>> I'D LIKE TO INTRODUCE ORLANDO FOR THOSE THAT DON'T KNOW HIM HE'S A WELL-KNOWN FIGURE IN DNA REPAIR AND GOT HIS SCIENTIFIC START AT IN ZURICH AS A CHEMISTRY UNDERGRADUATE AND MAYBE BEFORE THAT PLAYING WITH CHEMICALS AS A CHILD AND IN 1991 HE BECAME A GRADUATE STUDENT WHERE HE WORKED ON THE MECHANISMS OF DNA AND FOLLOWING THAT IN 1996 HE WORKED ON REPAIR OF GENETICS AND BUY OWE -- BIOCHEMISTRY AND ENDED UP BACK HOME AT ZURICH. IN 2005 HE WAS RECRUITED TO STONY BROOK AND WAS A TENURED ASSOCIATE PROFESSOR AND NOW A FULL PROFESSOR HERE AND I'M DELIGHTED TO INTRODUCE HIM TODAY. >> THANK YOU. LET ME SWITCH TO THE SLIDES AND CONFIRM YOU CAN SEE THE SLIDES. CAN YOU SEE THE SLIDES? >> YES, WE CAN. EVERYONE ELSE PLEASE MUTE. SO TODAY I'LL TELL YOU A LITTLE BIT ABOUT DUEL INCISION AND REPAIR SYNTHESIS THIS IS PART OF THE BROADER QUESTION WE LOOK AT WITH ENDONUCLEUSES AND THERE'S MANY IN DNA REPAIR AND THEY HAVE ONE THING IN COMMON THEY CUT DNA. SO UNLESS YOU DO THAT ONLY WHEN NEEDED THAT'S A DANGEROUS THINK. SO WE'RE INTERESTED IN UNDERSTANDING HOW IT IS THAT SUCH NUCLEUS IS CUT FOR CALCULATIONS AND MISMATCH REPAIR AND REPLICATION FORKS OR PERHAPS AT THE JUNCTION. AND THE TODAY I'LL FOCUS ON TWO AND THEY'RE KNOWN TO MANY OF YOU. SO THE QUESTIONS ARE FOR ONE THING HOW ARE THOSE TYPES OF ENZYMES RECRUITED SUCH THAT THEY ONLY CUT WHERE THEY NEED TO CUT. THE SHORT ANSWER IS THEY'RE REALLY LOUSY ENZYMES ON THEIR OWN SO THEY DON'T CUT WELL OR EVEN THE STRUCTURE OF DNA THAT RESEMBLES BUT ONLY BECOME ACTIVE WHEN THEY'RE BOUND BY OTHER ENZYMES AND PART OF MULTIPROTEIN COMPLEXES. THE SECOND QUESTION WE'RE INTERESTED IN IS WHAT HAPPENS ONCE THE CUT IS MADE? FOR EXAMPLE IT'S PERHAPS LESS DELETERIOUS SO WE'RE INTERESTED IN HAPPENING WHAT HAPPENS ONCE THE CUT IS MADE AND ENSURE THE DNA IS RESTORED AS QUICKLY AS POSSIBLE. WE'LL START SIMPLE AND MOST WILL KNOW A LITTLE BIT ABOUT THE PATHWAY SO NER TAKES CARE OF THE GENOME AND WE'LL FOCUS EXCLUSIVELY ON GLOBAL GENOME REPAIR FOR TODAY AND AFTER THE DAMAGE HAS BEEN RECOGNIZED ON THE SIDE BY THE XPG PROTEIN LEAVING A GAP OF ABOUT 30 NUCLEOTIDES AND TRADITIONALLY THE EXITION EVENT AND LIGATION HAVE BEEN CONSIDERED SEPARATE EVENTS AND WE THINK THEY'RE TIGHTLY CONNECT AND TO GET STARTED I'LL GIVE YOU AN OVERVIEW OF THE PATHWAY. NER TAKES CARE OF THE BULK AND IT RESTORES THE DNA HEALING. WE HAVE THE XPC PROTEIN IS THE FIRST TO RECOGNITION THE LESION A FINDS THE STRAND AND IT LATCHED ON TO THE NON-DAMAGED DNA STRAND. AND THE XPB PROTEIN ALLOWS IT TO TRANS LOCATE OPENING UP A BUBBLE IN THE IT HITS THE DAMAGE AND THEN XPD IS NOT ABLE TO GO PAST THE DAMAGE BECAUSE OF THE BULKINESS AND THAT SENDS THE SIGNAL THERE'S INDEED A LESION. THES THEIR STEP IS THE RECOGNITION OF THE BULKY CHEMISTRY OF THE LESION. IT GENERATES THE BPA PROTEIN AND THE RPA PROTEIN AND XPG PROTEIN. IT'S THE SINGLE STRAND PROTEIN SO IT COVERS THE NON-DAMAGED STRAND OF DNA. XPA IS PERHAPS THE GLUE OF THE WHOLE