>> SO WE'RE GOING TO GO TO DR. FORD WHO HAS SOME ANNOUNCEMENTS. >> HI, I FEEL I HOPE YOU FEEL BETTER, TOO. THE WEATHER IS NICE. ONE THING WE WANT TO SAY WAS WE WERE GOING TO TRY TO DO AN ADOBE CONNECT TEST RUN, NEXT TIME FOR MAY 20, RIGHT? SO INFORM ME AGAIN BY E-MAIL. WHAT WE'RE GOING TO DO IS JUST AFTER THE SESSION -- >> SURE. >> AFTER THE SESSION, WE WILL TRY TO CONNECT TO EVERYONE WITH ADOBEE CONNECT AND AND NIH- NIH-SPONSORED VIDEO VIA A SYSTEM THAT WHEN HE PRODUCED, WHICH IS SOMEWHAT SIMILAR TO THE FACILITIES THAT WE CAN MAINTAIN AND COPY THESE SESSIONS. AND WE WOULD -- INAUDIB[INAUDIBLE] ON THE TRANSMISSION. SO PLEASE LET US KNOW IF YOU CAN DO THIS BY THE AFTERNOON SESSION NEXT MONTH ON MAY 20TH. AND HAVE SOMEBODY BE AVAILABLE TO TEST IT AT 1:30 FOR YOUR CONFERENCE CALL. AND THEN I GUESS NEXT MONTH WE WILL HAVE A TALK ON HIGH, - -- HIV, TALKING ABOUT DNA REPAIR AND DNA REPAIR. AND THEN IN JUNE, WE HAVE OUR YOUNG INVESTIGATOR PRESENTATIONS PRESENTATIONS, WHICH WE HAVE LINED UP. AND THEN WE REMIND YOU ALL THAT WE DO LOOK FORWARD TO YOUR SUGGESTIONS FOR NEXT YEAR'S SPEAKERS IN THE SERIES. THANK YOU. BACK TO YOU, PAM. >> OKAY, THANK YOU. LET'S SEE IF WE CAN GET BACK. HERE I AM, THAT'S RIGHT. IN JUNE IT'S GOING TO BE JOHN FROM THE UNIVERSITY OF NORTH CAROLINA TALKING ABOUT -- INAUDIBLE INAUDIBLE AND ROBBIE SULLIVAN, THE INVESTIGATOR, AND WILL BE DR. KING AT N ASIEH S TALKING ABOUT HYPERMUTATION. OUR SPEAKER TODAY -- THE SPEAKER TODAY IS THE DR. MERRITT, WHO WAS AT TELL -AVIV UNIVERSITY AND HE WAS -- SHE WAS A RESEARCH FELLOW AND MOVED TO SCIENCE AND THEN CAME TO THE UNITED STATES IN LA JOLLAA HAS A POST DOC. AND IN 1999, SHE JOINED THE NCI IN THE LABORATORY OF MOLECULAR FAPHARMACOLOGY. IN 2007, SHE BECAME INVESTIGATOR AND SHE WILL BE TELLING US ABOUT DNA REPRESELICATION AND GENOME MAINTENANCE. LET ME SEE IF I CAN GET THIS THING TO WORK. HERE WE GO. >> GREAT. >> OKAY. >> THUM FOR THE INTRODUCTION. AND THANK YOU FOR INVITING ME TO PRESENT AT THIS CONFERENCE. THANK YOU. I HAVE BEEN WATCHING THEM REMOTE LY FOR MANY YEARS, AND I OFTEN LEARN VERY IMPORTANT INFORMATION FROM THOSE ON DNA REPRESELICATION. SO DNA REPRESELICATION IS TEHE PRIME PRIMARY TOP IC FOR MANY SIGNALING AND TO OUR STUDIES AT THE NCI, MANY ANTI-CANCER DRUGS IS DUE TO REPRESELICATION AND IT IS WELL-KNOWN THAT THE DISTINCT EFFECT ARE OFTEN UNKNOWN. AND THIS IS A VERY IMPORTANT POINT, BECAUSE CANCER SPECIFICALLY DEPENDS ON THE FOSS PHOSPHOR FER ARASE AND INHIBITED BY THE ANTI-CANCER DRUGS. SO OUR STUDIES THAT WASN'T RECENTLY MADE AVAILABLE AT THE BRANCH ASKED THE FOLLOWING QUESTION. WE ARE LOOKING AT HOW TO DECIDE WHEN AND WHERE REPRESELICATION WOULD START, AND WE ALSO ASK HOW DOES -- HOW DO DECISIONS CHANGE WHEN REPRESELICATION? OUR KNOWLEDGE IS HOW REPLICATION IS REGULATED IS STILL VAGUE ON THE MODEL THAT WAS PROPOSED MORE THAN HALF A CENTURY AGO. AND IN EUCAR YOUKARYOTES, WE KNOW THAT DUPLICATION ORIGIN ON CHROMATINS THAT THOSE ORIGINS CONTAIN SPECIFIC SEQUENCES THAT SERVE AS BINDING SITES FOR MULTIPLE. AND THOSE ARE THE INITIATORS THAT TARGET CHROMATIN TO STOP DNA REPRESELICATION. WE KNOW THAT THE MODEL IS THE OPERATING -- OPERATING THROUGH A RECOGNITION COMPLEX, OR ORC, WHICH BINDS CHROMATIN AND LOOKING AT THE REPRESELICATION COMPLEX. SO DURING THE G 1 PHASE, MANY ADDITIONAL COMPONENTS ARE ADDED TO FORM THE REPRESELICATION COMPLEX. REPRESELICATION COMPLEXES ARE NOTED IN AN ACTIVE FORM, AND DURING THE ESSENCE OF THE CELL SHOCK, THEY ARE ACTIVATED BY SPECIFIC EVENTS SUCH AS FOPHOSPHORYLATION WITH KINASES EXACTLY THROUGH THE INITIATION OF DNA REPRESELICATION. SO ALL THAT SUGGESTS TO YOU TO US THAT THE CHROMATIN POCKETS OF CELL CYCLE SIGNALING COULD BE IDENTIFIED BY REPRESELICATION ORIGIN AND WE ACOULD USE ALL THE SKENEITISS TO LOOK FOR REPRESENT REPLICATION ORIGINS. THAT IS TRUE. UNFORTUNATELY, IN OUR CELLS, WE DUPLICATE THE OTHER COMPONENTS OF PREREPRESELICATION COMPLEXES, WE CANNOT USE IN ORDER TO IDENTIFY REPRESELICATION ORIGIN, BECAUSE THOSE 14 COMPLEXES EXHIBIT NON- NON-SPECIFIC DNA BINDING. SO IN ORDER TO UNDERSTAND WHERE CHROMATIN REPRESELICATION MIGHT START, WE HAVE TO GO AND MEASURE DIRECTLY WHERE REPRESELICATIONS INITIATE. SO TODAY I AM GOING TO TELL YOU ABOUT OUR STUDIES ON REPRESELICATION ORIGIN AT THE NCI FOR 15 YEARS AND WE HAVE MADE SOME PROGRESS IN UNDERSTANDING OF DNA REPRESELICATION DURING THAT TIME. I AM GOING TO MENTION BRIEFLY SOME DNA INTEROAACTIONS THAT ARE DISTINCT FOR DNA REPRESELICATIONS, WHICH DICT AATE INITIATION OF DNA REPRESELICATION FOR SPECIFIC ORIGIN. I AM GOING TO TELL YOU ABOUT CHROMATIN MODIFICATION THAT ASSOCIATES THIS REPRESELICATION ORG ORIGIN AND ABOUT HOW REPRESELICATION CHANGES WHEN REPRESELICATION IS ACT ACTIVATED. AND FINALLY I AM GOING TO BRIEFLY MENTION DUPLICATION REPRESELICATION INITIATION EVENT TO MAINTAIN GENOME SPEC FIIFICITY. SO HOW DO WE DO ALL THAT? WE TAKE A TWO-PRONG APPROACH IN OUR LABORATORY BASED ON THE FACT THAT WE CAN IDENTIFY REPRESELICATION ORIGIN SKENEITISS IN HUMAN CELLS CELLS, AND WE CAN FOLLOW THE EXTENT