVEN NEARLY 80% OF
SUBSTRATE BECOME DUAL EXCISION
PRODUCT SHOW HERE SUBSTRATE 94
BASE PAIR PAIR AND DUAL EXCISION
RESULT SPREAD FROM 27 TO ABOUT
34. AND WITH CY 5 WE SEE GOOD
PRODUCT FORMATION, DOING
EXCISION WITH TWO BASIC SITE IS
MUCH WEAKERER. SO SOMEWHERE
SOMEHOW AFTER TFIIH XPA BINDING,
XPG AND RPA FURTHER DETERMINES
IN THE PRESENCE OF ATP THERE IS
A DIFFERENCE IN THE EXCISION
EFFICIENCY. SO OUR GOAL IS TO
REALLY FIGURE OUT HOW BULKY
LESION STALLS TFIIH AND HOW DNA
EXCISION REALLY -- SO WITH THIS
I WANT TO THANK THE REAL HEROES
OF THIS PROJECT. ERIC STARTED
THIS PROJECT MORE THAN TEN YEARS
AGO AND PHILLIP DID A LOT OF IN
VITRO HELOCASE ACTIVITY ASSAY
AND INITIAL EM CHARACTERIZATION,
JINSEOK CARRIED OUT FROM PHILLIP
TO COMPLETION AND ERIC IMAGING
WORKING TOGETHER OBJECT
FOLLOWING STRUCTURE WE HOPE TO
GET. AND (INDISCERNIBLE) WORKED
ON THE PROJECT BEFORE ERIC.
NADINE CLONED XPA XPA PURIFIED
THE PROTEIN RPA AND REALLY LAID
GROUND. WITH FELLOWSHIP OF THREE
YEARS, JUST NOT POSSIBLE TO
COMPLETE. AND TO WORK ON
SOMETHING WITH BARING RESULT
ENABLE TO MOVE ON AND NOW IS A
TENURE TRACK FACULTY AT NIH.
CRYOEM WORK CAN BE ACCOMPLISHED
WITHOUT (INAUDIBLE) HELP AND
WITH FACILITY AT NIDDK,
SUPERVISED BY (INDISCERNIBLE).
LAST BUT NOT LEAST,
(INDISCERNIBLE) APPROACH ME IN
EARLY 2010 AND STARTED THIRD
DEGREE PROJECT. IT TAKES A LONG
TIME BUT ACTUALLY IT IS
REWARDING. THANK YOU.
>> THANK YOU, DR. YANG FOR THE
WONDERFUL TALK. I BELIEVE
EVERYBODY AGREE THAT THE RESULT
IS VERY STRIKING AND VERY
IMPRESSIVE. CAN SEE THE
DEDICATED MOVEMENT OF THE
COMPRESSION TO HELP US
UNDERSTAND HOW IT WORKS. SO I
CAN TELL THERE ARE SOME
QUESTIONS TYPED IN THE CHAT BOX.
SO THE FIRST ONE IS BY CHERYL
WHICH IS MOSTLY ABOUT GENERAL
DNA REPAIR PATHWAY. IS ATR AND
ATM MEDIATED DNA DAMAGE RESPONSE
SOMEHOW PLAY A ROLE WITH TFIIH
RECRUITMENT? LIKEWISE HOW TFIIH
RESPOND TO PARP ACTIVATION?
>> SO AT THIS POINT, I CANNOT
SPEAK OF HOW ATR AND OTHER
PROTEIN GENERAL GENOMIC
INSTABILITY AFFECT TFI,H.
PROBABLY SHARING THOSE MORE THAN
I DO AND I'M ALL EARS. AND
RECRUITMENT OF TFIIH AS FAR AS I
CAN TELL WE CAN TEL
>> WE DO HAVE ONE COMMENT FROM
WILLIAM (INDISCERNIBLE) SAYING
BEAUTIFUL VISUALS, REALLY
ENJOYING THE PRESENTATION. I
THINK HE SPEAKS FOR ALL OF US.
THERE'S ALSO A QUESTION -- DO
YOU WANT TO GO AHEAD?
>> SURE. THERE IS A QUESTION
FROM DMITRI. WHAT WOULD HAPPEN
WITH BULKY LESION CLOSE TO A
DSBN? DO WE EXPECT HELOCASE
ACTIVITY CREATING A LONG SSDI
REGION?
>> IT IS POSSIBLE BECAUSE IT IS
(INDISCERNIBLE) WORK IN 2009, SO
BASICALLY ONCE TFI,H IS
UNLOADED, EVEN LOADED SAY BY A
MISMATCH BASE PAIR, XPD IS SUCH
A STRONG HELOCASE, ONCE LOADED
ON DOUBLE STRAND DNA IT CAN MOVE
ALONG THE DNA. DR.
