SO WELCOME TO THE DNA REPAIR GROUP. WE SEEM TO BE WORKING NICELY. NEXT MONTH WE'LL HAVE SUSAN WALLACE FROM THE UNIVERSITY OF VERMONT WILL BE SPEAKING ABOUT DNA IN BALTIMORE AND IN APRIL, ALEXANDRA WILL BE SPEAKING. AND THEN IN MAY WE WILL HAVE A SPECIAL HISTORY OF DNA REPAIR LECTURE FROM ALAN LEHMAN WHO WILL BE COMING FROM NEW ENGLAND TALKING ABOUT DNA REPAIR DISORDERS. IN JUNE WE WILL BE LECTURES BY DELAY YOUNG INVESTIGATORS AND PUTTING OUT THE REQUEST NOW THAT ANYONE HAS ANY NOMINEES FOR THAT WE WILL BE HAPPY TO GET THE NOMINATION AND GO FROM THERE. SO TODAY WE HAVE A LECTURE COMING FROM PITTSBURGH, KARA BERNSTEIN ASSURES ME SHE WILL BE DELIVERING A LECTURE TODAY BUT NOT A BABY AND WE WILL BE FOR HER TO SPEAK UNRAVELING MECHANISM FROM ERROR FREE DNA DOUBLE STRAND BREAK REPAIR. SO PLEASE EVERYONE ELSE MUTE AND WE WILL LET UNMUTE IN PITTSBURGH AND TAKE IT AWAY. >> THIS IS BEN, HELLO TO EVERYONE. IT'S MY PLEASURE TO INTRODUCE KARA BERNSTEIN TODAY. SHE GOT HER DEGREE IN BRYN MAWR IN PHILADELPHIA AND WENT ON TO GET HER MASTERS AND PH.D. AT YALE UNIVERSITY WORKING WITH DR. ZHU DR. -- SUSAN -- AND SHE WAS INTERESTED IN DOUBLE SOMAL DNA SPLICING. SHE DID A POST DOC AT COLUMBIA UNIVERSITY. SHE DID AN AMAZING JOB AND SERVE YEARS AGO WE WERE FORTUNATE TO RECRUIT KARA. THE COMMITTEE WAS EXCITED BECAUSE SHE BROUGHT DIFFERENT EXPERTISE. HER SAW SOME POWERS OF GENETICS AND IMAGING LOOKING AT DOUBLE STRAND BREAK REPAIR IN REAL TIME. AND THEN THE ABILITY TO DO LARGE SCALE DELETIONS. WE'RE VERY PLEASED, SHE CAME WITH K9 L00 AND GOT AN AWARD IN AGING. SHE BUILT HER LAB UP AND GOT A BEAUTIFUL PAPER IN NUCLEIC ACID RESEARCH. SO KARA. >> THANK YOU FOR THE INTRODUCTION. I STARTED MY LAB ABOUT A YEAR AND-A-HALF AGO AT THE UNIVERSITY OF PITTSBURGH CANCER INSTITUTE YOU CAN LEARN MORE ABOUT ME, AT MY LAB WWW.BERNSTEINLABS.COM. I'M GOING TO PRESENT A COLLABORATION WITH -- AT THE UNIVERSITY OF PITTSBURGH. TODAY'S TALK IS GOING TO BE DIVIDED INTO THREE PARTS. FIRST I'M GOING TO PROVIDE AN OVERVIEW OF CANCER AND ITS INTRINSIC RELATIONSHIP WITH GENETIC INSTABILITY. THEN I'M GOING TO TELL YOU ABOUT A NOVEL COMPLEX OF PROTEIN THAT WE FOUND TO BE AN IMPORTANT REGULATOR OF DNA RECOMBINATION. AND FINALLY I WILL TELL YOU HOW OUR DATA MEETS THIS NICE LITTLE MODEL IN CONCLUSION. CANCER IS A LEADING CAUSE OF DEATH IN THE UNITED STATES AND THIS IS ALSO TRUE WORLDWIDE. IN FACT IT'S THE SECOND LEADING CAUSE OF DEATH AFTER CARDIOVASCULAR DISEASE. THIS IS THAT CANCER DEVELOPMENT IS OF GLOBAL IMPORTANCE. IN THE UNITED STATES THE AMERICAN CANCER SOCIETY PREDICTS THAT THIS YEAR ALONE APPROXIMATELY 850,000 MEN AND 800,000 WOMEN WILL BE DIAGNOSED WITH CANCER. IMPORTANTLY, MANY OF THESE TYPES OF TUMORS ARE ASSOCIATED WITH MUTATIONS IN GENES UTILIZED IN A PAIR OF DOUBLE STRAPPED BREAKS. MY WORK HAS FOCUSED ON REPAIR OF DOUBLE STRAND BREAKS, ONE OF THE MOST LETHAL TYPES OF DAMAGE. HERE A DOUBLE STRAND BREAK IS ILLUSTRATED ON ONE END OF THE CHROMOSOME, THIS BEING THE CENTROMERE. EVERY DAY OUR CELLS NORMALLY REPAIR ABOUT 50 NATURAL DOUBLE STRAND BREAKS WHICH CORRELATES TO A FREQUENCY OF ONE DOUBLE STRAND BREAK FOR REPAIRS OF CELL CYCLE. SOME OF THE MOST COMMON CANCER THERAPEUTICS RELATE ON CELL DEATH SUCH AS RADIATION THERAPY WHICH CREATES DOUBLE STRAPPED BREAKS. SO WE ARE FACE THE WITH A THIS CONUNDRUM THAT MISREGULATION A COME STRAND BREAK REPAIR CAN BE USED, CAN BE A CAUSE OF CANCER ON THE ONE HAS NOT BUT ON THE OTHER HAND IT CAN ALSO BE USED AS A CURE FOR CANCER. IN FACT, GENOMIC INSTABILITY IS A HALLMARK OF TUMOR CELLS CAN WHICH LEAD TO -- THIS IS AN EXAMPLE OF A CARE OH TYPE OF A SUMER CELL WITH A EACH CHROMOSOME WILL BE PAINTED A SINGLE COLOR AND PRESENT IN TWO COPIES. HOWEVER, ANALYSIS OF CHROMOSOME ONE EXEMPLIFIES HOW THIS CHROMOSOME HAS UNDERGONE SIGNIFICANT CHROMOSOMAL REARRANGEMENTS. THERE ARE MULTIPLE COPIES OF THIS CHROMOSOME AS WELL AS INSERTIONS AND TRANSLOCATIONS OBSERVED. ACCURATE REPAIR DNA DAMAGE TO PREVENT THESE TYPE OF CHROMOSOMAL ABNORMALITIES WHICH ARE HALLMARKS FOR TUMORS. ONE IS A DIDN'T STRAND BREAK IS A MULTIPLE PATHWAY TO UTILIZE THE REPAIR BREAK AND THE TWO MAIN PATHWAYS ARE SHOWN HERE. DURING THE END JOINING, DIAGRAM ON THE LEFT ARE RELY GATED TOGETHER. ON THE RIGHT IS A HUH MALL COMBUST REBILLIONATION OF ILLUSTRATED. DURING THIS REPAIR PATHWAY