REACTION THAT MAKES INTERACTION WITH OTHER PROTEINS INNER AND BINDS TO THE DNA IN SOMEWHERE IN THE DAMAGED COMPLEX I'LL SHOW YOU MORE ABOUT THAT IN A MINUTE AND THEN THE XPG PROTEIN IS ONE OF THE TWO ENDONUCLEUSES THAT MAKES THE INCISION. NOTE AT THIS POINT IN THE PROCESS AS I'LL SHOW YOU THE XPG PROTEIN IS NOT CATALYTICALLY STRUCTURED. THE FIRST MODIFICATION TO THE DNA OCCURS UPON RECRUITMENT OF TO THE SIDE OF THE LESION THE XPA PROTEIN AND LET ME QUICKLY SHOW YOU TWO SLIDES OF WHAT'S GOING ON THERE. CAN YOU SEE THE SLIDES? >> WE CAN'T. >> LET ME GO BACK TO THIS? BETTER? NOW? >> NOW WE SEE IT. >> GOOD. ALL RIGHT. SO THE TWO FACTORS HERE XPA RECRUITS XPF TO THE SIDE OF THE LEGION AND A FEW WORDS ABOUT XPA IT'S A RELATIVELY SMALL PROTEIN 273 AMINO ACIDS AND WITH INTERACTION WITH OTHER PROTEINS. ERCC1 HAS A CENTRAL DOMAIN TO IN THE ACT WITH THE ERCCI AND BINDS WITH THE AMINO PROTEIN AND WITH THE LABORATORY WE SHOW THE TURE IN WHITE WITH THE HIGHLIGHTS THAT MAKE THE INTERACTION WITH XPA AND YOU CAN SEE THE PEPTIDE IN MAGENTA THAT AIN'T ACT WITH THE ERCC1. IT'S NOT STRUCTURED WHEN FREE IN SOLUTION BUT CAN INTERACT AND WE'VE SHOWN MANY WAYS THE INTERACTION IS RESPONSIBLE FOR RECRUITING IT TO THE COMPLEX AND IF YOU MAKE MUTATION IN XPA OR IN THE ERCC1 WE ABOLISHED THE INTERACTION AND ABOLISHED THE NER INTERACTION COMPLETELY. THAT LEAVES THE RECRUITMENT AND THIS IS THE REACTION TO THE LESION BY XPF. I'LL SHOW YOU REPAIR SYNTHESIS CAN BE INITIATED PRIOR TO INCISION BY THE PROTEIN LEADING TO THE COMPLETION OF THE PROCESS AND THIS REGULATION OF THE TRANSITION CREATES THE INABILITY AND COUPLES NER TO THE DAMAGE. ALL RIGHT. SO OUR STORY REALLY STARTS ABOUT 50 YEARS AGO AND THAT'S THE WORK DONE AND HE HAD TO DISCOVER THIS REPAIR SYNTHESIS WAS PART OF NER AND ARGUED THERE COULD BE TWO MODELS BY WHICH IT WOULD HAPPEN. YOU GET THE FIRST INCISION AND GET THE RELEASE AND REPLACEMENT AS THE POLYMER REJOINS AFTER THE LABORATORIES RECONSTITUTED THE PURIFIED FACTOR IT BECAME THE PREVAILING MODEL BECAUSE YOU COULD OBSERVE THE CORE FACTORS COULD RELEASE THE NUCLEOTIDE. I WAS UNCOMFORTABLE THAT YOU COULDN'T MAKE SURE OTHER THINGS WERE IN PLACE TO CONTINUE THE PROCESS AND INDEED IN 1996 HE PROPOSED YOU MAKE THE INCISION FIRST AND DO WHAT THE CALLS REPLACEMENT AND PEALING AND START REPAIR SYNTHESIS AND ONCE IT'S MORE OR LESS COMPLETED YOU MAKE THE INCISION AND DRAW IN THE DNAs AND I ARGUE TODAY THIS IS WHAT WE THINK IS HAPPENING. THE FIRST PATH WAS WHEN WE MADE CATALYTIC MUTANTS OF THE TWO PROTEINS TO KEEP THE WHOLE PROTEIN IN FACT BUT MESS UP THE BINDING OF THE ENZYME AND WHEN WE DID THIS AND I WANT TO SHOW YOU THE DETAILED DATA TO SUMMARIZE WE SAW WHEN WE HAVE A MUTATION IN THE CATALYTIC DOMAIN WE DON'T SEE IT HAPPENING AND XPG DOESN'T CUT. WHEN WE DO THE REVERSE NAMELY WE MAKE A MUTATION IN THE XPG PROTEIN SHOWN HERE THIS ACTIVE SIDE HAD BEEN DETERMINED BY THE LABORATORIES WHAT WE SEE THEN IS THAT INDEED THERE'S