1 00:00:06,602 --> 00:00:09,438 >> WE HAVE A PROUD RANGE OF TIME 2 00:00:09,438 --> 00:00:11,640 ZONES, SO GREETINGS TO YOU. 3 00:00:11,640 --> 00:00:13,509 BEFORE I INTRODUCE OUR SPEAKER, 4 00:00:13,509 --> 00:00:15,944 FOUR THINGS I WANT TO TELL YOU 5 00:00:15,944 --> 00:00:16,645 ABOUT. 6 00:00:16,645 --> 00:00:18,280 FIRST, I WANT TO THANK OUR 7 00:00:18,280 --> 00:00:21,216 ADVISORY BOARD FOR ALL OF OUR 8 00:00:21,216 --> 00:00:24,453 ADVICE AND GUIDANCE AND OUR A.V. 9 00:00:24,453 --> 00:00:26,455 PEOPLE WHO MAKE THINGS WORK 10 00:00:26,455 --> 00:00:26,755 SEAMLESSLY. 11 00:00:26,755 --> 00:00:28,056 SECONDLY AND VERY IMPORTANTLY, 12 00:00:28,056 --> 00:00:30,692 WE ARE COLLECTING NOMINATIONS 13 00:00:30,692 --> 00:00:34,463 FOR STELLAR YOUNG INVESTIGATORS 14 00:00:34,463 --> 00:00:36,498 TO SPEAK IN OUR ANNUAL YOUNG 15 00:00:36,498 --> 00:00:38,000 INVESTIGATORS SHOWCASE THIS 16 00:00:38,000 --> 00:00:38,200 YEAR. 17 00:00:38,200 --> 00:00:40,202 THOSE OF YOU WHO WANT TO 18 00:00:40,202 --> 00:00:41,637 NOMINATE SOMEBODY, PLEASE E-MAIL 19 00:00:41,637 --> 00:00:46,675 US WITH YOUR NOMINATIONS WITH A 20 00:00:46,675 --> 00:00:48,377 SHORT BIO WITH JUSTIFICATION, 21 00:00:48,377 --> 00:00:50,946 THE SOONER THE BETTER. 22 00:00:50,946 --> 00:00:53,882 THE REGISTRATION DEADLINE FOR AN 23 00:00:53,882 --> 00:00:55,584 UPCOMING DNA STRUCTURES IN 24 00:00:55,584 --> 00:00:57,653 BIOLOGY CONFERENCE THAT FOCUSES 25 00:00:57,653 --> 00:00:59,354 ON UNUSUAL DNA STRUCTURES AND 26 00:00:59,354 --> 00:01:02,324 THE EFFECTS OF TRANSCRIPTION, 27 00:01:02,324 --> 00:01:04,593 REPLICATION AND REPAIR, THE 28 00:01:04,593 --> 00:01:05,928 EARLY REGISTRATION DEADLINE IS 29 00:01:05,928 --> 00:01:08,263 PAST, BUT YOU CAN STILL MAKE IT 30 00:01:08,263 --> 00:01:09,765 IF YOU REGISTER SOON. 31 00:01:09,765 --> 00:01:11,667 LATE REGISTRATION IS COMING UP 32 00:01:11,667 --> 00:01:12,301 ON JULY 1. 33 00:01:12,301 --> 00:01:14,102 AND THEN FINALLY, I WANT TO 34 00:01:14,102 --> 00:01:16,505 MENTION THAT THE EARLY BIRD 35 00:01:16,505 --> 00:01:20,042 REGISTRATION FOR THE 55th ANNUAL 36 00:01:20,042 --> 00:01:22,544 EMGS MEETING ENDS ON JULY 15, SO 37 00:01:22,544 --> 00:01:26,048 GET IN THERE WHILE YOU CAN. 38 00:01:26,048 --> 00:01:29,251 NOW ON TO TODAY'S SEMINAR. 39 00:01:29,251 --> 00:01:31,019 OUR SPEAKER TODAY IS DR. SHARON 40 00:01:31,019 --> 00:01:32,721 BETH CANTOR. 41 00:01:32,721 --> 00:01:35,023 DR. CANTOR HAS BEEN ON THE 42 00:01:35,023 --> 00:01:36,725 FACULTY AT UMASS MEDICAL SCHOOL 43 00:01:36,725 --> 00:01:39,127 SINCE 2003 WHERE SHE IS THE GLAD 44 00:01:39,127 --> 00:01:41,163 SIS SMITH MARTIN CHAIR OF 45 00:01:41,163 --> 00:01:43,532 ONCOLOGY AND PROFESSOR OF 46 00:01:43,532 --> 00:01:44,833 MOLECULAR CELL AND CANCER 47 00:01:44,833 --> 00:01:45,067 BIOLOGY. 48 00:01:45,067 --> 00:01:49,004 SHE IS THE CO-DIRECTOR OF THE 49 00:01:49,004 --> 00:01:50,072 PROGRAM GENOME INTEGRITY WHICH 50 00:01:50,072 --> 00:01:51,907 SHE FOUNDED IN 2024. 51 00:01:51,907 --> 00:01:56,078 DR. CANTOR RECEIVED HER PH.D. IN 52 00:01:56,078 --> 00:01:57,279 BIOCHEMISTRY FROM TUFTS 53 00:01:57,279 --> 00:01:59,114 UNIVERSITY AND COMPLETED 54 00:01:59,114 --> 00:02:02,017 TRAINING AT DANA HARBOR CANCER 55 00:02:02,017 --> 00:02:03,752 MEDICAL INSTITUTE AND HARVARD 56 00:02:03,752 --> 00:02:05,687 MEDICAL SCHOOL. 57 00:02:05,687 --> 00:02:07,823 SHE IS KNOWN FOR HER RESEARCH IN 58 00:02:07,823 --> 00:02:10,459 BREAST AND OVARIAN CANCER GENES, 59 00:02:10,459 --> 00:02:17,165 RESEARCH FOCUSED ON BROICA 1, BA 60 00:02:17,165 --> 00:02:20,569 2, AND A GENE SHE IDENTIFIED AND 61 00:02:20,569 --> 00:02:20,802 CLONED. 62 00:02:20,802 --> 00:02:23,205 HER RESEARCH HAS CHALLENGED THE 63 00:02:23,205 --> 00:02:26,241 CONVENTIONAL UNDERSTANDING OF 64 00:02:26,241 --> 00:02:30,012 BRCANESS AND DNA REPAIR AND 65 00:02:30,012 --> 00:02:31,780 TRANSLATION SYNTHESIS AS A 66 00:02:31,780 --> 00:02:32,848 VULNERABILITY IN KEASHS. 67 00:02:32,848 --> 00:02:37,286 HER SEMINAR IS DEFINING THE GAP 68 00:02:37,286 --> 00:02:38,787 VULNERABILITY IN BRCA CANCER. 69 00:02:38,787 --> 00:02:40,355 I JUST WANT TO REMIND EVERYONE 70 00:02:40,355 --> 00:02:41,657 IN THE AUDIENCE TO PLEASE PUT 71 00:02:41,657 --> 00:02:44,860 YOUR QUESTIONS IN THE CHAT AND 72 00:02:44,860 --> 00:02:46,828 WE WILL, CHUN ZHANG AND I WILL 73 00:02:46,828 --> 00:02:48,764 POST THE QUESTIONS TO SHARON 74 00:02:48,764 --> 00:02:49,598 AFTER HER TALK. 75 00:02:49,598 --> 00:02:54,503 SHARON, WITHOUT FUR ADO. 76 00:02:54,503 --> 00:02:55,037 >> OKAY. 77 00:02:55,037 --> 00:02:56,905 I THINK I GOT THAT. 78 00:02:56,905 --> 00:02:57,773 ALL RIGHT. 79 00:02:57,773 --> 00:03:03,645 HOPEFULLY YOU CAN SEE IT OKAY. 80 00:03:03,645 --> 00:03:04,746 LET ME START SHARING. 81 00:03:04,746 --> 00:03:06,248 IS IT SHARING? 82 00:03:06,248 --> 00:03:06,815 >> NO. 83 00:03:06,815 --> 00:03:07,282 NOT YET. 84 00:03:07,282 --> 00:03:08,984 >> OH, THAT'S INTERESTING. 85 00:03:08,984 --> 00:03:11,019 >> IT IS SHARING, BUT NOT 86 00:03:11,019 --> 00:03:11,320 PRESENTING. 