A HOMOLOGOUS TEMPLATE SUCH AS A HOMOLOGOUS CHROMOSOME IS USED FOR REPAIR. OUR WORK IS FOCUSED ON THE HUH MALL GUST RECOMBINATION PATHWAY IN THE REPAIR OF DOUBLE STRAND BREAKS. ONCE THE CELLS ARE COMMITTED TO HUH MALL GUST REBILLIONATION A HOMOLOGOUS TEMPLATE IS USED FOR THE DAMAGE AND THEREFORE RESTORING INFORMATION AT THE BREAK SITE. THIS PATHWAY IS GENERALLY CONSIDERED AIR FREE AND IS USED WITH ENDOGENOUS DNA DOUBLE STRAND BREAK USED DURING DNA REPLICATION OR RESPONSE TO TOXIC CHEMICALS LIKE PHYSICAL AGENT RADON -- THE HUH MALL GUST RECOMBINATION PATHWAY WE CAN SEE AFTER A DOUBLE STRAND BREAK OCCURS, THE DNA ENDS ARE RESECTED GIVING IS RISE TO DNA OVERHANGS. THE SINGLE STRAND DNA -- THESE ARE RESOLVED IN THE MULTISTEP PROGRESSION. SO ALTHOUGH MANY OF THE GENES INVOLVED IN THE HR STEPS HAVE BEEN IDENTIFIED, IT'S LITTLE KNOWN ON HOW THESE PROTEINS ARE RECRUITED AND HOW HOMOLOGOUS RECOMBINATION IS REGULATED. SINCE WE DON'T UNDERSTAND THE FUNCTION OF ANY OF THESE GENES IN THIS PROCESS THESE ARE REALLY EXCITING TIMES TO STUDY HOMOLOGOUS RECOMBINATION. WE BELIEVE THESE PROVIDE AN EXCELLENT MODEL TO ADDRESS THESE QUESTIONS. SINCE THEY PREFERENTIALLY UTILIZE HUH MALL GUST PATHWAY TO REPAIR DOUBLE STRAND BREAKS. AFTER THE DOUBLE STRAND BREAK PROCESS THE SINGLE STRANDED DNA BINDING PROTEIN COATS THE SINGLE STRANDED DNA OVERHANG. THAT'S PROTECTING THE DNA SEGREGATION AND PERFORMING SECONDARY STRUCTURES. SUBSEQUENTLY RAD 52 AND ITS EPISTASIS PROTEIN INCLUDING THE RAD -- 55, 57 THIS YEAST OR RAD 51B, C, D, CC2 AND XRCC3 IN HUMANS HAVE PROMOTE -- FIRST BY DISPLACING RPA FROM THE SINGLE STRANDED DNA END AND SECONDLY BY ENABLING RAD 51 -- AND EXTENSION ALONG THE SINGLE STRANDED DNA ENDS. FORMATION OF RAD 51 FILAMENTS IS A KEY STAFF IN HUH MALL GUST RECOMBINATION BECAUSE THE RAD A 1 FILAMENTS ARE REQUIRED -- THEREFORE RAD 51 FILAMENT FORMATION IS VERY TIGHTLY REGULATED. THEY ARE BOTH PROTEINS THAT PROTEETH RAD 51 FILAMENT INFORMATION SUCH AS THE HIGH LIGHT OVER HERE. AS WELL AS PROTEINS THAT DISASSEMBLE RAD 51 FILAMENTS. OUR WORK AS I MENTIONED IS THE -- TO UNDERSTAND THE ROLE OF HOMOLOGOUS RECOMBINATION PROTEIN IN DOUBLE STRAND REPAIR. THERE ARE MANY ADVANTAGES TO USING YEAST AS A MODEL ORGANISM. FOR EXAMPLE THEY ARE EASILY MANIPULATED AND HAVE A SHORT DOUBLING TIME. THERE'S A LARGE NUMBER OF TYPES OF EXPERIMENTS THAT CAN BE DONE AND THERE'S A HUGE NUMBER OF RESOURCES AVAILABLE. FOR EXAMPLE WE HAVE ALL THE NON-ESSENTIAL GENES SYSTEMATICALLY DISRUPTED AND ERASED. AND MOST IMPORTANTLY IN THE MOST COMPELLING REASONS TO USE THESE TO STUDY DNA REPAIR IS THAT MOST OF THE DNA REPAIR PROTEINS IN YEAST ARE PRESERVED IN HUMANS WHICH MAKES IT OFTEN APPLICABLE TO THE -- CELLS. THIS IS AN EXAMPLE. FOR EXAMPLE THE RECQ IN HUMAN DISEASES IS CONSERVED IN ALL ORGANISMS. IN HUMANS THERE ARE FIVE DIFFERENT -- THERE'S ONE AND POSSIBLY A SECOND ONE HAS BEEN RECENTLY IDENTIFIED. SO THERE ARE THREE OF THE FIVE HUMAN GENES -- LEADS TO THESE HORRIBLE SYNDROMES. WARNER SYNDROME IS ONE I WILL HIGHLIGHT HERE. IT'S ILLUSTRATED IN THIS MIDDLE PANEL. THIS IS A VERY INTERESTING DISEASE BECAUSE THESE PATIENTS AGE PREMATURELY TO DEVELOP NORMALLY THROUGH EARLY ADOLESCENCE AND THEN THEY START TO TAKE ON THE PHENOTYPES OF AGING SUCH AS HAIR LOSS, OSTEOPOROSIS. AND THEY DIE PREMATURELY PRIMARILY FROM CANCER. THESE SYNDROMES ARE DIFFERENT FUNDAMENTALLY CHARACTERIZED AND TREATED FOR CANCER. INTERESTINGLY, THE HOMOLOGUE IN YEAST SGS1, DISRUPTION OF THIS GENE PHENOCOPIES MANY THAT ARE OBSERVED IN WARNER AND -- SYNDROME PATIENTS SUCH AS INCREASE CHROMATID EXCHANGE -- AND MEIOTIC -- IN FACT WHAT WE LEARNED IN YEAST ABOUT SGS1 HAS ENABLED US TO REALLY UNDERSTAND THE FUNCTION OF THESE OTHER PROTEINS IN DNA REPAIR. SO NOW FOR THE NEXT PART OF MY TALK, I'M GOING TO TELL YOU ABOUT A NOVEL COMPLEX OF PROTEINS CALLED THE SHOE COMPLEX AND HOW WE FOUND AN IMPORTANT REGULATORY ROLE FOR THIS COMPLEX DURING DNA REPAIR. SPECIFICALLY IN PROMOTING RAD 51 FILAMENTS. SO ONE OF THE KEY PROBLEMS IN THE FIELD IS THE UNDERSTANDING OF HOW DNA, HAS REPAIR DOUBLE STRAND BREAKS ARE REGULATED. AND THIS IS THE VERY IMPORTANT QUESTION BECAUSE MISREGULATION OF DOUBLE STRAND BREAK REPAIR IS ASSOCIATED WITH CANCER. THERE ARE MUTATIONS IN TUMORS THAT HAVE BEEN SHOWN TO BOTH YOU REGULATOR AND DOWN REGULATE