NO INCISION BY XPG PROTEIN IT'S CATALYTICALLY IN ACTIVE AND THE LESION HAD BEEN SHOWN BY THE XPF PROTEIN. THIS PROVIDED SOME INDICATION PERHAPS THE XPF PROTEIN CUTS BEFORE THE XPG PROTEIN. WE WANTED TO SEE WHAT THAT MEANS FOR REPAIR SYNTHESIS AND WE CAN LOOK AT REPAIR SYNTHET IS AND WE MADE A PLASMA WITH A STRAND INCI INC INCUEBATED WITH THE NUCLEOTIDES AND THEN FORMS THE XPG PROTEIN. WHAT WE CAN DO HERE ON THE RIGHT IS AFTER THE REACTION WE CUT THE PLASMIC REPAIR AND SEE IF IT TAKES PLACE AND WHEN CUT HERE IF YOU DON'T ADD PROTEIN YOU CAN SEE THE BACKGROUND BAND AND IT'S AT THE LEVEL OF THE OTHER BANDS. IF YOU ADD THE XPG PROTEIN YOU CAN SEE THE BAND IS MUCH INCREASED. IT IS NOT INCREASED IF YOU ADD THE CATALYTIC MUTANT PROTEIN. WHEN I COMBINE IT WITH THE CELL EXTRACT IT LEADS TO THE BACKGROUND SIGNAL BUT IT'S INCREASED IN THE XPG SITUATION WHEN YOU COMPARE THE UPPER BANDS WITH THE MAIN BAND OF NUCLEOTIDES. THAT WAS WHAT WE EXPECTED BUT AFTER LOOKING AT IT FOR A WHILE WHAT WE FOUND IS WHAT HAPPENS IN THE XPG MUTANT CASE IS THE FOLLOWING AND WE OVEREXPOSE WE SEE A SERIES OF BANDS AND WHEN WE MAP THEM ON EITHER SIDE OF THE DAMAGE WE CAN UNEQUIVOCALLY DETERMINE THESE ARE A PRODUCT OF PARTIALLY REPAIRED SYNTHESIS. IN FACTOR IT'S HALFWAY. CUT WITH XO1 YOU SEE THE SIGNAL AND IF YOU CUT ON THE OTHER SIDE YOU DO NOT SEE THE SIGNAL. WE CLEARLY COULD DETERMINE THIS IS PARTIAL REPAIR SYNTHESIS HAPPENING IN THE ABSENCE OF XPG INCISION SO THE XPF PRE -- PRO PROTEIN CAN CUT AND WE TURNED TO THIS RADIATION PROTOCOL WIDELY USED IN THE NER AND WE TAKE CELLS LOCALLY AND RADIATE THEM THROUGH A FILTER THAT ABSORBS MOST THE UV LIGHT BUT LEAVES SOME PORES AND FIXES CELLS AND LOOKS FOR THE ANTIBODY AGAINST THE DAMAGE AND DOWN HERE I SHOWED A TIME LINE OF WHAT WE EXPECT INNER SO XPG IS FIRST AND WE SHOW THE RECRUITMENT CUT BY ERCCIXPF AND THIS COULD THEN RECRUIT THE FACTOR. AND OUR XPG WOULD CUT LATER SO OUR PREDICTION WOULD BE THAT IN THE XPF INCISION DOES NOT TAKE PLACE WE WOULD NOT OBSERVE THE REPLICATION FACTORS BUT SEE THEM IN THE XPG DEFICIENT SITUATION. THIS INDEED TURNED OUT TO BE THE CASE SO WHAT A SHOWED FIRST IS THE EXPERIMENT IN XPF DEFICIENT CELL AND THE FIRST IS DEFICIENT CELLS AND TASE A SURROGATE FOR THE FACTOR WE LOOK AT XPC AND FOR REPLICATION WE LOOK AT PCNA. IF YOU DON'T HAVE ANY XPF WE IF YOU HAVE THE XPF PLACEBO, XPC AS WE'D EXPECT BUT YOU CAN APPRECIATE IF WE USE THE INACTIVE CELLS WE DO NOT SEE IT SUPPORTING THE MODEL. I'LL TURN TO THE XG CELLS AND DO THE SAME EXPERIMENT IF WE HAVE DEFICIENT XPG DEFICIENT CELL LINE WE SEE NO PCNA. IF YOU DO THIS WITH THE COMPLEMENT OF CELL LINE WE HAVE XPC AND PCNA ON THE SIDE OF THE LESION AND HAVE CATALYTICALLY INACTIVE CELLS AND IT'S RECRUITED IN THE SIDE OF DAMAGE IN THE ABSENCE OF XPG INCISION AND WE SOUGHT THE SAME RESULT AND DID SCHEDULED SYNTHESIS WHERE WE WOULD SEE IN THIS CONFIGURATION WITH THE XPF AND XPG CATALYTIC INACTIVE PROTEIN WE SEE 