87 00:03:11,320 --> 00:03:14,556 >> YES, THAT IS WHAT I MEAN. 88 00:03:14,556 --> 00:03:14,756 SORRY. 89 00:03:14,756 --> 00:03:16,591 YOU ARE NOT IN PRESENTER VIEW. 90 00:03:16,591 --> 00:03:18,293 >> I'M JUST TRYING TO GET MY 91 00:03:18,293 --> 00:03:24,666 CURSOR HERE. 92 00:03:24,666 --> 00:03:24,866 OKAY. 93 00:03:24,866 --> 00:03:25,901 ARE WE ALL SET? 94 00:03:25,901 --> 00:03:26,668 IS THAT WORKING? 95 00:03:26,668 --> 00:03:28,103 >> YES. 96 00:03:28,103 --> 00:03:31,306 >> THANK YOU SO MUCH, CHUN 97 00:03:31,306 --> 00:03:33,475 ZHANG, AND KAREN FOR THE 98 00:03:33,475 --> 00:03:34,609 INVITATION TO PRESENT TODAY. 99 00:03:34,609 --> 00:03:37,379 TO GET STARTED, I WILL JUST GIVE 100 00:03:37,379 --> 00:03:39,014 YOU AN OVERVIEW OF WHAT I HOPE 101 00:03:39,014 --> 00:03:40,449 TO COVER TODAY. 102 00:03:40,449 --> 00:03:44,186 I WILL GO THROUGH THE 103 00:03:44,186 --> 00:03:46,254 TRADITIONAL MOD OF BRCANESS AND 104 00:03:46,254 --> 00:03:48,390 COMPARE TO THE GAP MODEL. 105 00:03:48,390 --> 00:03:52,728 AND LOOK AT BOTH FOR THEIR 106 00:03:52,728 --> 00:03:55,464 RELEVANCE TO CHEMO THERAPY 107 00:03:55,464 --> 00:03:58,300 RESPONSE AND BETTER DEFINE THE 108 00:03:58,300 --> 00:03:59,668 GAP VULNERABILITY. 109 00:03:59,668 --> 00:04:02,237 SO WHEN WE THINK ABOUT 110 00:04:02,237 --> 00:04:03,705 CHEMOTHERAPY, MECHANISMS OF 111 00:04:03,705 --> 00:04:06,775 ACTION FOR THE LAST 30, 40 YEARS 112 00:04:06,775 --> 00:04:08,810 TYPICALLY IT EVOKES A DOUBLE 113 00:04:08,810 --> 00:04:11,013 STRANDED BREAK I'M SHOWING YOU 114 00:04:11,013 --> 00:04:12,381 THAT LEADS TO THE SENSETIZATION 115 00:04:12,381 --> 00:04:16,518 AND THE KILLING OF THE CANCER 116 00:04:16,518 --> 00:04:16,718 CELLS. 117 00:04:16,718 --> 00:04:17,452 THIS HAS BEEN WELL DESCRIBED 118 00:04:17,452 --> 00:04:18,920 OVER THE YEARS. 119 00:04:18,920 --> 00:04:22,657 THERE ARE SEVERAL REASONS, BUT 120 00:04:22,657 --> 00:04:25,761 HER REDTY BREAST/OVARIAN GENES 121 00:04:25,761 --> 00:04:28,230 WERE MISSING, HIGHLY SENSITIVE 122 00:04:28,230 --> 00:04:29,097 TO CHEMOTHERAPY. 123 00:04:29,097 --> 00:04:31,033 THE BEST KNOWN FUNCTIONS FROM 124 00:04:31,033 --> 00:04:32,634 THESE PROTEINS FROM THEIR 125 00:04:32,634 --> 00:04:35,003 ORIGINAL IDENTIFICATION FOR 126 00:04:35,003 --> 00:04:38,640 FUNCTIONS IN REPAIRING DNA 127 00:04:38,640 --> 00:04:42,677 DOUBLE STRAND BREAKS INVOLVED A 128 00:04:42,677 --> 00:04:45,914 RAD 51 RECOMBINATION PROTEIN. 129 00:04:45,914 --> 00:04:47,315 AND PROTECTING REPLICATION THAT 130 00:04:47,315 --> 00:04:50,652 STALL AND REVERSE WITH RAD 51, 131 00:04:50,652 --> 00:04:53,355 PROTECTING THEM FROM DEGRADATION 132 00:04:53,355 --> 00:04:58,393 THAT WOULD BE EXPECTED TO LEAD 133 00:04:58,393 --> 00:05:00,162 TO DOUBLE STEM BREAKS AND THE 134 00:05:00,162 --> 00:05:02,297 FORMATION THAT HAS BEEN THOUGHT 135 00:05:02,297 --> 00:05:04,933 TO BE THE FUNDAMENTAL NATURE OF 136 00:05:04,933 --> 00:05:07,035 BRCANESS THAT I'M DEPICTING HERE 137 00:05:07,035 --> 00:05:10,472 AS A CANCER CELL THAT CANNOT 138 00:05:10,472 --> 00:05:14,276 HANDLE DOUBLE STRAND BREAKS 139 00:05:14,276 --> 00:05:15,710 INDUCED BY CHEMOTHERAPY. 140 00:05:15,710 --> 00:05:17,612 COMING FULL CIRCLE WITH THIS 141 00:05:17,612 --> 00:05:20,082 MODEL, IT WAS SHOWN QUITE 142 00:05:20,082 --> 00:05:23,452 CLEARLY THAT CHEMO RESISTANCE 143 00:05:23,452 --> 00:05:27,355 WAS RESTORED HOOGEN COMBINATION 144 00:05:27,355 --> 00:05:28,723 OR FOR PROTECTION. 145 00:05:28,723 --> 00:05:29,958 WHY NOT CONSIDER THIS MODEL TO 146 00:05:29,958 --> 00:05:32,561 BE SOLID? 147 00:05:32,561 --> 00:05:34,196 WELL, IT HAS SEVERAL 148 00:05:34,196 --> 00:05:34,963 EXPECTATIONS. 149 00:05:34,963 --> 00:05:37,632 ONE OF THEM, OF COURSE, IS 150 00:05:37,632 --> 00:05:39,801 CHEMOTHERAPY SHOULD GENERATE DNA 151 00:05:39,801 --> 00:05:40,302 DOUBLE-STRANDED BREAKS. 152 00:05:40,302 --> 00:05:42,104 WE LOOKED AT WHAT THE LITERATURE 153 00:05:42,104 --> 00:05:44,306 REPORTED OVER THE YEARS AND WHAT 154 00:05:44,306 --> 00:05:46,741 YOU'LL NOTE IS THAT EVEN WITH A 155 00:05:46,741 --> 00:05:49,678 TOPSIDE AND IONIZING RADIATION, 156 00:05:49,678 --> 00:05:54,382 THERE ARE FAR GREATER 157 00:05:54,382 --> 00:05:54,983 SINGLE-STRANDED DNA THAN A 158 00:05:54,983 --> 00:05:56,017 DOUBLE STRAND BREAK. 159 00:05:56,017 --> 00:05:57,419 THE IDEA THAT THE DOUBLE STRAND 160 00:05:57,419 --> 00:06:02,057 BREAK IS THE PREDOMINANT 161 00:06:02,057 --> 00:06:05,060 DIFFICULT LESION HAS PERMEATED 162 00:06:05,060 --> 00:06:09,798 THE FIELD INCLUDING THIS 163 00:06:09,798 --> 00:06:12,134 RADIOLOGY TEXTBOOK WHO DISMISSED 164 00:06:12,134 --> 00:06:19,941 THE SINGLE STRANDED DNA BREAK 165 00:06:19,941 --> 00:06:21,309 NOT HAVING PROMINENCE. 166 00:06:21,309 --> 00:06:22,344 COMING BACK TO EXPECTATIONS, 167 00:06:22,344 --> 00:06:24,246 AGAIN, THE SOURCE OF THE DOUBLE 168 00:06:24,246 --> 00:06:25,914 STRAND BREAK AGAIN IS A LITTLE 169 00:06:25,914 --> 00:06:27,549 BIT MORE ELUSIVE. 