FOR COMBINATION. SO CLEARLY REGULATING RECOMBINATION IS A KEY FACTOR IN PROMOTING ERROR-FREE REPAIR. TODAY I'M GOING TO TOLD YOU ABOUT A NOVEL COMPLEX OF PROTEINS CALLED THE SHOE COMPLEX. THAT WAS CONSERVED FROM YEAST TO HUMAN. WE WERE SURPRISED TO DISCOVER TOGETHER IN OUR WORK AND FROM OTHER LABORATORIES THAT THE SHOE COMPLEX CONTAIN ADDITIONAL RAD 51 -- WE FIND THAT THE SHOE COMPLEX IS AN IMPORTANT REGULATOR OF THE COMBINATION THAT PROMOTES RAD 51 FILLMENT FORMATION AND THUS ERROR-FREE DNA REPAIR -- REPAIR MECHANISMS. THE SHU IS PREVIOUSLY IDENTIFIED BY A STUDENT IN MY LAB. SUPPRESS THE DNA DAMAGE SENSITIVITY OF SGS1 DISRUPTED CELLS -- I JUST TOLD YOU ABOUT. IDENTIFIED FOUR NOVEL GENES SHU 1, SHU 2, CSM2 AND CSY. THEY ARE COLOR CODED. ALTHOUGH THEY ARE IN THE SAME COMPLEX THEY EXHIBIT SOME PHENOTYPES. WHEN WE STARTED THIS PROJECT, WE KNEW THE COMPLEX FUNCTION IN A HOMOLOGOUS RECOMBINATION PATHWAY BUT WE HAD LITTLE UNDERSTANDING WHAT ITS FUNCTION MIGHT BE. CELLS WITH DISRUPTION OF THE SHU GENES ARE SENSITIVE TO THE DNA AGENT MMS WHICH CAN GIVE RISE TO DOUBLE STRAND BREAKS A REPLICATION ENCOUNTERS A LESION. HERE'S AN EXAMPLE OF A NICK ON ONE STRAND OF THE DNA THAT BECOMES THE DOUBLE STRAND BREAK UPON MOVEMENTS DURING REPLICATION. SO THESE ARE SERIALLY DILUTED ON TO RICH MEDIUM ON THE LEFT OR MEDIUM CONTAINING MMS AND YOU CAN SEE THE DISRUPTION OF ANY OF THESE COMPONENTS OR ANY COMBINATION OF THESE COMPONENTS LEADS TO SENSITIVITY OR TO A GROWTH DEFECT. DISRUPTION OF THE SHU GENES ALSO LEADS TO SENSITIVITY WHICH IS FLATTENED WHICH CAN ALSO RESULT IN DOUBLE STRAND BREAKS. THIS SENSITIVITY IS ALSO MUTATION OF ONE OF THE HUMAN OART LOGS AT STAR CC2. DISRUPTION OF THE SHU GENES LEADS TO A MUTATOR PHENOTYPE. WHEN RAISED TO RESPONSE TAIN ANNUAL MUTATION IN THE GENE ARE MEASURED AND THESE INCREASES TO ESSENTIAL DNA REPAIR PROTEIN RAD 52 IS DISRUPTED. CONSISTENT WITH THE MUTATOR PHENOTYPE DISRUPTION OF SHU 1 OR 2 LEADS TO GROWTH CHROMOSOMAL REARRANGE'S WHICH ARE CHARACTERISTIC OF TUMOR CELLS. SO THESE PROTEINS PHYSICALLY FORM A COMPLEX TOGETHER BY YEAST HYBRID AND -- THE SHU COMPLEX IS CONSERVED IN -- YEAST THIS IS WORK FROM PAUL RUSSELL'S GROUP AS WELL AS HUMAN CELLS. IN FACT TWO OF THE SHU OART LOGS RAD 51D AND -- ARE RAD 51 PARA LOGS. THESE ARE PROTEINS WHICH SHOWS STRUCTURAL SIMILARITIES TO RAD 51. INTERESTINGLY RAD 51D INTERACTS WITH THE HUMAN CHROME LOG OF SGS1 BLOOM SUGGESTING THAT THIS GENETIC INTERACTION BETWEEN THE SHU COMPLEX AND SGS1 MAY ALSO BE CON SERVED IN HUMANS. AND TOGETHER RAD 51D AND SRCC2 -- IN DESTRUCTION, IN DISRUPTION OF HOMOLOGOUS INTERMEDIATES. IMPORTANTLY SRCC2 AND ADD 52 HAVE MORPHISMS ASSOCIATED WITH CANCER PREDISPOSITION SUGGESTING A ROLE IN -- INTEGRITY. WE WANTED TO ADDRESS THE QUESTION OF HOW THE SHU COMPLEX PROMOTES RECOMBINATION. NOW I'M GOING TO SHOW YOU HOW WE FOUND THAT THE SHU COMPLEX -- YOUTHIZE IN THE HOMOLOGOUS RECOMBINATION PATHWAY THAT INTERACTS WITH RAD 51 THROUGH THE OTHER RAD 51 PARALOGS. AND FINALLY THE SHU COMPLEX IS A REGULATOR OF PROMOTING HOMOLOGOUS RECOMBINATION AT THE EXPENSE OF ERROR PRONE DNA REPAIR PATHWAYS. JUST THIS PAST YEAR TWO INDEPENDENT GROUPS CRYSTALLIZED TWO MEMBERS -- AND INDICATED PROTEINS ARE STRUCTURAL HOMOLOGUES TO RAD 51 -- JUST LIKE IN HUMANS. BOTH OF THESE PAPERS INDICATE THAT THE SHU COMPLEX BIND TO SINGLE STRANDED AND DOUBLE STRANDED DNA AND INTERESTINGLY THAT THE HETERODIMER IS CSM2 -- MEDIATES THE DNA BINDING ACTIVITY OF A COMPLEX. SO YOU CAN SEE THE STRUCTURE OF PSY3 AND PSM2. PART B IS AN OVERLAY OF THE TWO WITH STRUCTURAL SIMILARITY BETWEEN THESE PRO PROTEINS. ON THE BOTTOM LEFT IS -- OVERLAID WITH RAD 51 AND YOU CAN SEE THE STRUCTURAL SIMILARITY. AND ON THE BOTTOM RIGHT, CSM2 OVERLAID WITH RAD 51. AND SO SINCE THESE PROTEINS ARE VERY, HAVE A STRUCTURAL SIMILARITY TO RAD 51, THEY ARE INDEED LIKELY RAD 51 PARALOGS. SO ALTHOUGH CSM2 AND CSY3 HAD STRUCTURAL SIMILARITIES TO RAD 51 WE WANTED TO DETERMINE IF THE SHOE COMPLEX PAYER LOGS DOWN TO STRUCTURED DNA. TO DETERMINE THE PREFERRED BENDING SUBSTRATES FOR THE SHU COMPLEX WE -- HETEROGENEITY IN COLLABORATION WITH ADAM WIER FROM -- WE FOCUSED ON THESE TWO PROTEINS IN PARTICULAR BECAUSE SHU ONE AND SHU 2 ARE DISPENSABLE FOR DNA BINDING AND -- SHOWN HERE ON THE LEFT. TO TEST THE PREFERRED DNA SUBSTRATE OF THE