WELCOME TO THE VERSION OF WEBEX AT HOME DNA REPAIR INTEREST GROUP. WE'RE HAPPY THAT ALL OF YOU ARE ABLE TO TRY THIS WITH WEBEX AND THROUGH THE VIDEO STREAMING. ON WEBEX, YOU CAN TYPE IN QUESTIONS, AND AT THE APPROPRIATE TIME WE'LL READ THE QUESTIONS FOR A CHAT. BE SURE YOU'RE MUTED AND DON'T HAVE YOUR VIDEO ON. WE HAVE WITH THE VIDEO CONFERENCES DR. (INDISCERNIBLE) IN FRANCE WILL BE SPEAKING IN DECEMBER ROGER GREENBERG, UNIVERSITY OF PENNSYLVANIA WILL BE TALKING ABOUT OPPORTUNITIES AT THE INTERSECTION OF DNA REPAIR. IN FEBRUARY DR. JUDY CAMPISI, BUILDING UP THE REST OF THE (INDISCERNIBLE) YOU CAN FIND OUT ABOUT THAT BY GETTING ON THE DNA REPAIR INTEREST GROUP LISTSERV BY SENDING ME AN E-MAIL TO KRAMERK@NIH.GOV AND REQUESTING THAT YOU GET THERE. WE'LL INTRODUCE OUR SPEAKER. >> OKAY, THANK YOU. PLEASE REMEMBER TO TURN OFF THE MICROPHONE. IT'S A GREAT PLEASURE TO HAVE KEITH CALDECOTT FROM SUSSEX, UNIVERSITY OF SUSSEX, DEPUTY DIRECTOR OF THE GENOME DAMAGE CENTER, THEY HAVE A LONG HISTORY OF WORKING WITH DNA REPAIR. AND I HAVE KNOWN KEITH FOR A LONG TIME AND FOLLOWED HIS WORK WITH GREAT INTEREST. AND KEITH WAS A POSTDOCTORAL FELLOW WITH LARRY HANSON WHO WE ALL KNOW FOR CLONING THE EARLIEST HUMAN REPAIR GENES, AND THEN LATER ON HE SPEND A WHILE WITH THOMAS LINDALL, WORKED AT THE UNIVERSITY OF MANCHESTER AND MOVED TO UNIVERSITY OF SUSSEX, AND HE'S ELECTED TO (INDISCERNIBLE) IN EUROPE, IMPORTANTLY, AND ALSO KEITH HAS BEEN A LEADER IN THIS AREA OF SINGLE STRAND BREAKS AND REPAIRS AND CONNECTIONS TO NEUROSCIENCE, WHERE HE HAS DONE A LOT OF IMPORTANT WORK TO CLARIFY PATHWAYS, TO TRY TO UNDERSTAND THE IMPORTANT CONNECTIONS BETWEEN LACK OF DNA REPAIR AND NEURODEGENERATION. SO, KEITH, TAKE IT AWAY FROM HERE AND, AGAIN, WE'RE PLEASED TO HAVE YOU. WHAT I'M GOING TO TALK ABOUT GENETIC DISEASE AND SINGLE STRAND BREAKS, WHICH WAS MY PLAN, I WANTED TO TALK ABOUT A NEW STORY IN THE LAB, WHICH SORT OF RELATES TO SINGLE STRAND BREAKS AND THEIR RELATIONSHIPS TO GENETIC DISEASE AND PERHAPS COME BACK TO THE END IF I HAVE END. GIVEN THIS COMMUNITY IS STRONGLY INTERESTED IN BASIC MECHANISMS, DNA REPAIR, I WANTED TO TALK ABOUT THIS PROJECT THAT WE'VE BEEN INVOLVED IN, TRYING TO UNDERSTAND THE ROLE OF XRCC1. GOOD MONKEYS AND BAD MONKEYS CLEARLY RELATES TO THE PICTURE YOU CAN SEE IN THE BACKGROUND. THIS WAS ACTUALLY A PHOTO OF SOME WALL PAPER IN A THAI RESTAURANT IN LONDON, I'VE NEVER SEEN PICTURES OF SINGLE STRAND BREAKS IN A RESTAURANT, YOU CAN SEE THERE'S LARGE SINGLE STRAND BREAKS AND WEIRD DNA STRUCTURES OVER HERE. I IMMEDIATELY THOUGHT THE MONKEY IS BEING DNA REPAIR PROTEIN, THESE MONKEYS ARE PULLING ALONG, GOOD MONKEYS, THEY WERE GOING TO REPAIR THE BREAKS. ACTUALLY YOU SEE FROM THE CONTEXT OF WHAT WE'VE BEEN DOING IN THIS STORY I'M GOING TO TELL YOU ABOUT YOU CAN'T TELL FROM THE PICTURE LOOKING AT THEM, THERE'S NO CLUES WHETHER THE MONKEYS ARE GOOD OR BAD MONKEYS, ARE THEY GOOD MONKEYS REPAIRING THE BREAK OR ARE THEY ACTUALLY BAD MONKEYS THAT ARE INVOLVED IN THE CHAOS THAT YOU CAN SEE IN THAT BREAKAGE? SO THAT'S A BIT OF A STRANGE INTRODUCTION BUT HOPEFULLY YOU'LL UNDERSTAND AS I GO THROUGH IN A FEW MINUTES. PEOPLE HAVE DONE THIS WORK, A POSTDOC IN THE LAB HAS DONE PRETTY MUCH ALL THE WORK I'LL SHOW YOU. IT'S A COLLABORATIVE EFFORT, THE LAB IN CONJUNCTION WITH OTHERS HAVE SIMILAR DATA USING TK6 CELLS, SHE USES GENE TARGETING APPROACHES. ONCE WE REALIZE WE HAD DATASETS WE HOOKED UP AND FORMED A COLLABORATION, A MUCH BETTER WAY OF DOING THINGS THAN COMPETING DIRECTLY SO THIS IS A JOINT EFFORT FROM BOTH THE SUSSEX LABS AND THE KYOTO LABS, IF YOU LIKE. ANNIE WAS THE POSTDOC RESPONSIBLE FOR MOST OF THE DATA I'LL SHOW YOU. XRCC1, IT'S MY FAVORITE PROTEIN, OUR EFFORTS TO UNDERSTAND THE REPAIR XRCC1 IT HAS ELONGATED SHAPE, COMPRISED OF THREE STRUCTURAL DOMAINS, WELL UNDERSTOOD IN TERMS OF FOLDING AND THEIR PROPERTIES, N-TERMINAL DOMAIN, CENTRAL BRC 2 OH PAIN, THE THREE DOMAINS ARE SEPARATED BY TWO LARGELY FLOPPY, IF YOU LIKE, LINK REGIONS. YOU MIGHT IMAGINE FOR SCAFFOLD PROTEIN, A LOT OF FACTORS, THE DOMAIN BINDS TO DNA POLYMERASE, ALSO A PROTEASE DISCOVERED NOT AGO CALLED HYDRATE PEPTILASE. THE CENTRAL DOMAIN IS PARTICULARLY INTERESTING, IT HAS HIGH AFFINITY FOR BINDING, IMPORTANT, THE WAY XRCC1 GETS RETRUTHED TO BREAKS OR MAY NOT BE THE CASE IN BASIC REPAIR AND C-TERMINAL DOMAIN BINDS TO LIGASE 3, A STABLE COMPLEX WHICH IN THE ABSENCE OF XRCC1 RESULTS IN IT BEING DEGRADED SO YOU GET REDUCED ABILITY. THE LINK DOMAIN IS HEAVILY PHOSPHORYLATED BY CK 2 THAT SEEMS TO DRIVE INTERACTIONS WITH THE DNA REPAIR PROTEINS WHICH NKP 1 -- PKP IS THE ONE WE UNDERSTAND, TENDS TO BIND THE SAME SITES THROUGH THE INTERACTIONS, IT'S AN INTERESTING PROTEIN, USEFUL FROM REPAIR PERSPECTIVE BECAUSE OF THE VARIETY OF FACTORS IT CAN INTERACT. SO, THIS IS THE WAY OF XRCC1 STUDIED MOST IN TERMS OF SINGLE STRAND BREAK REPAIR, THE BREAKS HERE ARE A CARTOON OF SORTS OF BREAKS INTRODUCED BY DIRECT SINGLE STRAND BREAKS, LARGELY THE COMMON INSERTION BREAKS ARRIVING FROM OXIDATIVE ATTACKS OF DEOXY RIBOSE WHERE THE SUGAR MOIETY IS FRAGMENTED, THE SUGAR PHOSPATE BACKBONE, THE DIRECT BREAK, THESE BREAKS ARISING STOCHASTICALLY ACROSS THE GENOME, YOU MIGHT INDUCE