WE'RE GOING TO HEAR FROM PATTY O'PRESKO WHO HAS DONE BEAUTIFUL WORK IN BIOCHEMISTRY AND CHARACTERIZATIONS OF TELOMERES AND IT'S STARTED HERE AND GONE THROUGH HER OWN DEVELOPMENT IN PITTSBURGH WHERE SHE'S NOW BECOME PART OF THE CANCER GROUP AND HAS REALLY DEVELOPED A STRONG PROGRAM AND OF COURSE TELOMERES ARE EXTREMELY IMPORTANT. WE LEARN THAT FROM PATTY AND OTHERS AND TELOMERES ARE THE BEGINNING AND THE END OF EVERYTHING, THE BEGINNING AND THE END OF THE CHROMOSOME SO CLEARLY EXTREMELY IMPORTANT AND PATTY WE'RE VERY PLEASED TO HAVE YOU SO NOW KARA CAN SAY A WORD, TOO, AND WE WILL START THE LECTURE. >> ALL RIGHT, THANK YOU, KARA BERNSTEIN AT THE UNIVERSITY OF PITTSBURGH, I WILL GIVE A BRIEF INTRODUCTION OF PATTY, PATTY DID HER DOCTORATE AT PENN STATE UNIVERSITY AND SHE WAS A RECENT KEY NOTE FOR THE WONDERFUL SUCCESSFUL ALUMNI HER AND AFTER SHE FINISHED HER DOCTORATE IN INTO THE Ph.D. ON AGING AND THEN SHE STARTED HERE CAREER AT THE UNIVERSITY OF PITTSBURGH AND HAS BEEN HERE EVER SINCE WHICH HAS BEEN VERY FORTUNATE FOR US AND PATTY LAST YEAR, RECEIVED TENURE WHICH IS NOT EASY TO DO THESE DAYS. AND SO PATTY AS WELL MENTIONED HAS BEEN VERY IMPORTANT FOR THE TELOMERE FIELD LOOKING AT G-QUADRIPLEX TELOMERES, THE WORLD AND UNWINDING TELOMERES AND ALSO TYPES OF DAMAGE THAT HAPPEN TO TELOMERES AND SO IT GIVINGS ME GREAT HONOR AND PLEASURE TO INTRODUCE MY DEAR FRIEND AND COLLEAGUE PATTY. >> THANK YOU WILL AND KARAA FOR THE NICE INTRODUCTIONS. I'M NOT IRISH BUT TODAY I AM. TODAY EVERYBODY IS, RIGHT? >> MY DEPARTMENT AND AFFILIATION REMAINS THE SAME SO AN ENVIRONMENT OF THE ENVIRONMENTAL OCCUPATIONAL HEALTH DEPARTMENT. SO MY TALK WILL BE FOCUSED PRIMARILY ON TELOMERES, THEY ARE AS WE'LL MENTION, VERY IMPORTANT. [FEEDBACK ]--CAN YOU HEAR ME? >> YES. >> SOME KIND OF FEEDBACK GOING ON. >> I JUST TURNED IT OFF. >> OH, OKAY. SO THERE HAVE BEEN A WEALTH OF EPIDEMIOLOGY STUDIES IN RECENT YEARS, I THINK I DID A RECENT PUBMED SEARCH WITH TELOMERE AND EPIDEMIOLOGY AND THERE ARE OVER 600 PAPERS, THERE SEEMS TO BE ONE EVERY WEEK AND IT APPEARS TO GET THERE IS A CORRELATION WITH TELOMERES AND AGING RELATED DISEASES AS WELL AS VARIOUS LIFE CELL TYPE FACTORS. SO THE IDEA IS THAT ACCELERATED AGING MAYBE TO THE EARLIER ONSET OF DISEASE AND THE GOAL WOULD BE TO PRESERVE OUR TELOMERE LONGER SO THAT IN A WAY COULD PROMOTE HEALTHY AGING AND THE IDEA IS THAT GENOME STABILITY IN THE LOSS OF CELL VIABILITY THAT'S PRODUCED BY DYSFUNCTION CAN CONTRIBUTE TO DISEASE AND ORGAN DECLINE WITH AGE. THE LAB FOCUSED ON TRYING TO UNDERSTAND MECHANISMS OF TELOMERE LOSS AS WELL AS CLAR PATHWAYS THAT PRESERVE TELOMERES. WE BELIEVE THIS WORK IS SIGNIFICANT PRIMARILY FOR TWO REASONS, WE THINK THAT UNDERSTANDING MECHANISMS OF TELOMERE LOSS AND PRESERVATION COULD INFORM THE THERAPEUTIC STRATEGIES THAT ON THE ONE HAND PROTECT TELOMERES IN HEALTHY CELLS AND PREVENT TELOMERE FUNCTION DUE TO PATHOLOGY, THIS INCLUDES CELLS SENESCENCE THAT'S ASSOCIATE WIDE AGING AND GENOME INSTABILITY THAT CAN DRIVE CARCINOGENESIS. BUT ON THE FLIP SIDE, WE KNOW THE CANCER CELLS ARE HIGHLY DEPENDENT ON TELOMERE MAINTENANCE TO CONTINUE CELL PROLIFERATION. SO STRATEGIES THAT COULD ACCELERATE CANCER CELLS MAY HAVE THERAPEUTIC PEN FITS PARTICULARLY IN COMBINATION OF TELEMERACE INHIBI. SO THIS IS A BRIEF OUTLINE OF MY TALK, AND TELOMERE BIOLOGY IN THE DAMAGE AND REPAIR, AND I'LL TELL YOU BRIEFLY ABOUT THIS PROGS ECTOMYOSIN, SO THIS IS RECENTLY PUBLISHED LOOKING AT DNA CONSISTENT TELOMERES AND I WILL TELL YOU ABOUT UNPUBLISHED WORK FROM ANOTHER GRADUATE STUDENT IN THE LAB ASKING THE QUESTION IF BOTH OF THESE LESIONS AT TELOMERES ARE REPAIRED AND THEN I'LL FINISH WITH SUMMARY AND CONCLUSIONS. >> PATTY,--JUST THIS IS KEN, FOR A SECOND, THEY'RE SHOWING THE PICTURE OF YOU AND IT'S OVERLAPPING WITH YOUR SLIDES, SO, WE'RE SEEING TWO VIEWS AT ONCE, IF IT'S POSSIBLE, JUST TO SEE YOUR SLIDES AND NOT SEE YOU UNTIL THE END, THAT WOULD BE BETTER, I THINK, BECAUSE IT'S BLOCKING OFF PART OF THE SLIDES. >> NOW WE SEE JUST YOU. >> YOU SEE THE COMPUTERS? >> NOW WE SEE BOTH, THE COMPUTER AND YOU. YOU'RE UP IN THE CORNER. WE WOULD LIKE JUST THE SLIDES BECAUSE YOU'RE BLOCKING OFF THE--WHEN WE SEE YOU, IT BLOCKS OFF THE TOP CORNER OF THE SLIDE. >> OKAY. SO WE'RE TRYING TO GET AN I.T. PERSON HERE NOW TO HELP OUT. BECAUSE WE HAVEN'T HAD THIS ISSUE BEFORE, SO I'M NOT SURE WHY THAT IS. >> WHEN YOU SHOW THE SLIDES EARLIER, THAT WASN'T HAPPENING. >> I HAVEN'T HIT ANY BUTTONS. OKAY, WELL, SO I DON'T KNOW HOW TO FIX IT, I HAVE AN I.T. PERSON COMING. >> JUST GO ON, PATTY. >> WE DON'T MIND, WE DON'T