60% REPAIR INDICATING PARTIAL SYNTHESIS. SO IT LEADS TO ASSEMBLY AND IT SHOWS COMPLETION OF THE REPAIR SYNTHESIS. WHEN YOU THINK OF WHAT HAPPENS NEXT AND HOW IT'S REGULATED IT'S SIMPLY A CASE OF THE XPD BUMPING INTO THE COMPLEX LEADING TO A CONFIRMATIONAL CHANGE AND LEADING ACTIVATION OF THE XPG PROTEIN AND WHEN WE LOOKED CLOSER AT THE XPG PROTEIN WE THOUGHT MAYBE THERE'S MORE HERE THAN WHAT MEETS THE EYE. SO FOR EXAMPLE WE FOUND THERE WAS ONE PCNA DOMAIN AND IT WASN'T REALLY ESSENTIAL FOR NER AND IT MAKES SENSE HE WOULD INTERACT WITH THE DOMAIN IN THE SPACE AND THE N AND Y IN BLUE MAKE UP THE CATALYTIC DOMAIN AND IN THE MIDDLE IS THE DNA THE FUNCTION OF WHICH IS NOT CLEARLY KNOWN AND YOU HAVE THE DOMAIN THERE AND THE SECOND BINDING SO WHEN WE SAW THEM WE GOT SUSPICIOUS AND THE REASON WE DID IT IS THE FOLLOWING. SO THE REASON WE GOT SUSPICIOUS IS BECAUSE THIS LOOKED VERY MUCH LIKE THE DOMAINS FOUND IN TRANS LESION SYNTHESIS AND THAT'S AS MOST OF YOU KNOW TRANSMISSION SYNTHESIS ARE RECRUITED ASIDE THE DAMAGE AND AFTER THE PCNA PROTEIN AND ALL THESE ENZYMES UBM AND IT'S BEEN SHOWN THE TWO TYPES OF DOMAINS ARE IMPORTANT FOR RECRUITMENT TO THE SIDE OF THE LESION. SO WE WONDERED IF WE MIGHT BE FACING A SIMILAR SITUATION HERE WITH THE XPG PROTEIN. NAMELY AFTER REPAIR IS IN -- SYNTH SYNTHET COULD THIS LEAD TO THE PCNA AND SIGNAL TO XPG VIA THE UBM DOMAINS IT'S TIME TO MAKE THE INCISION. THIS COULD BE ANOTHER SAFEGUARD BEFORE MOVING AHEAD AND OUR LABORATORY SHOWS THAT PCNA HAS THE RECRUITMENT OF FULL CAPPA DEPENDS ON THE PCNA. IT WOULD BE POSSIBLE IT SILL -- SIGNALS THERE'S VARIOUS COMBINATIONS AND WE WANTED TO FOCUS ON THE BINDING DOMAINS OF XPG ARE IMPORTANT. SO BACK TO THIS SLIDE TO SHOW YOU INDEED XPG AND YOU CAN SEE HERE AND YOU CAN SEE THE UBIQUITIN DOMAIN AND SUSAN HAS MADE STRUCTURES OF THE XPG FAMILY PROTEIN AND THE CATALYTIC CORE OF XPG ITSELF AND WHEN YOU LOOK AT IT THIS ONE OF THE CHARACTERISTICS IN ORANGE IS THE CATALYTIC DOMAIN EMANATING FROM TWO HELIXES AND THEY'RE MADE UP OF TWO HELIXES AND THEY'RE CALLED GATEWAY HELIXES WAS THE NUCLEUS INTERACTS WITH THE RESIDUES AND THE UBIQUITIN WOUND BIND AT THE END OF THE HELIXES. SO THE BINDING WOULD LEAD TO A CONFIRMATIONAL CHANGE IN THE XPG P PROTEIN ALLOWING THE BINDING TO OCCUR AND I WOULD LOVE TO SHOW YOU THAT IS THE CASE FOR XPG AS WELL BUT WE'RE NOT ANYWHERE NEAR THAT YET. PART OF THE REASON BEING IT'S DISORDERED AND HARD TO CHARACTERIZE AT THE STRUCTURAL LEVEL. NONETHELESS GOING BACK TO THE UBM DOMAIN WE ASKED IF WE HAD A CELL LINE WHERE WE HAD MUTATIONS IN THE VARIOUS DOMAINS WOULD THEY STILL BE ABLE TO GO TO COMPLETION. AND WE HAVE THE PROTEIN AND DAMAGE PERSISTS FOR HALF AN HOUR AND THEN IT'S COMPLETE. IN THE XPG DEFICIENT CELL LINE THERE'S NO REPAIR. SHOWN HERE AS A REFERENCE POINT IS THE CATALYTICALLY INACTIVE PROTEIN THE SAME USED IN THE PRIOR STUDIES AND YOU CAN SEE HERE CELL REPAIR IS SLOWED DOWN. YOU MAY ASK WHY YOU STILL GET PARTIAL