INDUCE THOSE IN THE LAB USING RADIATION OR HYDROGEN PEROXIDE, THE SUGAR IS FRAGMENTS, OFTEN PHOSPATE AT THE SITE OF THE BREAK, AND THAT REQUIRES PROCESSING BEFORE REPAIR CAN OCCUR. WE UNDERSTAND THIS WELL, IN THE TERMS OF XRCC1, FIRST STEP IS DEPICTION BY RIBOSE 1 OR 2, SCANNING THE GENE FOR THESE BREAKS ARISING STOCHASTICALLY, IT BECOMES ACTIVATED, GENERATING CHAINS, ALSO PROBABLY ANOTHER PROTEIN, THESE CHAINS SERVE TO RECRUIT XRCC1, IF YOU REMEMBER I SHOWED YOU THAT LAST SLIDE, THE CENTRAL BRCT DOMAIN HAS THIS BINDING CAPACITY ALLOWS IT TO BE RECRUITED AND CAN BRING STRANDS WITH IT, IN PARTICULAR IN THIS PATHWAY PNKP IS MOST IMPORTANT, IT CAN REMOVE PHOSPHATES FROM THE THREE PRIME END, RESTORING IT AND CONDUCT GAP FILLING AND LIGASE 3 CAN SEAL THE NICK. IN THIS PATHWAY WE KNOW IN THE WORK THAT THE INTERACTION BETWEEN XRCC1 AND PRKP THAT'S THE MOST IMPORTANT IN TERMS OF ACCELERATING THE REPAIR PROCESS. I WANT TO TALK ABOUT A PATHWAY PROBABLY ALL OF YOU ARE FAMILIAR WITH, ALSO IMPORTANT, WE KNOW MUCH LESS ABOUT WHY XRCC1 IS IMPORTANT. BASIC REPAIR, WE HAVE A DAMAGE BASE HERE. TYPICALLY IN THE CANONICAL FORM OF THE PATHWAY THIS WOULD BE REMOVED BY DNA GLYCOSYLASE, GENERATING FIVE PRIME SUGAR PHOSPATE, THAT AND THE NUCLEOTIDE CAN BE REPLACED BY DNA POLYMERASE, DUAL ACTIVITIES, WHICH IS VERY USEFUL. IT BINDS, CAN BRING ALONG XRCC1 HETERODIMER AND LIGASE 3, THERE'S A LOT OF REDUNDANCY IN THE PATHWAYS, IN THE LIGASE 1 MAY BE ABLE TO COMPENSATE LOSS OF LIGASE 3. IN ACTUAL FACT, WE DON'T REALLY UNDERSTAND WHY XRCC1 NEEDS TO ASSEMBLE THIS TRIMERIC COMPLEX TO SUPPORT BASIC REPAIR. XRCC1 IS IMPORTANT. IF YOU DON'T HAVE IT, THE IMPACT ON CELLS IS MUCH HIGHER FOR REPAIRING BASE DAMAGE THAN IT IS REPAIRING THE DIRECT BREAKS THAT I'VE SHOWN ON THE LAST, BOTH IN TERMS OF THE ACCUMULATION, ALSO IN TERMS OF THE SENSITIVITY AS WELL. WHICH OF THESE ARE THE IMPORTANT ONES, IF YOU PREVENT THE INTERACTION, YOU REDUCE ABILITY TO SUPPORT RAPID DER, YOU DON'T ABLATE WITH LIGASE 3, PARTIAL EFFECTS, IT'S NOT CLEAR WHAT THE FUNCTION OF THE COMPLEX IS, THAT'S THE QUESTION THAT WE'VE BEEN TRYING TO ADDRESS THAT I WANT TO TALK ABOUT TODAY. ONE OF THE POINTS OF INTEREST IS THE ROLE OF PARP IN THIS PATHWAY. THIS IS EXCITE CONTENTIOUS, SO FOR A LONG TIME IT'S BELIEVED PARP1 AND/OR 2 WERE ENGAGED DURING, WHICH THEY CLEARLY ARE, ENZYMES ARE ACTIVATED IF YOU TREAT CELLS (INDISCERNIBLE) FOR EXAMPLE. FOR A LONG TIME IT WAS THOUGHT THEY DON'T PLAY A POSITIVE ROLE. SOME INITIAL WORK SUGGESTED THEY DO SUPPORT BER AND PLAY AN ACTIVE ROLE, AND ANOTHER LAB PRESENTED MORE RECENTLY, NICE DATA SHOWING THAT AS WELL. YOU HAVE TO REMOVE THE PARP1 AND PARP2 TO SEE THAT ROLE. PARP1 AND PARP2 PLAYING THE SAME ROLE AS IN DIRECT BREAKS, SO ONE OF THE MAJOR ROLES IN DIRECT BREAKS IS RECRUITMENT OF XRCC1. THERE'S NO NEED TO INVOTE PARP ACTIVITY BECAUSE IT COMES WITH BETA, WHICH COMES BY BINDING (INDISCERNIBLE) AND SO THIS IDEA THAT INTERMEDIATE TO BASIC REPAIR CAN SHUFFLE ENZYMES IN THE PATHWAY, THE IDEA OF THE MODEL BY JOHN AND SAM WILSON SOME TIME AGO, YOU DON'T NEED TO INVOKE PARP1, IT'S NOT CLEAR THE ROLE IN THE PATHWAY. VERY RELEVANT TO THE STORY BUT NOT FROM THE PERSPECTIVE OF POSITIVE ROLE BUT FROM PERSPECTIVE OF IT BEING ONE OF THOSE BAD MONKEYS I WAS TALKING ABOUT AT THE VERY BEGINNING. SO, THE QUESTION I WANT TO ASK, ADDRESS TODAY, WHAT IS THE ROLE OF THE COMPLEX, IMPORTANT TO ASSEMBLE THESE PROTEINS TOGETHER IN THE FIRST CLUE CAME WHEN WE STARTED LOOKING AT THE SURVIVAL OF THE DIFFERENT CLONOGENIC SURVIVAL OF DIFFERENT CELLS LACKING COMPONENTS OF THE REPAIR PROCESS, MADE EASY NOW BECAUSE OF CRISPR/CAS9 DELETION. WHAT YOU SEE FOR RESPONSE, THE BEST WAY IN MY MIND OF INDUCING SIMPLE BASE DAMAGE, YOU CAN SEE IT'S THE WILDTYPE RESPONSE HERE, CLONOGENIC SURVIVAL OF RP CELLS TO THESE DOSES, YOU CAN SEE DELETE PARP 2 OR PARP1 ALONE SMALL EFFECT, IF YOU DELETE BOTH TOGETHER YOU GET PROFOUND SENSITIVITY, IMAGINE IT THE SHOW THIS IN A NICE PAPER A COUPLE YEARS AGO, SIMILAR THINGS, YOU CAN ALSO SEE IF YOU DELETE XRCC1 YOU GET A SIMILAR SENSITIVITY SPECTRUM AS WELL. THIS IS SHOWING BOTH PARP1 AND PARP2 COLLECTIVELY AT LEAST PROVIDING POSITIVE ROLE IN BER. WE DON'T KNOW WHAT THE ROLE IS YET. XRCC1 IS DOING THAT AS WELL. WHAT'S REALLY STRIKING WITH THIS EXPERIMENT WHERE WE LOOK AT XRCC 1 STRAIN, YOU CAN SEE NOW THE EXCESS OF SENSITIVITY HAS GONE AWAY. SENSITIVITY OF THE MUTANT IS SIMILAR TO PARP1 SINGLE. THAT'S IMMEDIATELY SAYING THE SENSITIVITY THAT YOU SEE LACKS SCCC1 IS INVOKED BY THE PRESENCE OF PARP1. PARP1 IS HAVING A TOXIC EFFECT. SO THIS IS BY NO MEANS A GENERAL PHENOMENON. IF YOU LOOK AT DIFFERENT DAMAGE, INDUCES BREAKS, YOU SEE A MORE CLASSIC EPISTATIC RELATIONSHIP BETWEEN XRCC1 AND PARP1, YOU NEED BOTH OF THEM FOR SURVIVAL, IF YOU DELETE BOTH YOU HAVE THIS REACTION, SHOWING THEY ARE FUNCTIONING TOGETHER, GOOD FRIENDS, BOTH DRIVING THE SAME PATHWAY, BUT THAT'S NOT THE CASE IN BER SEE WHAT'S GOING ON THERE? WHAT IS SURPRISING TO ME IS WHEN WE LOOKED AT THE LEVEL OF DNA DAMAGE THAT WAS ACCUMULATED IN XRCC1. THESE ARE ASSAYS WE USE A LOT, SIMPLY MEASURE DNA STRAND BREAKAGE, THESE ARE SINGLE STRAND OR BASIC SCIENCE, WE KNOW THEY ARE SINGLE STRAND