OF DUPLICATION, AS WELL AS THOSE REPRESELICATION ORIGIN SEQUENCES GENETICALLY. SO ONCE WE HAVE INFORMATION ABOUT DNA REPRESELICATION, WE TAKE THOSE REPRESELICATION ORIGINS AND USE THEM AS BROWNIES TO CAPTURE INTERACTING PROTEINS AND IDENTIFY ORIGIN-SPECIFIC PROTEIN DUPLICATION ON ONE HAND. ON THE OTHER HAND, SO THIS IS INFORMATION FOR VERY DISTINCT REPRESELICATION ORIGIN. WE ARE ALSO INTERESTED IN MAPPING ORIGIN GENOME-WIDE JOB, WE CREATE GENOME-WIDE REPRESENT REPLICATION MAPS AND THEN LINE THOSE MAPS ARE WITH CHROMATIN DATA TO IDENTIFY ORIGIN-SPECIFIC INTEROAACTIONS THROUGH THE ENTIRE GENOME. WE CAN THEN COMBINE THAT INFORMATION TO DETERMINE THE SPECIFIC INTEROAACTION IN REGULATE REGULATING THE GROWTH AND MAINTAINING GENOMIC STABILITY. SO OUR ORIGINAL STUDIES FOCUSED ON A SINGLE REPRESELICATION ORIGIN, WHEN WE HAVE DONE AN STENEXTENSIVE GENETIC SECTION AND WHEN WE START WITH THOSE INTERACTIONS, THE FIRST THING WE NEED, OF COURSE, IS A MODEL. AND THIS IS MY MODEL, THE HUMAN HEMODMROEBIN MODEL. AND CODED GENES IN CELLS. THEY CALL ENCODE FOR THE DIFFERENT HEMOGLOBINS IN EM EMBRYONIC CELLS AND SICK LLE DEVELOPMENT. REPRESELICATION IN ALL THOSE CELLS, AS FAR AS WE KNOW, STARTS AT THE THIS REGION, THE INITIATION REGION. AND THAT'S THE LEP INDICATION ORIGIN. AND WE HAVE IDENTIFIED WITH THIS REGION. AND THE GENETIC REQUIREMENT FOR INITIATION WITHIN THAT REGION, WE HAVE IDENTIFIED PARTICULAR LOCATIONS AND PARTICULAR SKENEITISS OF OUR EXTENSION FOR INITIATION. WE HAVE CARRIED THAT ANALYSES THROUGH THE SINGLE BASE AND WE NOW HAVE A VERY GOOD IDEA OF WHAT IT TAKES TO START DNA REPRESELICATION. WE HAVE ALSO IDENTIFIED SOME PROTEIN INTEROAACTIONS WITHOUT EXTENSION FOR INITIATION. BESIDES THE INTEROAACTIONS, WE ALSO FOUND THAT THERE IS A REGION OF INTEROAACTION WITH OTHER PROTEINS THAT IS ESSENTIAL FOR INITIATION. WE KNOW THAT IT IS ESSENTIAL BECAUSE OF THE INTEROAACTION SITES SITES. AND WHEN THE MUTANTS DONE BIND, IT ALSO DOES NOT INITIATE DNA REPRESELICATION. AND THOSE STUDIES ARE SUBMITTED AND IF YOU ARE INTERESTED, I WOULD BE VERY HAPPY TO TELL YOU MORE ABOUT THAT. THE QUESTIONS THAT I WOULD LIKE TO SHOW NOW IS WHETHER IT'S SPECIFICALLY WITH THE HEMOGLOBIN MODELS OR IT IS A REGULATOR OF REPRESELICATION INITIATION GENOME- GENOME-WIDE. IN ORDER TO DO THAT, WE WERE INTERESTED WHAT WOULD HAPPEN IF WE -- AND WHAT WILL HAPPEN THROUGH REPRESELICATION PROFILES THROUGHOUT THE GENOME. SO HOW ARE WE GOING TO LOOK FOR THE REPRESELICATION PROCESS THROUGHOUT THE GENOME? YOU SEE A FIBER ANALYSIS, ALSO KNOWN AS COMBINE, IN WHICH WE TAKE CELLS AND WE LABEL THEM WITH TWO SUBSEQUENT NUCLEOTIDES ANALOGUES IN THIS CASE IDU AND WE HAVE SPECIFIC ANTIBODIES THAT CAN RECOGNIZE EITHER ONE OF THOSE HOMOLOGS. SO ONCE THE LABELING IS COMPLETE COMPLETE, WE CAN ISOLATE DNA TRANSFER AASE CELLS AND STRETCH THEM ON THE SLIDES IN A WAY THAT WE FORM A UNIFORM STRETCHING. WHEN WE DO THAT AND IDENTIFY THE LOCATION S S IN WHICH ANALOGUE IS INKRORCORPORATED, WE CAN FOLLOW DNA REPLICATION. SO FOR EXAMPLE, WE CAN IDENTIFY REPRESELICATION ORIGIN IN THIS STAGE STAGE, INITIATED REPRESELICATION DURING THE FIRST PHASE. SO WE TAKE THIS IN GREEN, FLAGGED BY TWO RED SIGNALS ON THOSE SITES AND REPRESELICATION IS INITIATED FROM THIS ORIGIN. IF REPRESELICATION ONLY STARTS DURING THIS SECOND PERIOD, WOULD BE AN ISOLATED WEB SIGNAL AND THE REPRESENT -- IF REPRESELICATION STARTS BEFORE THE BEGINNING OF THE LABELING PERIOD, WE COULD SEE A REPRESELICATION ORIGIN IN THE REGION THAT FORM ON THOSE SITES. SO THIS WAY, WE CAN TAKE THOSE REPRESELICATION ORIGINS. WE CAN MEASURE THE DISTANCES BETWEEN THOSE REPRESELICATION ORG ORIGINS, AND WE CAN ALSO MEASURE THE EXTENT OF DNA REPRESELICATION AND VESSLOCITY OF REPRESENLICATION FOSS PHOSPHORYLATION SIMPLY BY MEASURING THE EXTENT OF LADIES AND GENTLEMEN. WE CAN ALSO PROVIDE THAT ANALYSIS WITH INSIGHT INTO VISUALIZATION. AND HERE AGAIN I AM SHOWING A VISUALIZATION WITH MY FAVORITE LOGGING, AND YOU SEE THAT IT MIGHT BE HARD TO SEE ON THE VIDEOCONFERENCE, SO IN BLUE, WE CAN ACTUALLY SEE THE REPRESELICATION PROCESS IN THE VICINITY OF OUR FOLD. AND THIS WAY, WE CAN DETERMINE REPRESELICATION INTO THE ORIGIN DISTANCES GENOME-WIDE AND ALSO AT SPECIFIC SITES. SO IF WE USE THIS PARTICULAR TECHNIQUE, ALL THE CELLS THAT EITHER CONTAIN ID OR DERIVES FROM ANY MODES THAT HAS A DELETION F ID, WE KNOW THAT IT WAS IDENTIFIED BASED ON THE SITE SITE. AND IF WE MEASURE THE INTERORG INTERORIGIN DISTANCES IN THE CELLS, WE FIND THAT THE INTERORG INTERORIGIN DISTANCE IS LONGER, SUGGESTING THAT SOME ORIGINS MIGHT NOT INITIATE DNA REPRESENT REPLICATION. THEY MIGHT HAVE FEWER INITIATION EVENTS. ON THE OTHER HAND, IT IS IMPORTANT TO NOTE THAT THOSE MICE ARE GROWING, SUGGESTING THAT IT'S NOT ESSENTIAL FOR INITIATION OF DNA REPRESELICATION IN ALL REPRESELICATION ORIGINS.