170 00:06:27,549 --> 00:06:30,685 AGAIN, THE SINGLE STRANDED DNA 171 00:06:30,685 --> 00:06:38,994 BREAK BEING THE MORE COMMON 172 00:06:38,994 --> 00:06:39,227 LESION. 173 00:06:39,227 --> 00:06:41,596 IF YOU HAD THESE FUNCTIONS, IF 174 00:06:41,596 --> 00:06:43,031 YOU DIDN'T HAVE THEM YOU SHOULD 175 00:06:43,031 --> 00:06:46,301 BE SENSITIVE AND IF YOU DID 176 00:06:46,301 --> 00:06:49,070 RESTORE THEM CONFIRM RESISTANCE. 177 00:06:49,070 --> 00:06:50,305 THE GENES THAT REGULATE THESE 178 00:06:50,305 --> 00:06:53,875 RESPONSES WERE NOT OVERLY 179 00:06:53,875 --> 00:06:57,112 PREDICTIVE NOR IS THE HRD SCORE, 180 00:06:57,112 --> 00:06:58,613 PERFECT BIOMARKER OF RESPONSE. 181 00:06:58,613 --> 00:07:01,249 AND SO WE STARTED TO FOCUS ON 182 00:07:01,249 --> 00:07:04,019 THIS OTHER FUNCTION OF THE BRCA 183 00:07:04,019 --> 00:07:06,655 PATHWAY IN WHAT WE ARE CALLING 184 00:07:06,655 --> 00:07:07,489 REPLICATION GAP SUPPRESSION OR 185 00:07:07,489 --> 00:07:08,523 GAP SUPPRESSION. 186 00:07:08,523 --> 00:07:10,158 AND SO HERE WE ARE NOT ALONE. 187 00:07:10,158 --> 00:07:12,994 THIS AREA HAS REALLY EXPLODED. 188 00:07:12,994 --> 00:07:14,362 MANY LABS HAVE CONTRIBUTED HERE 189 00:07:14,362 --> 00:07:22,304 THROUGHOUT THE YEARS. 190 00:07:22,304 --> 00:07:23,572 INCLUDING LOOKING AT THESE 191 00:07:23,572 --> 00:07:25,740 PATHWAYS IN FROG EXTRACTS AND 192 00:07:25,740 --> 00:07:27,209 REALLY ACROSS THE WHOLE SPECTRUM 193 00:07:27,209 --> 00:07:30,145 OF THE PATHWAY THERE IS ROLES 194 00:07:30,145 --> 00:07:31,279 FOR GAP SUPPRESSION. 195 00:07:31,279 --> 00:07:33,949 SO WHAT REALLY CONVINCED US THIS 196 00:07:33,949 --> 00:07:35,383 WAS AN IMPORTANT PATHWAY WAS A 197 00:07:35,383 --> 00:07:38,186 TABLE LIKE THIS WHERE WE WOULD 198 00:07:38,186 --> 00:07:40,121 LOOK AT A SERIES OF CANCER CELL 199 00:07:40,121 --> 00:07:45,927 LINES WELL CHARACTERIZED IN THE 200 00:07:45,927 --> 00:07:47,696 FIELD FOR HR -- WAS SUPPORTED. 201 00:07:47,696 --> 00:07:50,398 WE LOOKED THAT FULLY ALIGNED 202 00:07:50,398 --> 00:07:52,834 WITH RESISTANCE AND LOSS OF GAP 203 00:07:52,834 --> 00:07:53,802 SUPPRESSION OR THE PRESENCE OF 204 00:07:53,802 --> 00:07:59,207 GAPS FULLY ALIGNED WITH 205 00:07:59,207 --> 00:08:05,447 CHEMOTHERAPY RESPONSE. 206 00:08:05,447 --> 00:08:10,485 THIS OFFERS FOR CISPLATIN AND 207 00:08:10,485 --> 00:08:14,089 THIS MODEL HELD UP. 208 00:08:14,089 --> 00:08:15,724 EVEN IF PEOPLE DIDN'T LIKE THIS 209 00:08:15,724 --> 00:08:18,393 MODEL, YOU HAVE TO AGREE GAPS 210 00:08:18,393 --> 00:08:19,327 ARE HIGHLY PREDICTIVE OF 211 00:08:19,327 --> 00:08:23,865 RESPONSE. 212 00:08:23,865 --> 00:08:26,101 SO THIS TOGETHER LED US TO PUT 213 00:08:26,101 --> 00:08:28,203 TOGETHER THIS NEW FRAMEWORK THAT 214 00:08:28,203 --> 00:08:29,971 A HEALTHY CELL IS ABLE TO 215 00:08:29,971 --> 00:08:31,306 WITHSTAND CHEMOTHERAPY BECAUSE 216 00:08:31,306 --> 00:08:32,807 IT HAS THE ABILITY TO SUPPRESS 217 00:08:32,807 --> 00:08:38,413 AND REPAIR REPLICATION GAPS 218 00:08:38,413 --> 00:08:40,048 WHEREAS A BRCA WAS COMING FROM 219 00:08:40,048 --> 00:08:44,085 INABILITY TO TAKE CARE OF THIS 220 00:08:44,085 --> 00:08:45,820 AND CHEMORESISTANCE WAS RESTORED 221 00:08:45,820 --> 00:08:48,189 GAP SUPPRESSION AND REPAIR. 222 00:08:48,189 --> 00:08:49,591 OTHERS HAVE, OF COURSE, LOOKED 223 00:08:49,591 --> 00:08:52,560 AT THIS AND HAD DIFFERENT 224 00:08:52,560 --> 00:08:52,927 INTERPRETATIONS. 225 00:08:52,927 --> 00:08:54,963 I WANT TO SHARE THAT WITH YOU 226 00:08:54,963 --> 00:08:56,298 NOW IN A BRIEF SUMMARY. 227 00:08:56,298 --> 00:08:59,034 FOR EXAMPLE, OTHERS HAVE 228 00:08:59,034 --> 00:09:03,538 DETECTED GAPS FOLLOWING 229 00:09:03,538 --> 00:09:05,006 CHEMOTHERAPY SUCH AS SHOWN HERE, 230 00:09:05,006 --> 00:09:08,910 BUT THEY ARGUED IT IS THE SECOND 231 00:09:08,910 --> 00:09:10,812 S FACE WHERE THESE DOUBLE STRAND 232 00:09:10,812 --> 00:09:19,020 BREAKS FORM IS THE REAL KILLING 233 00:09:19,020 --> 00:09:19,254 LESION. 234 00:09:19,254 --> 00:09:21,956 WE ARE SAYING THIS IS THE 235 00:09:21,956 --> 00:09:23,425 CRITICAL JUNCTION WHERE BRCA 1 236 00:09:23,425 --> 00:09:25,994 PROTEINS ARE PROTECTING FROM 237 00:09:25,994 --> 00:09:26,628 CHEMOTHERAPY. 238 00:09:26,628 --> 00:09:31,266 WE WOULD AGREE THESE DRUGS MAKE 239 00:09:31,266 --> 00:09:32,801 DOUBLE STRAND BREAKS. 240 00:09:32,801 --> 00:09:36,738 THAT IS TRUE AND EASY TO SEE. 241 00:09:36,738 --> 00:09:41,276 THEY ACTIVATE APOPTOSIS. 242 00:09:41,276 --> 00:09:50,018 IF WE INHIBIT APOPTOSIS, YOU SEE 243 00:09:50,018 --> 00:09:51,853 BREAKS IN KEERM THERAPY. 244 00:09:51,853 --> 00:09:52,954 I SEE DOUBLE STRAND BREAKS 245 00:09:52,954 --> 00:09:54,556 THEREFORE THAT IS THE 246 00:09:54,556 --> 00:09:56,458 SENSITIZING LESION, SO I THINK 247 00:09:56,458 --> 00:09:58,093 THERE IS MORE WORK TO DO TO 248 00:09:58,093 --> 00:09:59,794 SOLVE THIS MYSTERY. 249 00:09:59,794 --> 00:10:04,799 WE HEARD FROM ANOTHER GROUP THAT 250 00:10:04,799 --> 00:10:07,769 BRCA'S PRIMARY ROLE IS IN HR. 251 00:10:07,769 --> 00:10:11,539 SHE, OF COURSE, HAS PIONEERED 252 00:10:11,539 --> 00:10:14,409 REPLICATION FWAPS THROUGHOUT HER 253 00:10:14,409 --> 00:10:14,642 STUDIES. 