BREAKS BECAUSE THEY WERE TRIGGERING PARP ACTIVITY. ON THE LEFT UNTREATED CELLS, A VARIETY OF RPs, GENOTYPES, BLAH, BLAH, BLAH. AND THEN AFTER TREATMENT YOU SEE CELLS UNDER THESE CONDITIONS RELATIVELY SMALL NUMBERS, RELATIVELY LOW LEVEL OF ACCUMULATION OF BER INTERMEDIATE. MOST ARE BEING RAPIDLY REPAIRED, YOU CAN SEE BECAUSE YOU GET ELEVATED LEVELS OF BREAKS. I SHOULD SAY IF THESE LOOK STRANGE I'VE PLOTTED RAW DATA HERE, EACH OF THESE PARALLEL -- EACH OF THE VERTICAL STRIKES IS AN INDIVIDUAL BIOLOGICAL REPEAT EXPERIMENT, THE INDIVIDUAL CELLS HAVE BEEN PLOTTED, USING SOME OF THESE STATISTICS. SOME IDEAS OF THE BIOLOGICAL REPRODUCIBILITY AS WELL. THERE'S HIGH LEVELS OF BREAKS IN THE KNOCKOUT, IT HAS REDUNDANCY WITH PARP 2, PARP 2 KNOCKOUT, IF YOU DELETE PARP1 AND PARPYOU SEE ACCUMULATION OF BREAKS, NOWHERE NEAR THE LEVEL WHEN YOU ACCUMULATE -- AND LACK XRCC1. THE STRIKING UNIQUE THING TO FOCUS ON THIS IN BLUE, DELETED PARP1 AND BREAKS ARE GONE. SO, THIS IS SAYING THAT NOT ONLY IS THE SENSITIVITY OF XRCC 1 KNOCKOUT CELLS DUE TO PRESENCE OF PARP1 BUT THE SINGLE STRAND BREAKS ACCUMULATE IN THE ABSENCE OF XRCC 1, ALSO IMPOSED BY PARP1, GENERATING THE DAMAGE DURING BER, BUT ACCUMULATES, AND THAT WAS A BIG SURPRISE TO ME ACTUALLY. SO I DON'T HAVE TIME TO SHOW YOU, WE CAN DO S RNA INSTEAD OF CRISPR, NICE DATA USING TK 6 CELLS, USING CLASSIC GENE KNOCKOUTS, YOU GET THE SAME RESPONSE, ANOTHER ARTIFACT OF GENE EDITING IN ANY WAY AT ALL. SO HOW CAN YOU HAVE A SITUATION WHERE PARP IS INDUCING DNA DAMAGE? THIS IS REMINISCENT OF WHAT WE NOW UNDERSTAND TO BE HAPPENING IN THE SITUATION WHERE YOU TREAT WILDTYPE CELLS WITH PARP INHIBITOR AND TRAP PARP ON DNA BREAKS AND PREVENT MOST BREAKS FROM BEING REPAIRED PROPERLY AND ACCUMULATE THOSE SINGLE STRAND BREAKS, INTERMEDIATES, TO PREPARE THE SITUATION IN XRCC1 KNOCKOUT CELLS, THE SITUATION IN WILDTYPE CELLS WHERE WE TREAT THE CELLS WITH PARP INHIBITOR, THAT'S IN THIS SLIDE, YOU CAN REALLY FOCUS ON THE RIGHT. SO, THE FOUR CELL LINES I'M SHOWING HERE, THIS IS THE SITUATION ESSENTIALLY REPEAT SET OF EXPERIMENTS, HIGH LEVEL OF SINGLE STRAND BREAKS, INDUCEED DURING BASIC EXCISION REPAIR AND XRCC 1 AND KNOCKOUT, THE RIGHT IS THE SAME BUT IN THE PRESENCE OF PARP INHIBITOR, PARP INHIBITOR RESULTS IN THE BLOCKAGE OF BASIC EXCISION REPAIR IN WILD TYPE CELLS, THE SAME SORT OF LEVEL IN XRCC1 KNOCKOUT. ADDING PARP INHIBITORS TO DEFECTIVE BACKGROUND, THE RED ONES, DOES INCREASE SINGLE STRAND BREAKS BUT NO HIGHER THAN PARP INHIBITOR TO WILDTYPE CELL, THAT'S SUGGESTING INDEED XRCC1 KNOCKOUT AND PARP INHIBITOR TO SOME EXTENT COPYING EACH OTHER SUGGESTING XRCC1 MAY BE INVOKING THE MODELS, THE SAME KIND OF PARP TRAPPING IF YOU TAKE A WILDTYPE CELL AND THAT PARP INHIBITOR. THAT'S THE CONCLUSION OF THAT SLIDE. XRCC1, SO THE QUESTION IS DOES PARP1 CONTRACT DURING BASIC EXCISION REPAIR UNDER NORMAL ENDOGENOUS CIRCUMSTANCES, NOT PRESENT, I'M GOING TO TRY TO CONVINCE YOU THAT'S THE CASE. TO STEP BACK, FOR THOSE NOT FAMILIAR, SOME IDEA OF THE CONCEPT OF PARP TRAPPING, CERTAINLY MY TAKE ON THIS, BOUND TO SINGLE STRAND BREAKS, WE CALL THIS ENGAGED. IN THE PRESENCE OF NAD, BECOMES ACTIVATED, MODIFIES WITH SPIKE-LIKE LEGS, CHAINS OF RIBOSE, THE LEVEL IS NOT CLEAR TO WHAT EXTENT THE CHAINS HAVE TO GROW, PARP1 WILL DISENGAGE FROM DNA SO YOU HAVE THIS DISENGAGED STATE. THE ENZYME PARP, POTENT CATABOLIC ENZYME, MAIN PROTEIN DEGRADING, THAT STARTS TO ACT ON THESE CHAINS, ACTUALLY THE LONGER THE CHAINS ARE, THE FASTER YOU LIKE PARP DEGRADES THEM. AT SOME POINT CHAINS GET REGRADED, PARP REENGAGES WITH SINGLE STRAND BREAK. DOESN'T HAVE TO BE THE SAME PARP MOLECULE, CAN BE, THERE'S AND EVENT, NOT THE BREAK, THAT COULD BE THE MOLECULE, MODIFIED MOLECULE OF PARP TO THE CELL. SO, PARP INHIBITOR, THE IDEA IS YOU BLOCK OR REDUCE THE RATE OF CHANGE EXTENSION, YOU TEND NOW TO HAVE GREATER ENGAGEMENT PARP1 ON THE BREAK, THINK ABOUT TRAPPING AS BEING ONE OF THE PARP BEING IN THE BREAK, DOESN'T HAVE TO BE THE CASE, A NICE PAPER SESSION IT COULD BE THE ON-RATE THAT RESULTS IN INCREASED ENGAGEMENT. FOR EXAMPLE, CHAINS SHORTER TAKE LESS TIME TO FEE GREAT THEM, THEREFORE PARP WILL REENGAGE MORE QUICKLY, PEOPLE SHOULD BE FLEXIBLE THINKING ABOUT TRAPPING. THE PURPOSE OF THE TALK THINK OF TRAPPING SIMPLY MEANING INCREASED ENGAGEMENT WITH PARP1 AT ANY GIVEN SINGLE STRENGTH BREAK, ENGAGING, DISENGAGING, REENGAGING OR COULD BE ANOTHER PARP. AND THE KEY THING ABOUT THE SLIDE HOW YOU MEASURE TRAPPING, THAT'S WHAT WE WANT TO DO. SO I'VE TAKEN THIS SLIDE FROM A PAPER, WHERE THEY ACTUALLY USED CHROMATIN RETENTION AS A MEASURE OF MEASURING TRAPPING INDUCED ALAPARIB, INCREASING AMOUNTS OF PARP 2, THIS IS TRAPPED PARP, NOT IN A PRODUCTIVE FASHION, IT'S PARP1 DISENGAGED BECAUSE ACTIVITY HAS BEEN SUPPRESSED BY PARP INHIBITOR. SO, WE USE THIS APPROACH LOOKING AT THE AMOUNT OF CHROMATIN BOUND PARP1 TO ASK COULD WE FIND ABSENCE IN THE XRCC 1, TRAPPED IN EXIST REPAIR, DOES PARP1 CONTRACT IN BASIC EXCISION REPAIR IN ABSENCE OF XRCC1 USING CHROMATIN RETENTION ASSAY AT LEAST, THE ANSWER IS YES, TAKES WILDTYPE CELLS, WE TREATED THEM WITH MMS, UP TO 60 MINUTES, WE'VE LOOKED AT THE LOCATION OF PARP1 IN TERMS OF WHETHER IT'S IN SOLUBLE FRACTION OR