REMOVAL OF THE DAMAGE AND THAT WILL BECOME CLEAR TOWARDS THE END OF THE TALK I HELP BUT LET'S JUST SAY IT'S DRAMATICALLY REPAIRED DEFICIENT FOR NOW. WHEN WE LOOKED AT THE MUTATIONS IN THE PCNA BINDING DOMAINS AND THE ONES DETERMINED PRIOR LOOKS LIKE THE PROTEIN. THERE'S A SLIGHT DELAY NOT LIKE THE CATALYTIC MUTANT IF YOU LOOK IT INDICATES THE ROLE HERE AND WHEN YOU COMBINE THE MUTATION YOU CAN SEE WE GET INTO THE RANGE OF THE CATALYTICALLY INACTIVE XPG PROTEIN INDICATE IN THE ABSENCE OF THE BINDING IT DOESN'T GO TO COMPLETION. WE WANTED TO GET A LITTLE MORE INSIGHT INTO WHAT'S GOING ON WITH THE DOMAIN AND WE WANTED TO ASK IF WE GET MUTATIONS WOULD THAT AFFECT RECRUITMENT OF PCNA. FOR THIS EXPERIMENT WE'LL LOOK AT PCNA AND THE ERCC1. IF YOU HAVE THE PCNA AS YOU'D EXPECT AND THEY GO TO 100% WITH THE MUTATION HOWEVER, WHEN WE LOOK AT THE MUTATION WE DON'T FIND THE PCNA AT THE SITE OF THE DAMAGE. WE CAN'T RULE OUT IT'S RECRUITED BUT IT DID SHOW IT'S NOT ASSOCIATED AS STABLY ASSOCIATED WITH THE MUTATION AS IN THE OTHER CELLS. FOR CONTROL IF YOU MAKE THE UBM MUTATION TO THE INTERNAL PEPTOMAIN SO YOU CAN SEE THAT IN THE MUTATION WE'RE SEWING IN RED AND RESPECTIVELY DAMAGE ACCUMULATES AND THEN IS REPAIRED WITH THE EXPECTED TIME SCALE NOTE THAT PCNA DISENGAGEMENT FROM THE LESION IS SLOWER THAN PNA REMOVAL. PCNA HANGS AROUND IN THE CATALYTIC XPG MUTANT AND YOU CAN SEE THE PROTEIN GETS THERE. IT'S NOT REALLY LOCATED AND NOT HAPPY THERE AND THEN DISAPPEARS. SINCE WE'RE SEEING THE DAMAGE IS REPAIRED ALBEIT MORE SLOWLY IN THIS SITUATION WE'D SAY THAT PCNA IS AT THE SITE OF THE LEGION BUT THE ASSOCIATION WITH THE COMPLEX IS WEAKENED BECAUSE THE INTERACTION OF XPG IS ALSO WEAKENED. SO AT THIS POINT ID I'D MAKE THE ARGUMENT AS THE DUE TO LATE BINDING OF NER THERE COULD BE ALTERNATIVE EXPLANATIONS FOR EXAMPLE HERE IN THE CONTEXT OF THE CELLS AND IT'S DEFICIENT AND THEN BOTH CELLS RAISE THE PROTEIN AND THAT IS REQUIRED FOR FURTHER PROGRESSION TO THE PATHWAY. IT IS PRESENT AT THE LESION AND LOOK WHAT HAPPENS NEXT IS NAMELY WHEN XPG COMES IN AND XPC LEAVES XFG TAKES OVER THE GEOGRAPHIC LOCATION INDICATING IT COULD BE POSSIBLE THE INTERACTION BETWEEN XPC AND XPG COULD BE IMPORTANT. IF THAT WERE THE CASE WE CAN EXPECT IF WE HAVE A MUTATION MAYBE XPC WOULD PERSIST AT THE LESION AND HERE I SHOW YOU THIS IS NOT THE CASE. I HAVE SHOWN HERE OF THE RECRUITMENT AND DISENGAGEMENT OF XPC OVER TIME AND HERE IN READ YOU CAN SEE IT'S THERE 30 MINUTE AND THEN LEAVES AND IS COMPLETELY GONE BY THREE HOURS. IN THE XPG DEFICIENT CELL WHERE REPAIR DOESN'T COME TO COMPLETION AND THE COMPLEX IS NOT ASSEMBLED WE DON'T ONLY LACK THE CATALYTIC COMPONENT AND XPC REMAINS HOWEVER, SHOWN IN GREEN IN THE OTHER MUTATIONS YOU CAN SEE XPC GETS THERE AND LEAVES RIGHT AWAY IN THE SITUATION WHERE THE UBM DOMAIN IS MUTATED ARGUING AGAINST THE FACT THE INTERACTION BETWEEN THE UBIQUITIN AND XPG MAY HAVE TO DO WITH THE PATHWAY SO I THINK WE CAN