254 00:10:14,642 --> 00:10:17,545 SHE IS ABLE TO SEPARATE THIS 255 00:10:17,545 --> 00:10:19,814 THROUGH A BRCA 2C2 TERMINATION 256 00:10:19,814 --> 00:10:21,783 IN MOUSE OR IN HUMAN WITH A 257 00:10:21,783 --> 00:10:22,817 SLIGHTLY DIFFERENT NUMB. 258 00:10:22,817 --> 00:10:25,820 HERE USING THIS SORT OF STANDARD 259 00:10:25,820 --> 00:10:29,190 DNA FIBER ASSAY, HER WILD TYPE 260 00:10:29,190 --> 00:10:31,659 CELLS ARE NOT SHOWING ANY 261 00:10:31,659 --> 00:10:36,898 CUTTING WHEN YOU ADD S1NUCLEAS. 262 00:10:36,898 --> 00:10:41,069 WHEN SHE ADDS THE S1 IN THE BRCA 263 00:10:41,069 --> 00:10:45,607 2 MUTANT, SHE IS SEEING S1 264 00:10:45,607 --> 00:10:47,008 CUTTING SUGGESTING THIS MUTANT 265 00:10:47,008 --> 00:10:48,476 ACROSS A SERIES OF CELL LINES 266 00:10:48,476 --> 00:10:49,944 HAS REPLICATION GAPS. 267 00:10:49,944 --> 00:10:52,013 SO THE ISSUE HERE IS WE ACTUALLY 268 00:10:52,013 --> 00:10:54,649 DIFFER AND WE LOOKED AT THIS AS 269 00:10:54,649 --> 00:10:54,916 WELL. 270 00:10:54,916 --> 00:11:00,155 WE DID NOT PERFORM S1 ANALYSIS. 271 00:11:00,155 --> 00:11:01,856 WE PERFORMED A DIFFERENT STUDY, 272 00:11:01,856 --> 00:11:08,363 BUT WE DIDN'T SEE GAPS IN THIS 273 00:11:08,363 --> 00:11:08,596 MUTANT. 274 00:11:08,596 --> 00:11:14,436 SHE LOOKED HYDROXYREA. 275 00:11:14,436 --> 00:11:18,239 THIS SHOWS DISPLAY RESISTANCE 276 00:11:18,239 --> 00:11:20,608 AND HR PROFICIENCY AND FOR 277 00:11:20,608 --> 00:11:21,976 PROTECTION DEFICIENCY, THE GAP 278 00:11:21,976 --> 00:11:31,019 STILL REMAINS TO BE FULL YVETTED 279 00:11:31,019 --> 00:11:34,355 -- FULLY VETTED. 280 00:11:34,355 --> 00:11:36,891 THIS CAME OUT COVERING THESE 281 00:11:36,891 --> 00:11:38,760 OUTSTANDING ISSUES IN THIS GAP 282 00:11:38,760 --> 00:11:38,960 SPACE. 283 00:11:38,960 --> 00:11:40,395 SO IF YOU ARE INTERESTED. 284 00:11:40,395 --> 00:11:43,031 SHE HAS CONCLUDED THERE IS TOO 285 00:11:43,031 --> 00:11:44,466 MUCH CLOSELY INTERTWINED 286 00:11:44,466 --> 00:11:45,867 FUNCTIONS THAT WAS GOING TO BE 287 00:11:45,867 --> 00:11:47,302 DIFFICULT TO TAKE THESE APART. 288 00:11:47,302 --> 00:11:48,403 SO THIS IS, OF COURSE, WHERE WE 289 00:11:48,403 --> 00:11:52,173 ARE TRYING TO WORK AND SETTLE 290 00:11:52,173 --> 00:11:52,740 SOME ISSUES. 291 00:11:52,740 --> 00:11:56,644 WE ARE TRYING TO DEVELOP A MORE 292 00:11:56,644 --> 00:11:58,680 ROBUST AND UNIVERSALLY 293 00:11:58,680 --> 00:12:00,348 ACCESSIBLE STANDARDIZED GAP 294 00:12:00,348 --> 00:12:01,149 DETECTION ASSAY. 295 00:12:01,149 --> 00:12:03,284 BECAUSE WE SEE UP WITH OF THE 296 00:12:03,284 --> 00:12:06,154 ISSUES THAT PEEL APPLY DIFFERENT 297 00:12:06,154 --> 00:12:09,457 AMOUNTS OF PARP INHIBITOR AND 298 00:12:09,457 --> 00:12:11,593 DIFFERENT AMOUNTS OF TIME AND 299 00:12:11,593 --> 00:12:13,661 HOW THEY PORTRAY THE DATA. 300 00:12:13,661 --> 00:12:16,231 SO IT IS TRICKY TO COMPARE 301 00:12:16,231 --> 00:12:17,098 ACROSS PAPERS. 302 00:12:17,098 --> 00:12:18,533 WE ARE WORKING ON SOMETHING THAT 303 00:12:18,533 --> 00:12:20,635 WE HOPE IS EASIER AND COMING 304 00:12:20,635 --> 00:12:23,104 ONLINE IN THE FUTURE. 305 00:12:23,104 --> 00:12:25,974 WE WILL EXAM THIS AND LOOK AT 306 00:12:25,974 --> 00:12:28,843 OTHER HR MODELS AND SEPARATE 307 00:12:28,843 --> 00:12:31,012 FUNCTION AND GET A SENSE OF 308 00:12:31,012 --> 00:12:32,013 UNDERLYING THERAPY RESPONSE. 309 00:12:32,013 --> 00:12:33,815 WE HOPE TO BETTER DEFINE THE 310 00:12:33,815 --> 00:12:35,350 GAPS, WHERE THEY COME FROM AND 311 00:12:35,350 --> 00:12:37,886 THEIR ULTIMATE FATE AND HOW THEY 312 00:12:37,886 --> 00:12:38,119 KILL. 313 00:12:38,119 --> 00:12:41,856 FOR THIS TALK I WANTED TO START 314 00:12:41,856 --> 00:12:44,626 BY ASKING WHERE DO YOU, SO DO 315 00:12:44,626 --> 00:12:48,196 GAPS EXPLAIN OTHER MODELS LINKED 316 00:12:48,196 --> 00:12:49,564 TO RESTORED HR? 317 00:12:49,564 --> 00:12:51,399 THIS IS A FAMOUS SPACE HERE. 318 00:12:51,399 --> 00:12:55,470 WE HAVE LOTS OF PAPERS HAVE 319 00:12:55,470 --> 00:12:58,006 DESCRIBED THIS ANTI-RESECTION 320 00:12:58,006 --> 00:13:00,608 PATHWAYS PROPOSED TO DRIVE THE 321 00:13:00,608 --> 00:13:03,778 SENSITIVITY TO DRUGS BECAUSE 322 00:13:03,778 --> 00:13:06,981 THEY INTERFERE WITH DNA M 323 00:13:06,981 --> 00:13:07,448 PROCESSING. 324 00:13:07,448 --> 00:13:10,952 AND SO WE WONDER THEN, COULD WE 325 00:13:10,952 --> 00:13:14,622 TEST THE ESSENTIALLITY OF HR FOR 326 00:13:14,622 --> 00:13:19,027 RESISTANCE IN CELLS WHERE BRCA 1 327 00:13:19,027 --> 00:13:26,367 IS MISSING AND TAKE OUT 50BP1. 328 00:13:26,367 --> 00:13:28,536 WHAT IF WE DEPLETE C TIP-IN THIS 329 00:13:28,536 --> 00:13:29,237 SCENARIO. 330 00:13:29,237 --> 00:13:31,773 IF HR IS ESSENTIAL THE DOUBLE 331 00:13:31,773 --> 00:13:33,274 KNOCKOUT CELL SHOULD BECOME 332 00:13:33,274 --> 00:13:33,541 SENSITIVE. 333 00:13:33,541 --> 00:13:37,812 HERE WE DEPLETED C TIP, WE SEE 334 00:13:37,812 --> 00:13:39,914 IN OUR WILD TYPE AND DOUBLE 335 00:13:39,914 --> 00:13:40,848 KNOCKOUT CELLS. 