CHROMATIN FRACTION. YOU DON'T REALLY SEE IN THIS INSTANCE OF ACCUMULATION OF PARP1 AND CHROMATIN, BUT YOU CAN SEE A DIFFERENT FERENT SITUATION IN THE KNOCKOUT, ALMOST ALL OF THE CELLULAR PARP1 IS NOW PRESENT IN CHROMATIN, YOU COULD ARGUE IF IT'S JUST PARP1 BEING ENGAGED IN REPAIR AND SINGLE STRAND BREAKS WHICH ARE ELEVATED IN XRCC1 BUT THAT'S NOT THE CASE. I SAID ALREADY PARP1 IS CAUSING SINGLE STRAND BREAKS DURING BASIC EXCISION REPAIR AND XRCC 1 KNOCKOUTS. IF YOU CONDUCT COMET ASSAY, LOOK AT THE LEVEL OF SINGLE STRAND BREAK DURING THE SAME TIME COURSE YOU CAN SEE XRCC 1 IS ACCUMULATING AT THE POINT WHICH YOU SEE ALL OF THE PARP1 TRAPPED IN CHROMATIN, THE AMOUNT OF SINGLE STRAND BREAK IS NOT SO DIFFERENT BETWEEN WILD TYPE AND XRCC1. THE PARP WE'RE SEEING HERE IN THE CHROMATIN IN XRCC1 IS NOT A READOUT, IT'S SOMETHING DIFFERENT IN THE PARP INDIVIDUAL BREAKS, IN THE ABSENCE OF XRCC1, SO THIS IS TRAPPED. PARP TRAPPING BY PARP INHIBITORS ARISES BECAUSE YOU REDUCE ACTIVITY OF PARP, AS I MENTIONED ON THE PREVIOUS SLIDE REDUCED OSCILLATION, WE WANTED TO LOOK CAREFULLY IN THE ACTIVITY OF PARP IN WILDTYPE VERSUS XRCC 1 CELLS, THAT'S INTERESTING. IT IS THE SAME TIME COURSE, THESE EXPERIMENTS WERE CONDUCTED TOGETHER, IN PARALLEL AND SO COULD GET A SENSIBLE IDEA OF WHAT WAS HAPPENING WITH DIFFERENT PARAMETERS. YOU CAN SEE HERE THAT IN WILDTYPE CELLS THE ACTIVITY OF PARP BEHAVES SENSIBLY, STARTS LOW, INCREASES, THAT PARALLELS THE FORMATION OF SINGLE STRAND BREAKS, THIS IS THE SINGLE STRAND BREAK I SHOWED ON THE PREVIOUS SLIDE, REFLECTED IN CELL LINE, BLOCKS OF CELL LINE RATHER THAN TIME COURSE. YOU CAN SEE SINGLE STRAND BREAKS INCREASING IN WILDTYPE, CORRESPONDING ACTIVITY OF PARP1 INCREASING IN TERMS OF OSCILLATION, SO WE'RE MEASURING WESTERN PLOT SO LOOKING AT THE MODIFIED VERSION OF PARP1, PARP1 PROTEIN, A MEASURE OF ACTIVITY. YOU SEE A DIFFERENT SITUATION IN XRCC 1 KNOCKOUT CELLS. EARLY ON IN THE REACTION, PARP1 IS HYPERACTIVATED, IN THE ABSENCE OF XRCC 1, QUITE DRAMATICALLY SO, COMPARING LANE 7 WITH LANE 2. MASSIVELY HYPERACTIVATED, THE NUMBER OF SINGLE STRAND BREAKS ARE NOT SO DIFFERENT BETWEEN WILD PIPE AND XRCC1 KNOCKOUT CELLS, THEY CAN'T ALLOW FOR THE LEVEL OF HYPERACTIVATION. THE SECOND THING, TIME COURSE PERCEIVED, YOU SEE ACTIVITY OF PARP1 CONTINUES TO INCREASE IN LINE WITH NUMBER OF BREAKS, ACTUALLY DECLINES IN XRCC 1 KNOCKOUT CELLS, BY THE END OF THE TIME COURSE OF 60 MINUTES IT'S LESS ACTIVE THAN IN WILDTYPE CELLS, SORT OF THE PREDICTION YOU WOULD EXPECT FROM PARP1 ENTRAPPED, THIS IS THE TIME COURSE, ALL OF THIS PARP1 HERE, KNOCKOUT CELLS IS TRACKING CHROMATIN, RETRACTED CHROMATIN. FANTASTIC, THIS DIFFERENCE, IT'S WELL KNOWN ONE OF THE IMPORTANT THINGS IS THE EXTENT OF CHAIN LENGTH, YOU CAN SEE THIS IS QUITE COMPACT, IN XRCC 1 COMPARED TO WILDTYPE, SUGGESTING IT'S SHORTER IN THE KNOCKOUT, YOU CAN LOOK BY EXPENDING ELECTROPHORESIS PERIOD AND DOING SO YOU CAN STRETCH OUT THE PARP MOLECULES BASED ON CHAIN LENGTH, YOU CAN SEE YOU'VE GOT A BROAD SPECTRUM, THAT DOESN'T REALLY HAPPEN IN THE KNOCKOUTS. THE KNOCKOUT CELLS, SHORT CHAINS IN ABSENCE OF XRCC 1. WE'RE USING POLIATED RIVAL SPECIFIC ANTIBODIES. WE'RE LOOKING AT SHORT CHAINS OF RIBOSE, NOT MONO RIBOSE. SO, AS ANOTHER TEST, ANOTHER WAY OF TESTING WHETHER THIS TRULY IS TRAPPED PARP SEEN DURING BASIC EXCISION REPAIR, WE ASK THE QUESTION CAN THE INACTIVE OR CHROMATIN TRAPPED PARP IN THESE CELLS BE REACTIVATED BY SECOND BURST OF SINGLE STRAND BREAKS, IF PARP1 IS TRAPPED IN THE CELLS AND IF YOU NOW TAKE THOSE CELLS AND ADD ANOTHER DIFFERENT SINGLE STRAND BREAKS, PARP1 SHOULDN'T BE ABLE TO DO ANYTHING BY DEFINITION TRAPPED IN CHROMATIN. THIS IS THE EXPERIMENT ANNIE CONDUCTED TO TEST THIS. SHE TOOK WILDTYPE, BLUE LINES ARE DNA, GREEN CIRCLES ARE ACTIVE PARP IF YOU LIKE OR FUNCTIONAL PARP, RED ONES ARE TRAPPED PARP. ANNIE TOOK WILDTYPE CELLS, KNOCKOUT CELLS, TREATED THEM, ONE HOUR, SHOWED YOU PREVIOUSLY, NOW WE SHOULD HAVE MOST OF THE PARP TRAPPED, KNOCKOUT SITUATION, WE SHOULDN'T BE TRAPPED IN WILDTYPE. NOW TREAT THESE CELLS. PEROXIDE IS GOOD AT ACTIVATING PARP, WHICH ACTUALLY MUCH BETTER AT TRIGGERING PARP ACTIVATION, BREAKS ACROSS THE GENOME, A WAY OF INDUCING A LARGE NUMBER OF STOCHASTIC BREAKS TO TEST WHETHER THE PARP IS SUPPOSEDLY TRAPPED ON THE BER INTERMEDIATES IS ABLE TO JUMP OFF. MEASURING PARP OSCILLATION AFTER THIS PERIOD, THIS IS THE RESULT, THESE ARE WILDTYPE CELLS UNTREATED, NO SURPRIE, JUST TREAT WITH PULSE OF PEROXIDE, LOTS OF ACTIVITY, INDEED IF YOU TREAT THE CELLS WITH MMS, YOU CAN SEE THE AMOUNT OF OSCILLATION UNDER THESE CONDITIONS IS TINY COMPARED TO EFFECT YOU GET WITH PEROXIDE, AS I SAID PEROXIDE IS FAR BETTER AT INDUCING PARP ACTIVITY THAN ANY OTHER AGENT WE'VE LOOKED AT. IF YOU DO SEQUENTIAL REACTION YOU HAVE THIS SITUATION, YOU HIT WITH PEROXIDE, THEY LOOK LIKE YOU HIT THEM WITH PEROXIDE ALONE. IN PARP AND WILDTYPE CELLS FOLLOWING 60 MINUTES OF BER IS NO LESS ABLE TO BE ACTIVATED BY A SECOND BURST OF BREAKS, IF IT DOESN'T ENGAGE IN BER IN THE FIRST PLACE, NOT THE CASE IN THE KNOCKOUT CELLS. SO PEROXIDE CAN ACTIVATE PARP AND XRCC KNOCKOUT CELLS AS WELL AS WILD TYPE, THESE ARE NAIVE CELLS, COUNTER BREAKS, PARP IS THERE. IF