MIND. >> OKAY, SO IF SHE COMES IN, SHE CAN FIX IT BUT IN THE MEAN TIME I'LL GO ON AND HOPEFULLY WE CAN FIX IT BEFORE I GET TO THE EXCITING DATA. SO THIS IS A BRIEF OVERVIEW OF THE TELOMERES WE KNOW THEY SHORTEN AS A FUNCTION OF CELL DIVISION AND IT'S SHORTENED AS A FUNCTION OF AGE AND TO THE POINT WHERE THEY'RE SHORT LENGTH AND IT ACTIVATES THE DNA DAMAGE RESPONSE AND RECOGNIZED BY THE DOUBLE STRAND BREAK AND IT ELICITS PATHWAY LEADS TO CELLULAR SENESCENCE AND IT'S DUE TO AGING AND HOWEVER THE P53 OR RB-GENES ARE MUTATE THAD THEY'RE NOT FUNCTIONAL, THE CELL CONTINUES TO DIVIDE AND THE TELOMERES WILL CONTINUE TO SHORTEN, THIS CAUSES A CYCLE OF BRIDGE FUSION AND BREAKAGE AND RAMPANT CHROMOSOME INSTABILITY, AND MOST OF THE CELLS WILL DIE IN THIS PERIOD OF CRISIS, BUT THESE SURVIVORS ARE ABLE TO EMERGE HAVE FOUND A WAY TO UPREGULATE TELEMERACE OR THE TELOMERE--ALTERNATIVE LENGTHENING OF THE PATHWAY TO MAINTAIN TELOMERES AND THIS OF COURSE IS VERY DANGEROUS AS IT CAN DRIVE CARCINOGENESIS, SO JUST TO REMIND YOU THOSE DRAMATIC CELLS DO NOT EXPRESS DETECTABLE LEVELS OF TOLL-LIKE RECEPTOR MERACE AND THEN OVER NIGHT, 85-90% CANCERS EXPRESS TYPES. SO OUR TELOMERES WILL CONSIST OF BOTH PROTEIN COMPONENT, BOTH ESSENTIAL, THE DNA COMPONENT IS ABOUT FIVE-10 KILOBASES OF TTAGT REPEAT OF HUMAN CELLS. THEY END IN THE THREE SINGLE STRAND OVER HEAD THAT CAN WRAP AROUND AND STABILIZE. THE PROTEIN COMOPPOSITE BEHAVIORIAL PHENOTYPE SENT A COMPLEX OF SIX PROTEINS, AND THREE OF THE MEMBERS HERE THAT BIND SPECIFICALLY TO TELOMERE SEQUENCE ARE SHOWN IN THE LITTLE CARTOON. SO LOSS OF EITHER THE DNA COMPONENT OR THE PROTEIN COMPONENT CAN LEAD TO A PERSISTENTLY UNTAPPED FELL MERE THAT IS RECOGNIZED AS A DOUBLE STRAND BREAK BY THEMSELVES AND AS A MENTIONED BEFORE THIS, CAN LEAD TO CELL SENESCENCE OR CHROMOSOME STABILITY, SO WITH MY TALK, THERE ARE REPORTS EARLIER OF WORK IN COLLABORATION WITH EITHER [[INDISCERNIBLE] ] THE NUCLEACE IS PARTICULARLY CHANGEEROUS AT THE TELOMERES BECAUSE IN THE ABSENCE IT CAN REPRISE IT OVERHEAD THAT'S ESSENTIAL FOR TELOMERE FUNCTION. OKAY SO WE HAVE I.T. SUPPORT HERE THAT MIGHT BE ABLE TO HELP OUT. SO THIS IS JUST A DRAMATIC PICTURE AND HOPEFULLY YOU CAN SEE THIS. IF YOU STRIP OFF THE PROTEIN COMPONENT OF THE TELOMERES, YOU CAN GET CHROMOSOME FUSION AND CAN YOU SEE MY SLIDES NOW? >> WE SEE YOUR SLIDES NOW, JUST LET IT BE. >> IT'S OKAY, WE CAN STILL SEE THE SLIDES. >> ALL RIGHT, WE'RE MOVE ON HOPEFULLY. >> HOPEFULLY YOU WILL BE ABLE TO FOLLOW WITHOUT BEING ABLE TO SEE IT ALL. SO AS I MENTIONED AGAIN WHEN I WAS A POST DOCTORAL FELLOW IN HIS LAB, I SAID YOU'VE ALL HEARD ABOUT THE PREMATURE AGING DISORDER WHICH THE INDIVIDUAL'S EXHIBIT EARLIER ON TO NUMEROUS FEATURES THAT ARE NORMALLY SEEN IN THE ELDERLY POPULATION AND THIS INCLUDES CANCER AND HEART DISEASE, IT'S CAUSED BY MUTATIONS AND THE THE HELIXCASE, AND IN MY LAB THEY APPEAR TO HAVE IA I CRITICAL ROLE IN MAINTAINING THE TELOMERES, THE LAB SHOWED THAT THESE INDIVIDUALS WHEN YOU LOOK AT THE CHROMOSOMES, EXHIBITED INCREASE IN THE TELOMERE LINE. SO MY MOUSE IS--YEAH, SO YOU NOTICE HERE THAT THE CHROMOSOME, INSTEAD OF SEEING FOUR PINK SPOTS, YOU ONLY SEE THREE. SO WORK FROM HIS LAB AND OTHERS, FOUND THAT THESE CELLS EXHIBIT THE TELOMERE LAW, SENESCENCE AND PREMATURE CHROMOSOME THAT COULD BE PLACING [INDISCERNIBLE] BACK IN THE CELLS WHICH YOU COULD THINK OF THE SPECIFIC PARASITES. SO THIS IS REALLY IMPORTANT BECAUSE IT TURNS OUT ALTHOUGH AVERAGE TELOMERE LENGTH AND MORE COMMONLY MEASURED, IT'S REALLY THE SHORTEST TELOMERES THAT ARE THE CRITICAL DETERMINANT OF CELL VIABILITY AND GENOME STABILITY. SO ROGER AYODELE'S LAB SHOWED THAT FIVE OUT OF 92 TELOMERES IN ONE CELL WAS SUFFICIENT TO INDUCE CELLULAR SENESCENCE. SO WE'RE INTERESTED IN MECHANISMS THAT CAN LEAD TO THIS IT TELOMERE LOSS TYPE PHENOTYPE. SO THE MODEL THAT HAVE EMERGED FROM THE FIELD BASED ON THE WORK FROM NUMEROUS LABORATORIES, TOO MANY TO REALLY REVIEW IN THIS TALK HAS LED TO THE MODEL THAT SPECIFIC TELOMERE LOSS COULD RESULT FROM A PROBLEM WITH REPLICATING THE TELOMERES, SO IT TURNS OUT THE TELOMERES ARE HYPER SENSITIVE TO REPLICATION STRESS, TREATMENT FASTER SUCH AS INHIBITS POLYMERASES OR ONCOGENIC SIGNALING OR LIGANDS THAT WILL STABILIZE THESE FOUR STRANDED STRUCTURES THAT CAN FORM AND THE SEQUENCE OF TELOMERES, THESE QUADRIPLEXS LEAD TO BOTH TELOMERE PERTILITY AND TELOMERE LOSS AND A NUMBER OF LABS INDICATE THAT THE DEFICIENCIES AND SEVERAL HELIXES, WERE IN BLOOM, SHELTER PROTEINS AS WELL AS WORK [INDISCERNIBLE] SHOWED DEFICIENCIES IN BASIC EXCISION REPAIR AND CAN LEAD TO TELOMERE LOSS. SO