^ IT MIGHT BE IMPORTANT FOR INITIATION OF A PARTICULAR GROUP OF A PARTICULAR ORIGIN. WITH THAT -- [INDISCERNABL[INDISCERNIBLE] THERE IS AN ANALYSIS AND WE ALSO HAVE SHOWN IN HUMANS. SO THIS OBSERVATION IN MICE REMINDED US OF A FACT THAT WE HAVE KNOWN FOR A VERY, VERY LONG TIME, SINCE THIS REPRESELICATION ORG ORIGIN WAS ORIGINALLY IDENTIFIED IN THE LAB OF SEED ER, WHERE THEY HAVE IDENTIFIED A DELETION OF THE ENTIRE I RCHR IN THE HUMAN GENOME. AND WHEN -- THIS IS A DELETION THAT OCCURS IN PATIENTS. THOSE PATIENTS HAVE PROBLEMS WITH THE HEMODMROEBIN PRODUCTION PRODUCTION. BUT THEY DONE HAVE ANY ISSUES WITH REPRESELICATION. REPRESELICATION STOPS AT THE GENOME. THE ENTIRE REPRESELICATION -- THERE IS NO INITIATION WITHIN THE IR. BUT WE DO NOT SEE ANY SUBCYCLES AND FOPHOSPHORYLATION IS TEHE GOING VERY WELL. AND THAT IS CONSISTENT WITH OTHER OBSERVATIONS, PRIMARILY OBSERVATIONS IN YEAST IN WHICH REPRESELICATION ORIGINS CAN SPREAD THAT SUGGEST THAT DELETINGS OF INDIVIDUAL DUPLICATION ORIGIN DOES NOT AFFECT PROGRESSION. SO ONE OF THE QUESTIONS IS IF WE REALLY DON'T NEED ALL THOSE ORG ORIGINS, WHAT ARE THEY GOOD FOR? I WILL RETURN TO THAT QUESTION LATER. SO THE CURRENT VIEW OF THE INITIATION WITH REPRESELICATION ORG ORIGIN IN CEUCAKARYOTES IS THAT ONLY A FRACTION OF ORIGIN AND THAT FRACTION VARIES BETWEEN 10 PERCENT AND 20% ACTUALLY INITIATE REPRESELICATION THROUGH A NORMAL GROWTH. AND THAT FRACTION IS SUFFICIENT TO REPRESELICATE THE ENTIRE GENOME UNDER NORMAL CONDITIONS. SO THE MODELS WERE ORIGINALLY SUGGESTED BY DE PONTE HERE AT NIH WAS THAT REPRESELICATION ORIGIN WAS DURING THE G 1 CYCLE. HOWEVER, ONLY A FEW OF THE REPRESELICATION ORIGINS ARE ACTUALLY SELECTED FOR INITIATION ON EACH CHROMOSOME, EACH CELL CYCLE. SO ONCE REPRESELICATION STARTS REPLICATING AT PARTICULAR LOCATIONS AND HERE WE SEE TWO REPRESELICATIONS, THOSE REPRESENLICATION BUBBLES WILL EXPAND AND THOSE WILL CONTINUE AND THEY MIGHT OVERRUN ANOTHER INACTIVE POTENTIAL REPRESELICATION ORIGIN. AND SIN-- UNTIL THE REPLICATION OF THE ENTIRE GENOME IS COMPLETED. SO SOME ORIGINS ARE SIMPLY NOT UTILIZED THROUGH EACH SUBCYCLE. -- CELL CYCLE. SO WHY DO WE NEED THOSE ORIGINS? ALSO IS THAT CRYPTIC OR DORM AANT ORIGINS FIRE PARTICULAR CYCLE MAY BE REALLY IMPORTANT WHEN REPRESELICATION IS PRESERVED. AND THAT IS BECAUSE IF THOSE REPRESELICATIONS COMING FROM TWO DIRECTIONS ARE INHIBITED AT A PARTICULAR REGION AND THERE IS NO REPRESELICATION ORIGIN IN THE MIDDLE, THOSE CELLS CANNOT EASY EASILY RECOVER FROM DAMAGE. THEY HAVE TO RESORT TO A GENE TYPIC EVENT IN SOME CASES. AND IF THERE IS READY REPRESENT REPLICATION ORIGIN, THAT COULD START REPRESELICATION IF REPRESENLICATION FOR ANY REASON IS REGISTERED AT AJANIT ORIGIN. SO THOSE DORM AANT ORIGINS CAN ACT AS BETA AND CAN BE USED WHEN CELLS RECOVER FROM REPRESELICATION. SO KEEPING THAT FACT IN MIND, IT WILL BE REALLY USEFUL TO KNOW WHERE THE ADJACENT ORIGINS ARE IN OUR SYSTEMS. AND FOR THAT, WE CHOSE TO LOOK AT WHERE THE OTHER ORIGINS ARE THROUGHOUT THE GENOME. SO AS I'VE MENTIONED BEFORE, A SECOND PART OF OUR STRATEGY IS CREATING GENOME-WIDE REPRESELICATION MAPS AND HOW ARE WE DOING THAT? SO WE ARE DOING THAT BY DIRECTLY ISOLATING NEWLY REPRESENLICATED DNA STRANDS ON CELLS THAT ARE GROWING. SO THE ISOLATION IS BASED ON SIZE, AND WE ACTUALLY HAVE TWO WAYS TO IDENTIFY -- REPRESELICATION. ONE WAY IS DEMONSTRATED HERE, WHERE IT IS TAKING ADVANTAGE OF THE FACT THAT EACH NEWLY REPRESELICATED STRAND HAS A TAIL. SO BASICALLY WHAT WE DO IS ISOLATE IT ON SITE AND WE KEPT THEM BY USING A NUCLEASE THAT WILL ELIMINATE DNA FRAGMENTS OF THE DNA END BUT WILL KEEP THE DNA FRAGMENTS THAT HAVE RNA PHASE. THE OTHER METHOD SIMPLY INVOLVES ANALOGUES, IN THIS CASE WE USE REDOX IN ISOLATING THOSE INTER INTERMEDIATE SELECTIONS. AND IN BOTH YIELD MORE OF THE SAME DATA. AND WHAT WE CAN DO WITH THOSE DNA FRAGMENTS IS WE CAN TEND SEND IT TO SEQUENCING, OBVIOUSLY AND THEN ALIGN WITH THE ENTIRE GENOME. SO HERE AGAIN I AM SHOWING A LOBING AND THIS IS ON A GENOME VIEW AND WE CAN SEE THAT WHEN WE HAVE ACCUMULATION OF STRANDS CORRESPOND TO THE REPRESELICATION ORIGIN. WE CAN THEN TAKE THESE DATA AND ALIGN THEM WITH OTHER DATA. FOR EXAMPLE, PRECIPITATION OR DATA OF PARTICULAR DUPLICATION AND OF COURSE DNA INTEROAACTION. WHEN WE DO THAT, WE CAN ACTUALLY TAKE ALL THOSE LINES OF THE VARIOUS PARTS OF THE GENOME AND ALIGN THEM, SUCH AS AS I'M SHOWING HERE. SO EACH LINE HERE CONTAINS THE E EQUIVALENT OF 50 RAND AOMLY SELECTED GENOMIC REGIONS ON A REPRESELICATION-ONLY GENE AND THEY -- THE EXTENT OF COLORING HERE CORRESPONDS TO THE LEVEL OF DUPE DUPLICATION. IN THIS CASE THIS IS THE AR TIC ARTICULATION OF 3 ON LINE 27. AND ATS YOU CAN SEE, THERE IS A VERY STRONG CORRELATION OF THAT PARTICULAR REPRESELICATION WITH A REPRESELICATION ORIGIN. THIS IS FOR ALL REPRESELICATION AND FOR ALL THE REPRESELICATIONS. SO HERE IS A REPRESELICATION THAT IS LOCALIZED, AND WE CAN TAKE ALL THIS DATA AND WE CAN QUANTITFY THIS. WE CAN TAKE THE AREAS UNDER THE GRAPH, AND WE CAN CLUSTER THOSE DATA AND WE CAN ACTUALLY ARRIVE THROUGH A POPULATION OF CHROMAT CHROMATIN MODIFICATIONS LOCAL LOCALIZED, EITHER WITH ALL REPRESELICATION ORIGINS OR WITH GROUPS OF ORGIGES -- ORIGINS. WE DID NOT FIND A REPRESELICATION PU UNIQUE LY MARKED BY WE DO