(INDISCERNIBLE) PLAYS A REAL
BULKY LESION, 100 BASE PAIR AWAY
AND XPD STILL CAN FIND IT AS
LONG AS IT IS LOADED ON THE
RIGHT STRAND. IF IT IS LOADED ON
THE OTHER -- MISMATCH YOU CANNOT
TELL WHICH STRAND IT IS BUT IF
YOU HAVE AN A ASIC SITE, A BASIC
IS THE LESION AND IF YOU
DOWNSTREAM THERE IS A BULKY
LESION, IT WILL BE RECOGNIZED SO
IT CAN TRAVEL AND I DON'T KNOW
HOW LONG IT CAN TRAVEL. AND ALSO
I HAVE TO GIVEN THAT THERE IS A
NUCLEOSOME ON THE DNA, SO THERE
IS A -- PROBABLY REMOVAL OF
NUCLEOSOME LOCALLY THROUGH THE
DDBE 1, DDB 1, DDB 23 BECAUSE
THEY ACTIVATE THE
(INDISCERNIBLE) DISSOCIATE THE
NUCLEOSOME BUT PROBABLY NOT LONG
DISTANCE BECAUSE IT DOESN'T DO
GOOD FOR THE GENOME.
>> A QUESTION FROM BOB, HAVE YOU
BEEN ABLE TO EXCLUDE THE
POSSIBILITY THAT XPD STALLED BY
THE LESION AT A STRAND SWITCHES
TO THE OTHER SIDE OF THE NEW
BUBBLE? OR ANOTHER XPD MOLECULE
LOADS TO THE NON-LESION STRAND
TO EXPAND THE BUBBLE?
>> YEAH, IT IS POSSIBLE BUT I
FEEL LIKE THAT IS VERY MUCH A
SETUP IN VITRO. WE CAN SEE IT.
AND THE REASON IS I GUESS I CAN
USE THIS SLIDE. IN THE PRESENCE
OF XPC THE LOADING IS REALLY
DICTATED BY XPC IN THE LESION.
AND THE SAME WITH RNAP AND TFIIH
RECRUITMENT. WITHOUT THOSE
RECRUITERS, IF YOU JUST HAVE A
BUBBLE, TFI,H CAN LOAD ON EITHER
SIDE, EITHER XPD ON THE LESION
STRAND OR NON-LESION STRAND IF
YOU GIVE A BUBBLE. RIGHT? BUT IF
YOU HAVE THE XPC OR RNAP TO
REPORT WHERE THE PROBLEM STRAND
IS, THEN THE LOADING IS
ABSOLUTELY ONLY ONE WAY. SO THAT
IS WHAT I LEARNED, WE KNOW AS A
BIOLOGIST WE DIVIDE AND CONQUER,
EVEN TODAY WE STILL DIVIDE AND
CONQUER, WE DON'T STUDY WHOLE
ANIMAL, HAVE TO GET TO TISSUE
AND ANIMAL GETS YOU CERTAIN AM
OF READING. AT READING OF
MECHANISM WE GET SMALLER PIECE
AND CRYOEM IS A SAVIOR FOR OUR
STRUCTURAL BIOLOGISTS BECAUSE
ALL THOSE STRUCTURES WE WOULD
NEVER BE ABLE TO CRYSTALLIZE IF
WE HAVE THE BIG -- THERE'S
CONFIRMATIONAL CHANGE SO THIS IS
THE INTACT ONE AND I'M NOT
SHOWING THE XPC AND RNAP. THIS
IS THE WHOLE ENZYME, WHEN YOU
HAVE RNAP OR XPC TFI,H IS LOADED
CORRECTLY. WE ALREADY HAVE SOME
DATA TO INDICATE THE
CONFIRMATIONAL CHANGE I SHOW YOU
THE BIG MOVIE, SURE IT IS KIND
OF HARD TO GET YOUR HEAD AROUND.
IT IS NOT THE END. WITH ATP
PRESENCE, WITH RPA BINDING
SINGLE STRAND DNA AND TWOWE ALWO
KEEP IN MIND WHAT WE SEE IS
MAYBE JUST THE LEG OR EAR OF
ELEPHANT.
VERY TRUE.
WELL, THANK YOU, I THINK
THAT'S FOR ALL THE QUESTIONS.
AND FOR ALL THE AUDIENCE, PLEASE
JOIN ME TO THANK AGAIN DR.
YANG'S WONDERFUL TALK. THANK
YOU, EVERYBODY'S PARTICIPATION.
DR. YANG, THANK YOU AGAIN.
REALLY GREAT TO HAVE YOU HERE.
WE WILL KEEP IN TOUCH.
>> THANK YOU VERY MUCH.
LOVELY STORY, THANK YOU SO
MUCH.
THANK YOU. GOODBYE.
THANK YOU, BYE-BYE.