336 00:13:40,848 --> 00:13:43,885 THE ABILITY OF RPA TO LOAD ON TO 337 00:13:43,885 --> 00:13:46,287 AN END IS GREATLY CURTAILED BY 338 00:13:46,287 --> 00:13:49,357 THE LOSS OF C TIP PRESENTING THE 339 00:13:49,357 --> 00:13:52,060 SINGLE STRANDED DNA FOR RP 340 00:13:52,060 --> 00:13:53,061 LOADING. 341 00:13:53,061 --> 00:13:59,033 WE DISRUPTED RECEPTION. 342 00:13:59,033 --> 00:14:02,270 IF YOU LOOK AT THIS, WE DID NOT 343 00:14:02,270 --> 00:14:04,439 SEE A MASSIVE GAIN OR MUCH OF A 344 00:14:04,439 --> 00:14:07,008 CHANGE WITH C TIP DEPLETION, WE 345 00:14:07,008 --> 00:14:09,177 SAW A MILD ENHANCEMENT IN WILD 346 00:14:09,177 --> 00:14:10,445 TYPE CELLS. 347 00:14:10,445 --> 00:14:13,348 WE WENT FURTHER TO SAY WHAT IF 348 00:14:13,348 --> 00:14:17,685 WE DISTRACT RAD 51 WITH SEVERAL 349 00:14:17,685 --> 00:14:18,319 INHIBITORS. 350 00:14:18,319 --> 00:14:23,458 HERE IT WAS STRIKING THE DOUBLE 351 00:14:23,458 --> 00:14:25,526 KNOCKOUT CELLS WERE MORE 352 00:14:25,526 --> 00:14:26,628 RESISTANT THAN THE WILD TYPE 353 00:14:26,628 --> 00:14:27,128 CONTROL. 354 00:14:27,128 --> 00:14:28,262 THIS DOUBLE KNOCKOUT CELL 355 00:14:28,262 --> 00:14:30,898 REGAINED HR IS NOT HIGHLY 356 00:14:30,898 --> 00:14:35,637 DEPENDENT ON THIS HR FOR ITS 357 00:14:35,637 --> 00:14:38,973 BASIC FITNESS OR INHIBITOR 358 00:14:38,973 --> 00:14:39,273 RESISTANCE. 359 00:14:39,273 --> 00:14:42,243 WE CAN SUM THIS IN THE TABLE. 360 00:14:42,243 --> 00:14:44,045 THESE CELLS DID NOT HAVE 361 00:14:44,045 --> 00:14:46,014 PROTECTION AND WE CAN SEE GAPS 362 00:14:46,014 --> 00:14:47,615 WERE STILL SUPPRESSED. 363 00:14:47,615 --> 00:14:51,352 NOW, IF YOU WANT TO TAKE A BRCA 364 00:14:51,352 --> 00:14:55,490 1 DEFICIENT CELL AND MAKE IT 365 00:14:55,490 --> 00:14:57,625 SENSITIVE TO THERAPY, YOU CAN 366 00:14:57,625 --> 00:14:59,694 GET RID OF LIGASE3. 367 00:14:59,694 --> 00:15:01,863 HE WAS SURPRISED TO SEE HR 368 00:15:01,863 --> 00:15:03,331 REMAINED INTACT, BUT HE COULD 369 00:15:03,331 --> 00:15:05,700 SEE GAPS WERE PRESENT AGAIN. 370 00:15:05,700 --> 00:15:08,503 SO, AGAIN, THIS SUGGESTS THAT 371 00:15:08,503 --> 00:15:11,039 GAPS WERE CENTRAL TO THE THERAPY 372 00:15:11,039 --> 00:15:11,406 RESPONSE. 373 00:15:11,406 --> 00:15:12,907 AND WE WENT AHEAD AND JUST 374 00:15:12,907 --> 00:15:14,342 CONFIRMED THAT. 375 00:15:14,342 --> 00:15:17,345 IT IS QUITE STRIKING LIAGE3, 376 00:15:17,345 --> 00:15:20,515 DEPLEGS WITH TWO DIFFERENT SR 377 00:15:20,515 --> 00:15:24,252 RANNE REAGENTS CONFERS HIGH 378 00:15:24,252 --> 00:15:27,955 SENSITIVE TO THESE CELLS PARPI 379 00:15:27,955 --> 00:15:28,222 INHIBITOR. 380 00:15:28,222 --> 00:15:33,094 WHAT IS THE SOURCE OF GAPS AND 381 00:15:33,094 --> 00:15:36,297 HOW DOES BP1 CREATE THESE GAPS? 382 00:15:36,297 --> 00:15:38,466 THOSE ARE TWO QUESTIONS WE ARE 383 00:15:38,466 --> 00:15:39,734 LOOKING AT QUITE STRONGLY. 384 00:15:39,734 --> 00:15:41,602 SO I WILL START WITH THE FIRST 385 00:15:41,602 --> 00:15:42,336 ONE. 386 00:15:42,336 --> 00:15:44,839 WHEN WE LEARNED THIS BEAUTIFUL 387 00:15:44,839 --> 00:15:47,975 WORK THAT PARP HAS A CRITICAL 388 00:15:47,975 --> 00:15:50,511 FUNCTION IN UNPRETESCHED ASSAYS 389 00:15:50,511 --> 00:15:53,414 AND BASICALLY IN A BACKUP PATH 390 00:15:53,414 --> 00:15:56,551 FILLS IN GAPS THAT THE 391 00:15:56,551 --> 00:15:57,652 CANNONICAL PATHWAY COULDN'T TAKE 392 00:15:57,652 --> 00:15:58,486 CARE OF. 393 00:15:58,486 --> 00:16:02,523 YOU HAD TO KILL THIS PAR G 394 00:16:02,523 --> 00:16:04,792 ENZYME WHICH WILL ACTSIVELY 395 00:16:04,792 --> 00:16:05,860 REMOVE THESE. 396 00:16:05,860 --> 00:16:07,795 YOU HAVE TO INHIBIT AND NOW YOU 397 00:16:07,795 --> 00:16:10,364 CAN SEE THIS IN THE REPLICATION 398 00:16:10,364 --> 00:16:12,867 ON A REGULAR S PHASE. 399 00:16:12,867 --> 00:16:15,503 SO WE PONDERED WHETHER BRCA 1 400 00:16:15,503 --> 00:16:18,172 WAS CONTRIBUTING TO LAGGING 401 00:16:18,172 --> 00:16:18,539 STRAND BIOLOGY. 402 00:16:18,539 --> 00:16:22,343 TO ADDRESS THAT WE SIMPLY ASKED 403 00:16:22,343 --> 00:16:23,077 IN ABSENCE OF BRCA 1 WHAT 404 00:16:23,077 --> 00:16:25,513 HAPPENS TO PARP ACTIVITY? 405 00:16:25,513 --> 00:16:26,681 IT WENT UP. 406 00:16:26,681 --> 00:16:28,416 IT WAS NOTHING LIKE GETTING RID 407 00:16:28,416 --> 00:16:32,687 OF PHEN 1, WHERE YOU GET MASSIVE 408 00:16:32,687 --> 00:16:36,858 PARP ACTIVITY, BUT IT WAS HIGHER 409 00:16:36,858 --> 00:16:38,292 IN BRCA PATHWAY. 410 00:16:38,292 --> 00:16:42,330 THERE COULD BE A ROLE FOR BRCA 1 411 00:16:42,330 --> 00:16:50,071 IN BASIC BIOLOGY AND UNPRETERBE 412 00:16:50,071 --> 00:16:52,974 AND PARP. 413 00:16:52,974 --> 00:16:55,009 WE RECENTLY ASKED WHY THOSE WERE 414 00:16:55,009 --> 00:16:58,045 DEFICIENT, NOT SENSITIVE TO PARP 415 00:16:58,045 --> 00:16:58,780 INHIBITORS. 416 00:16:58,780 --> 00:17:00,982 WE UNCOVERED IT IS CRITICAL TO 417 00:17:00,982 --> 00:17:03,851 GET PARP ACTIVE IN THE S PHASE. 