FIND REPRESELICATIONS THAT ASSOCIATE WITH GROUPS OF ORIGINS. SO IN ORDER TO -- BECAUSE THIS WORKS IN A COMPLEX AND IN ORDER TO SIMPLY IIFY THAT AND TO MAKE IT ACCEPTABLE TO EXPERIMENT, WE HAVE DEVELOPED A WEBSITE THAT A ALLOWS UTSERS TO INPUT THEIR FILES, AND WE ARE WORKING WITH REPRESELICATION ORIGINS THAT OTHER PEOPLE CAN USE THE SAME WEBSITE IN ORDER TO PUT ANY COLLECTION OF DNA SEQUENCES THAT THEY ARE INTERESTED IN. AND THEN WE MEASURE THE EXTENT OF LOCALIZATION WITH PUBLICLY AVAILABLE CHROMATIN MODIFICATION DATA, SUCH AS THE DATA THAT WE HAVE TAKEN FROM THE USC BROWS ER AND SOFROM OTHER DATA SETS. WHEN ONE ESTIMATES THE SITES FOR ANALYSIS, THEN THE OUTPUT IS -- AND WE CAN ALSO GET STABLE THAT COMPUTES THE EXTENT OF LOCAL LOCALIZATION. SO THAT HELPS TO CYCLE HOW OTHERS COORDINATE WITH OTHER FACTORS, SUCH AS TRANSCRIPTION AND DNA FOPHOSPHORYLATION BASED ON THOSE DUPLICATIONS. AND THIS IS A PUBLIC WEBSITE, AND I WILL BE VERY INTERESTED IN SHARING. AND SO WITH THIS, WE COULD LOOK AT GENOME-WIDE REPRESELICATION DINE DYNAMICS AND ASK QUESTIONS, SUCH AS LOCALIZATION TO IDENTIFY PROTEINS THAT ACTIVATE WITH GROUPS OF ORIGINS GENOME-WIDE. IF THOSE INTEROAACTIONS CHANGE WHEN REPLICATION IS PERFORMED AND HOW REPRESELICATION INITIATION FOR -- TO ORG YOU NATE WITH TRANSCRIPTION AND WITH OTHER TRANSACTIONS THAT HAVE IMPORTANTLIED CONCOM TANT LY ON CHROMOSOMES. SO AS AS AN EXAMPLE, THIS IS AN ANALYSIS OF THE EXTENT OF TRANSCRIPTION ON ONE SIDE AND THE EXTENT OF REPRESELICATION INITIATION ON THE OTHER SIDE, IN WHICH OF THE GENOMES THAT ARE CLUSTERED BASED ON THE EXTENT OF GENE INITIATION. AND AS YOU CAN SEE, METHYLATION CORRELATES WITH INITIATION, WHEREAS IT CORRELATES WITH TRANSCRIPTION, SUGGESTING THAT REPRESELICATION INITIATION EVENTS DO NOT OCCUR AT EXACTLY THE SAME LOCATION AS TRANSCRIPTION INITIATION EVENTS. WE CAN LOOK AT IT DIRECTLY THIS WAY. SO HERE WHAT WE DID WAS WE TOOK THE GENOMIC REGIONS OF CHROMATIN AND ALIGNED THEM SO THAT THEY ARE ON THE TRANSCRIPTION START SITE AND THEN LOOKED AT THE EXTENT OF REPRESELICATION INITIATION EVENTS. WHAT WE CAN SEE IS THAT IN THE TRANSCRIBED GENES, WE SEE A MIX TO THE TRANSCRIPTION START SITE, SUGGESTING THAT REPRESELICATION ORG ORIGINS ARE DEPLETEED FROM THE ACTUAL TRANSCRIPTION START SITE. THIS HAPPENS IN TRANSCRIBED REGIONS BUT NOT IN UNTRANSCRIBED REGIONS. AND OBVIOUSLY, IN CELLS WE HAVE GENE-SPECIFIC TRANSCRIBED, AND THAT TRANSCRIPTABILITY MIGHT EXPLAIN WHY WE NEED ADDITIONAL ORIGINS AND THE LOCATIONS OF INITIATION EVENTS SHOULD BE FLEXIBLE. SO THERE ARE TWO POSSIBILITIES. SO WE KNOW THAT REPRESELICATION INITIATION EVENTS, PARTICULARLY WITH ORIGIN, INHIBIT DUPLICATION FROM ADJACENT ORIGINS. WHAT WE THINK I DON'T KNOW IS WHETHER THESE EVENTS ARE FLEXIBLE SO THAT DURING NORMAL GROWTH, EACH PROMSOME MIGHT START AT A DIFFERENT LOCATION. OR IF CHROMOSOME CONTAINS DOM DOMINANT ORIGINS. AND THE ORIGINS ARE MENTION EARLIER ARE DOORNLT, THE CHROME CHROMOSOMES ARE NEVER ACTIVATED UNLESS REPLICATION IS PRESERVED. IF REPRESELICATION ORIGINS ARE FLEXIBLE AND EACH ORIGIN STARTS AT A DIFFERENT CHROMOSOME, UNDER CONDITIONS OF STRESS, WHEN ADDITIONAL ORIGINS ARE ACTIVATED ACTIVATED, WE WILL NOT MAP MORE REPRESELICATION ORIGINS. WE WILL MAP THE SAME LOCATIONS A DUPLICATION ORIGINS. THEY WILL SIMPLY STOP DUPE DUPLICATION AT A HIGHER FREQUENCY. ON THE OTHER HAND, IF WE HAVE DORM AANT GENES THAT ARE CONSISTENT LY REPLAYCING OTHER GENES, WE WILL START THE LOCAL ORIGINS IF WE START DNA REPRESELICATION ENOUGH TO CAUSE A CELL CYCLE DELAY BUT NOT ENOUGH FOR TRANSCRIPTION. SO WE WERE INTERESTED IN DOING THIS EXPERIMENT. AND ONE OF THE CHALLENGES WAS TO FIND CONDITIONS THAT WOULD SLIGHTLY PRESERVE DNA REPRESENT REPLICATION. IT WAS OBVIOUSLY OBVIOUS. LET'S TAKE AND INHIBIT DNA POLY POLYMERASE. WHEN WE TRIED TO DO THE EXPERIMENT, WE HAVE ENCOUNTERED AN INTERESTING FEN APHENOMENON. NOT ALL CELLS COULD JUST SLOW DOWN DUPLICATION. SO WHAT I AM SHOWING HERE. I AM SHOWING A COPY OF DNA FIB FIBERS FROM CELLS THAT HAD THE LABEL FIRST WITH ONE IDU AND AFTERWARDS THEY WERE LABELED WITH A SECOND ANALOGUE CONCOM TANT LY WITH THE ADDITION FOR A HALF-HOUR. AND WHAT HAPPENS IS THAT IF THE CELLS WERE NOT FOLLOWING, WE SEE LABELING WITH BOTH ANALOGUES, EACH SPECIFICALLY IS ADDED IN ONE SITE CELLS. WE SEE THAT THE GRAY SIGNALING IS SHORTER THAN THE RED SIGNALING, SUGGESTING REPRESENT REPLICATION SLOWS DOWN OR REPRESELICATION STOPS. HOWEVER, THIS IS WHAT HAPPENS IN WILD TYPE CELLS. THERE IS A SERIES OF CELLS THAT CONTAINS MUTANTS IN DNA REPAIR PROCESSES, AND THOSE INCLUDE MUTANTS IN -- THAT DNA CELLS AND CELLS THAT DO NOT CONTAIN 81 NUKE AASE, AS SHOWN HERE. IN THOSE CELLS, REPRESELICATION IS COMPLETE, SUGGESTING THAT THAT PARTICULAR ADAPTATION FROM PAST REPRESELICATION TO SLOW REPRESENLICATION REQUIRES A PATH WWAY THAT INCLUDES ATP KINASE AND GLOIKS AND THIS ANALYSIS WAS PUBLISHED A FEW YEARS AGO. AND WE ALSO FOUND FROM OUR BRANCH THAT THE SAME RESPONSE OCCURS FOR POLYMERASE. SO IT SEEMS THERE IS A PARTICULAR PATH WWAY THAT HELPS CELLS