WITH CONFIDENCE SAY IT HAS SOMETHING TO DO WITH THE NER. SO WE WANTED TO GET A LITTLE BIT FURTHER EVIDENCE, HARD EVIDENCE WHAT HAPPENS TO THE DAMAGE AND THE GAPS PERSIST. SO TO DO THAT WE TEAMED UP WITH MIT AND YOU CAN LOOK FOR BREAKS IN DNA IN GENOMIC DNA SO IN THE CONTEXT OF NER WE MIGHT EXPECT THE FOLLOWING BEFORE INCISION TAKES PLACE IN THE XPF CATALYTIC LESION WE EXPECT TO SEE NO BREAK IN THE DNA SO IT SHOULD BE INTACT. AS SOON AS WE HAVE THE INCISION AND UP TO ANY POINT BEFORE THE LIGATION TAKES PLACE DURING NER WE WOULD EXPECT TO SEE THE MIX WHICH IS REPRESENTED FROM WHICH THIS GETS ITS NAME AND UPON COMPLETION ON NER EXPECT AND TURNS OUT WE HAVE TO DO OPTIMIZING TO GET IT RIGHT BECAUSE THE VARIOUS TREATMENT YOU CAN SEE IN THE NER SITUATION IT'S A DYNAMIC PROCESS SO THIS IS SOME OF THE DATA WE GOT AND SHOWN HERE IS THE TAIL END AND YOU CAN SEE THERE'S NO INCREASE IN TAIL AND IT GOES UP DRAMATICALLY AFTER ONE HOUR AND GOES BACK TO BACKGROUND LEVELS SLOWLY AFTER FOUR HOURS. IT SHOWS INCISION HAS TAKEN PLACE. I SHOULD TELL YOU IF WE DO THE EXPERIMENT ON XPF CELLS WE DO NOT SEE A SIMILAR INCREASE IN THE FORMATION OF THE XPF CELLS EXPRESSING THE CATALYTIC PROTEIN WE DO NOT SEE A SIMILAR INCREASE. YOU NOTICE NOW I SWITCHED SCALES BECAUSE THE RESULT IS THERE BUT NOT QUITE AS IMPRESSIVE. I THINK YOU CAN APPRECIATE ZERO IN RED AND ONE HOUR AND IT SHOWS THE INCREASE AND THE AUBM YOU SEE IT PERSIST OVER FOUR HOURS IN A SIGNIFICANT WAY INDICATING IT PERSISTS IN AUBM AND CELLS EXPRESSING AUBM EXPRESSION INDICATING THIS IS REQUIRED FOR THE COMPLETION OF REPAIR SYNTHESIS IT'S NOT STATISTICALLY SIGNIFICANT WHEN WE LOOK AT THE DOMAIN AND THE SITUATION IS WHEN WE HAVE NO XPG AND WE SEE AN INCREASE AND THINK IT'S DUE TO ANOTHER EFFECT NOT NER PROPER SHOWN WITH VARIOUS MUTANTS SO PERHAPS IT'S OXIDATED DAMAGE IS PROCESSED IN SOME WAY AND REMEMBER XPG HAS ROLES OUTSIDE OF NER AS WELL. SO I'LL LEAVE YOU WITH THE FOLLOWING MODEL SHOWING HOW REPAIR SYNTHESIS IS INITIATED BY THE PROTEIN AND THINK IT LEADS TO THE PCNA AND XPG. IF ALL GOES WELL, FINE, WE DO COMPLETE NER AND EVERYBODY'S HAPPY. HOWEVER, IF THERE'S IN ANY WAY THIS COMPLETION CAN BE INHIBITED AND THE COMPLETION MAY BE BECAUSE WE ADD DNCP AND REPAIR IS IN -- SYNTHESIS AND MAYBE IT DOESN'T ALLOW REPAIR SYNTHESIS TO GO TO COMPLETION OR PREVENTING THE INCISION FROM HAPPENING AND WHAT IS SHOWN THEN IS SHOWN THE XPG PROTEIN IS REPLACED BY THE NUCLEUS 1 AND HOW THAT HAPPENS IS NOT KNOWN YET AND ACTIVATED THE DAMAGE IN G1 SO IN THE ABSENCE OF THE REM INDICATION LEADING TO THE FORMATION OF A LONG SINGLE STRAND PATH COVERED BY RPA AND ACTIVATES THE INDUCED DAMAGE VERY MUCH LIKE IT DOES IN THE CONTEXT OF REPLICATION STRANDS BUT IT CAN BE MEDIATED BY NER. WE THINK THE XPG PROTEIN INCISION IS A KEY TRANSITION POINT WHETHER WE GO COMPLETE REPAIR SYNTHESIS AND THE CELL GOES ON WITH WHATEVER IT HAS TO DO OR USE AS A FULL PROPER DNA DAMAGED RESPONSE TO GIVE THE CELL