418 00:17:03,851 --> 00:17:06,854 WHAT WE FOUND IN A SUMMARY FORM 419 00:17:06,854 --> 00:17:09,757 HERE IN THE ABSENCE OF FENG J, 420 00:17:09,757 --> 00:17:12,927 DANK CASE, PARP WAS GENERALLY 421 00:17:12,927 --> 00:17:15,029 SEQUESTERED ON A G4 STRUCTURE 422 00:17:15,029 --> 00:17:16,798 AND THEREFORE IT WAS BASICALLY 423 00:17:16,798 --> 00:17:19,033 HELD IN INACTIVE POSITION. 424 00:17:19,033 --> 00:17:21,702 SO YOU HAD LOW S PHASE PARP 425 00:17:21,702 --> 00:17:22,336 ACTIVITY. 426 00:17:22,336 --> 00:17:23,504 THIS WAS SIMILAR TO HAVING NO 427 00:17:23,504 --> 00:17:25,773 PARP OR USING THE PARP 428 00:17:25,773 --> 00:17:26,040 INHIBITOR. 429 00:17:26,040 --> 00:17:28,676 ALL OF THESE SITUATIONS WE FOUND 430 00:17:28,676 --> 00:17:32,814 TO BE TOXIC WITH BRCA DEFICIENCY 431 00:17:32,814 --> 00:17:35,683 WE SAW ELEVATED PARP ACTIVITY. 432 00:17:35,683 --> 00:17:38,085 THIS SUGGESTS IT WAS THIS COMMON 433 00:17:38,085 --> 00:17:40,154 LESS OF S PHASE PARP ACTIVITY 434 00:17:40,154 --> 00:17:44,325 THAT WAS DRIVING THE PARP 435 00:17:44,325 --> 00:17:46,694 SYNTHETIC LETHALITY WITH BRCA 436 00:17:46,694 --> 00:17:46,894 LOSS. 437 00:17:46,894 --> 00:17:48,596 I WOULD SUGGEST TRAPPING HAS A 438 00:17:48,596 --> 00:17:51,799 MORE MINOR ROLE BASED ON THESE 439 00:17:51,799 --> 00:17:52,333 FINDINGS. 440 00:17:52,333 --> 00:17:52,700 OKAY. 441 00:17:52,700 --> 00:18:00,041 SO NOW COMING BACK TO HOW DOES 442 00:18:00,041 --> 00:18:01,509 CREATING GAPS IN CELLS? 443 00:18:01,509 --> 00:18:05,446 WE HAD LOOKED AT THE ACTIVATION 444 00:18:05,446 --> 00:18:07,548 OF THIS PARP IN THE BRCA 445 00:18:07,548 --> 00:18:09,584 DEFICIENT CELLS, WHICH WE COULD 446 00:18:09,584 --> 00:18:15,056 SEE WAS RELATED TO 50BP1, IF YOU 447 00:18:15,056 --> 00:18:19,026 GOT RID IT OF IN DOUBLE KNOCKOUT 448 00:18:19,026 --> 00:18:22,230 CELLS, IT WENT DOUNT. 449 00:18:22,230 --> 00:18:25,967 IF WE GOT RID OF THE 50BP1 WE 450 00:18:25,967 --> 00:18:31,038 COULD SEE IT SOMEWHAT REDUCED 451 00:18:31,038 --> 00:18:32,907 AND CRITICAL DOWNSTREAM TARGETS 452 00:18:32,907 --> 00:18:34,642 OF THIS PARP ACTIVITY NOW SEEM 453 00:18:34,642 --> 00:18:40,448 TO COME BACK INTO THE CHROMATIN. 454 00:18:40,448 --> 00:18:44,252 THIS SUGGESTS WHEN YOU HAVE BOTH 455 00:18:44,252 --> 00:18:45,686 PROTEINS MISSING THIS BACKUP 456 00:18:45,686 --> 00:18:49,190 PATHWAY WAS WORKING BETTER IN AN 457 00:18:49,190 --> 00:18:50,091 UNEREGULATED MANNER. 458 00:18:50,091 --> 00:18:53,961 SO THIS FIT WELL WITH WHAT 459 00:18:53,961 --> 00:19:00,968 YONKERS FOUND, LIGASE THREE AND 460 00:19:00,968 --> 00:19:03,804 XRCC1 ARE TARGETS TO KNOCK OUT 461 00:19:03,804 --> 00:19:04,105 WITH. 462 00:19:04,105 --> 00:19:09,410 OTHERS FOUND FEN-1 INHIBITOR WAS 463 00:19:09,410 --> 00:19:11,512 SENSITIZING TO BRCA CELLS. 464 00:19:11,512 --> 00:19:15,750 YOU COULD RESCUE WITH LOSS OF 465 00:19:15,750 --> 00:19:17,952 BP1 AND THE BACKUP PATHWAY WAS 466 00:19:17,952 --> 00:19:18,920 HIGHLY COMPENSATING HERE. 467 00:19:18,920 --> 00:19:25,993 MORE RECENTLY WE LOOKED AT 468 00:19:25,993 --> 00:19:30,765 LIGASE1 AND RESCUE BY LOSS OF 469 00:19:30,765 --> 00:19:33,668 53BP1. 470 00:19:33,668 --> 00:19:39,440 IT WAS SHOWN BY SYLVIA 471 00:19:39,440 --> 00:19:41,142 NORDAMER'S GROUP, THIS MAKES 472 00:19:41,142 --> 00:19:44,512 SENSE TO US THROUGH THIS BACKUP 473 00:19:44,512 --> 00:19:44,745 PATHWAY. 474 00:19:44,745 --> 00:19:48,149 AND I ALSO WANT TO POINT OUT 475 00:19:48,149 --> 00:19:52,753 THERE'S MANY EXAMPLES TO 476 00:19:52,753 --> 00:19:53,988 SYNTHETICALLY VALUE LOSS OF PARP 477 00:19:53,988 --> 00:19:57,224 WHICH ARE NON-RH GENES. 478 00:19:57,224 --> 00:19:59,193 WE DON'T INVOKE DOUBLE STRAND 479 00:19:59,193 --> 00:20:02,129 BREAK WHEN WE WANT TO WHEN WHEN 480 00:20:02,129 --> 00:20:04,565 WE HAVE A BRCA GENE MUTATION. 481 00:20:04,565 --> 00:20:06,968 LAGGING STRAND DEFECTS ARE 482 00:20:06,968 --> 00:20:09,770 SENSITIZED TO PARP INHIBITOR AND 483 00:20:09,770 --> 00:20:11,038 AGAIN, LEADING THE POSSIBILITY 484 00:20:11,038 --> 00:20:12,139 THAT THE DOUBLE STRAND BREAK IS 485 00:20:12,139 --> 00:20:16,110 NOT REALLY REQUIRED HERE. 486 00:20:16,110 --> 00:20:16,310 OKAY. 487 00:20:16,310 --> 00:20:18,112 BUT WHAT IS REQUIRED FOR THIS 488 00:20:18,112 --> 00:20:21,782 MODEL TO HAVE ANY LEGS IS THAT 489 00:20:21,782 --> 00:20:24,919 53BP1 HAS TO GET TO REPLICATION 490 00:20:24,919 --> 00:20:26,821 FORKS AND IN CELLS THERE IS NO 491 00:20:26,821 --> 00:20:30,224 DNA DAMAGE. 492 00:20:30,224 --> 00:20:32,727 THIS GROUP HAD SHOWN THROUGH 493 00:20:32,727 --> 00:20:36,897 PROXIMITY LIGATION ASSAY, THAT 494 00:20:36,897 --> 00:20:39,300 50BP1 WAS RIGHT WITH NASCENT DNA 495 00:20:39,300 --> 00:20:41,068 IN UNCHALLENGING CONDITIONS. 496 00:20:41,068 --> 00:20:43,771 IT WAS RECENTLY SHOWN BY 497 00:20:43,771 --> 00:20:46,207 WAGNER'S GROUP THAT 50BP1 IS ON 498 00:20:46,207 --> 00:20:48,909 THE OKAZAKY FLAP. 499 00:20:48,909 --> 00:20:52,947 IT SITS AS A BINARY BINDING 500 00:20:52,947 --> 00:20:54,782 PROTEIN. 501 00:20:54,782 --> 00:20:56,350 THAT FITS IT INTO THE THE FLAP. 502 00:20:56,350 --> 00:20:58,219 SO WE WANTED TO SEE IF WE COULD 503 00:20:58,219 --> 00:21:01,389 SEE A SIMILAR RELATIONSHIP. 