RECOVER FROM AN INHIBITION OF DNA REPRESENT SATION AND CONTINUE REPRESELICATION AT A SLOW PACE. SO ONE QUESTION ABOUT 81 NUKE ASE WAS AN ACCESS PATHWAY. SO THAT ACTUALLY REQUIRES THE NUKE AASE ITSELF, OR IS IT JUST 81 PART OF A LARGE COMPLEX INVOLVING METHYL 4 IN IMMEDIAMEDIATING THE RESPONSE THAT LEADS TO RECOVERY FROM AN INHITIAN -- IN INHIBITION? SO IN ORDER TO ANSWER THAT QUESTION, FROM MY LAB HAD MUTATE MUTATED 81 TO CREATE A NUKE AASE AND USE THAT PARTICULAR MUTANT TO COMPLEMENT CELLS THAT ARE 81. AND THEN WE MEASURED AGAIN THE EXTENT OF RECOVERY FROM THE IN INHIBITION OF DNA REPRESELICATION. SO AS YOU CAN SEE HERE, WHEN WE TAKE CELLS THAT ARE COMPLEMENTED WITH THE NUCLEASE, WE SEE THAT THOSE THAT ARE ABLE TO RECOVER FROM INHIBITION, AND THEY CAN REPRESELICATE A NORMAL PHASE, WHERE WHEREAS CELLS THAT DO NOT EXHIBIT NUCLEIC ACTIVITY, ARE UNABLE TO RECOVER, SUGGESTING THAT THE INTERNUCLEAR ACTIVITY OF 81 IS NORMAL FOR DIVISION. THEN WE LOOKED AT THE SLOW REPRESELICATION AND AT THE INTERORG INTERORIGIN DISTANCE S S IN THOSE CELLS. SO WHAT I AM SHOWING HERE AGAIN IS AN ANALYSIS OF THE CHROMATIN UNDER SANGS CONDITION S S IN WHICH WE DO NOT ADD -- INAUDIB[INAUDIBLE]. AND TO OUR SURPRISE, WE THOUGHT THAT THOSE CELLS NOT ONLY NEED THE NUCLEARLEASE ACTIVITY IN ORDER TO RECOVER FROM DNA REPRESELICATION, WE ALSO SEE THAT THOSE CELLS REPLICATED AT A SLOWER PACE, EVEN WITHOUT ANY PRIOR XERNL DNA DAMAGE OR REPRESELICATION. SO REPRESELICATION SPEED IS LOWER, AND THE INTERORIGIN DISTANCE IS SHORTER, SUGGESTING THAT THOSE CELLS REPRESELICATE AT A SLOWER PACE AND THEY COMPENSATE FOR THE SLOWER PACE BY ACTIVATION OF ADDITIONAL REPRESELICATION ORIGINS. SO FINALLY, WE FOUND A SYSTEM WHERE WE COULD DO DUPLICATION WITHOUT HAVING ANY EXPERIMENTS REPRESELICATION AND WE COULD LOOK AT THE ACTIVATION OF THOSE ORIGINS. AND AS I MENTIONED, THE BIG QUESTION WAS WHETHER WE WILL SEE ANOTHER ORIGIN OR IF WE WILL SEE A PATTERN OF REPRESELICATION THAT WE STILL HAVE THIS -- THE SAME REPRESELICATION ORIGIN SIMPLY EXHIBIT FLEXIBLE INITIATION. AND ATS YOU CAN SEE HERE, THE ANSWER TO THAT QUESTION IS THAT WE DO NOT OBSERVE NOVEL INITIATION SITE. AND THE PERCENTAGE OF COMMON PEAKS IS VERY, VERY HIGH. WAS WE ACTUALLY CAME AT THE CONCLUDESIVE ANALYSIS IS AT LEAST WE'RE -- WHEN WE ARE SLOWING DOWN REPRESELICATION WITHOUT A VERY STRONG CONNECTION FOR A PREFERENTIAL, WE SEE THE REPRESELICATION ORIGIN FROM A CONSISTENT AND THEY DO THAT AT A HIGHER FREQUENCY IN 81 CELLS AND IN OTHER CONDITIONS THAT CAN ALSO SLOW DOWN REPRESELICATION. SO IF WE GO BACK TO OUR MODEL, WE HAVE THE MODEL AND WE CAN -- WE END UP WITH A FLEXIBLE MODE OF REPRESELICATION, WHEREBY WE HAVE A REPRESELICATION-ONLY GENE. EACH ONE ACTIVATED IN A DIFFERENT CHROMOSOME IN A DIFFERENT CELL DURING NORMAL GROWTH AND WHEN REPRESELICATION SLOWS FOR ANY REASON, THIS SLOW- SLOW-DOWN IS NOT SUSPICIOUS TO ACTIVATE A NORMAL GENE BUT THE SAME ORIGIN FROM THE SAME INITIATE REPRESELICATION AT A HIGHER FREQUENCY. SO WE DON'T NEED REPRESELICATION -- PARTICULAR REPRESELICATION ORIGINS IN ORDER TO MAINTAIN -- WE CAN DELETE ALMOST ANY INDIVIDUAL ORG ORIGIN AND SUBCYCLE WILL COP RATE AT THE SAME PACE. WE CAN BASICALLY REPLACE REPRESELICATION ORIGIN WITH EACH OTHER. AND EVEN THOUGH DUPLICATION SLOWS DOWN, WE HAVE BACKUP TO MAINTAIN ORIGIN STABILITY. SO CAN WE FIND ANY INTEROAACTION AT DUPLICATION ORIGIN THAT WOULD HELP MAINTAIN GENOMIC STABILITY? SO IN ORDER TO ANSWER THAT QUESTION, WE WENT BACK TO OUR ORIGINAL MAPPING AND LOOKED AT SOME INTEROAACTION.^ AND ONE OF THE INTEROAACTION THAT'S CAUGHT OUR ATTENTION WAS THE INTEROAKS INTERACTION WITH THIS PARTICULAR MODIFICATION OF SYSTEM H 3. THIS IS THE MANIPULATION OF LYIIGANS THAT EXHIBIT A VERY HIGH ASSOCIATION WITH DUPLICATION ORG ORIGINS. AND WE ASKED WHETHER WE CAN SEE -- SO THIS SUGGESTS TO US THAT THAT MODIFICATION SOMEHOW ASSOCIATES WITH INITIATION EVENTS AND WE COULD ASSESS THAT A FUNCTION GENETICALLY BY GOING BACK TO OUR FAVORITE MAP. BECAUSE AS I HAVE MENTIONED EARLIER, WE HAVE MUTANTS THAT START DNA REPRESELICATION. SO WE CAN CHECK WHETHER THOSE MUTANTS ASSOCIATE WITH THE MAN MANIPULATION OF GENOME-WIDE. AND THIS IS A CHROMATIN PRECIPITATION ANALYSIS THAT SHOWS THAT WILD TYPE NON-MUTANTS I RCHR ASSOCIATE WITH MANIPULATION. WHERE AS THE MUTANT DOES NOT INITIATE DNA REPRESELICATION, ALSO RESULTS IN METHYLATION AT THAT LOCATION. AND THIS SUGGESTS TO US THAT THIS MODIFICATION ASSOCIATES WITH INITIATION. NOW THERE IS AN ENZYME THAT CAT ALIESS THE MODIFICATION VERY SPECIFICALLY WITH ENZYME 1 L AND CANCER THERAPY IN THE CLINICAL TRIALS. AND THAT ENZYME, LUCKILY FOR US, CAN CAT ALLIES THE MANIPULATION ONLY OF THAT PARTICULAR LIYSINE ON H 3. SO IT COULD ACTUALLY PERFORM SOME OTHER ANALYSIS, SUCH AS ASKING WHETHER THIS ENZYME IS ESSENTIAL FOR THE INITIATION OF DNA REPRESELICATION GENOME-WIDE. WHEN WE DO SUCH ANALYSES, WE DO NOT SEE THAT THIS METHYLATION IS ESSENTIAL FOR INITIATION. WE DONE SEE A CHANGE IN REPRESELICATION INITIATION IN THE FREQUENCY OR IN REPRESELICATION. HOWEVER, WE