HOPEFULLY YOU CAN SEE THIS BUT THE FRAGILE TELOMERES ARE WHEN THE ONE CHROME TID INSTEAD OF SEEING A SINGLE SPOT, YOU SEE A DOUBLET OR 200 SIGNAL. THE EXACT NATURE OF MOLECULAR NATURE OF THE STRUCTURE IS NOT CLEAR, IS THE RESULT FROM THE CHROMEITIZATION OF SINGLE STRANDED DNA THAT ACCUMULATING THE TELOMERES WITH AN INABILITY TO REPLICATE THAT REGION. >> JAY. >> THE TELOMERE LOSS FOR THE RESULT OF POSSIBLY FROM A COLLAPSE REPLICATION, SUCH THAT THE TELOMERE IS REPLICATED BUT THE TELOMERE FROM THE OTHER IS NOT RESULTING IN THIS LOSS PHENOTYPE YOU CAN SEE UPTOP. SO, WE WERE REALLY INTERESTED, TELOMERES HAVE A PROBLEM WITH STRUCTURES THAT LOST THE REPLICATION THEN WHERE IS THAT DNA DAMAGE, WE'RE INTERESTED IN WHETHER OR NOT BOTH LESIONS THAT COULD POTENTIALLY FORM ATLET TELOMERES COULD INTERFERE WITH TELOMERE REPLICATION AND LEAD TO THE FRAGILE TELOMERES OR TELOMERE LOSS PHENOTYPE. SO IT TURNS OUT THE THAT TELOMERES ARE NOT IDENTICAL AND THERE ARE SEVERAL FACTORS THAT CAN INFLUENCE DNA DAMAGE AND WILL TELOMERES, ONE IS THAT THE SHELTER AND COMPLEX HAS BEEN FOUND TO INTERACT WITH AND REGULATE ENZYMES IN EVERY KNOWN DNA REPAIR PATHWAY. AND OF COURSE, WE ALSO HAVE THE APERIENT STRUCTURE FORM AT THE TELOMERES AND THE ALL OF THEM, MAY INFLUENCE THE REPAIR, SO OF COURSE TO THIS GROUP, I DON'T MEAN TO OVER THE DIFFERENT DNA REPATHWAYS, WORK COULD REALLY USE FOR THE LABS, AMONG OTHERS TO WORK CERTAINLY THAT WE'VE DONE IN COLLABORATION WITH THE LAB, INDICATE THAT AXIDATIVE DAMAGE IF REPAIRED AT TELOMERES AND BASIC EXCISION REPAIR AND OPERATIONAL, IT'S NOT QUITE AS CLEAR IF THE V. E. R. IS SUFFICIENT AS TELOMERES COMPARED TO ELSEWHERE IN THE GENOME. NUCLEOTOTIC EXCISION HAS BEEN FAR LESS EXAMINED, ONE OTHER REPORT FROM THE LAB AND MORE RECENT REPORT FROM THE LAB INDICATES THAT TELOMERES ARE SUSCEPTIBLE TO USE THE INDUCED DIMER FORMATION. HOWEVER, THE LEVEL OF REPAIR WAS NOT CLEAR, SO, REPAIR WAS DETECTED IN EARLIER PAPER FROM WILL'S LAB IN 95 AND THEN DOUG'S GROUP REPORTED MORE RECENTLY THAT THEY WERE TELOMERES WERE REFRACTORY TOLT REMOVAL OF CBD, SO VERY LIMITED WORK IN THIS AREA. THERE HAS BEEN A LOT OF WORK LOOKING AT DOUBLE STRAND BREAK REPAIR PATHWAYS OF TELOMERES AND THE EVIDENCE INDICATES THAT THE COMBINATION AND PATHWAYS ARE NORMALLY SUPPRESSED AT TELOMERES AND THIS MAKES SENSE BECAUSE IT'S THE JOB OF THAT SHELTER AND COMPLEX TO MASS THE CHROMOSOME END FROM THE DOUBLE STRAND BREAK REPAIR MACHINERY. OF COURSE THAT CAN BE A PROBLEM WHEN YOU ACALLY HAVE A BONA FIDE BREAK OR A COLLAPSE REPLICATION FORK AND THEN TRANSLATION DNA SENDS A VERY IMPORTANT FOR MAINTAINING THE GENOME, THE PRESENCE OF LESIONS THAT NORMALLY BLOCK REPLICATIONS BUT TRANSLESION DNA SYNTHESIS HAD NOT BEEN EXAMINED FOR TELOMERES YET. SO THIS SLIDE IS REALLY JUST TO REMIND YOU THAT BOTH THE ABILITY TO REMOVE THE BULL BULKY LESIONS, AND ULTIMATE BYPASS DURING REPLICATION IS CRITICALLY IMPORTANT FOR PRESERVING THE GENOME AND PREVENTING GENE CANCER AND GENE PHENOTYPES. THERE'S SOME ABOUT HOW IT MIGHT EFFECT TELOMERES AND THERE'S INTERESTING RECENT REPORTS THAT OBSERVE SHORTENED TELOMERES IN SKIN TISSUE FROM HUMANS AND MICE FOR THE SHORTEST TELOMERES WERE OBSERVED IN THE SUN LIED LIGHT OR THE EXPOSED AREA, COMPARED TO THE NONSUN LIGHT EXPOSED AREAS. THEY WERE JUST LOOKED AT. SO THIS IS HANNAH POPE, A GRADUATE STUDENT FROM MY LAB WHO IS NOW THE EPA, AND SHE SET OUT TO EXAMINE THE IMPORTANCE OF TRANSLESION D NA SYNTHESIS IN PRESERVING TELOMERES AFTER DNA DAMAGE, SO THE MODEL WAS THAT IF LESIONS ARE NOT EFFICIENTLY REMOVED FRIDAY THE TELOMERES THEY CAN POTENTIALLY INTERFERE WITH REPLICATION AND COULD THEY LEAD TO THE TELOMERE LOSS OR FRAGILE PHENOTYPES I TOLD YOU ABOUT, SO I KNOW THAT DNA POLYMERASE IS CRITICALLY IMPORTANT FOR BEING ABLE TO ACCURATELY SYNTHESIZE AND BYPASS THESE THAT ARE PRODUCED BY U. V. LIGHT. HANNAH LOOK AT THOSE AND ONE IS AN EXAMPLE OF ACUTE PHYSICAL DAMAGE AND THE OTHER IS AN EXAMPLE OF CHRONIC CHEMICAL DAMAGE. SO THIS IS JUST A BRIEF REVIEW OF THE U. V. ED PHOTOPRODUCTS. CAN YOU GET THIS FROM THE DIMERS AND THE PRODUCTS IN--SO BOTH OF THESE LESIONS ARE RENEWED IN THE EXCISION REPAIR AND BOTH CHROMIUM AND U. V. PRODUCTS LOCK, POLYMERASES, AND CAUSE REPLICATION DEPENDENT DNA BREAKS SO THEY DON'T DIRECTLY BREAK THE GENOME BUT THEY CAN INDUCE BREAKS DUE TO FAILURE AND REPLICATION. OF COURSE WE KNOW THAT THIS IS IMPORTANT FOR BYPASSING PHOTOPRODUCTS BUT EVIDENCE IN YEAST SUGGESTED IT WAS ALSO IMPORTANT FOR BYPASSING THESE CHROMIUM DNA ADICS AND HAD A LOT OF INTEREST IN ENVIRONMENTALLY TOXIC METALS. SO HER WORK IS PUBLISHED. I