SHU COMPLEX, WE GENERATE A SERIES OF DNA SUBSTRATES SUCH AS A FOUR SUBSTRATE, A DNA WITH A THREE TIME OVERHANG, FIVE TIME OVERHANG, DNA DIMER AND A DNA DIMER AND WE TESTED HOW -- BIND TO THE SUBSTRATE BY ELECTROMOBILITY SHIFT ASSAY. THE SINGLE STRANDED DNA AND FOURTH SUBSTRATE -- IN THE MIDDLE LANE, YOU CAN SEE WHERE THE -- WITHOUT BEING BOUND TO CSM THE AND PSY3. IF WE NOW ADD THEM, WE OBSERVE A GEL CHECK INDICATING THAT THEY BIND TO THIS LABELED DNA. IF WE ADD AN UNLABELED VERSION OF THE FORK, THE UNLABELED FORK EFFECTIVELY COMPETES FOR CSM2 AND PSY3 BINDING RESULTING IN THE SHIFT OF -- TO DETERMINE IF THE BINDING OF CSM2 AND PSY3 IS SPECIFIC FOR SUBSTRATES WE NEXT TESTED OTHER DNA SUBSTRATES FOR BINDING TO CSM2 AND PSY3 HETERODIMER SUCH AS A FIVE TIME DNA OVERHANG. THE FIVE TIME OVERHANG DOES NOT COMPETE NEARLY AS WELL FOR CSM2 AND PSY3 AS THE FORK. THE THREE TIME OVERHANG COMPETES SIGNIFICANTLY BETTER FOR THE FOURTH SUBSTRATE AND THE FIVE TIME OVERHANG BUT NOT AS WELL AS THE FOURTH SUBSTRATE. AND NEITHER THE DIMER. DNA DIMER OR SINGLE STRANDED DNA COMPETES FOR CSM2 AND PSY3. SO AL TOGETHER WE FIND THAT CSM2 AND PSY3 BIND TO A FOURTH DNA AS SHOWN HERE AND HERE. OVER THE THREE PRIME DNA OVERHANG. U.S. AND WE WERE REALLY EXCITED ABOUT THIS BECAUSE THE DNA ARE HR SUBSTRATES SO THEREFORE THE SHU COMPLEX MAY BE RECRUITED TO HOMOLOGOUS RECOMBINATION INTERMEDIATES THROUGH CSM2 AND PSY3. SO THIS IS THE -- SUBSTRATE. OKAY. SO TO QUANTITATE THE AMOUNT OF CSM2 AND PSY3 BOUND TO THE FOURTH DNA WE GENERATED DNA BINDING CURVE USING FLUORESCENCE AND -- THIS WORK WAS DONE BY A GRADUATE STUDENT IN MY LABORATORY, STEVEN GOLDEN IN COLLABORATION IN COLLABORATION WITH ADAM WIER AND THE WORK WAS PRIMARILY DONE BY STEPHEN. IN THIS ASSAY WE DETERMINED THAT CSM2 AND PSY3 PREFERENTIALLY BIND TO A FOURTH DNA WITH AN APPARENT KD OF 360 NANOMOLAR. WE FOUND IT BIND TO DNA WITH SUBSTRATES SHOWN HERE IN THE TOP RIGHT. AGAIN WE FIND DNA 4-S THE BEST COMPETITOR FOR CSM2PSY3 BINDING FOLLOWED BY THE THREE TIME OVERHANG SHOWN IN GREEN. AND THEN THE FIVE PRIME OVERHANG. THESE RESULTS CONFIRM OUR PREVIOUS OBSERVATIONS THAT THEY PREFERENTIALLY BIND TO DMA SUBSTRATES UTILIZED BY THE HOMOLOGOUS RECOMBINATION PATHWAY. NOW THAT WE KNOW THAT THE SHU COMPLEX BINDS TO THREE PRIME DNA OVERHANG, WE WANTED TO DETERMINE CSM2 OR PSY3 INTERACT WITH OTHER PROTEINS THAT WERE KNOWN TO FUNCTION AT THAT SAME PROCESSING HR SPECIFICALLY RAD 51 WHICH IS THIS YELLOW, LITTLE YELLOW DOTS AND THE RAD 51 PARALOGS RAD 55 AND 57 SHOWN. TO DO THIS WE USE THE HYBRID ASSAY. IN THIS ASSAY -- WE SUBSEQUENTLY INTRODUCED TWO PLASMIDS INTO THE STREAM. ONE THAT EXPRESSES THE -- PROMOTER BINDING DOMAIN FUSED TO CSM2 OR PSY3. THE OTHER PLASMID EXPRESSES THE PROMOTER ACTIVATING DOMAIN FUSES TO EITHER RAD 51, RAD 55 OR RAD 57. IF THE TWO PROTEINS INTERACT TO THE ACTIVATING DOMAIN AND BINDING DOMAIN WILL BE BROUGHT TOGETHER AS SHOWN HERE. AND THE REPORTER GENE WILL BE EXPRESSED WHICH ALLOWS GROWTH IN THE HISTIDINE. BY THESE TWO HYBRID WE FIND THAT CSM2 SPECIFICALLY INTERACTS WITH RAD 51. AND THE OTHER, AND THE RAD 51 PARALOGS, RAD 55 AND RAD 57. THERE ARE CSM2 AND PSY3 LIKELY BRING THE SHU COMPLEX TO HOMOLOGOUS RECOMBINATION THAT INTERACTS WITH RAD 51 AND THE RAD 51 AWE LOGS. WE WONDER IF CSM2 INTERACTED WITH RAD 55 VIA RAD 51 OR IF CSM2 INTERACTED WITH RAD 51 TO RAD 55. TO ADDRESS THIS QUESTION, WE DISTRUST THE EITHER RAD 51 OR RAD 55 AND -- CSM2 INTERACTION WITH RAD 55, RAD 57 OR RAD 51, RAD 57 RESPECTIVELY. WHEN RAD 51 IS DISRUPTED, WE STILL OBSERVE THE TWO HYBRID INTERACTION WITH RAD 55 SHOWN HERE. IN CONTRAST, WHEN RAD 55 IS ENTRUSTED, WE NO LONGER SEE INTERACTION BETWEEN CSM2 AND RAD 55. OR RAD 51. THEREFORE THE INTERACTION IS LIKELY MEDIATED BY RAD 55 SHOWN HERE. CURRENTLY WE ARE NOW VERIFYING THE RESULTS. SINCE BOTH OF THESE THE SHU COMPLEX AND RAD 55, RAD 57 FUNCTIONING TO PROMOTE HOMOLOGOUS RECOMBINATION WE ASK IF THEY FUNCTION AT THE SAME PATHWAY OR IF THEY REDUNDANT FUNCTIONS TO ONE ANOTHER. ONE OF THE DEFINING CHARACTERISTICS OF RAD 55 IS THEIR COLD SENSITIVITY. WHEN RAD 55 IS DISRUPTED AND DILUTED THE CELLS WERE -- OR AFTER EXPOSURE TO MNS OR IONIZING MEDIATION. THE SENSITIVITY OF RAD 55 DISRUPTIVE CELLS ARE THOUGHT TO REFLECT THE ROLE THAT RAD 55 PLAYS IN STABILIZING THE RAD 51 