DID FIND THERE IS AN INTERESTING FEN APHENOMENON THAT IS ASSOCIATED WITH REPRESELICATION KINASE. SO WITH THIS TYPE OF EXPERIMENT, WE TOOK ADVANTAGE OF THE FACT THAT EACH GENOMIC CLOCK REPRESELICATES A PARTICULAR TIME WINDOW AND REPRESELICATION CHROME CHROMOSOMES EXHIBIT AN AYE DISTINCT NUCLEAR DISTRIBUTION. SO HERE I AM SHOWING AN IMAGE, LIVE IMAGE OF DNA AT THE LOCATION WHERE REPRESELICATIONS HAPPEN. AND ALL THIS IMAGE IS ONE NUCLEASE, AND WE LOOKED AT THE PATTERN OF REPRESELICATION THAT REPRESELICATES EARLY. IF WE TAKE A SINGLE NUKE AASE AND LOOK, WE SEE A VERY DIFFERENT PATTERN. SO WE COULD ACTUALLY LOOK AT THOSE UNDER THE MICROSCOPE AND DETECT WHETHER THEY ARE IN EARLY ASSAYS OR IN LATE ASSAYS. O SO WITH THIS KIND OF TOOL, WE CAN TAKE THOSE CELLS AND LOOK AT THEM UNDER THE MICROSCOPE AND CORRELATE REPRESELICATION PATTERNS WITH DNA CONCEPTS AND ASK IF THE ORGANIZATION OF REPRESELICATION DO DOMAIN CHANGED DURING REPRESENT REPLICATION? SO WE WOULD LIKE TO KNOW WHETHER WE CAN HAVE A DIFFERENT PATTERN OF INITIATION OF DNA REPRESELICATION REPLICATION. AND HERE IS WHAT WE FIND. SO WHEN WE DID 1 A, WE LOSE THE METHYLATION OF 59, AS EXPECTED. AND IF WE LOOK AT CELLS THAT WERE NOT MUTATED, WE FIGURED EARLIER REPRESELICATION HAPPENED IN CLUSTERS. WHEREAS A REPRESELICATING PATTERN AT ABOUT TWICE AS MUCH DNA CONTENT. AND THEN WHEN WE LOOK AT CELLS THAT WERE DELETED AT PHASE 1, WE SEE THE SAME PATTERN BUT WE ALSO SEE A POPULATION OF CELLS THAT EXHIBIT AN EARLY PATTERN THAT THEY CONTAIN EITHER LATE ASSAY DNA CONTENT OR MORE THAN THAT. SO THESE CELLS CAN COMPLETE THEIR REPRESENT SATION AND INITIATE DNA REPRESELICATION WITHOUT UNDER GOING MIGHT OTOSIS. AND THIS SUGGESTED TO US THAT PERHAPS THE A 3 KINASE MAN MANIPULATION ASSOCIATES REPRESELICATION AND PREVENTS RE REREPRESELICATION DURING A SINGLE SUBCYCLE. SO WHEN WE USE THE LIPIDS, THESE ORIGINS THAT START AREPRESENLICATION AFTER MIGHT OTOSIS, WOULD REINITIATE THE REPRESELICATION. AND WE CAN ALSO SEE THAT USING AN ANALYSIS. SO IF WE LOOK AT CELLS THAT ARE SUFFICIENT, WE SEE A CELL CYCLE PATTERN. WITH G 1, S PHASE. THIS IS A G 2 PATTERN. AND WE DO SEE THAT PATTERN. WE ALSO SEE A POPULATION WITH MORE AND WE SEE MANY CELLS THAT HAVE UNDERGONE, SUGGESTING THAT FUNCTION OF H GCHG PCHLP IS TO PREVENT AUTOREPRESELICATION. SO TO SUMMARIZE WHAT I HAVE SHOWN TODAY, WE HAVE SEEN DNA FREQUENCY SCOMBRIZ EPGEIGENETIC INTEROAACTIONS COMBINE TO DETERMINE WHEN IT OCCURS FIRST AND WE START ED ED TO DECIPHER WHICH PROTEINS ARE INVOLVED, AT SPECIFICALLY OUR REPRESELICATION ORG ORIGIN. AND THOSE CAN AFFECT REPRESELICATION FACTORS. PROTEINS INVOLVED WOULD -- CAN ALSO HELP DNA REPRESELICATION WITHOUT EXTERNAL DAMAGE AND MODIFY THE FREQUENCY OF REPRESELICATION INITIATION EVENTS AND INTERRUPTIONS AT REPRESELICATION ORIGIN CAN PLAY TWO ROLES. IT CAN FACILITATE INITIATION OF DNA REPRESELICATION, OR IT CAN PREVENT REREPRESELICATION. SO TO SUMMARIZE THAT IN A GRAPHIC WAY, WE HAVE REPRESELICATION ORIGINS SIGNALING THROUGH THE REPRESELICATION ORIGINS. AND THOSE INTEROAACTIONS ARE ALSO MODULATED BY ID-TYPE PROCESSING THAT ARE FACILITATING INITIATION AT THE RIGHT TIMES AND BY COPYING MODIFIERS SUCH AS HISTO HISTOMODIFICATIONS AND RESPONSES TO REPRESELICATIONS PROBABLY THROUGH INTEROAACTIONS WITH CHROMATINS THAT MODULATE THE FREQUENCY OF INITIATION EVENTS. AND THOSE INTEROAACTION SPECIFICATION ORIGINS CAN ORG ORIGINATE REPRESELICATION OR THROUGH ANTI-REINITIATION. AND ALL THOSE INTEROAACTIONS PROVIDE THE SUSCEPTIBILITY TO A ADAPT SCOMBRIECHLT LIKE TO END BY THANKING MY COLLABORATORS AND COLLEAGUES FROM THE DNA REPRESELICATION GROUP, ESPECIALLY CIYNTHIA, WHO HAS BEEN MY ASSISTANT FOR THE LAST FEW YEARS YEARS. INVOLVED IN THE ID WORK SCAAND CAN DEVELOPING NORMAL WAYS TO LOOK AT THE DNA REPRESELICATION. I WOULD ALSO LIKE TO THANK THE FORMER MEMBERS OF MY GROUP WHO ARE NOW ENGAGED IN OTHER EXPERIMENTS AND MY COLLABORATORS FROM GNCI AND FROM THE NIH. THANK YOU VERY MUCH FOR YOUR ATTENTION. APPLAU [APPLAUSE] APPLAU[APPLAUSE] >> THANK YOU. >> OKAY, SO NOW LET'S TRY -- WE'LL DO ONE QUESTION FROM EACH SIDE. AND SINCE WE'RE ALL -- WHY DON'T WE DO THAT FIRST? >> THANK YOU. THAT WAS A VERY INTERESTING TALK TALK. I WAS THINKING ABOUT IF YOU COULD SAY ANYTHING ABOUT THE QUANTITY OF DNA DAMAGE OR THE TYPES OF DNA DAMAGE THAT IS REQUIRED TO TURN ON ALL THESE REPRESELICATION SITES. CAN YOU SHOW -- YOU SHOWED SAMPLES OF THIS. BUT CAN YOU SAY THAT YOU TREAT CELLS WITH SOME TYPES OF THESE THAT TURN ON THE -- INAUDIB[INAUDIBLE] >> [INDISCERNABL[INDISCERNIBLE] [HE CECHO]. >> OKAY, SO WE HAVE NOT PERFORMED AN STENEXTENSIVE ANALYSIS OF THE DOSES OF DNA DAMAGE AGENTS. WE KNOW THAT THOSE ORIGINS CAN BE ACTIVATED BY PREFERENTIAL IN CHROMATINS AND WE HAVE SOME WORK TO DO ON KH INHIBITOR THAT ALSO ACTIVATE SOME DORM AANT ORIGINS. WE HAVE LOOKED AT A LINK PRIME PRIMARILY. AND WE ALSO HAVE SOME DATA SUGGESTING THAT SHOULD -- JUST LOWERING THE LEVELS OF ISOMERASE 1 IN SOME CELLS SLOWED DOWN REPRESELICATION AND ACTIVATE DUPE DUPLICATION ORIGIN. SO OTHER ANALYSIS