504 00:21:01,389 --> 00:21:05,192 SO WE PERFORMED THESE PROXIMITY 505 00:21:05,192 --> 00:21:06,193 LIGATION ASSAYS AND WE COULD SEE 506 00:21:06,193 --> 00:21:10,398 THAT AS COMPARED TO 50BP1 507 00:21:10,398 --> 00:21:12,033 KNOCKOUT CELLS. 508 00:21:12,033 --> 00:21:14,702 CLEARLY 50BP1 WAS SHOWING UP AT 509 00:21:14,702 --> 00:21:16,804 THE REPLICATION FORK, PRESENT AT 510 00:21:16,804 --> 00:21:19,040 THE FORK AND BEHIND THE FORK WE 511 00:21:19,040 --> 00:21:20,608 COULD SEE THROUGH A 20-MINUTE 512 00:21:20,608 --> 00:21:21,242 CHASE PERIOD. 513 00:21:21,242 --> 00:21:23,110 WHAT IS INTERESTING IN THE 514 00:21:23,110 --> 00:21:26,614 ABSENCE OF BRCA 1 THIS PRESENCE 515 00:21:26,614 --> 00:21:27,782 WAS ENHANCED DRAMATICALLY, AS 516 00:21:27,782 --> 00:21:31,052 YOU CAN SEE HERE, AND THOSE 517 00:21:31,052 --> 00:21:36,590 IMAGES, AND QUANTIFIED AND THE 518 00:21:36,590 --> 00:21:37,658 GREATEST ACCUMULATION WAS COMING 519 00:21:37,658 --> 00:21:40,594 BEHIND THE REPLICATION FORK 520 00:21:40,594 --> 00:21:43,264 POSSIBLY BECAUSE 50BP1 WAS 521 00:21:43,264 --> 00:21:46,867 HANGING OUT ON LAGGING STRAND 522 00:21:46,867 --> 00:21:49,203 FLAPS, NOT PROPERLY REMOVED. 523 00:21:49,203 --> 00:21:55,042 BRCA 1 RESTRICTS 50BP1, IT COULD 524 00:21:55,042 --> 00:21:58,579 BE RESTRICTING THROUGH ITS 525 00:21:58,579 --> 00:22:01,482 ABILITY TO PROMOTE PROCESS AND 526 00:22:01,482 --> 00:22:02,950 REPLICATION GAP SUPPRESSION. 527 00:22:02,950 --> 00:22:09,523 IT IS CONFOUNDED BECAUSE WHILE 528 00:22:09,523 --> 00:22:11,025 BPR1 LOSS WILL -- STILL LEADING 529 00:22:11,025 --> 00:22:13,060 US TO NOT REALLY KNOW WHICH ONE 530 00:22:13,060 --> 00:22:15,463 IS DRIVING THE RESISTANCE, EVEN 531 00:22:15,463 --> 00:22:17,331 THOUGH WE CAN TARGET THIS AND 532 00:22:17,331 --> 00:22:20,468 STILL SEE RESISTANCE. 533 00:22:20,468 --> 00:22:21,836 ULTIMATELY, IT IS NOT GOING TO 534 00:22:21,836 --> 00:22:23,037 BE FULLY CLEAR. 535 00:22:23,037 --> 00:22:25,072 WHAT WE HAVE BEEN TRYING TO DO 536 00:22:25,072 --> 00:22:28,409 IS FIND THINGS LIKE 53BP1 537 00:22:28,409 --> 00:22:32,746 ELEVATED IN A BRCA DEFICIENT 538 00:22:32,746 --> 00:22:36,450 SELL BUT PARTICIPATE IN NHR OR 539 00:22:36,450 --> 00:22:37,585 REPLICATION GAP SUPPRESSION BUT 540 00:22:37,585 --> 00:22:38,052 NOT BOTH. 541 00:22:38,052 --> 00:22:41,288 SO CAN WE FIND THAT? 542 00:22:41,288 --> 00:22:51,565 53BP1 IS ELEVATED IN A BRCA 543 00:22:51,565 --> 00:22:52,032 DEFICIENT CELL. 544 00:22:52,032 --> 00:22:56,737 WE ARE LOOKING FOR ANOTHER 545 00:22:56,737 --> 00:23:00,508 PROTEIN THAT IS JUST LIKE 53BP1 546 00:23:00,508 --> 00:23:04,578 THAT IS ENRICHED AND DIRECTING 547 00:23:04,578 --> 00:23:06,814 THERAPY RESPONSE SIMILARLY. 548 00:23:06,814 --> 00:23:08,582 HOW TO DO THAT? 549 00:23:08,582 --> 00:23:13,387 HERE WE SET UP A COMPARISON 550 00:23:13,387 --> 00:23:19,026 ACROSS ISOGENIC CELLS. 551 00:23:19,026 --> 00:23:20,194 WE SUGGESTED JUST GRAP 552 00:23:20,194 --> 00:23:23,564 EVERYTHING AND SEE WHAT WE CAN 553 00:23:23,564 --> 00:23:23,998 FIND. 554 00:23:23,998 --> 00:23:28,102 THIS WORK WAS RUN BY SYLVIA 555 00:23:28,102 --> 00:23:31,038 AMIN, A GREAT GRADUATE STUDENT 556 00:23:31,038 --> 00:23:33,240 ABOUT TO DEFEND HER PH.D. AND 557 00:23:33,240 --> 00:23:34,642 THEN -- WHO HAS BEEN WORKING 558 00:23:34,642 --> 00:23:36,510 WITH ME OVER 20 YEARS. 559 00:23:36,510 --> 00:23:39,780 WHAT DID THEY FIND? 560 00:23:39,780 --> 00:23:44,185 THEY FOUND PARP AND 53BP1 WERE 561 00:23:44,185 --> 00:23:46,020 HIGHER IN THE DEFICIENT CELLS, 562 00:23:46,020 --> 00:23:48,322 WHICH WE WOULD EXPECT. 563 00:23:48,322 --> 00:23:52,393 WE FOUND HL THE F, THIS PROTHAT 564 00:23:52,393 --> 00:23:55,996 IS INVOLVED ARE REVERSAL AND 565 00:23:55,996 --> 00:23:58,032 SHOWN TO DRAW RESPONSE IN BRCA 566 00:23:58,032 --> 00:23:59,800 DEFICIENT CELLS. 567 00:23:59,800 --> 00:24:02,436 DEPLETION OF HL THE F REDUCED 568 00:24:02,436 --> 00:24:04,371 FITNESS SOMEWHAT, BUT THE CELLS 569 00:24:04,371 --> 00:24:05,539 THAT SURVIVED WERE MORE 570 00:24:05,539 --> 00:24:07,908 RESISTANT TO A PARP INHIBITOR. 571 00:24:07,908 --> 00:24:09,710 AND SO THAT WAS SATISFYING THAT 572 00:24:09,710 --> 00:24:13,581 WE WERE FINDING THINGS IN THE 573 00:24:13,581 --> 00:24:15,382 CHROMATIN THAT WERE CONTRIBUTING 574 00:24:15,382 --> 00:24:17,551 TO BRCA RESPONSE IN A DEFICIENT 575 00:24:17,551 --> 00:24:17,851 BACKGROUND. 576 00:24:17,851 --> 00:24:21,855 WE ALSO FOUND THIS ENZYME HERE, 577 00:24:21,855 --> 00:24:23,757 EEPD1, WHICH YOU HAD ALSO BEEN 578 00:24:23,757 --> 00:24:27,428 FOUND BY YONKERS GROUP AS A GENE 579 00:24:27,428 --> 00:24:30,931 WHOSE DEPLETION WOULD CONFER 580 00:24:30,931 --> 00:24:32,066 RESTORED SENSETIZATION IN PARP 581 00:24:32,066 --> 00:24:33,267 INHIBITOR IN DOUBLE KNOCKOUT 582 00:24:33,267 --> 00:24:35,102 CELLS AND, AGAIN, WE COULD 583 00:24:35,102 --> 00:24:36,237 CONFIRM THAT AS WELL. 584 00:24:36,237 --> 00:24:38,772 WE THOUGHT THIS PROCEDURE HAD 585 00:24:38,772 --> 00:24:40,341 THE MOBILITY TO FIND INTERESTING 586 00:24:40,341 --> 00:24:43,010 FACTORS THAT WERE DRIVING 587 00:24:43,010 --> 00:24:44,545 THERAPY RESPONSE. 