WILL SHOW YOU TWO PIECES OF DATA BRIEFLY, AND HOPEFULLY YOU CAN SEE THIS, I'LL NOT SURE HOW WELL IT'S PROJECTING BUT WHAT YOU'RE LOOKING AT ARE CELL NUCLEI, THESE ARE AX DATA PROTECTIONED AFTER THE EXPOSURE OR CHRONIC TREATMENT WITH THE HEXAYODELE CHROMIUM AND, WE WERE ABLE TO RECAPITULATE THE RESULTS THAT AFTER THIS FOCI THAT YOU CAN SEE HERE, YOU IT TURNS OUT CHROMIUM EXPOSURE DOES THE SAME THING AND HAD A QUANTITATIVE FOCI THAT CO LOCALIZED WITH TELOMERES AND SO SHE OBSERVED THAT BOTH U. V. AND CHROMIUM INDUCED IN AND INCREASED IN LOCALIZATION TO TELEMERRIC AND SHE OBSERVED IN THESE CELLS IN THE CONCEPT ABOUT 10% OF THE TELOMERES WERE SHOWING COLOCALIZATION OF PAULETA, SO PAULET WAS GOING THERE BUT WOULD IT DO ANYTHING AT THE TELOMERES? AND TO DO THIS, SHE EXAMINED CELLS, ISOGENIC CELLS SHE OBTAINED FROM THE LAB, THESE WERE CELLS FROM EX-TB PATIENTS TAT WERE COMPLEMENTED BACK WITH POLARIZED EDA AND WHAT SHE OBSERVED WAS THAT IN THE WILD-TYPE CELLS, U. V. EXPOSURE DID INDUCE A MINOR INDUCTION OF TELOMERE LOSS. YOU CAN SEE HERE. AS WELL AS FRAGILE TELOMERES THAT CAN YOU SEE HERE, AND THEN SHE SAW A MINOR INDUCTION OF THE CHROMATID BREAKS WHICH HAVE BEEN REPORTED IN SOME PAULETA DEFICIENT CELLS. IN BOTH SHE AWE DECREASE IN THE ABERRATION SAYS AS WELL AS CHROMATID BREAKS. HERE SHE REPEATED THE EXPERIMENTS AND THE RESULTS ARE MORE DRAMATIC WHERE SHE FOUND THAT THE ABSENCE LED TO A DRAMATIC RELEASE OF THE FRAGILE TELOMERES. SO, JUST TO SUMMARIZE HER WORK, WE'VE UNCOVERED NEW EVIDENCE THAT WILL PROTECT AGAINST CHROMIUM INDUCED CYTOTOXICITY AND STRESS IN HUMAN CELLS, THIS HAD ONLY BEEN INVESTIGATED IN THESE PREVIOUSLY, AND HANNAH SHOWED THAT THE INDUCTION OF BULKY DNA GENERATES TELOMERE DEFECTS CONSISTENT WITH FAILURES AND TELOMERE REPLICATION, AND THE LOSS IS FRAGILITY CAUSED BY BOTH U. V. AND CHROMIUM EXPOSURES AND THESE ABERRATIONS WERE GREATLY INCREASED IN PAULETA DEFICIENT CELLS. SO HALF THE TELOMERES MAY BE HIGHLY DEPEBBLEDDENT ON THE DNA DAMAGE BYPASS MECHANISMS DUE TO DEFICIENCIES IF THE REPLICATION GOES INTO KAY BREAK, IT MAY BE DIFFICULT TO REPAIR THAT AT A TELOMERE, BUT ALSO POSSIBLY DUE TO DEFICIENCIES IN DAMAGE REMOVAL AT THE TELOMERES. SO WE THOUGHT IT WAS CRITICALLY IMPORTANT TO ADDRESS THIS QUESTION, HOW WELL, OUR PHOTOPRODUCTS REMOVED FROM THE TELOMERE END BECAUSE IF THEY'RE NOT REMOVED VERY WELL, YOU COULD EXPECT THEY WOULD ACCUMULATE AND THAT WOULD NEED THOSE LINES IN TRANSLATION DNA SYNTHESIS. SO THIS SAY QUESTION THAT A GRADUATE STUDENT IN MY LAB DID AND THIS WORK IS NOT YET PUBLISHED SO AS I MENTIONED, WE ALSO WANT TO LOOK AT THIS FOR PHOTOPRODUCTS BECAUSE WE KNOW THEY'RE REPAIRED VERY RAPIDLY. AND I JUST WANT TO REMIND YOU OF WHAT I TOLD YOU EMERGINGER AND THEY'RE SHOWING TEAR TWO INHIBIT SPF, E. R. 61 ABILITY TO CHIEF THAT OVERHEAD BUT WEATHER APPEARS TO INFLUENCES, THE PR61 IN THE CONTEXT OF THE REPAIR, WERE UNKNOWN. IT POTENTIALLY COULD. SO TO ADDRESS THE EFFICIENCY OF THE INFORMATION AND REMOVAL OF THE TELOMERES WE TRIED TO COME UP WITH THE MOST DIRECT APPROACH WE COULD THINK OF, AND WE BORROWED AN IDEA THAT WAS PUBLISHED A FEW YEARS BACK IN WOODY WRIGHT'S LAB. SO THEY ISOLATED TELOMERES BY FIRST CHOPPING UP THE GENOME WITH THE RESTRICTION ENZYMES THAT ARE CUT OVERWHERE, THAT PARTICULAR TELOMERE FRICTION ASSAY, SO INSTEAD OF RUNNING THESE ON A GEL, THEY REPORTED THE TELOMERE TYPE TWO-THREE TIMES OVERHEAD AND WE USED THIS TO FISH THE TELOMERES OVER THE CELLS AND WE NEEDED A LOT OF GENOMIC DNA AND A LOT OF CELLS BECAUSE IT TURNS OUT THE TELOMERES ARE LESS THAN .025% OF THE GENOME. SO FROM 100 MICROGRAMS TO GENOMIC DNA, I BET WE COULD HOPE TO RECOVER 25 NANO GRAMS OF TELOMERES AND THEN WE COULD PUT T ON A BLOB AND GO WITH ANTIBODIES AGAINST THESE PRODUCTS. SO SHE HAD TO SEE HOW WELL HER ASSAY WAS WORKING AND IT TOOK HER TWO YEARS TO DEVELOP THIS ASSAY. SHE WAS A VERY HARD WORKING PERSISTENT STUDENT. HERE'S AN EXPERIMENT WHERE SHE DID NOT ADD A CAPTURE OLIGIO NUCLEOTIDE. SHE ADD A NONCAPTURE OLIGO, AND YOU CAN SEE THAT IN THIS SHE SAW STRONG ENRICHMENT DNA, SO SHE RECOVERED A THIRD BIT GREATER THAN THE THIRD OF THE TELOMES AND THAT WAS AS GOOD AS WE COULD GET AND THESE ARE SO SENSITIVE WE DIDN'T NEED A LOT OF DNA TO CONTINUE THOSE PRODUCTS SO SHE WANTED TO MAKE SURE THE FRAGMENTS WERE INTACT AND WERE NOT DEGRADED THROUGH THE PURIFICATION PROCESS, SO WE RAN THE FRAGMENTS, BEFORE PURIFICATION AND AFTER PURIFIC ON THE POLYCHROME LINE GEL AND CAN YOU SEE THEY'RE NOT MULTIPRODUCTS DOWN HERE, IF ANYTHING, THEY MIGHT BE A LITTLE BIT SMALLER AND WE THINK THIS COULD POTENTIALLY BE DUE