PROTEIN FILAMENTS. TO ADDRESS THIS CSM2 AND RAD 55 ARE FUNCTION IN THE SAME PATHWAY, WE ASK CSM2 WERE EVEN STATIC TO RAD 55. WE CREATED CSM2 AND RAD 55 SINGLE AND DOUBLE AND TESTED THEIR SENSITIVITY TO DNA DAMAGE AT 23 OR 30 DEGREES CELSIUS. AT FIRST IS YEAST WERE DILUTED ON TO RICH MEDIUM AS A LOADING CONTROL. SUBSEQUENTLY WE ANALYZED THE GROWTH OF THESE EXPOSED TO MEDIUM CONTAINING MMS AN -- AT 23 DEGREES CELSIUS. WHAT WAS UNEXPECTED IS IT IS ALSO COLD SENSITIVE SO YOU CAN SEE CSM2 IS MORE SENSITIVE AT LOWER TEMPERATURES. INTERESTINGLY, CSM2 RAD 55 DOUBLE IS NOT MORE SENSITIVE TO MMS THAN A RAD 55 SINGLE MUTANT. INDICATING THAT RAD 55 IS EVEN STATIC TO CSM2 IN RESPONSE TO MMS. SIMILARLY, CSM2 RAD 55 DOUBLE IS NOT MORE SENSITIVE TO IONIZING RADIATION THAN A RAD 55 SINGLE. AGAIN INDICATING AN EPI STATIC RELATIONSHIP. SINCE RAD 55 IS EVEN EPI STATIC -- C IF THEY HAD DONE IN FUNCTION. TO ADDRESS THIS QUESTION WE ASKED IF RAD 55 OVER EXPRESSION COULD RESCUE THE MMS SENSITIVITY OF A CSM2 MUTANT. WE INTRODUCED MP PLASMIDS EXPRESSING RAD 55 AND RAD 57 INTO WILD TYPE OR CSM2 DISTRUSTED CELLS IN WILD TYPE CELLS EXPRESSION OF ANY OF THE PROTEINS FROM THE PLASMIDS HAD NO EFFECT ON GROWTH. EITHER ON RICH MEDIUM OR MMS. IN CSM2 DISRUPTED CELLS OVER EXPRESSION OF RAD 55, RAD 57 DOES NOT RESCUE THE MMS SENSITIVITY TO THESE CELLS. IN CONTRAST WITH CSM2 OVER EXPRESSION OF RAD 55, RAD 57 IN A RAD 55 MUTANT PARTIALLY RESCUES THE GROWTH EFFECT OF A RAD 55 MUTANT. THESE RESULTS SUGGEST THAT RAD 55, RAD 57 DO NOT LIKELY HAVE REDUNDANTANT FUNCTIONS OF CSM2 EVEN THOUGH THEY FUNCTION THE SAME PATHWAY. SINCE SCM2 AND RAD 55 WERE TO PROMOTE RAD FILAMENT INFORMATION WITHIN THE SAME EPISTASIS GROUP WE ASKED THE CSM2 OR PSY3 MY ALTER THE RECRUITMENT TO THE DNA DAMAGE SITES. FOLLOWING TREATMENT WITH DNA DAMAGING AGENTS LIKE IONIZING RADIATION FLUORESCENTLY -- LIKE RAD 51 OR RAD 55 WILL CONCENTRATE AT A BREAK SITE FORMING VISIBLE -- RAD 55 WITH YOLE OWE FLUORESCENT PROTEIN IN A WILD TYPE CSM2 OR PSY3 LACK GROUND WE CAN EVALUATE RAD 55 TO FOCUS INFORMATION UNDER UNTREATED OR RADIATED CONDITIONS. UNDER WILD TYPE CONDITIONS SHOWN HERE IS UNTREATED, RAD 55 IS EXTREMELY RARE IN ALL THREE STRAINS. FOLLOWING TREATMENT WITH IONIZING RAIDATION, RAD 55 IS RECRUITED TO THE BREAK SITE SHOWN BY THIS WHITE ARROWHEAD FORMING A DISCREET FOCUS IN THE NUCLEUS OF TREATED CELLS. THIS HAPPENS IN WILD TYPE CELLS THAT WAS SIGNIFICANTLY REDUCED IN CSM2-PSY3 INFECTED CELLS. WE ANALYZED THE RECRUITMENT OF RAD 55 TO DNA OVER TIME IN EITHER WILD TYPE OR CSM2 OR PSY3 AFTER THE CELLS WERE -- IONIZING RADIATION AND THAT IS DOUBLE BRAND BREAKS. AFTER 60 MINUTES THERE'S A POKE OF RAD 55 OBSERVED AND A SUBSEQUENT DECREASE OVER TIME LIKELY RING REPAIR AND RESOLUTION OF THESE DOUBLE STRAND BREAKS. IN CONTRAST, CSM2 AND PSY3 DO NOT HAVE A LARGE INCREASE IN RAD 55 AT ANY TIME POINT. WE NEVER WERE ABLE TO OBSERVE ABOVE 4% OF THE CELLS RAD 55 FOCUS. SO THE INABILITY OF RAD 55 FORM SUGGESTS THAT CSM2 AND PSY3 ARE NEEDED FOR EFFICIENT RECRUITMENT TO THE DNA DAMAGE SITE. SINCE THE SHU COMPLEX PROMOTES RAD 51 REMEDIATED RECOMBINATION WE WANTED TO KNOW IF -- TO ADDRESS THIS QUESTION UNDERGRADUATE IN THE LABORATORY -- UTILIZED A DIRECT REPEAT RECOMBINATION ASSAY TO DETERMINE THE RATE OF RAD 51 REPAIR. THIS ASSAY IS COMPRISED OF A FUNCTIONAL -- WHICH ALLOWS THESE TO GROW WITHOUT ADDED CELLS SURROUNDING BY HETEROALLELES. ON THE LEFT-HAND SIDE IS A GENE REQUEST THE THREE PRIME MUTATION BY INSERTION OF -- WHEREAS ON THE RIGHT-HAND SIDE THE GENE HAS A FIVE TIME MUTATION BY INSERTION OF THE -- FOLLOWING A SPONTANEOUS DOUBLE STRAND BREAK IN EITHER THESE TWO ALLELES. HUH MALL GUST RECOMBINATION CAN OCCUR BETWEEN THE CHROMATID WHERE THE MUTATION OF ONE ALLELE IS REPAIRED BY THE WILD TYPE PORTION OF THE ALLELE. SO THIS FIVE TIME WILL BE REPAIRED BY USING THIS. THIS IS SHOWN IN THE BOTTOM LEFT HEAR AND IS ABSOLUTELY DEPENDENT ON RAD 51. ALTERNATIVELY THE BREAK REPAIR USING THE RAD 51 INDEPENDENT PATHWAY SUCH AS A -- WHERE THE ENDS OF THE BREAK ARE RESECTED EXPOSING HOMOLOGY BETWEEN THE TWO ALLELES. THIS RESULTS IN LOSS OF THE INTER-- WILD TYPE ALLELE WHICH NOW DETERMINES HOW THE BREAK WAS REPAIRED BY ASKING FOR THE PRESENCE OR ABSENCE OF THE GENE. USING THIS ASSAY A WILD TYPE STRAIN HAS APPROXIMATELY -- AMOUNTS