TIME TO DEAL WITH WHATEVER TROUBLE LIES AHEAD. I LEAVE YOU WITH THAT AND SO I SHOWED YOU THE NUCLEAR ACTIVITY ARE TIGHTLY REGULATE TO AVOID INAPPROPRIATE INCISION AND I'VE SHOWN YOU XPG IS A LATENT ENDONUCLEUS AND XPG INCISION ACTIVITY IS REGULATED BY AIN'T ACTION OF XPG WITH PCNA AND UBIQUITIN AND REGULATION OF XPG INCISION MAY BE KEY SWITCH BETWEEN THE NER AND THE DNA DAMAGE SIGNAL. I'D LIKE TO THANK THE PEOPLE WHO CONTRIBUTED TO THIS WORK IN MY LABORATORY AND THANK YOU FOR THE DATA I SHOWED YOU AND THOSE CONTINUING TO WORK ON THE PROJECT AND OUR COLLABORATORS AND ALSO AND THE EARLIER LABORATORIES AND ONE VERY LAST POINT STARTING EARLY 2017 WE'LL BE MOVING TO SOUTH KOREA WITH KJ MYUNG TO START A CENTER AND FOR THOSE WHO THINK IT'S AN OPPORTUNE TIME TO SPEND A COUPLE YEARS MAYBE FOUR YEARS ABROAD WE HAVE PLENTY OF FACULTY APPLICATIONS AVAILABLE SO IF THAT'S OF INTEREST PLEASE GET IN TOUCH WITH ME AND LASTLY THANK YOU ALL FOR YOUR ATTENTION. >> THE INCOMING AUDIO IS NOT SO GREAT HERE. >> OK. WELL, THANK YOU ORLANDO FOR AN EXCELLENT TALK AND A CONGRATULATIONS TO MOVING TO SOUTH KOREA. IT'S A VERY BEAUTIFUL PLACE AND A FUN PLACE FOR PEOPLE TO VISIT. NOW WE'LL ASK SOME QUESTIONS FROM THE DIFFERENT CENTERS HERE. KENTUCKY YOU GO FIRST AND EVERYBODY ELSE MUTE. IN KENTUCKY DO WE HAVE QUESTIONS FOR ORLANDO? >> VERY NICE TALK. CAN YOU MAYBE ELABORATE ON WHAT YOU'RE TALKING ABOUT AS THE BACKUP PATHWAY THAT INVOLVES NER SIGNALLING AND HOW IT RELATES TO XPG DEFICIENT CELLS? >> THAT'S A GOOD QUESTION AND IT'S NOT CLEAR TO ME THE TWO ARE CONNECTED. SO IF YOU SEE SOMETHING SIMILAR IN XPG PATIENTS AND ALL THE PATIENTS I'M AWARE OF DON'T HAVE THE SURGICAL MUTATION WE INTRODUCES TO ABOLISH CATALYSTIC ACTIVITY BUT LEAD TO THE ASSEMBLING THE COMPLEX SO WE DON'T HAVE A PRECISE EVENT HAPPENING I THINK IN THE XPG PATIENTS NORMALLY YOU WOULD NOT GET TO THE POINT WHERE YOU SEE THE XGF MUTATION. THERE'S ONE ADJACENT SO WHERE WE MAKE THE CATALYTIC MUTATION BUT THAT PROTEIN IS STABLE SO THE SITUATIONS DO NOT CORRESPOND ONE TO ONE. I'M NOT SURE THAT WE CAN POPULATE THAT INCREASED DAMAGE SIGNAL THAT WOULD BE A FUTURE OF XPG PATIENTS. >> OK. THANKS. THAT MAKES SENSE. >> OK, MUTE IN KENTUCKY. GO ON TO NIUHL. >> GREAT TALK. I HAVE A QUESTION [INDISCERNIBLE] >> SO YOU'RE ASKING IF DNA1 OR 2 COULD BE A BACKUP MECHANISM. >> THEY CAN BUT IT'S NOT KNOWN -- IT'S NOT CLEAR TO ME. WE DID AN EXPERIMENT WHERE WE HAD A CATALYTICALLY INACTIVE AND THE EXPLANATION IS IT'S SO TIGHTLY INVOLVED IT'S HARD TO SWITCH IN AND OUT OF NUCLEUS AND MY FAVORITE MODEL AND THAT REMAINS TO BE SHOWN WOULD BE THAT BY SOME MECHANISM TO OTHER PROTEIN INTERACTIONS THAT IS XO1 THAT IS NOT DNA TOO BUT SHOWING THAT CONATIVELY CONCLUSIVELY REMAINS TO BE DONE. >> THANK YOU. >> THANK YOU, WE MOVE ON TO NCI TO KEN. >> THAT WAS A WONDERFUL TALK. I HAVE A QUESTION. WE HAVE XPG PATIENTS AND SOME OF THEM HAVE NEUROLOGIC DEGENERATION AND OTHERS DON'T AND THE MUTATIONS