TO AND WE ALLUDE AT 65-DEGREES SO YOU COULD GET QUADRIPLEX BOLDING DURING THE HEATING OF THESE FRAG -FRAGMENTS THROUGH THE DILUTION PROCESS. SO WE PROBED BOTH VARIOUS AMOUNTS OF GENOMIC DNA, WITH A PROBE THAT BIND TO PT-DNA, AND THIS IS USED IN CHROMATIN IMMUNOPRECIPITATION EXPERIMENTS TO COMPARE SPECIFICITY OF PROTEIN BINDING FOR TELOMERE D NA COMPARED TO RANDOM GENOMIC DNA AND YOU CAN SEE THAT THE DNA IS COMPARED TO ONE NANO GRAM OF DNA AND INDICATING THE TELOMERES ARE 90% PURE. SO, THE OTHER THING THEY HAD TO WORRY ABOUT WAS WE WANTED TO MAKE SURE THAT DURING THE REPAIR TE, THE PHOTOPRODUCTS ARE NOT BEING DISCIPLINARY LEWDED OUT DURING CELL DIVISION. FORTUNATELY A NUMBER OF THIS HAS SHOWN THAT U. V. EXPOSURE DOES INDUCE THE CHECK POINT SO THAT THE PHOTOMIGHT BE REPAIRED BEFORE THE CELLS CONTINUE TO REPLICATE THE GENOME. AND IN FACT, THAT'S EXACTLY WHAT WE SAW. THE DOUBLING TIME FOR THE CELLS WAS ABOUT--WAS ABOUT 25 HOURS, BUT EVEN AFTER U. V. EXPOSURE, AFTER U. V. EXPOSURE, EVEN AFTER 72 HOURS, YOU COULD SEE THIS RED LINE HERE, THE VJ LINE STILL DID NOT DOUBLE. SO WE CAN BE CONFIDENT THAT ANY PHOTOPRODUCT WE SEE OVER TIME IS NOT DUE TO CELL DIVISION. SO THESE ARE SPA DISCIPLINARY FICENT CELLS AND THEY'RE MORE SENSITIVE TO THE EXPOSURE COMPARED TO THE VJ TURN. SO THE CELLS THAT SHE ISOLATED THAT SHE HARVESTED FOR ISOLATION OF TELOMERES AREN'T ALIVE, AT LEAST AT THE TIME OF HARVEST AND SHE CAN CONFIRM THIS BY TYPING UP BLUE STAIN. SO THIS IS HER FIRST REPAIR ASSAY. WHAT YOU'LL SEE UP HERE IS EIGHT NANO GRAMS OF GENOMIC DNA COMPARED TO EIGHT NANO GRAMS OF THE TELOMERES. THIS IS AN ANTIBODY AGAINST THE BUTANE DIMERS WHICH COULD FORM BOTH ON THE G-STRAND AS WELL A--BITS AS C-STRAND. AND YOU WILL NOTICE THERE ARE FEWER PRODUCTS AT THE TELOMERES COMPARED TO THE BULK GENOME. WE SAW THE SIGNAL WAS ABOUT 2.5 FOLD LOWER BUT YOU'LL SEE THAT THE SIGNAL DOES FIT DISAPPEAR OVER TIME AND SO THE PRODUCT AT 48 HOURS DID NOT--WAS NOT BECAUSE THEY WERE LESS TELOMERES LOADEDOT BLOKE BLOCK, HE DID IT THE HATTER WAY, YOU CAN'T TRIP THESE AND FOUND THEM, YOU HAD TO DO SEQUENTIAL PROBING AND KEPT IT PROBE WIDE A RADIO LABEL PROBING AND YOU CAN SEE STRONG ENRICHMENT FOR TELOMERES FACTIONS BUT THERE IS EQUAL LOADING AND ALL THE RECOVERY TIME POINTS AND THEN SHE PROBED WITH THE REPEAT DNA JUST TO CONFIRM EQUAL LOADING OF THE GENOMIC DNA. SO ANOTHER THING SHE NOTICED AFTER WE QUANTITATED THE DATA WAS THAT THE TELOMERE FACTION WAS BEING REPAIRED SLIGHTLY FASTER, 1.5 FASTER COMPARED TO THE BULK GENOME. WE KNOW THOSE ARE REPAIRED MUCH MORE RAPIDLY BUT THEY'RE ALSO FORMED AT A LOWER FREQUENCY SO HERE SHE HAD TO ISOE AND LOAD 15 NANO GRAMS OF TELEGRAMS SO THIS REQUIRES 22 DISHES, AND THEN SHE LOADED 15 AND 7.5 NANO GRAMS IN GENOMIC DNA, YOU WILL NOTICE THAT THE RATE RAPIDLY REPAIRED AND AGAIN, PROBING WITH THE TELOMERE PROBE, CAN YOU SEE EQUAL LOADING AND SHEER SHE OBSERVED SIMILAR RATES FOR THE PRODUCT REMOVAL AND GENOMIC DNA AND TELOMERE DNA. N WE HAD TO PICK A HIGHLY PROLIFERATIVE CELL BECAUSE WE MENTIONED WE NEED BUCKETS OF CELLS FOR THESE TELOMERES SO WE CHOSE THE BJ'S FIBER BLAST BINE LIEN THAT HAD BEEN ENGINEERED TO SO THAT THE TELOMERES ARE LONG AND INTACT AND THEY EXPRESS TELEMERACE, SO WE WONDER IF IT'S REQUIRED FOR PRODUCT REMOVAL. SO WE ALSO DAMAGE TELOMERES FOR THE FORMATION AND REMOVAL AND AND THIS IS A CELL LINE THAT IS TELEMERACE NEGATIVE THAT USES THE ALTERNATIVE LENGTHENING OF THE PATHWAY TO MAINTAIN THEIR TELOMERES AND AGAIN YOU CAN SEE THAT THE PHOTOPRODUCTS ARE REPAIRED BOTH IN GENOMIC AND TELOMERE DNA, THE TELOMERE BLOCKS ENRICHMENT FOR THIS AND THE RATES ARE SIMILAR. SO TELEMERACE IS NOT REQUIRED BUT ALL WE'RE SEEING HERE IS PHOTOPRODUCT REMOVAL, ARE THESE REALLY BEING REMOVED BY THE NUCLEOTIDE EXCISION REPAIR? TO ADDRESS THAT WE CHOSE A CELL LINE FROM AN [INDISCERNIBLE] AND HERE LOOKING AT THE PHOTOPRICE FORMATION THERE'S NO SIGNIFICANT PHOTOPRODUCT REMOVAL, OVER TIME EVEN UP TO 12 HOURS SO HERE YOU SEE THE PLOT, REPEATED THE EXPERIMENTS, AGAIN, AND EXTENDED THE TIME ALL THE WAY UP TO 12 HOURS TO MAKE SURE THERE WASN'T ANY REPAIR, AND WE DON'T SEE ANY SIGNIFICANT DIFFERENCE. SO THIS IS REALLY TO OUR KNOWLEDGE, THE FIRST EVIDENCE THAT THE EXCISION REPAIR CAN FUNCTIONAL TELOMERIC ADATOMS. SO WE PREDICTED THAT THE PHOTOPRODUCTS ARE NOT REMOVED FROM THE TELOMERES AND THEY COULD ACCUMULATE AND ONE CONSEQUENCE OF THAT COULD BE THE SHELTER SO WHEN I WAS IN A POST DOC IN THE LAB, I DID A STUDY WITH DAVE WILSON LOOKING AT HOW [INDISCERNIBLE] MIGHT EFFECT TRF ONE AND TWO BINDING TO TELOMERE DNA AND WE SAW A