IN OTHER GROUPS HAVE LOOKED AT IT AND BASICALLY ANYTHING THAT THOSE REPRESELICATIONS, REGARDLESS OF WHETHER THIS IS A DNA-DAMAGING AGENT OR A CHROMATIN MODIFIER OR OTHER SEQUENCE THAT ONE CAN THINK OF WOULD ACTIVATE A GROUP OF REPRESELICATION ORIGINS THAT, IN OUR ANALYSIS, WE HAVE PERFORMED. WE THINK THAT THIS ORIGIN BELONG TO THE SAME AS THE ACTIVE ORIGIN ORIGIN. SO IT'S NOT A GENETICALLY IDENTIFIED SEQUENCE. IT'S SIMPLY THE SAME ORIGIN THAT DO AT THE HIGHER FREQUENCY IN THE POPULATION. AND WE ARE NOW TRYING TO DO IS TO TRY TO SEE IF THE -- THERE ARE SITUATION S S IN WHICH WE CAN ACTIVATE A SEPARATE DUPLICATION ORIGINS AND I HOPE TO BE ABLE TO ANSWER THAT QUESTION. >> THANK YOU. >> GOOD, THANK YOU. PLEASE BE SURE TO MUTE. AND LET'S GO TO N ASIEH S. >> THANK YOU VERY MUCH FOR THE TALK. IT WAS DELIGHTFUL TO WATCH. WE DO NOT HAVE ANY QUESTIONS TODAY. >> OKAY, GREAT. LET'S MUTE AT -- WE'LL GO TO THE UNIVERSITY OF PITTSBURGH. >> CAN YOU HEAR ME? >> YEAH. >> I HAVE A QUESTION. VERY NICE TALK. ONE QUESTION IS REGARDING THE CELLS. YOU SAY THAT THEIR REPRESELICATION IS SLOWER AND THEREFORE PROBABLY AS A RESULT YOU HAVE MORE ORIGIN BINDING. THIS IS IN THE ABSENCE OF DAMAGE DAMAGE. DO YOU THINK THAT THE DEFICIENCY LEADS TO A SLOWER REPRESELICATION? >> OKAY. >> PLEASE MUTE, NOW PITTSBURGH AND WE'LL ANSWER THE QUESTION. >> SO THERE ARE POSSIBILITIES. ONE POSSIBILITY IS THAT YOU NEED REPRESELICATION AND IN ITS ABSENCE THERE IS A COMPONENT THAT IS MISSING FROM THE MACHINERY THAT NORMALLY DICTATES THE REPRESENT REPLICATION FOR VESSLOCITY. THERE ARE SOME IMPLICATIONS THAT THEY ARE A PART OF THE REPRESENT REPLICATION. THE OTHER POSSIBILITY IS THAT THERE ARE REGIONS IN THE GENOME THAT ARE NORMALLY -- TEND TO REPRESELICATE. AND WE KNOW THAT THERE ARE SUCH REGIONS AND THAT THOSE REGIONS MIGHT, DURING NORMAL REP REPLICATION, THEY MIGHT ENCOUNTER SOME SITUATIONS WHERE LEGIONS ARE FORMED AND 81 ESSENTIALLY WITHIN THOSE REGIONS REGIONS. SO WHAT HAPPENS IN OUR CELLS IS DURING EXTREME DNA DAMAGE REPRESELICATION REPEATS AND COUNTERS THE AGENTS IN VARIED FREQUENCY AND THOSE MUTANTS HAVE TO BE RE RESOLVED BY KIND OF A INAUDIBLE [INAUDIBLE] STRUCTURE. AND THIS HAS TO COME THERE AND CATCH THE REPRESELICATION. SO THERE IS SOME EVIDENCE THAT THAT IS WHAT HAPPENS FROM SOME WORK FROM A LAB THAT SUGGEST THAT THOSE REPRESELICATIONS ARE MUCH MORE FREQUENT THAN ORIGINALLY THOUGHT. WE HAVE NOT LOOKED DIRECTLY AT THAT. WE HAVE LOOKED AT A PROCESSING, BECAUSE ONE OF THE PROCESSES WAS THAT 81 MIGHT PLAY A ROLE IN THE PRODUCTION OF FRAGMENTS PROCESS PROCESSES AND IT MIGHT SPEED THE PROCESS. IN OUR ANALYSIS, WE DID NOT SEE ANY DIFFERENCES. SO WE TEND NOW TO FAVOR THE HYPOTHESIS THAT THERE ARE VERY FREQUENT EVENTS THAT HAVE TO BE RESOLVED DURING NORMAL REPLICATIONS AND THOSE EVENTS HAVE TO BE RESOLVED BY 81. AND IN SOME STUDIES THAT I HAVE NOT SHOWN YOU TODAY BUT I'D BE SHA HAPPY TO SHARE FU WOULD LIKE, WE HAVE ALSO DONE SOME ANALYSES IN CELLS AND WHAT WE SEE IS THAT WHEN ONE DOES NOT HAVE -- THE FEPHENOTYPE IS MUCH MORE SEVERE, SUGGESTING THAT THEY CAN AT AH ALLIES THAT TYPE OF INITIAL RES RESOLUTION. SO THAT IS MY HYPOTHESIS. I DON'T HAVE ALL THE BIOCHEMICAL DATA TO BE SURE IT'S ONE WAY OR THE OTHER. BUT THE EMERGING DATA SUGGESTS THAT WE ARE LOOKING AT RESULTING VISIONS BUT THEY ARE NOT AS FREQUENT AS ORIGINALLY THOUGHT. >> OKAY. THANK YOU. PLEASE MUTE AT PITTSBURGH. AND LET'S GO TO PORTLAND, ORGANIOREGO OREGON. UNMUTE. >> THANK YOU VERY MUCH FOR THE PRESENTATION. I THINK I ALREADY KNOW THE ANSWER TO THE QUESTION I WAS GOING TO ASK, BUT YOU DIDN'T SPECIFICALLY MENTION. [INDISCERNABLEIBLE] STEPHENLOID, SORRY. IF YOU SLOWED DOWN DNA REPRESENT REPLICATION BY DEPLETING OR RE REDUCING DNTP POOLS, BY USERACIL THAT THE POINT AT WHICH YOU ARE SLOWING DOWN REPRESELICATION ENDS UP BEING A DIFFERENT ONE ATHAN SOME OF THE ONES THAT YOU PREVIOUSLY MENTIONED. SO IS THE EXPECTATION THAT SIMPLY A SEVERE DNTP POOL DE DEPLETION WOULD ALSO RESULT IN SIMPLY A SLOWING OF THE PRE-LOAD PRE-LOADED COMPLEXES OR WHETHER OR NOT SOMETHING LIKE THAT MIGHT BE ENOUGH TO SPARK INITIATION AT THOSE THAT ARE POTENTIALLY PRIMED BUT HAVE NOT FIRED YET? >> PLEASE MUTE IN ORGANIEGON. >> SO ACTUALLY, THE DIRECT ANSWER TO YOUR QUESTION IS THAT WE HAVE NOT TRIED TO USE THE AGENTS THAT ARE MORE -- RESULT RESULTING MORE SEVERE. WE ARE INTERESTED IN REPRESELICATING INITIATION IN ALL THOSE ORIGINS IN CONDITIONS WHERE REPRESELICATION IS SEVERELY LIMITED BECAUSE WE WERE LOOKING AT THE MATERIALS. HOWEVER, WE SHOWED AN EXPERIMENT LIKE THAT, WHERE SHE LOOKED AT A PARTICULAR MODEL IN CANCER CELLS AND SHE USED NUCLEIC REPRESENLICATION AND MODIFIED ORIGINS THAT WERE NOT INITIATE BID DNA REPRESELICATION THROUGH DNA REPRESELICATION WHEN THOSE CONDITIONS WERE ACT VAUGHT VAUGHTED. SO WE -- ACTIVATED. SO WE KNOW THAT BECAUSE THE CONDITION IS AT A DIFFERENT STAGE, WE ARE LOOKING DIRECTLY AT THE INHIBITION OF A DNA POLY POLYMERASE. SO WE THINK THERE MIGHT BE ASSOCIATION WITH THE INHIBITION AND POLYMERASE. OR IT COULD BE SIMPLY THAT