YOU TREAT WITH NNS, YOU SEE THE CONTRAST IS TURNED DOWN. THE KEY THING TO KNOW IF YOU TREAT WITH MMS AND LIT WITH SECOND DOSE OF PEROXIDE YOU BARELY INCREASE THE ACTIVITY, YOU GET A LITTLE BIT BUT NOTHING LIKE THE ACTIVITY YOU SEE IF THESE CELLS WERE NAIVE WHEN YOU TREATED THEM WITH PEROXIDE. THIS IS CONFIRMING THE PARP THAT'S GENERATED IN XRCC 1 KNOCKOUT CELLS TRAPPED ON CHROMATIN IS NOT ABLE TO BE REACTIVATED, IF YOU SWAP THE CELLS A WHOLE NEW SET OF STOCHASTICS AND SINGLE STRAND BREAKS. DYNAMICS OF PARP ACTIVATION I'VE SHOWN YOU, THIS INITIAL HYPERACTIVITY EARLY ON IN BER, FOLLOWED BY PROGRESSIVE DECLINE IN ACTIVITY, THAT HAS TO BE RELATED SOMEHOW TO THE TRAPPING EFFECT. AND SO UNDERSTANDING THE CAUSE OF THE HYPERACTIVITY AND SUBSEQUENT ACTIVITY IS GOING TO BE IMPORTANT TO UNDERSTANDING WHAT'S HAPPENING HERE. ALSO WHETHER THEY ARE CONNECTED WHICH OF COURSE THEY ARE. SO THIS IS OUR MODEL FOR WHAT'S HAPPENING WITH HYPERACTIVITY. I HOPE I'VE CONVINCED YOU THE HYPERACTIVITY IN THE XRCC1 KNOCKOUT CELLS DURING BER HAS NOTHING TO DO WITH NUMBER OF SINGLE STRAND BREAKS, THE EXTRA BREAKAGE IS IMPOSED BY PARP1, IT'S NOT -- THE BINDING IS NOT A CONSEQUENCE OF INCREASED BREAKAGE. FOR EVERY STRAND BREAK DURING BER THE IDEA IS THAT PARP IN WILDTYPE SETTING WE BELIEVE PARP1 IS IN COMPETITION WITH THIS COMPLEX XRCC1 LIGASE. THERE'S OTHER INFORMATION THAT WOULD SUPPORT THE IDEA THAT EACH OF THESE PROTEINS AS INDIVIDUAL UNITS COMPETED WITH PARP1, THE CANONICAL SUBSTRATE IT'S BEEN WAITING FOR, IT WANTS TO TAKE OFF THE SUGAR PHOSPATE, POL BETA HAS AN AFFINITY FOR THIS, COMPETING WITH PARP1. LIGASE 3 WE KNOW FROM THE MID-90S, IF YOU MIX THESE, LIGASE 3 WITH TITRATE PARP1 AND SINGLE STRAND BREAKS, IN PART BECAUSE IT ACTUALLY HARBORS ZING FINGER MOTIF SIMILAR TO PARP1 THAT BIND BREAKS. WE NEVER UNDERSTOOD WHY PARP 3 HAD THE LIGASE FINGER. WE KNOW IN ADDITION TO SCAFFOLD PROTEINS CAN BIND DNA, IT'S NOT GOOD BUT THE DOMAIN HAS ABILITY TO BIND DNA. THE IDEA WE THINK THIS COMPLEX WORKS QUITE WELL, PARP1 ENGAGEMENT OF INTERMEDIATES. IN THE BE ABSENCE OF XRCC 1 NOT SO GOOD AT COMPETING PARP1, PARP1 IS GETTING ENGAGED TOO MUCH, CONSEQUENTLY HYPERACTIVE, AND GENERATING LOTS OF OSCILLATED PROTEIN, WHAT WE SEE AT EARLY STAGES OF BER. I'LL TALK ABOUT SUBSEQUENT ACTIVITY IN ONE OR TWO SLIDES. TO TEST THIS IDEA THE COMPLEX, ASSEMBLY IS IMPORTANT LIMITING PARP ENGAGEMENT WE TOOK ADVANTAGE OF SOME XRCC 1 CELLS, USING RPEs WITH THIS EXPERIMENT, EASY TO TRANSFECT, WHAT ANNIE AND MACK IN THE LAB GENERATED WAS XRCC1 KNOCKOUT HARBORING (INDISCERNIBLE) OR A TRUNCATED VERSION OF XRCC1, SO ONE OF THE NICE THINGS, YOU CAN LOP OFF A BIT WITHOUT HARMING THE OTHER BITS, IF YOU LIKE. WE CAN EASILY LOP OFF N-TERMINAL, LOP OFF THAT BINDS THE PROCESS, PNKP IS NOT REQUIRED, NOT AN IMPORTANT COMPONENT. YOU'RE LEFT WITH ONE-THIRD OF INTACT PROTEIN, CENTRAL BER DOMAIN WITH BINDING MOTIF, CRITICAL FOR EVERYTHING XRCC1 DOES. HAS DNA BINDING MOTIF. (INDISCERNIBLE) THAT MAY OR MAY NOT BE IMPORTANT. WE CAN GENERATE STABLE CLONES EXPRESSING PROTEINS, THIS IS THE KNOCKOUT ANTI-VECTOR, (INDISCERNIBLE). THIS BRCT DOMAIN IS GOOD AT FOLDING ITSELF, THIS DOESN'T SEEM TO DO ANY HARM, SO THE QUESTION MANY OF YOU HAVE IS THIS FRAGMENT COMPARED TO THE WILD FILAMENT PROTEIN, AT CONTROLING PARP1 ACTIVITY ENGAGEMENT AND ACTIVITY DURING BER. SO, A LOT OF DATA HERE. I'M GOING TO GO THROUGH IT SLOWLY AND HOPEFULLY NOT CONFUSE YOU ALL. SO WHAT WE'RE DOING HERE, WE'VE GOT THE KNOCKOUT CELLS WITH VECTOR, WE'RE DOING INDUCED TIME COURSE, A DIFFERENT TIME COURSE, NOT SO DIFFERENT FROM THE ONES I'VE SHOWN PREVIOUSLY, LOOKING AT THE PARP ACTIVATION OR MODIFICATION DURING THE TIME COURSE BY WESTERN BLOT. WE ASSURINGLY SINGLE KNOCKOUT BEHAVES, THIS PERIOD OF HYPERACTIVITY EARLY ON, RAPID DECLINE IN ACTIVITY DURING THE REST OF THE TIME COURSE. WILDTYPE XRCC1 THIS GOES AWAY, WE LOSE THE PERIOD OF HYPERACTIVITY AND DON'T SEE THE DECLINE, BALANCED OUT PARP ACTIVATION THROUGHOUT THE TIME COURSE OF THE BASIC EXCISION REPAIR REACTION. ON THE FRAGMENT HAS THIS PERIOD OF HYPERACTIVITY, ACTUALLY ALSO RESULTS IN BEING RELATIVELY INACTIVE BY THE END OF THE TIME COURSE. IT'S NOT AS DEFECTIVE AS THE ANTI-VECTOR HARBORING CELLS, THERE IS WEAK ACTIVITY IN THIS FRAGMENT, WE'VE SEEN THAT IN OTHER ASSAYS, SINGLE STRAND BREAK AND ACTIVITY, FOR EXAMPLE, WE THINK IT MAY DUE TO THE FRAGMENT STILL RETAINS THE DNA BINDING ACTIVITY, CLEARLY WITHOUT HAVING BEEN ABLE TO ASSEMBLE COLLECTIVELY. OH, YES. THIS IS A DIFFERENT EXPERIMENT, LOOKING FOR PARP TRAPPING. IT'S JUST SHOWING IN THE ABSENCE OF THAT COMPLEX ASSEMBLY, WE STILL HAVE TRAPPED PARP BY THE END OF THE TIME COURSE, CHROMATIN PARP1 NOW, ACTIVITY IS PARP1 ITSELF, PRETTY MUCH ENTIRELY TRAPPED IN CHROMATIN. FRAGMENT IS WILD TYPE XRCC1, I'LL CONVINCE YOU HOPEFULLY BECAUSE THE ABILITY OF FRAGMENT TO SUPPORT NORMAL PARP1 ACTIVATION KINETICS IS NOT DUE TO INSTABILITY OF THE FRAGMENT. THE FRAGMENT ITSELF ACCUMULATES NICELY DURING BER AS DOES WILD TYPE. SO, OKAY. SO THAT'S HYPERACTIVATION, WE THINK THE COMPLEX XRCC 1 INTO TRIMERIC COMPLEX DIVIDES MORE COMPETITIVE UNIT, LIMITING THE ENGAGEMENT ACTIVITY OF PARP1 DURING BER. IF YOU DON'T HAVE THAT INTACT