ARE DIFFERENT. WE HAVE A SUBSTITUTION IN SOME AND -- [AUDIO DIFFICULTIES] . >> I HAVE LOOKED AT PATIENTS FROM CELLS WITH NEUROLOGIC DISEASE AND SOME THAT DON'T. >> TO CLARIFY YOU'RE TALKING ABOUT XPG NOT XPGCS CORRECT? >> WELL, SOME HAVE THE SYNDROME AS WELL. IT'S A QUESTION OF WHAT THE NEUROLOGIC DEGENERATION IS. >> THERE'S A LOT MORE GOING ON THAN OUTSIDE OF NER. IT MAY BE INTERESTING TO LOOK AT THE ONES THAT ARE NOT CLEAR XPGCS BUT XPG WITH NEUROLOGICAL ABNORMALITIES. [AUDIO DIFFICULTIES] >> IT'S NOT TRIVIAL TO DISTINGUISH BETWEEN XP NEUROLOGIC DE GENERATION. >> IT'S HARD TO HEAR. >> FOR EVERYONE ELSE PLEASE MUTE. >> IT WOULD BEING WORTH THINKING ABOUT I DON'T KNOW I HAVE AN EASY ANSWER TO LOCK AT THAT. -- TO LOOK AT THAT WHETHER WE'D PICK UP THE CLEAR DIFFERENCES. >> WE'LL HAVE IT TALK LATER ABOUT THAT. >> OK. OK. THANK YOU AGAIN AND PLEASE MUTE AND MOVE ON TO SOUTH ALABAMA. DO WE HAVE ANYBODY THERE? ROB? . WE HAVE TWO QUESTIONS HERE. ONE FROM MIKE. >> YOU MENTIONED IN YOUR MODEL IT'S POSSIBLE [INDISCERNIBLE] >> THAT WOULD BE INTERESTING. THERE'S EXPERIMENTS FROM A NUMBER OF LABS TO SHOW YOU GET THE ACTIVATION OF DAMAGE SIGNALLING BUT WE HAVEN'T LOOKED AT THE CELL WITH THE REAL IMBALANCE IT WOULD BE NICE TO LOOK AT TO SEE THE DIFFERENCE. >> THANK YOU. REALLY INTERESTING AND I JUST WANT TO ASK ABOUT THE UBIQUITIN LIGASE? >> WE HAVEN'T BEEN ABLE TO SHOW THE REPAIR AND HAVEN'T BEEN ABLE TO RESULT THAT. IT WOULD INDICATE THAT ALSO TRIGGER THE XPG SIGNAL OR THERE'S ADDITIONAL LIGASIS WE DON'T KNOW YET. >> WE HAVE A QUESTION IN PITTSBURGH. NICE TALK, ORLANDO. CONGRATULATIONS ON YOUR MOVE. IS IT KNOWN WHETHER THE HYDROXY UREA TREATMENT SLOWS DOWN THE REPAIR? >> DO YOU SEE A DECREASED REPAIR RATE? >> IT'S A GREAT SUGGESTION. I THINK IT HAS NOT BEEN DONE BUT WE'D HAVE TO LOOK AT IS AND OUR MODEL WOULD SUGGEST IT SHOULD. IT SHOWS REPAIR DAMAGE IS INDUCED UNDER THE SITUATION BUT REPAIR HASN'T BEEN MEASURED AT THE SAME TIME BUT THAT WOULD BE A GREAT EXPERIMENT. >> OK. GREAT. PLEASE MUTE IN PITTSBURGH. IS THERE ANYONE ELSE I'VE NOT CALLED UP? OK WE STILL HAVE TIME FOR A QUESTION OR TWO. ANYONE WITH A QUESTION? >> PEOPLE WERE ASKING IF YOU CAN SHOW RECRUITMENT OF THE DNA2. DO YOU THINK SUCH AN ANTIBODY MAY EXIST? >> XO1 IS A PROBLEM AND PEOPLE HAVE LOOKED AT RECRUITMENT AND IN THE CATALYTIC XPG MUTANT XO1 IS RECRUITED SPECIFICALLY AND YOU ALWAYS HAVE TO OVEREXPRESS THE PROTEIN BECAUSE IT'S AT SUCH LOW LEVELS NORMALLY IT'S HARD TO SEE. I ASSUME THERE'S AN ANTIBODY AND WE HAVEN'T LOOKED UNDER THESE SPECIFIC CONDITIONS BUT IT COULD BE DONE OF COURSE. >> THANK YOU, MATT. >> WE HAVE A QUESTION FROM HERE, STONY BROOK. >> THIS IS A GENERAL KINETICS QUESTION. IT SEEMS WHEN SCORED AS AVERAGE NUMBER FOR CELLS GO OVER SEVERAL HOURS BUT THE REPAIR PROCESS IS ALMOST CERTAINLY FASTER THAN THAT. WHAT IS THE LIFE TIME OF AN INDIVIDUAL FOCUS? >> WHEN YOU HAVE EXPERIMENTS IT APPEARS -- THANK YOU FOR AN EXCELLENT TALK