TWO-THREE FOLD REDUCTION IN BINDING. THIS TIME WE ENGINEERED IN SO THIS PRODUCT HAD A DRAMATIC EFFECT, THIS IS A GEL SHIFT ASSAY, AND YOU CAN SEE IN THE LINEAR RANGE, THE BINDING CURVE ABOUT A 13 FOLD INCREASE IN BINDINGS OF TRF ONE PROTEIN TO THESE THAT HAVE A SINGLE PHOTOPRODUCT COMPARED TO THE TWO THAT DID NOT SO WE THOUGHT THIS WOULD BE EVIDENCE THAT IF THE PHOTOPRODUCTS ARE NOT REMOVED EXCEPT FOR THE BINDING. WE ALSO WANT TO DO A PROEFFICIENT CELL, SO HERE AGAIN, WE TOOK XPA CELLS FROM PATIENTS THAT HAD BEEN COMPLEMENTED BACK WITH XPA PROTEIN AND THESE ARE AVAILABLE FROM THE REPOSITORY. AND THEN LOOKED AT TELOMERES AND LOOKED FOR EVIDENCE OF THE TELOMERE LUNCHING THERE WAS VIRTUALLY NO DIFFERENT. SO THIS REALLY SURPRISED US, SO WE WEREN'T SURE WHY WE WERE RECEIVING SIMILAR RESULTS BUT OUR DATA AND OUR ANALYSIS FOR THE CELLS CLEARLY SHOWED THAT THE UV PHOTOPRODUCTS COULD INTERFERE WITH PHOTOREPLICATION. SO WHAT'S GOING ON? SO THE XTA COMPLEMENTED ITSELF AND DO HAVE THE PROTEIN IN THEM. --THESE, A POST DOC IN THE LAB DID PREPARED ASSAYS ON GENOMIC DNA AND SHE OBSERVED THAT YES, THE COMPLEMENTED CELLS CAN REPAIR THE DNA BUT THE REPAIR IS VERY SLOW. SO IT'S NOT OPTIMAL. YOU CAN SEE EVEN AFTER 48 HOURS, THE PHOTOSLIDES ARE PRES SENT, NOW AFTER ALL THIS WORK WE THINK THIS IS NOT THE IDEAL CELL LINE TO ANSWER OUR QUESTION: IS NUCLEOTIDE EXCISION IMPORTANT FOR PRODUCING ABERR DENT TELOMERES, AND INSTEAD WE'RE TAKING THE CELLS AND HAS USED AND USED SiRNA TECHNOLOGY TO KNOCK DOWN THE EXPRESSION OF THE PROTEIN, AND WE'RE ALSO LOOKING THE TECHNOLOGY TO KNOCK IT OUT AS WELL. AND THE WILD-TYPE FOR ANY N. A. R. AND DEFICIENCIES FOR N. A. R. WE HAVE OBSERVED THAT U. V. INDUCED THE BUTANE DIMERS HAVE BEEN OBSERVED PREVIOUSLY AND AND WE CAN ADD TO THAT, WHERE WE ALSO SEE SIX MORE PHOTOPRODUCTS FORM AT TELOMERE SAYS AS WELL. SO THIS IS TWO LEFT COMPARED TO GENOME, AND WE OBSERVED THAT CPDs ARE REPAIRED, ABOUT 1.5 FOLD FASTER AT TELOMERES, COMPARED TO THE BULK GENOME. SO WE WERE TRYING TO UNDERSTAND WHY THIS IS, IT TURNS OUT THAT TELOMERES ARE TRANSCRIBED, THEY'RE TRANSCRIBED INTO THESE NONCODING RNAs, VERY LONG NONCODING RNAs AND ABOUT 25% OF THEM HAVE FOUND TO BE TRANSCRIBED SO WE'RE WONDER FIGURE IT'S A FASTER RATE OF CPD REMOVAL THAT WAS DETECTED COMPARED TO THE BULK GENOME WHICH WOULD BE REPAIRED PRIMARILY MAYBE DUE TO TRANSCRIPTION REPAIR OF THE TELOMERES SO WE'RE INTERESTED IN WEATHER OR NOT TRANSCRIPTION REPAIR IS HAPPENING AT THE TELOMERE AGAIN. WE FIND THAT SIX PHOTOPRODUCTS PERSISTENT TELOMERES AND IN THE BULK GENOME AND SPA PHYSICIAN CELLS, SO AS I MENTIONED TO OUR KNOWLEDGE THIS IS THE FIRST EVIDENCE THAT IS REREQUIRED TO PRESERVE THE TELOMERE INTEGRITY. WE START THAT TELOMERES ARE SIMILAR TO THE BULK GENOME AND THE TERMERACE POSITIVE BJ-ATURIC CELLS AS WELL AS TELEORDER OF MICRONSER ACE NEGATIVE CELLS. SO IN OTHER WORDS TELEO--METABOLIZEDDER ACE IS NOT REQUIRED FOR THE PRODUCTS. AND FINALLY WE HAVE THE EVIDENCE THAT THIS THAT ARE CONSISTENT WITH FAILURES AND THE TELOMERE REPLICATION, THESE ARE INCREASE INDEED TRANSLESIONS AND SUFFICIENT FOR TRANSLESION DNA SYNTHESIS, FOR THE CELLS BUT WE REALLY DIDN'T ADDRESS WHETHER OR NOT THAT WE HAD FOR THE U. V. INDUCED DEFECTS IN TELOMERE REPLICATION. SO FOR THIS WE NEED A CELL LINE THAT'S WILD-TYPE FOR ANY EXCISION REPAIR AND AVOID IT, OKAY, SO, GOOD, I HAVE TIME FOR QUESTIONS. WITH THAT AND I WAS ALWAYS TRYING TO PAD IN TIME FOR ANY TECHNICAL ISSUES. SORRY WE WERE NOT ABLE TO RESOLVE THE ISSUE WITH THE LITTLE BOX IN THE CORNER. I DID WANT TO MENTION HANNAH WHO'S NOW AT THE EPA AND SHE'S WORKING ON DNA SYNTHESIS WITH TELOMERES AND JUAN WORKED ON THE NUCLEOCISION TELEOR MERES AND THEY WERE BOTH WORKING ON WITH POST DOCS AND I'M GRATEFUL TO THE COLLABORATORS AND AND NOW THE TELOMERE BIOLOGY WAS [INDISCERNIBLE]. SO THAT THE GENOME STABILITY GROUP HERE HAS BEEN VERY INSTRUMENTAL IN GET THANKSGIVING WORK DONE AND WE ALSO HAVE WONDERFUL COLLABORATION WITH THE CARNEGIE MELON, AND I WOULD ALSO LIKE TO NOTE THAT I LOST CONNOR, AND DHVANI, WHO GRADUATED SO WE HAVE POST DOC POSITIONS AVAILABLE, SO ANYONE WHO WANTS TO WORK IN DNA DAMAGE REPAIR AND TELOMERE BIOLOGY, THIS MIGHT BE A GOOD PLACE FOR YOU. SO WITH THAT I'M HAPPY TO TAKE ANY QUESTIONS, WE WILL GO AROUND THE DIFFERENT SITES. [ APPLAUSE ] >> THANK YOU FOR AN EXCITING TALK. IT WAS GREAT. AND NOW WE WILL ALLOW SOME SITES, SO LET'S START WITH--NIEHS, ONE QUESTION EACH SITE AND THEN MORE AT THE END IF THERE ARE. NIEHS, EVERYBODY ELSE MUTE, PLEASE. >> MIKE REZONING NICK --REZNIK, HERE, AND LONG OR SHORT TELEOR MERE