COMPLEX, THEN PARP1 RAPIDLY HYPERACTIVATES. WHAT'S THE CAUSE OF THE PROGRESSIVE DECLINE HERE, DOWNSTREAM HYPERACTIVATION? I'M GUESSING MANY OF YOU HAVE INCLUDE ON -- CLUED ON. A COLLABORATION, WE PROVIDED JIM WITH THE XRCC 1 CELLS I WAS WORKING WITH, MARY THOMPSON GENERATED, JOE BATTLES WAS INVOLVED, TRYING TO GENERATE ASSAY YOU COULD USE TO MEASURE KINETICS. NAD IS RAPIDLY DEPLETED, YOU CAN SEE THAT HERE IN MINUTES. IF YOU COMPLEMENTED THEM WITH WILDTYPE HUMAN XRCC 1, THE CELL LINES WE PROVIDED YOU PREVENT OR LIMIT THAT DEPLETION. FOR A LONG TIME BASIC EXCISION REPAIR AND XRCC 1 CELLS IS IN RAPID DEPLETION OF NAD, THE CO-FACTOR OF PARP ACTIVITY SO IT'S NOT SO DIFFICULT TO IMAGINE THAT THE -- AFTER THE INITIAL PERIOD OF HYPERACTIVATION WAS SEEING DEFECTIVE CELLS, SUBSEQUENT PERIOD OF REDUCTION AND ACTIVITY DUE TO NAD DEPLETION, SO IF PARP ACTIVITY DECLINED DURING BER AND XRCC 1 BECAUSE OF NAD. I'LL SHOW YOU AN EXPERIMENT ANNIE DID TO TEST THIS. AGAIN, IT'S A BIT -- NOT SO COMPLEX, NOT SO EASY TO DESCRIBE. I'M GOING THROUGH SLOWLY. THE SCHEMATIC IS SIMILAR TO THE ONE PREVIOUSLY. WILDTYPE EXCEPTION, KNOCKOUT CELLS, DNA, BLUE LINES, ACTIVE OR FUNCTIONAL PARP1 IN THE GREEN CIRCLES, TRAPPED PARP1. WITH MOST OF PARP1 BEING TRAPPED NOW IN THE KNOCKOUT, NOT IN THE WILD TYPE, BUT NOW INSTEAD OF DOING ANYTHING ELSE TO THEM, ANNIE GENTLY LYSES THEM WITH DETERGENT, AND ASKED THE QUESTION, IF THE TRAPPED PARP1 IS INACTIVE BECAUSE NAD IS EXHAUSTED DO YOU KNOW INCUBATE THE LYSED CELLS WITH NAD, CAN YOU REACTIVATE TRAPPED PARP, THAT WOULD PROVE NAD WAS A FACTOR, OF COURSE YOU USE WILDTYPE CELLS AS CONTROL. AT THE END OF THE 45-MINUTE BIOCHEMICAL INCUBATION WITH OR WITHOUT NAD, YOU BLOCK THEM, BLOCK OSCILLATION. IT'S COMPLICATED TO DESCRIBE. SO I'M NOT GOING THROUGH ALL, BASICALLY PLUS OR MINUS, JUST GOING TO GO THROUGH THE CELLS THAT WERE TREATED WITH MMS BEFORE THEY WERE LYSED. THIS IS A LEVEL OF PARP, ANNIE DID A CONTROL, ANNIE DID A CONTROL, INCUBATE EXTRACTS IN THE PRESENCE OF NAD, ALSO IN PRESENCE OF INHIBITORS OF PARP AND PAR TO STOP ANYTHING FROM HAPPENING. BASELINE LEVEL OF OSCILLATION, BEFORE YOU DO THE IN VITRO, YOU ALLOW BIOCHEMICAL PARP AND PAR ACTIVITY TO OCCUR. INTERESTINGLY, NOW IF YOU NOW TAKE THE WILDTYPE EXTRACT IT'S, INCUBATE 45 MINUTES, ABSENCE OF NAD AND ABSENCE OF PARP OR PAR INHIBITOR, YOU SEE INCREASING LEVEL OF ULTRA OSCILLATION. THAT'S TELLING ME SUFFICIENT NAD IN WILDTYPE TO SUPPORT CONTINUED EXTENSION OF THE RIBOSE CHAINS INTRODUCED BY BASIC INTERMEDIATES. DUE TO BREAKS IN LYSINE, BIOCHEMICAL INCUBATION, IF YOU TAKE EXTRACTS FROM CELLS YOU DON'T SEE ANYTHING. YOU'RE SEEING INTO THESE LANES DUE TO ACTIVITY OF PARP1, SO WILD TYPE EXTRACTS, SUPPLEMENTED EXTRACTS WITH NAD, YOU SEE FAR MORE PARP ACTIVITY AS YOU WOULD EXPECT, BECAUSE YOU NOW ARE SUPPLYING CO-FACTOR, EVEN WILDTYPE CELLS, NAD IS LIMITING. WHAT YOU SEE WITH THE KNOCKOUT EXTRACTS, FIRST TAKE THE KNOCKOUT EXTRACTS AFTER THE 60-MINUTE INCUBATION WITH MMS, SUBSEQUENT INCUBATION IN THE PRESENCE OF PARP AND PARG INHIBITOR, THIS IS THE SIGNAL, LOOKS MORE LIKE WILDTYPE BECAUSE THIS IS LOWER CONTRAST, WHEREAS WILD TYPE IS EXTENDED OVER BIGGER CHAINS, IT'S COMPACT IN THE KNOCKOUT. INTERESTINGLY IF YOU INCUBATE THOSE EXTRACTS NOW IN THE ABSENCE OF NAD BUT IN ABSENCE OF PARP OR PARG INHIBITOR, ALLOWING ENDOGENOUS PARP AND PARG TO DO WHAT IT WANTS TO DO, YOU REDUCE THE SIGNAL, THE SIGNAL GOES AWAY. OUR INTERPRETATION IS PARG IS STILL THERE, BIOCHEMICAL INCUBATION, DEGRADING SIGNAL IN ABSENCE OF PARP ACTIVITY. PARP IS THERE BUT HAS NOT NO NAD, YOU LOSE THE SIGNAL IN CONTRAST, INCREASED OSCILLATION, EXPERIMENTAL OBJECTIVE TO SUPPLEMENT TRAPPED PARP MOLECULES WITH NAD AND YOU CAN SEE MEETLY THE LEVEL OF PARP ACTIVATION IS ACTUALLY HIGHER THAN THE LEVEL OF PARP ACTIVATION IN THE WILDTYPE EXTRACT. THIS IS ENTIRELY REACTIVATION OF PARP1 MOLECULES, TRAPPED, BECAUSE IF YOU TAKE THE CELL EXTRACTION MUTANT CELLS IN A NAIVE STATE, THEY NEVER SAW MMS, YOU SEE NO ACTIVATION. ESSENTIALLY SHOWING YOU THAT THE ABSENCE OF PARP ACTIVITY LATER STAGES OF BER, THE KNOCKOUT CELLS PARP1 IS TRAPPED IN CHROMATIN BECAUSE NAD HAS BEEN EXHAUSTED. THE LAST QUESTION IN THIS CONTEXT, I WANT TO ADDRESS HOW DOES A TRAPPED PARP BLOCK BER? THIS IS SIMPLER TO UNDERSTAND I THINK. THE MODEL ON THE LEFT REALLY SHOWS HOW PEOPLE THINK ABOUT PARP TRAPPING, PARP INHIBITOR, PARP OF COURSE IS EXCESSIVELY ENGAGED, THIS IS NO DIFFERENT THAN THE ARGUMENT EARLIER, BOUND TO XRCC1 EFFECTIVE COMPETITORS WITH PARP1, IN THE ABSENCE THE SINGLE ENZYMES BECOME LESS EFFECTIVE, SO PARP1 IS TRAPPED, BLOCKING ACCESS TO THE BREAK BY REPAIR ENZYMES, AGAIN ON THE RIGHT THIS SHOWS THAT'S THE CASE. I DON'T REALLY WANT TO GO THROUGH EVERYTHING. I WILL SHOW YOU IF YOU LOOK AT THE AMOUNT OF PARP1 IN CHROMATIN, WILD TYPE CELLS, THESE ARE CELLS THAT WE TREATED WITH MMS, YOU CAN SEE PARP1 AND CHROMATIN, NOT MUCH COMPARED TO TREATED CELLS, VERY TYPICAL. YOU GET WHAT EVERYBODY HAS KNOWN AS BLOCKING PARP1 AND CHROMATIN, TRAPPING AT PARP1, WILDTYPE SECOND. WHAT'S INTERESTING IF YOU LOOK AT THE AMOUNT OF XRCC 1 IN CHROMATIN, YOU SEE IT START TO ACCUMULATE WHEN WE TREAT WILDTYPE CELLS WITH MMS, ALREADY YOU'RE SEEING