OF RAD 51 DEPENDENT AND INDEPENDENT REPAIR. SO RAD 51 DEPENDENT REPAIR BEING GENE -- AND INDEPENDENT REPAIR BEING THE SING UG STRANDED ALLELE SHOWN ON THE RIGHT. HOWEVER, A CSM2 MUTANT HAS AN ELEVATED LEVEL OF RAD 51 INDEPENDENT SINGLE STRANDED AND A DECREASE LEVEL IN RAD 51 DEPENDENT GENES CONVERSION. A RAD 55 IS VERY LITTLE RAD 51 DEPENDENT GENE CONVERSION AND AN INCREASE IN THE RATE OF SINGLE STRANDED EVENTS. THE DOUBLE CSM2 RAD 55 MUTANT HAS NO ADDITIONAL EFFECT ON GENE CONVERSION OR SINGLE STRAND -- COMPARED TO A RAD 55 SINGLE MUTANT AGAIN SUGGESTING THAT RAD 55 IS EVEN STATIC TO CSM2. AND SO TO PUT ALL THIS DATA TOGETHER IN A MODEL, WE THINK THAT AFTER A DOUBLE STRAND BREAK OCCURS, CSM2 AND PSY3 MEDIATE BINDING OF THE SHU COMPLEX TO THE BREAK SITE. THIS IS THROUGH THE DNA BINDING ACTIVITIES PREFERRED DNA BINDING ACTIVITY FOR A THREE TIME DNA OVERHANG OR REPLICATION INTERMEDIATES. ONE DOUBLE DNA THE SHU COMPLEX LIKELY STABILIZES RAD 51 FILAMENTS, PERHAPS THROUGH INTERACTING WITH RAD 51, VIA RAD 55, RAD 57 AND THROUGH A SECOND INDEPENDENT MECHANISM WHICH I DO HAVE TIME TO TALK TO YOU ABOUT IS THROUGH INHIBITING -- WHICH IS KNOWN TO DISASSEMBLE RAD 51 FILAMENTS. AND SO WE THINK THAT THE SHU COMPLEX IN ESSENCE PROMOTES HOMOLOGOUS RECOMBINATION WHILE SUPPRESSING REPAIR SO IT SHIFTS THE BALANCE OF DNA REPAIR TOWARDS AN ERROR-FREE DNA PATHWAY WHILE AT THE SAME TIME SUPPRESSING -- SO WITH THAT, I'D LIKE TO THANK THE MEMBERS OF MY LABORATORY, PARTICULARLY STEPHEN GODDEN -- WHO ALSO CONTRIBUTED TO THIS PROJECT. THEY ARE BOTH VERY TALENTED UNDER GRADUATES. I WOULD ALSO LIKE TO THANK ANDREW -- AND ADAM -- AND DR. BEN VAN HOUTON WHO HELPED US WITH THE -- EXPERIMENTS AND A LOT OF THE BIOCHEMICAL ANALYSIS AND I WOULD LIKE TO THK MY FUNDING MECHANISM ESPECIALLY THE NATIONAL INSTITUTE OF HEALTH FOR THEIR FUNDING MY PROJECT. [APPLAUSE] >> THANK YOU VERY MUCH FOR THAT NICE TALK. AT THIS POINT WE'LL GO AROUND AND ASK QUESTIONS. THE RULES ARE WE GET ONE QUESTION PER SITE. WE'LL GO AROUND FIRST. WE HAVE A FAIR AMOUNT OF TIME, THAT'S WONDERFUL. IF THERE ARE ADDITIONAL QUESTIONS WE CAN GO AROUND A SECOND TIME. BE SURE THAT ALL OF YOUR SITES ARE MUTED. AND DON'T WE GO TO CHAPEL HILL FIRST, UNIVERSITY OF NORTH CAROLINA. >> THAT WAS A WONDERFUL TALK, THANK YOU VERY MUCH. THIS IS SOMETHING THAT I HAVE BEEN THINKING ABOUT, I'VE BEEN STUDYING -- AND I'M TRYING TO UNDERSTAND THE MECHANISMS OF TRANSLOCATIONS BETWEEN CHROMOSOMES. >> COULD YOU PLEASE IDENTIFY YOURSELF. >> I'M SORRY? I'M SORRY [INDISCERNIBLE] THE QUESTION THAT I HAVE IS IN THIS DECISION TO BETWEEN USE OF HOMOLOGOUS RECOMBINATION PATHWAY OR STINGAL STRAND ALLELING MEDIATED BY THE SHU COMPLEX, COULD IT BE THAT WHEN THE SHU COMPLEX IS EFFECTIVE AND THING ARE BEING SHUNTED TO SINGLE STRANDED -- THAT THAT PATHWAY ISN'T MORE ERROR-PRONE SUCH THAT IT WILL TAKE DIFFERENT CHROMOSOMES SEARCH FOR HOMOLOGY BETWEEN SEGMENTS OF THOSE BREAKS AND MUST GENERATE RECOMBINED CHROMOSOMES AS OPPOSED TO ONE CHROMOSOME BREAK THAT IS JUST RELIGATED BETWEEN MATERIALS IN THE END -- COULD BE CONTRIBUTING TO EXCHANGES BETWEEN -- HOMOLOGOUS CHROMOSOME. >> I THINK THAT'S ABSOLUTELY A POSSIBILITY. >> KARA, COULD YOU PLEASE SWITCH THE CAMERA SO WE CAN SEE YOU AND NOT THE SLIDE. THERE WE GO. >> YOU CAN SEE THE BOTTOM OF MY HEAD. I THINK THAT'S ABSOLUTELY A POSSIBILITY AND I THINK PERHAPS AN EVIDENCE FOR THAT IS THE FACT THAT IT DISRESULTS SHU ONE OR SHU 2. RICHARD'S GROUP HAS SHOWN -- IT'S POSSIBLE THAT THERE MAY BE A ROLE FOR THESE GENES IN [INDISCERNIBLE] MELANOMA TUMORS. >> THANK YOU. >> THANK YOU. LET'S BE SURE TO MUTED IN CHAPEL HILL. LET'S MOVE TO UNIVERSITY OF KENTUCKY. >> YES. CAN YOU HEAR US? >> YES. >> OKAY. YES, WE ENJOYED THE TALK AND I HAD A QUESTION. YOU SHOWED THAT THE SHU COMPLEX PREFERENTIALLY BINDS -- OVERHANG. DID YOU CHECK FOR ANY OTHER SUBSTRATES LIKE OTHER THREE STRANDED OR FOUR STRANDED STRUCTURES THAT MAY BE POTENTIALLY LIKE RECOMBINATION INTERMEDIATES OR SOMETHING TO LIKE WIDEN THE, YOU KNOW, WINDOW OF WHAT IT WOULD BIND TO. >> COULD YOU PLEASE IDENTIFY YOURSELF WHEN YOU ASK A QUESTION. >> THIS IS -- FROM THE UNIVERSITY OF KENTUCKY, I'M AN ASSISTANT PROFESSOR HERE AND THAT'S OUR QUESTION. >> THAT'S A VERY GOOD QUESTION. SO WE DID NOT TEST FOR THINGS THAT RESEMBLE HOLIDAY JUNCTION. SO WE