SYSTEM THERE ANYWAY TO ADDRESS THE REPAIRS FOR THE LONGER AND SHORTER TELOMERES AND SAY THE TRANSFORMED CELLS VERSES NONTRANSFORMED CELLS. >> CAN YOU HEAR ME NOW? >> WE CAN HEAR YOU. >> OKAY. >> THAT'S A GREAT QUESTION. SO WE THOUGHT A LOT ABOUT WHETHER TELOMERE LENGTH MIGHT INFLUENCE REPAIR AND ONE GREAT MODEL SYSTEM WE THOUGHT ABOUT USING WAS THERE ARE VARIOUS STRINGS OF HELIX CELL LINES, THERE'S ONE STRING THAT HAS VERY LONG TELOMERES BUT IT'S MAYBE 30 SOMETHING KILOBASES AND ANOTHER THAT HAS FIVE KILOBASES. SO IT MIGHT BE INTERESTING TO USE THAT MODEL SYSTEM TO COMPARE RATES OF--PHOTOINFORMATION TO THE TWO CELL LINES. AS FAR AS NONTRANSFORMED, TELEMERACE CELLS ARE NOT REALLY TRANSFORMED, AND PUT THEM IN A EXTEN O GRAPH MODELS FOR TUMORS THEY'RE NOT NORMAL EITHER. THEY'VE BEEN ENGINEERED TO OVEREXPRESS TELEMERACE SO WE THOUGHT A LOT ABOUT COULD WE GROW UP ENOUGH TO THE PRIMARY FIBER BLAST TO ADDRESS THIS QUESTION. WE MIGHT BE ABLE TO FOR PHOTOPRODUCT REPAIR, BECAUSE THE PREPARES SO RAPIDLY, BUT WE ALSO OBTAIN A CELL LINE IN WHICH THE TELEMERACE HAS BEEN CREE LOCKED IN. SO POTENTIALLY WE COULD GROW UP A BUNCH OF THESE CELLS AND THEN BEFORE WE IRRADIATE THEM WE PRELOCK OUT TELEO--METABOLIZEDDER ACE AND LOOK FOR PHOTOREPAIR THERE SO WE THOUGHT A LOT ABOUT IT. >> THANKS. >> SORRY WE KEEP SEEING ME. YOU'RE SUPPOSED TO SEE PATTY, I DOPE KNOW HOW WE CHANGE THIS. I MUTED BUT ANYHOW, LET'S MOVE TO KENTUCKY, DAVE? >> YEAH, THIS IS KENTUCKY, MAYBE YOU CAN CLARIFY, I THINK YOU JUST ALLUDED TO IT BUT I DIDN'T CATCH WHETHER THE EFFICIENCY OF SIX-FOUR REPAIR IS MUCH HIGHER OR SIMILAR TO WHAT YOU WOULD SEE IN THE DIFFERENCE BETWEEN THE OVERALL GENOME AND TRANSCRIPTION COUPLED REPAIR COMPARED TO CPD REPAIRS? I DIDN'T CATCH THAT--PROBABLY WAS IN THE GRAPHS BUT I WASN'T ABLE TO SEE THAT KIND OF DETAIL SO MAYBE YOU CAN CLARIFY HOW THIS REPAIR AS FAST IN TELOMERES AS IT WOULD BE IN THE NORMAL GENOME. >> CAN YOU HEAR ME? >> CAN YOU HERE ME NOW HOPEFULLY? >> SO THE RESULT WAS THAT CPDs ARE REPAIRED ABOUT ONE AND HALF FASTER THAN TELOMERES COMPARED TO BUMK GENOME BUT FOR THE PHOTOPRODUCT WEEST OBSERVE THE REPAIR WAS SIMILAR SO WE THINK WE THINK THAT THE PHOTOPRODUCTS ARE REMOVED VERY RAPIDLY. SUCH THAT THERE'S NOT MUCH OF A DIFFERENCE IN REPAIR RATE WITH TRANSCRIPTION COUPLED AND GLOBAL GENOME REPAIR SO THERE WE WOULDN'T NECESSARILY PICK UP THE TRANSCRIPTION AT THE TELOMERE THAT WOULD NOT BE OBVIOUS WITH THE PHOTOPILOT. BUT THE PSYCHOBUTANE DIMERS ARE REPAIRED SLOTLY BY GENOME RPAIR, WE SAW EVIDENCE FOR THEM AFTER 48 HOURS. SO IF THERE IS TRANSCRIPTION REPAIR HAPPENING AT THE TELOMERES, THAT MIGHT EXPLAIN WHY WE SEE SLIGHTLY HIGHER RATE AT C PD REMOVAL BECAUSE IT'S TRICKY BECAUSE NOT ALL OF THEM ARE TRANSCRIBED, IT'S A MIX OF GLOBAL GENOME AND TCR. >> OKAY, THANK YOU. >> THANK YOU DAVE AND ON TO STONY BROOK. >> EVERYBODY ELSE MUTE. OTHER THAN. >> HI, HI, PATTY, FANTASTIC TALK. I WAS WONDERING IF THE DIMER PROCESS FOR THE TELOMERE IS PARTICULARLY DEPENDENT ON POLL-TELOMERE OR THE SEQUENCE IS ERROR FREE OR HAVE YOU USED THE CELL LINE THAT IS DEPLETED OF OTHER TRS TELOMERES FOR [INDISCERNIBLE] FOR INSTANCE? >> YOU COULD MAKE A CASE FOR OTHERS--WE HAVE BANGING GOING ON, CONSTRUCTION--SO OF COURSE IT MIGHT DEPENDOT TYPE OF DAMAGE, YOUR OTHER QUESTION WAS--OH IF WE SEQUENCED THE TELOMERE, WE HAVE NOT WE'RE VERY INTERESTED IN THE ACCUMULATION OF THESE IN THE TELOMERE SEQUENCE MIGHT ALSO END UP EFFECTS [INDISCERNIBLE] HAVEN'T FIGURED OUT HOW TO DO THAT TO, SEQUENCE THESE HIGHLY COMPETITIVE REGIONS TO THE GENOME BUT YEAH, WE'RE HOPING TO GET THERE AT SOME POINT. >> THANK AND YOU ON TO NCI, KEN? >> THANK YOU THAT WAS A VERY NICE PRESENTATION, PATTY AND HAPPY ST. PATRICK'S DAY. I HAD A COUPLE QUESTIONS, WHERE ARE WE? HERE WE ARE AND ONE IS A TECHNICAL ONE. YOU WANT ME TO USE THE ANTIBODY FOR TO LOOK FOR THE CPD PHOTOPRODUCT AND YOU WONDER IF THERE MIGHT BE SOME PROBLEM WITH THE ANTIBODY HAVE YOU THOUGHT ABOUT USING ANOTHER APPROACH LIKE THE C-PROTEIN NUCLEACE? >> RIGHT, WE ONLY USE FEDERAL FOR THE PHOTOPRODUCTS AND THE CPD, THE PSYCHE ON CLOUGH BUTANE DIMERS AND, AND THIS IS A GREAT IDEA AND THIS IS ESSENTIALLY WHAT WAS DONE IN WILL'S PAPER BY PATRICIA CRUMS IN 19 SNIFF WE LOOKED FOR THE FRAGMENTS TO BE CLEARED IN THE T-FOUR NUCLEACE AND SAW OVER TIME THE RECOVERY TIME THEY OBSERVE LESS CLEAVAGE. SO, GREAT EXPERIMENTS. ALMOST--IT WOULD BE NICE IF WE COULD THE PHOTOPRODUCT REMOVAL USING A SIMILAR APPROACH. >> YEAH, I WAS WONDERING ABOUT THE LOWER FREQUENCY OF CPDs IN THE TELOMERES, THE OTHER IS IF YOU CHECK FOR CELLS IN PATIENTS