EVIDENCE THAT PARP IS EFFECTIVELY -- YOU TRAP IT, IT'S ABLE TO STOP ACCESS. YOU SEE LOTS OF PARP TRAPPING, IN FACT AT THIS TIME POINT VERY MUCH ALL OF THE PARP1 IS CONSTANT CELLULAR FRACTION, TRAPPED CHROMATIN, LOOK AT THE AMOUNT, THERE'S ALMOST NOTHING THERE. NOT ONLY XRCC 1 IS FAR WORSE IN TERMS OF ABILITY TO JOIN CHROMATIN, THE PARP INHIBITOR TO WILDTYPE CELL. IF YOU LOOK AT PARP KNOCKOUT, YOU SEE THE REVERSE, YOU SEE IN PARP1 KNOCKOUT, MMS TREATMENT, MORE XRCC 1 ACCUMULATES IN THE WILDTYPE, MOVE POL BETA, IN ITSELF PARP1 IS COMPETING WITH XRCC 1 POL BETA COMPLEX. IF YOU TAKE XRCC 1 CELLS IN THREES TO PARP1, YOU SEE POL BETA COMES BACK TO NORMAL LEVELS ALMOST. SO YOU CAN SEE HERE ALMOST NO POL BETA IN CHROME IF YOU DON'T HAVE XRCC 1. IF YOU TAKE PARP1 POL BETA COMES BACK AND STRUGGLES TO GET INTO CHROMATIN IF XRCC1 IS NOT THERE, PARP1 IS PRESENT. SO, HERE IS THE MODEL. I'M NOT SURE I HAVE TIME. I CAN STOP THERE IF WE DON'T HAVE TIME. IF NOT I'VE GOT A FEW SLIDES ON THE GENETIC DISEASE SIDE TO MAKE IT INTERESTING. THE CENTRAL CORE ASPECT, WE HAVE BASE DAMAGE, GLYCOSYLATED DAMAGE, (INDISCERNIBLE) THE REASON IT'S IMPORTANT FOR XRCC1 TO ASSEMBLE ENZYMES, THIS COMPLEX THEY PROVIDE SUFFICIENT COMPETITIVE ABILITY TO PRESENT PARP FROM BINDING INTERMEDIATES BOOKING ENGAGED AND HOPEFULLY ACTIVE, THIS IS A COMPETITIVE, AND INTERACTION I'M SHOWING YOU DATA, THE INTERACTION OF XRCC 1S PARP IS AN IMPORTANT COMPONENT. THESE ARE FRIENDS, PARP1 AND XRCC1 ARE FRIENDS, THEY LIKE TO BE TOGETHER. AT THE SAME TIME XRCC1 IS CHAPERONING AND STOPPING IT FROM BEING OVERLY ENGAGED WHICH CAUSES PROBLEMS. THE COMPLEX IS COMPETING WITH PARP1, GETS ACCESS TO THE BREAK, BER OCCURS. THIS COMPETITION IS NOT HAPPENING, PARP1 IS OVERLY ENGAGED AT THE BREAK, HYPERACTIVATED. THIS IS A FORM OF TRAPPING, SOMETHING I WANT PEOPLE TO TAKE HOME. I DON'T THINK WE NECESSARILY HAVE TO HAVE PARP1 STOP CHROMATIN TO CONSIDER THAT TRAPPING, SIMPLY PARP1 ENGAGING AND DISENGAGING OR REENGAGING IS TRAPPING. THE LACK OF COMPETITIVE ELEMENT HERE IS WITH STRAPPING BUT IF YOU ALLOW THE SITUATION TO GO ON LONGER, HYPERACTIVATION, AGGRESSIVE EXHAUSTION, TIGHTER TRAPPING OF PARP TO THE BREAK AND YOU END UP WITH ALL THE PARP1 EVENTUALLY ON THE CHROMATIN, ALL THESE THREE SITUATIONS COMPETITIVE ELEMENTS OR COMPLETELY TRAPPED VERSION YOU'RE BLOCKING ACCESS, NOW IF YOU TAKE PARP1 IN THE CELL THE BREAK ALMOST RETURNED AS FAR AS (INDISCERNIBLE) RETURN BACK TO NORMAL. IN THE ABSENCE OF PARP1 YOU DON'T NEED XRCC 1, YOU DON'T NEED THE XRCC1 COMPLEX, POL BETA, NOW ACCESS THE GRAPE PERFECTLY HAPPILY, THE CELL IS CONTENT, AT LEAST IN TERMS OF THE PAIR RATE. I'VE SHOWN YOU THE PARP INHIBITORS AND HOW THEY BLOCK REPAIR. HOW AM I DOING? >> ANOTHER FIVE MINUTES. >> PERFECT. THIS IS SUPPOSED TO BE ABOUT SINGLE STRAND BREAK AND DISEASE. HOW DOES THIS RELATE TO DISEASE? SINGLE STRAND BREAK REPAIR PROTEINS ARE STRONGLY ASSOCIATED IN NEUROLOGICAL DISEASE, YOU'VE HEARD IN THE INTRODUCTION, SO ALL OF THESE PROTEINS IF MUTATED GIVE RISE TO CEREBELLAR ATAXIA, INTERESTING BECAUSE IT GIVES RISE TO (INDISCERNIBLE) DEVELOPMENTAL PROBLEMS UP TO PROGRESSIVE NEURODEGENERATION, TEMPORAL MRIs OF THE PATIENT. XRCC 1 IS RELEVANT TO THIS PARTICULAR TALK, THE QUESTION IS TO WHAT EXTENT IS THIS FOUND IN REPAIR, ACCOUNT OR IMPACT ON THE PHENOTYPE OF THE PATIENTS? WE CAN'T SAY ABSENT BECAUSE WE DON'T (INDISCERNIBLE) CAUSING NEURODEGENERATION, TO ME IT COULD BE TOP1 BREAKS, OXYLATED BREAKS. THE MASSIVE LOSS OF INTERNEURONS AT THE CEREBELLUM IN THE ABSENCE OF XRCC1, THIS MOUSE HAS A LOT OF PHENOTYPES OF PATIENTS IN SENSE OF CEREBELLAR ATAXIA AND ALSO SHOWS SEIZURES AND NOW MORTALITY BUT I'M JUST SHOWING THE NEURONS AND CEREBELLUM, YOU CAN SEE THIS MASSIVE LOSS OF INTERNEURONS IS RESCUED, IF YOU DELETE PARP1. CLEARLY IN THIS MODEL PARP1 IS ALSO INTERESTING A TOXIC IMPACT, RESCUE THE ABILITY OF THE XRCC1 TO BALANCE, YOU SUPPRESS ATAXIA. YOU ALSO RESCUE THE -- SUPPRESS JUVENILE MORTALITY, THIS IS THE AVERAGE LIFESPAN, THE KNOCKOUT, DELETE PARP1, BOTH ALLELES, GET SUBSTANTIAL RESCUE. IF YOU DO ONE YOU GET ELONGATED RESCUE, MEDIAN LIFESPAN OF THE ANIMALS GOES UP 70-FOLD. SO, WE CAN SEE IN THE MOUSE MODEL PARP1 IS HAVING A TOXIC IMPACT, WE DON'T KNOW YET IF THE NATURE OF THAT TOXICITY IS DUE TO PARP1 TRAPPING, IT COULD BE DUE TO PARP1 TOXICITY OR TYPES OF BREAKS, WE DO THINK THAT THE ROLE WE FOUND FOR XRCC 1 COMPLEX, EXCESSIVE ENGAGEMENT, LIKELY TO IMPORTANT IN NEUROLOGICAL DISEASES, PARTICULARLY XRCC 1. SO THAT'S SUMMARY. ESSENTIAL ROLE OF XRCC 1 DURING BER TO ASSEMBLE BER PROTEIN COMPLEXES THAT CAN COMPLETE WITH PARP1. LIMITING ACTIVITY, ENGAGEMENT AND ACTIVITY OF PARP1 DURING BER. EXCESSIVE PARP1 ENGAGEMENT CAN LEAD TO NAD EXHAUSTION, PARP1 ACCUMULATION, BLOCKAGE OF BER INTERMEDIATES SEEMS A PARTICULAR PROBLEM TO POL BETA, CONSEQUENTLY ACCUMULATION OF SINGLE STRAND BREAKS AND TOXICITY, WE'RE THINKING OF ENDOGENOUS ANTI-TRAPPER, TRAPPING IS NOT JUST -- NOT JUST SOMETHING INVOKED BY PARP INHIBITORS, IT'S CLEAR NOW, THERE ARE CONDITIONS IN CELLS BY PARP1, CONTRACT ENDOGENOUS SETTING SEEMS TO BE THE ROLE OF XRCC1 TO STOP THAT HAPPENING DURING BER BY ASSEMBLING COMPLEXES THAT CAN REPEAT. YOU MIGHT WONDER, I'M