DON'T KNOW IF THAT WAS A POTENTIAL SUBSTRATE. THE ONLY THING THAT WE TESTED FOR SINGLE AND DOUBLE STRAND DNA IS A FIVE TIME OVERHANG AND A FOURTH SUBSTRATE BUT THAT'S WHERE WE, THE BIOCHEMISTRY WAS ALL NEW TO ME -- SPECIAL HELP FROM BEN LAB AND --'S LAB. >> OKAY, THANK YOU. IT WAS A GREAT TALK. THANK YOU. >> THANK YOU. >> THANK YOU. PLEASE MUTE IN KENTUCKY. AND LET'S GO TO BALTIMORE. >> I WOULD LIKE TO REITERATE IT WAS A VERY ELEGANT AND CLEAR PRESENTATION. I HAVE SORT OF A GENERAL QUESTION RELATED TO THE COLD SENSITIVITY. IS THERE -- >> ABSOLUTELY. WHAT WE THINK IS THAT COLD SENSITIVITY IS OFTEN ASSOCIATED WITH STABILIZATION OF A LARGER COMPLEX. AND SO WE THINK THAT FOR AT LEAST IN THE CASE OF RAD 55 OR RAD 57, THAT THEY ARE STABILIZING RAD 51 NUCLEAR PROTEIN FILAMENTS. EVIDENCE FOR THAT IS THAT YOU CAN REST DO THIS COLD SENSITIVITY BY OVER EXPRESSING A RAD 51, RAD 51. WE DID NOT SEE WHEN WITH A OVEREXPRESSED RAD 51 IN OUR MUTANT STRAIN, ALTHOUGH IT'S POSSIBLE THAT MAYBE WE DIDN'T HAVE ENOUGH RAD 51 BEING OVEREXPRESSED SO THAT'S SOMETHING THAT WE'RE CURRENTLY TESTING. WE THINK THAT LIKELY THE SHU COMPLEX IS ALSO STABILIZING A LARGER PROTEIN THAT EITHER THE PROTEINS ON TO THE RAD 51 FILAMENT OR THE RAD 51 FILAMENT ITSELF AND THAT'S SPECULATIVE AND WE DON'T HAVE DIRECT EVIDENCE FOR THAT RIGHT NOW. >> THANK YOU VERY MUCH. >> OKAY, WONDERFUL. PLEASE MUTE IN BALTIMORE AND LET'S GO TO BROOK HAVEN. YOU HAVE A QUESTION. IDENTIFY YOURSELF FIRST. OKAY. IT MAY NOT BE THERE. LET'S GO DOWN THE I'LL AISLES. >> -- IT LOOKS LIKE THE STUDY WERE PERFORM WITH -- PSY3 HETERODRIERM. DO YOU KNOW WHETHER THE OTHER TWO SUBUNITS INFLUENCE THE BINDING SPECIFICITY. >> SO WE FOCUSED ON THESE TWO PROTEINS SPECIFICALLY BECAUSE IT WAS FOUND BY TWO OTHER GROUPS WHERE THEY DID THE CRYSTALLIZATION ANALYSIS THAT THE DNA BINDING WAS MEDIATED BY -- SPECIFICALLY. AND I DON'T REMEMBER ANY EVIDENCE SUGGESTING THAT THERE WAS FURTHER OR ENHANCED BINDING WITH SHU 1 OR SHU 2. ALTHOUGH IN BOTH THESE STUDIES THEY ONLY LOOKED AT SINGLE STRANDED DNA OR DOUBLE STRANDED DNA. AND SO WE DIDN'T LOOK WITH THE STRUCTURED DNA AND SO THAT IS I GUESS STILL POTENTIALLY A POSSIBILITY. >> ONE OTHER POINT ABOUT THIS BINDING. I NOTICE YOU HAD A HILL COEFFICIENT GREATER THAN 2. DO YOU HAVE SOME IDEA THAT PERHAPS THERE'S MORE THAN ONE COMPLEX BOUND TO EACH MOLECULE? >> GOOD QUESTION. I DON'T THINK THAT WE THOUGHT THAT THERE WAS -- YES, HE'S NAWGD HIS HEAD. AT THE PRESENT TIME WE DON'T THEY ARE THERE'S MORE THAN ONE COMPLEX BINDING ALTHOUGH IT IS POSSIBLE THAT AS THE RAD 51 FILAMENT FORMS THERE MIGHT BE MULTIPLE SHU COMPLEXES THAT ARE INCORPORATING THE FILAMENT. ALTHOUGH WE HAVEN'T TESTED THAT MODEL YET. IT'S SOMETHING THAT WE WERE LOOKING TO DO BY PERHAPS SINGLE MOLECULE. >> OKAY, THANKS. >> OKAY, THANK YOU. PLEASE MUTE IN STONY BROOK AND LET'S GO TO NIAID RESEARCH TRIANGLE IN NORTH CAROLINA. >> THIS IS MIKE -- I HAVE A QUESTION ABOUT THE NATURE OF THE -- ENDS. FIRST OF ALL -- AND SECOND OF ALL IS THERE ANY EVIDENCE OF THIS COMPLEX IN TERMS OF THE RESECTION. AND FINALLY -- WHAT ABOUT WHAT WOULD HAPPEN AFTER THE -- TUMORS. >> OKAY. SO -- >> THAT WAS A TRIPLE QUESTION. >> YES, THAT'S OKAY. WE DEFINITELY, WE HAVEN'T TESTED GAS MOLECULES. I DO NOT KNOW ABOUT THAT. IN TERMS OF TELOMERES, I DON'T, WE HAVE NEVER LOOKED AT TELOMERES SPECIFICALLY. SO I DON'T KNOW IF THERE'S ANY FUNCTION AT THE TELOMERE. AND I HAVE DONE A LOT OF WORK ON RESECTION AND I HAVEN'T REALLY FOUND, NOT REQUEST MORE WITH RESPECT TO SGS1 AS OPPOSED TO THE SHU COMPLEX. WE HAVEN'T FOUND ANY EVIDENCE THAT SUGGESTS THAT PERHAPS THESE PROTEINS ARE INVOLVED IN RESECTION. THEY'RE NOT NUCLEASED AND THEY ARE NOW -- LIKE SOME OF THE OTHER RESECTION, SOME OF THE OTHER PROTEIN THAT ARE INVOLVED IN THOSE INITIAL LIKE -- OR DNA2 OR XL1 AND SO I DON'T KNOW IF THERE'S ANY COMPELLING EVIDENCE RIGHT NOW BUT WE HAVEN'T TESTED THAT DIRECTLY. >> THANK YOU. >> I DID USE MIKE'S PAPER TO FIGURE OUT HOW MANY DOUBLE STRAND BREAKS THERE ARE PER IR. SO THAT'S A REALLY GREAT CONTRIBUTION. >> GREAT, THANKS, MIKE. PLEASE MUTE AT RESEARCH TRIANGLE. LET'S GO TO PORTLAND ORGANIZE. >> THIS IS AARON JACOBS FROM -- VERY NICE TALK, THANK YOU FOR THAT. I HAD A VERY. WHAT HAPPENS TO THE RAD 51 FILAMENTS AFTER STRAND INVASION. YOU MENTIONED THAT THEY -- TURNOVER