WITH [INDISCERNIBLE] CONGENERATEDITTA OR OTHER FORMS OF [INDISCERNIBLE] COMPLEX [INDISCERNIBLE]? >> YEAH, THAT'S A GREAT QUESTION. WE HAVE NOT ADDRESSED ANY OF THAT YET LOOKING IN SOME OF THESE CELL LINES PARTICULARLY CELL LINES FROM PATIENTS THAT HAVE MUTATIONS IN THE 10-TWO SO IT'S NOT CLEAR YET HOW IT MIGHT INFLUENCE PHOTOPRODUCT FOR REAL OF TELOMERES, AND WHETHER OR NOT SHELTERING MIGHT BE HELPING TO SHIELD THE TELOMERES, FROM UV DAMAGE AND PERHAPS ONE REASON WE SEE LESS PHOTOPRODUCT INFORMATION, AND SHELTER IN COMPLEXES ALTERED IN SUCH A WAY, SUCH AS WITH THE CONTINUED MUTATIONS AND PATIENTS WITH THE TELOMERE SHORTENING SYNDROME, YOU MIGHT SEE A DIFFERENCE IN PHOTOPRODUCT FORMATION, RATES AS WELL AS REPAIR. SO, GREAT QUESTION. >> OKAY, THANKS, KEN WE GO TO N. I. A. HERE, AND I ALLOW MYSELF TWO QUICK QUESTIONS, WE MIGHT HAVE MORE, MY FIRST IS THAT'S REALLY COOL, PATTY IT'S REALLY NEAT AND I WONDER IF YOU'RE LOOKING AT C. S., CALK 18 18--CAUCANE CELLS AND PLUS THE CSP PROTEIN INTERACTING WITH THE TRANSCRIPTS. >> YES, SO WE'RE VERY INTERESTED IN WHETHER OR NOT UVs MIGHT EFFECT THE LEVELS OF TRANSCRIPTION AT TELOMERES AND CERTAINLY HAVE THOUGHT ABOUT LOOKING IN CELLS DEFICIENT FOR XPC, CFA, CSB AND WE SAW IT APPROACH THAT WE MIGHT USE THAT WE CAN LOOK AT ISOGENIC CELL LINES AND WE'RE TRYING TO MANIPULATE THEM NOW FOR THE TECHNOLOGY FOR THE VJ CELLS AND KNOCK OUT XTC, KNOCK OUT, IF THEY KNOCK OUT CSV, WE HAVE A BIT OF TROUBLE LOOKING AT TELOMERES IN SOME OF THESE TRANSFORM CELLS BECAUSE WHEN WE LOOK AT THE TELOMERE ABNORMALITIESOSHATION, THE FRAGILE TELOMERES AND THE TELOMERE LOSS, BOTH WITH THE TELOMERES BECAUSE THOSE HAVE GONE THROUGH CRISIS AND THE TELOMERE VS SHORTENED AND THEN THEY TELEMERACES IS UPREGULATED WHERE WE LIKE THE CELLS FOR STUDYING DNA DAMAGE AND REPAIR THE UV TOAD O PROJECT BECAUSE THOSE CELLS WERE EXPRESSED IN AN EARLY PASSAGE BEFORE THEY'RE TELOMERES EVER WENT THROUGH ANY CRISIS. SO THAT'S THE APPROACH THAT WE'RE TRYING TO TAKE NOW. >> YEAH. >> OF COURSE WITH THIS TECHNIQUE, CAN YOU GO FOR ANY ANTIBODY YOU WANT TO FOR VARIOUS NATIONS AND LOOK AT OTHER PATHWAYS. I WAS JUST WONDERING, SO, YOU SAY THAT YOU DETECT 30% OF THE TELOMERES, RIGHT? AND SO, IS THERE ANY BIAS IN THAT 30%? MIGHT IT BE THAT IF YOU HAVE A LESION SLOWEST TO THE BIOTIN BINDING REGION OR SOMETHING YOU COULD SWAP THAT OR SOMETHING, IS THAT IN REGARD TO WHAT'S BEEN PREPARED OR NOT PREPARED? >> AM I ON? OKAY. YEAH, THAT'S CLEARLY SOMETHING WE WORRY ABOUT. WE DID SEE THAT THAT 30% RECOVERY DIRECTLY BETWEEN 30-40% DID NOT CHANGE AFTER U. V. IRRADIATES THE CELLS SO THE RECOVERY EFFICIENCY IS ABOUT THE SAME, FROM UNTREATED CELLS TO CELLS THAT HAVE BEEN USED. BUT, CERTAINLY, WE CAN'T--AND THE OLIGIO BINDS FOR THE THREE PRIME OVERHAG WOULD BE A SMALL TARGET FOR RADIATION BUT CAN'T RULE OUT THE POSSIBILITY THAT PERHAPS NOT ALL THE TELOMERES ARE REPAIRED. THERE MAY BE A FRACTION THAT ARE NOT REPAIRED AND OUR ASSAY MIGHT BE BI ASSED FOR BEING ABLE TO PICK UP THE ONES THAT ARE REPAIRED, PARTICULARLY WE MIGHT HAVE, YOU KNOW A SENTENCATIVITY ISSUE THAT WE'RE LOOKING AT FALSE TELOMERES AND A FEW OF THEM REALLY ARE NOT REPAIRED, THE REPAIR IS VERY, VERY SLOWLY, WE WOULD NOT BE ABLE TO TAKE THAT UP IN OUR SARKS A. VERY GOOD. SO THANK YOU AND NOW, I WANT TO SEE IF THERE ARE ANY OTHER CENTERS, ANY PLACES THAT HAVE NOT HAD AN OPPORTUNITY TO ASK QUESTIONS? THEN IT'S TIME TO SPEAK UP NOW. IF NOT THEN IT'S OPEN FOR QUESTIONS FROM ANYWHERE. >> KEN,-- >> WE DIDN'T REALLY HAVE ANY MAJOR QUESTIONS, OF COURSE WE'VE SEEN THESE STORIES UP IN THE PAST YEAR, WE'RE GLAD SLEEP APNEA AND OBESITY SEE YOUR UP AND RUNNING FROM YOUR NEW PLACE. >> FROM MY MEMORY, IT'S A GREAT PLACE. >> SHE HAS MY OLD SPACE THERE SO IT LOOKS REALLY GREAT, PATTY AND AGAIN, WE DON'T HAVE ANY QUESTIONS HERE, WE JUST WANTED TO SAY, HI, THANKS. >> OKAY, THANKS AND NOW, EVERYBODY MUTE UNLESS SOMEONE HAS ANOTHER QUESTION? IT'S OPEN FOR QUESTIONS FROM ANYWHERE. >> WHAT ABOUT MISMATCH REPAIR? >> DAVE WILSON ASKS ABOUT MISMATCH REPAIR? >> GREAT QUESTION. THERE'S NOT MUCH KNOWN ABOUT MISMATCHED REPAIRS EITHER, THERE'S SOMETHING IN THE LITERATURE THAT MISMATCHED REPAIRS IN CHRIST ARE MORE PRONE TO TURNING ON THE ALTERNATIVE LENGTHENING WITH THE YEAST BUT WIDE OPEN AREA. YOU NOTICE I LEFT'RE LEFT THAT OFF MY REPAIR SLIDE. I CUT OFF MISMATCHED REPAIR AND PUT ON TRANSLATION DNA CENT SIS. >> WE NOTICED. >> OTHER QUESTIONS, ANYWHERE? >> OKAY, ANY FINAL WORK FROM YOU, KEN? >> NO, THANK YOU VERY MUCH, AND HAPPY ST. PATRICK'S DAY. >> GREAT TO SEE PATTY AND EVERYONE ELSE. [ APPLAUSE ]