WONDERING WHY BER EVEN USING PARP1. PARP2 IS A MUCH BETTER ENZYME DRIVING THE PATHWAY, CAN PROVIDE THE OSCILLATION, AND COULD -- IT'S NOT CLEAR WHY PARP1 GETS ENGAGED IN BER, SEEMS QUITE DANGEROUS TO HAVE AROUND. SO I'LL STOP. ANNIE WAS CONDUCTING THE EXPERIMENTS. MAREK, AND JAN, RICH, WILL, HANA. LIMEI, EMILIA AND JACK DRIVING THE WORK, THE LAST THREE SLIDES. AND I THANK THE OTHERS, COLLABORATIVE EFFORT WITH THESE GUYS, THE KYOTO GROUP, SIMILAR DATA, COMBINED FORCES AND I THINK IT STRENGTHENED WORK HUGELY AND THANK THE PEOPLE WHO FUND THE WORK, AND THE ERC WHO FUNDED AN ADVANCE GRANT. THESE ARE THE GUYS IN THE LAB, THIS IS ONE OF OUR RETREATS, IN PRAGUE ACTUALLY. WITH THAT I'LL STOP AND TAKE ANY QUESTIONS. >> THANK YOU. VERY NICE START. WE HAVE TIME FOR A FEW QUESTIONS. DO YOU WANT TO READ OR SHOULD I GO? >> I CAN DO IT. VERY INTERESTING. WE JUST HAVE VERY LITTLE TIME TO ADDRESS QUESTIONS, BUT THERE ARE A LOT OF QUESTIONS, VERY INTERESTING. IN REGARDS TO -- HERE IS ONE, IN REGARDS TO THE TRAPPED PARP HYPOTHESIS, COULDN'T THESE RESULTS OCCUR BECAUSE OF NAD DEPLETION? WOULD EXPLAIN LOSS OF CORRELATION AT THE TIME AND ALSO WHY A SECOND MMS TREATMENT WOULD NOT INCREASE? >> YEAH, THAT'S EXACTLY WHAT WE'RE SAYING, THE LOSS OF -- PROGRESSIVE LOSS OF THAT ACTIVITY IS EXACTLY THAT, DUE TO NAD. >> THAT'S WHAT YOU SAID. THIS TRAPPING THAT YOU SEE IN THE PRESENCE OF NAD COMPLEMENTATION WHAT HAPPENS TO THE CHAIN LENGTH OF PARP? YOU SHOWED SOME TRAPPING ALSO C MINUS, DEPLETION, RIGHT? >> YES, PRESENCE OF NAD, THE CHAINS EMPLOYEE -- GROW HUGELY TO AT LEAST THE WILDTYPE. >> (INDISCERNIBLE). >> YES, YOU TAKE A WILDTYPE CELL THAT'S BEEN TREATED WITH MMS DOESN'T HAVE ANY TRAPPED PARP1, THE PARP1 IS FULLY PRODUCTIVE. IF YOU TAKE THOSE AND INCUBATE FOR A FURTHER PERIOD IN VITRO WITH NAD YOU GET MORE PARP ACTIVITY BECAUSE YOU ARE PROVIDING MORE CO-FACTOR. THE KEY IS ALSO THE KNOCKOUT, ALL THE PARP1 IS TRAPPED ON BER INTERMEDIATES, SUPPLY WITH NAD, AND NOW THE INTERACTION YOU'VE GOT BIGGER CHAINS AND MORE (INDISCERNIBLE) BECAUSE WHAT YOU'VE DONE IS TAKE ALL OF THAT STALLED TRAPPED PARP1, GIVEN IT NAD, BANG. >> THIS IS VIVIC FROM PORTLAND, OREGON. WHAT HAPPENS IF YOU DELETE PARP2? DOES THAT ALSO RESCUE THE SIGNAL XRCC1 KNOCKOUT? >> YEAH, NO, IT DOESN'T. WE'VE DONE THAT EXPERIMENT. I DIDN'T SHOW YOU THE DATA BUT TAKEN PARPAWAY, WITH CONTROLS TO SO S RNA WAS SUCCESSFUL. AND IT DOES NOT RESCUE. THIS IS ENTIRELY -- I THINK THE ROLE OF PARP AND BER, THAT'S A GENETIC THING BUT THE TOXIC ROLE OF PARP1 XRCC1 COMPLEX TO COMBAT RESTRICTED TO PARP1. >> ANOTHER QUESTION, KNOCKOUTS, HAVE YOU BEEN TREATING THEM WITH PARP INHIBITORS? >> YEAH, THAT'S A REAL BONE OF CONTENTION. IT'S TYPICAL. WE'RE TRYING TO DO THAT EXPERIMENT. THERE ARE SEVERAL PROBLEMS, THE FIRST PROBLEM IS THAT TO GET PARP INHIBITOR INTO THE ANIMALS TO SEE IF WE CAN RESCUE THE INTERNEURON DEFECT, WE HAVE TO FEED THE INHIBITOR, YOU CAN'T CONTROL, THE TROUBLE IS GETTING TO THEM. THE SECOND PROBLEM IS PARP1 INHIBITORS EARLY IN DEVELOPMENT, PARP1 AND PARP2, THAT'S ANOTHER FACTOR. I DO NEED DO THAT EXPERIMENT. IF WE CAN GET A LATER TIME POINT, LOOK AT PROGRESSIVE DEGENERATION, WE CAN HAVE SENSIBLE EXPERIMENT IN THE CURRENT CROP OF INHIBITORS, CURRENTLY WITH THE INHIBITORS WE HAVE IT'S DIFFICULT TO ASK THOSE QUESTIONS. WE NEED IDEALLY PARP1-SPECIFIC INHIBITOR, IT'S NOT BY TRAPPING, GENETIC DELETION. I CAN'T ANSWER THE QUESTION WITH PARP INHIBITORS THAT TRAP, HAVE PROPENSITY TO RESCUE. ESSENTIALLY I'M SAYING NOT HAVING XRCC 1 IS THE SAME THING, TRAPPING. WE HAVE DATA, I DON'T HAVE TIME TO TALK ABOUT TODAY, I WILL TALK AT THE COLUMBIA SERIES IN A FEW WEEKS, WHICH SHOWS THE CONSEQUENCE OF THE TRAPPING, IN TERMS OF TOXICITY IS NOT BLOCKING REPAIR OF THE ENTER INTERMEDIATE, MORE TO DO WITH PARP1 IMPACTING ON TRANSCRIPTION, THE DATA I'LL SHOW YOU ALONG THOSE LINES WOULD ARGUE PARP INHIBITORS IF WE COULD GET THEM IN THE ANIMALS EARLY ENOUGH MIGHT RESCUE, YOU HAVE TO TUNE IN TO SEE THAT DATA. >> THERE'S A QUESTION (INDISCERNIBLE) XRCC 1 INVOLVED (INDISCERNIBLE). >> YEAH, THERE'S BEEN A FEW REPORTS, XRCC 1 INTERACTS, GLYCOSYLASE, WE'RE NOT SEEING THAT, WE'VE LOOKED HARD, (INDISCERNIBLE) KNOCKOUT [ BACKGROUND NOISE, INAUDIBLE ] >> THAT XRCC 1 HAS IMPACT WITH FACTORS UPSTREAM, WE'RE NOT SEEING THAT. IN MY REVIEWS WHEN I SHOW PROTEIN (INDISCERNIBLE) TO VERIFY CONVINCING DATA FROM MULTIPLE LABS, AND THEY ALL TEND TO BE DOWNSTREAM, WITH THE POSSIBLE EXCEPTION OF (INDISCERNIBLE) VERY CONVINCING, OTHER FACTORS TEND TO BE DOWNSTREAM. >> MITOCHONDRIAL QUESTIONS THEMSELVES (INDISCERNIBLE) EFFECT OF XRCC 1? >> WE HAVEN'T LOOKED AT THAT. WE HAVE COLLABORATED WITH A GROUP IN NEWCASTLE, AND THEY HAVE NOT SEEN ANY OVERT OBVIOUS PROBLEMS IN MITOCHONDRIAL DNA IN THE XRCC 1 DISEASED CELLS OR ANY OTHER DISEASED CELLS SENTINEL BUT YOU CAN'T RULE OUT A MITOCHONDRIAL (INDISCERNIBLE) BIOCHEMICAL PROBLEMS YOU MIGHT PUT DOWN TO MITOCHONDRIAL DEFECT BUT WE DON'T HAVE THE EXPERTISE SO WE RESTRICT IT OURSELVES TO NUCLEAR. >> OKAY. I THINK THAT DOES IT. THANK YOU SO MUCH. WE REALLY APPRECIATE THAT. (INDISCERNIBLE) SO EVERYBODY KEEP SAFE. MORE ABOUT DNA REPAIR ON LISTSERV, SEND E-MAILS TO KRAEMERK@NIH.GOV.