IS NOT MUCH OF AN ISSUE OF DOUBLE STRAND BREAK REPAIR BUT IS THERE ANY TYPE OF DISASSEMBLY THAT GOES ON? >> YES, THERE IS. SO THERE ARE ALSO POSTINGS THAT ARE INVOLVED IN DISASSEMBLING RAD 51 FILAMENTS. IN YEAST, THERE'S SRS2 ALTHOUGH I THINK SRS2 MOSTLY DISASSEMBLED BEFORE THE STRAND [INDISCERNIBLE] RIGHT, RAD 54. AND RDH54 PERHAPS ARE ALSO INVOLVED. AND THEN IN HUMANS IT'S A LITTLE BIT MORE COMPLICATED. THERE'S A NUMBER OF DIFFERENT PROTEINS SUCH AS -- L WHICH IS ANOTHER RESCUE HOMOLOGUE AND RTELL WHICH ARE ALL INVOLVED IN EITHER REMOVING RAD 51 BEFORE OR AFTER STRANDED LESION. IN HUMANS IT SEEMS LIKE THERE'S SOME PROTEINS THAT HAVE SPECIFICALLY INTERACTIVELY PERFORMED AT THESE DIFFERENT -- BUT I THINK IT'S STILL A LITTLE UNCLEAR. >> THANK YOU. >> OKAY, THANK YOU. PLEASE MUTE IN PORTLAND. IS THERE ANYONE THERE IN ANN ARBOR? ARE YOU THERE? >> YES, WE'RE HERE, THANK YOU VERY MUCH KARA, IT WAS A NICE TALK. ALL OF THE QUESTIONS HAVE BEEN ANSWERED, SO WE ARE DEFERRING ANY QUESTIONS. THANK YOU VERY MUCH. OKAY, THANK YOU. PLEASE MUTED IN MICHIGAN. AND WE HAVE A QUESTION HERE. PLEASE IDENTIFY YOURSELF. >> HI, I'M [INDISCERNIBLE] SO HOW LONG ARE THE SUBSTRATES THAT YOU USED WHEN YOU WERE SHOWING ONE OF THE EARLIER SLIDES, THE FORK AND THE OVERHANGS BINDING TO THE PROTEIN. >> MM-MM. TO BE HONEST, I DON'T REMEMBER OFFHAND. >> THE REASON WHY I'M ASKING IS I'M ACTUALLY COMING FROM U.S. CHAPEL HILL AND I WAS IN -- LAB THERE AND ONE OF THE THINGS HE DOES THERE IS LOOKING AT THINGS YOU ARE DOING USING EM. IF THE SUBSTRATES ARE LONG ENOUGH AND THE PREY TEEN COMPLEX IS LARGE ENOUGH, YOU MAY BE DO IMAGING THAT YOU MIGHT MAY NOT BE ABLE TO ADDRESS SUCH AS DOES THE PROTEIN BIND AT THE FORK BUT LIKE YOU SHOWED, BUT AFTER BINDING, DOES IT SLIDE. AND IF IT SLIDES, WHERE MIGHT IT GO. OR DOES IT BIND TO THE FORK AND STOP NUCLEASING -- DNA. ALSO, ONE OF THE EARLIER QUESTIONS WAS DOES MORE THAN ONE COMPLEX BIND. SO THAT ALSO WOULD BE POSSIBLE QUESTION TO TACKLE USING TECHNIQUE SUCH AS EM. >> SO HOW LONG DO THOSE SUBSTRATES HAVE TO BE? >> IT'S EASILY ABOUT A KB BUT SHORTER LIKE 500 WOULD ALSO BE POSSIBLE. >> YES. I THINK WE'RE IN THE 20-50 RANGE. WE'RE WAY OFF ON THAT. >> BUT IT CAN BE ENGINEERED TO BE LONGER OF COURSE SO THAT'S USUALLY -- TO REENGINEER THEM FOR MIBLGING. >> THAT'S AN INTERESTING POINT. THANK YOU. >> VERY NICE TALK, THANKS. >> I'VE GONE AROUND ONCE. I HAVE A QUESTION RELATED TO YOUR INTRODUCTION. YOU WERE POINTING OUT THAT THE DOUBLE STRANDED BREAK REPAIR IS IMPORTANT BOTH FOR CERTAIN DISEASES RESCUE RE RE-- AND FOR CANCER. DO YOU HAVE ANY SPECULATION HOW THE INSIGHTS IN THE MECHANISM OF DOUBLE STRAND BREAKS REPAIR MIGHT BE USED EITHER FOR CANCER TREATMENT OF TREATMENT OR TO ALLEVIATE SOME OF THE SYMPTOMS IN THE PATIENTS. >> SURE. SO IT'S SOMETHING THAT WE'VING THINKING A LOT ABOUT, SO WE BELIEVE THAT THE SHU COMPLEX IS REGULATING RECOMBINATION AND SO MAYBE PERHAPS IN CASES WHERE YOU'RE HAVING TOO MUCH RECOMBINATION YOU COULD PERHAPS TARGET REGULATORS OF RECOMBINATION TO HELP TO ALLEVIATE THAT PROBLEM. AND SO YOU KNOW WE HAVEN'T DONE ANY WORK, MORE CLINICAL WORK ON APPLYING IT. I THINK MOSTLY WE DIDN'T KNOW ANY OF THESE PROTEINS WERE DOING SO I THINK WE'RE JUST TRYING TO SHED SOME LIGHT ON TO HOW THEY MIGHT ACTUALLY BE REGULATING THE RECOMBINATION AND THEN WE CAN THINK MORE CAREFULLY ABOUT HOW THIS KNOWLEDGE CAN BE USED OR APPLIED THERAPEUTICALLY. >> IT MIGHT BE USED IN CANCER TREATMENT. >> IF YOU HAD WAYS TO EITHER UP REGULATE OR DOWN REGULATE RECOMBINATION, THAT COULD BE REALLY IMPORTANT FOR LIKE I MENTIONED BEFORE IN MY INTRODUCTION, CREATING DOUBLE STRAND BREAKS IS USED AS A MECHANISM TO -- THE TUMOR CELLS SO MAYBE YOU COULD CREATE MOLECULES THAT WOULD MAKE THEM MORE SUSCEPTIBLE TO KILLING BY DISRUPTING AND CHARGING, I DON'T KNOW, SPECIFICALLY IN THOSE TUMOR CELLS. I GUESS THAT'S ONE POSSIBILITY. I MEAN THERE'S OTHER THING THAT YOU MIGHT IMAGINE. >> YES, THANK YOU. ARE THERE QUESTIONS OF ANY OF THE OTHER SITES. HOW ABOUT IN PITTSBURGH, ANYONE IN THE AUDIENCE HAVE QUESTION THERE? >> I DON'T BELIEVE THERE ARE ANY QUESTIONS. I THINK THEY'VE HEARD THIS BEFORE. >> ARE THERE ADDITIONAL QUESTIONS AT ANY OF THE SITES YOU CAN JUST UNMUTE AND JUMP IN. NO. IF NOT, WELL THANK YOU. [APPLAUSE] >> THANK YOU VERY MUCH FOR AN EXCELLENT TALK.