THESE ARE DIFFERENT CELLS. SO WE'VE NEVER DONE EXACTLY THE EXPERIMENT JOB, I AM NOT SURE THAT IT SEEMS LIKE THERE ARE CONDITIONS LIKE THAT AND THEY MIGHT BE MORE SEVERE THAN THE CONDITIONS THAN WE ARE USING. SO USUALLY IT SEEMS TO ME THAT AS LOANING AS THE ORIGINS FROM THE APOOL ARE SUFFICIENT, THEN WE INITIATE DNA REPRESELICATION AND WE NEED TO MANIPULATE THE REPRESENT REPLICATION COMPLEXES IN ORDER TO ACTIVATE ORIGINS THAT ARE MAYBE YOU COULD HOLD CRYPTIC ORG ORIGINS AND NOT DORM AANT ORIGINS THAT DO NOT DUPLICATE REPRESENT REPLICATION. >> THANK YOU VERY MUCH. >> OKAY. PLEASE MUTE PORTLAND AND LET'S GO ON TO CHAP EL HILL, UNIVERSITY OF NORTH CAROLINA. >> ARE YOU THERE? >> OKAY, LET'S GO ON TO SUNNY SUNNYBROOK. >> YES, HELLO. THIS IS BRUCE DIMPLE. THE QUESTION I HAVE CONCERNS THE MECHANISM OF REPRESSION OF NEARBY ORIGINS ONCE ORIGIN IS ACTIVATED. CAN YOU COMMENT WHETHER -- WELL, LET'S TALK ABOUT THE D RE REPRESSION ACTUALLY. CAN YOU COMMENT WHETHER THAT'S THE LOSS OF A REPRESSIVE SIGNAL OR THE ACTIVATION DOCTOR THE -- OR THE GENERATION OF AN ACTIVATE ACTIVATED SIGNAL? >> PLEASE MUTE THERE. >> IT'S A GREAT QUESTION. AND IF YOU ARE LOOKING AT A HIST HISTOMODIFICATION, THERE SEEMS TO BE ASSOCIATED WITH A GROUP OF ORIGINS. AND TO PREVENT REINITIATION, ONE MIGHT CONSIDER THE POSSIBILITY THAT THERE IS A -- WHAT HAPPENS IF WHEN WE LOSE THE MODIFICATION ON THAT HIST OOME, WE ARE MAKING THE CHROMATINS MORE ACCEPTABLE THROUGH A FACTOR THAT WOULD INITIATE DNA REPRESELICATION. SO ONE MIGHT ENVISION A SITUATION WHERE SOME OF THE COMPONENTS OF THE COMPLEXES LEADS. AND SO MY THEORY WOULD BE THAT THE MODIFICATION SUCH AS THOSE MANIPULATIONS WITH 589 ARE A PART OF THE MECHANISM THAT PREVENTS REASSOCIATION OF THE FACTOR. WE HAVE NOT YET IDENTIFIED THAT FACTOR, BUT WE ARE FAR WAY FROM THAT. >> AND ONE MORE THING. CAN YOU SAY WHETHER THAT SAME D REPRESSION IS OBSERVED FOR THE LATE ORIGINS AS FOR THE EARLY ONES? >> SO EARLIER STUDIES SUGGEST THAT FOR THE AK 379 REPLICATION, WE SEE ASSOCIATION WITH EARLY ORIGIN. SO IN THIS CASE, IT SEEMS TO US THAT IT IS ASSOCIATED WITH GENES GENES. THEY ARE NOT -- AND THIS IS WHERE THAT MODIFICATION -- SO THESE ARE THE EARLY ORIGINS. THERE MIGHT BE SITUATIONS WHERE THERE MIGHT BE DIFFERENT MODIFICATION THAT'S ASSOCIATE WITH THE LATE ORIGINS AND WE HAVE NOT BEEN ABLE TO FIND IT. >> OKAY, THANK YOU. >> THANK YOU. >> OKAY, THANK YOU. PLEASE MUTE SUNNYBROOK. AND LET'S TRY UNIVERSITY OF KEN TUCK Y. >> HI. THIS IS DAVE. I WANT TO THANK YOU FOR A VERY INTERESTING TALK. IS THERE ANY ASSOCIATION WITH DOWNREGULATION OF THE STOP 1 L IN CANCER CELLS? MAYBE THOSE THAT ARE PARTICULARLY SUSCEPTIBLE TO -- INAUDIB[INAUDIBLE] >> THERE IS A CLINICAL TRIAL ON A PARTICULAR TYPE OF LYMPHOMA, WHERE 1 L ESSENTIALLY RECORD THOSE CELLS WITH ANALYSIS OF MODIFIERS. SO THOSE CELLS ARE VERY, VERY -- 1 L INHIBITION, BUT THAT FOR NOW -- INAUDIB[INAUDIBLE] IS ALSO ALTERED. AND THEN THOSE BECOME DEPENDENT ON THOSE TYPES OF MODIFICATIONS. IN OUR CELLS WHAT WE SEE IS THAT THE CELLS OVERREPRESELICATE AND UNDERGO ISOSELLES. ONE OF OUR PLANS IS TO DO CANCER CELLS AND COMBINE THE DOT 1 L IN INHIBITION WITH SOME OTHER HISTO HISTOMODIFIER DRUGS THAT TARGET HIST OMODIFICATIONS. IT'S NOT -- TO DO THAT BECAUSE MOST REPRESELICATIONS ARE IMMEDIATMEDIATED BY MORE THAN ONE ENZYMES AND THE ENZYMES ARE NOT VERY SPECIFIC. SO WHEN WE CAN ACTUALLY INHIBIT ONE PARTICULAR MODIFICATION. IN MOST CASES WE HAVE MORE THAN ONE. SO WE ARE LOOKING AT THAT. ANOTHER THING YOU MENTIONED -- WE ARE TRYING TO SEE WHETHER WE CAN LOOK AT THE CELLS THAT UNDER GO REPRESELICATION AND SEE IF WE DO SEE. FOR NOW THE ANSWER IS NOT YET. WE ARE SEEING A SLIGHT INCREASE IN DNA CONCEPTS BUT WE DONE SEE THAT ALL THE INDICATCASES OR PARTICULAR CHROMOSOMES HAVE AN AMPLIFIED. IT COULD BE THERE IS A CHECKPOINT THAT REPRESENTS STENEXTENSIVE REPRESENLICATION. INAUDIB[INAUDIBLE] >> OKAY, THANK YOU. >> OKAY, THANK YOU. PLEASE MUTE IN LEXINGTON. >> CHAP EL HILL, GIVING YOU ANOTHER CHANCE. IT SEEMS LIKE THEY'RE HAVING SOME TECHNICAL PROBLEMS. WE HAVE A QUESTION HERE. WHAT ABOUT THOSE CELLS THAT HAVE SPONTANEOUS CHROMOSOME BRIDGE, LIKE LOOM SYNDROME? HAVE YOU STUDIED THEM? >> SO WE DID -- THOSE CELLS -- GLUCOSE -- INAUDIB[INAUDIBLE] CELLS. DNA REPRESELICATION FOR A SHORT TIME -- OKAY. [INDISCERNABLEIBLE] SO WHEN YOU IN INHIBIT DNA REPRESELICATION, WE SEE THAT IN NORMAL CELLS WE SEE A SURGE OF DNA BREAKAGE. BLOOM CELLS DO NOT UNDERGO THAT BREAKAGE, AND THAT LEADS TO CELL BREAKAGE. WE HAVE NOT TRIED THAT ON YOUR END. AP M-DEFICIENT CELLS DONE DO THAT. APR-DEFICIENT CELLS GO BACK BACK TO 81. WE THINK THAT BASICALLY WHEN REPRESELICATION STARTS AND SUDDENLY SLOWS DOWN, THIS IS NOT A CONTINUOUS PROCESS. THOSE LESIONS HAVE TO BE RE REPAIRED AND THAT REPAIR GOES TO NUKEATESS, ESPECIALLY 81. SO THEY MIGHT BE ABLE TO COMP COMPENSATE. [INDISCERNABLEIBLE]. AND WE ARE DOING SOME MORE WORK IN THAT ASPECT. >> GREAT, THANK YOU. >> DOES ANYONE ELSE HAVE A REPEAT QUESTION? >> NO. >> IF NOT, THANK YOU VERY MUCH. >> THANK YOU. APPLAU