588 00:24:44,545 --> 00:24:46,480 LIKE 53BP1. 589 00:24:46,480 --> 00:24:47,815 NOW, TURNING TO GOING A LITTLE 590 00:24:47,815 --> 00:24:51,385 FURTHER, WE MOVED ON TO WORK 591 00:24:51,385 --> 00:24:55,022 WITH JUST NOT DAN DEROACHER 592 00:24:55,022 --> 00:24:57,725 CELLS, BUT THESE EMBRYONIC FIBER 593 00:24:57,725 --> 00:25:01,829 CELLS WHICH HAS SIMILAR 594 00:25:01,829 --> 00:25:02,096 GENOMICS. 595 00:25:02,096 --> 00:25:06,533 WHAT WAS ENRICHED ACROSS CELL 596 00:25:06,533 --> 00:25:08,369 LINES IN THE BRCA BACKGROUND 597 00:25:08,369 --> 00:25:10,604 DEFICIENT OF 53BPR1. 598 00:25:10,604 --> 00:25:12,840 WE SAW THE ONE THAT STOOD OUT 599 00:25:12,840 --> 00:25:14,775 THE MOST WAS MDC1. 600 00:25:14,775 --> 00:25:18,612 THIS HAS BEEN RECENTLY SHOWN BY 601 00:25:18,612 --> 00:25:21,282 ANOTHER GROUP TO DIRECT THERAPY 602 00:25:21,282 --> 00:25:23,017 RESPONSE IN BRCA DEFICIENT 603 00:25:23,017 --> 00:25:23,350 CELLS. 604 00:25:23,350 --> 00:25:24,752 SO THAT MADE IT QUITE 605 00:25:24,752 --> 00:25:26,787 COMPELLING. 606 00:25:26,787 --> 00:25:28,889 WE COULD SEE THE LEVELS DID NOT 607 00:25:28,889 --> 00:25:32,259 CHANGE IN THE OVERALL EXTRACTS 608 00:25:32,259 --> 00:25:36,597 IN THE CELLS UNLESS IT WAS 609 00:25:36,597 --> 00:25:36,930 LEADED. 610 00:25:36,930 --> 00:25:39,033 WHEN WE DID WESTERN BLOT 611 00:25:39,033 --> 00:25:41,201 ANALYSIS, BRCA WAS UP. 612 00:25:41,201 --> 00:25:44,705 WE COULD SEE THAT THROUGH A 613 00:25:44,705 --> 00:25:46,573 CHOMATIN EXPERIMENTS WHERE IT 614 00:25:46,573 --> 00:25:49,376 WAS HIGHER ONLY WITH A 53BP1 WAS 615 00:25:49,376 --> 00:25:51,345 PRESENT IN THESE MAPS AS WELL 616 00:25:51,345 --> 00:25:54,148 AND IN ANOTHER CELL LINE WE HAD 617 00:25:54,148 --> 00:25:55,849 FROM KNEEL JOHNSON'S GROUP WHERE 618 00:25:55,849 --> 00:26:01,055 THE RESISTANCES SHOW LOW 53BP1 619 00:26:01,055 --> 00:26:04,191 LEVELS GO DOWN. 620 00:26:04,191 --> 00:26:09,496 SIMILAR TO ANOTHER GROUP, WE 621 00:26:09,496 --> 00:26:13,734 DEPREET, AND OUR FITNESS IS NO 622 00:26:13,734 --> 00:26:13,967 REDUCED. 623 00:26:13,967 --> 00:26:15,402 BRCA DEFICIENT CELLS ARE NOT 624 00:26:15,402 --> 00:26:17,171 ONLY SENSITIVE TO PARP 625 00:26:17,171 --> 00:26:19,807 INHIBITOR, BUT SENSITIVE TO WHEN 626 00:26:19,807 --> 00:26:22,109 YOU INHIBIT TRANSLESION 627 00:26:22,109 --> 00:26:22,810 SYNTHESIS. 628 00:26:22,810 --> 00:26:24,978 WE WANTED TO CONFER RESISTANCE 629 00:26:24,978 --> 00:26:28,315 TO THAT DRUG AS WELL. 630 00:26:28,315 --> 00:26:35,189 SO QUICK STRIKINGLY WE COULD 631 00:26:35,189 --> 00:26:36,690 RECAPITULATE THAT THE BRCA CELLS 632 00:26:36,690 --> 00:26:38,792 ARE SENSITIVE, BUT THE DOUBLE 633 00:26:38,792 --> 00:26:41,695 KNOCKOUT CELLS ARE HIGHLY MORE 634 00:26:41,695 --> 00:26:43,030 RESISTANT THAN THE WILD TYPE AND 635 00:26:43,030 --> 00:26:47,634 THE SINGLE KNOCK OUT. 636 00:26:47,634 --> 00:26:50,671 MBC ONE DEFICIENT -- HL T F 637 00:26:50,671 --> 00:26:52,906 DEFICIENCY DID NOT PROVIDE ANY 638 00:26:52,906 --> 00:26:54,007 RESISTANCE HERE. 639 00:26:54,007 --> 00:26:59,346 SO TOGETHER WE FOUND MDC1 640 00:26:59,346 --> 00:27:01,915 SATISFIED REQUIREMENTS, IT 641 00:27:01,915 --> 00:27:04,585 DIRECTS RESPONSE SIMILAR TO 50 642 00:27:04,585 --> 00:27:06,920 3W BP1. 643 00:27:06,920 --> 00:27:10,023 WILL MDC1 SEPARATE FOR 644 00:27:10,023 --> 00:27:10,824 REPLICATION GAP SUH PRESENTATION 645 00:27:10,824 --> 00:27:11,458 VERSUS HR. 646 00:27:11,458 --> 00:27:13,494 TO BEGIN TO ADDRESS THAT WE 647 00:27:13,494 --> 00:27:15,262 STARTED WITH LOOKING AT 648 00:27:15,262 --> 00:27:17,398 REPLICATION GAPS BY THE DNA 649 00:27:17,398 --> 00:27:18,732 FIBER ASSAY. 650 00:27:18,732 --> 00:27:20,601 WE COULD SEE THE BRCA 1 KNOCKOUT 651 00:27:20,601 --> 00:27:27,074 CELLS ARE CUTTING WITH THE 652 00:27:27,074 --> 00:27:28,942 S1NUCLEASE, WE DEPLETE IT, THAT 653 00:27:28,942 --> 00:27:31,145 CUTTING WAS SUBSTANTIALLY 654 00:27:31,145 --> 00:27:31,445 REDUCED. 655 00:27:31,445 --> 00:27:33,147 MOREOVER, WHEN WE WORKED WITH 656 00:27:33,147 --> 00:27:35,048 OUR DOUBLE KNOCKOUT CELLS WHICH 657 00:27:35,048 --> 00:27:39,453 IS 53BP1 DELETED WE COULD 658 00:27:39,453 --> 00:27:41,255 RECAPITULATE WHAT WE PREVIOUSLY 659 00:27:41,255 --> 00:27:41,789 SHONED. 660 00:27:41,789 --> 00:27:44,057 A DOUBLE KNOCK OUT WITH MDC1 WE 661 00:27:44,057 --> 00:27:46,660 COULD FIND A SIMILAR FINDING AS 662 00:27:46,660 --> 00:27:46,960 WELL. 663 00:27:46,960 --> 00:27:50,431 IT LOOKED LIKE MDC1 WAS DRIVING 664 00:27:50,431 --> 00:27:52,032 GAP IN BRCA DEFICIENT CELLS. 665 00:27:52,032 --> 00:27:54,735 WE FURTHER VALIDATED THIS 666 00:27:54,735 --> 00:27:59,573 LOOKING AT RPA LEVELS IN 667 00:27:59,573 --> 00:28:07,781 PARALATION CONTRIBUTED BY 53BP1. 668 00:28:07,781 --> 00:28:11,018 WE COULD SEE IT WAS GOING DOWN 669 00:28:11,018 --> 00:28:13,620 WE DEPLETION AND INHIBIT WITH 670 00:28:13,620 --> 00:28:14,688 THE -- ENZYME. 671 00:28:14,688 --> 00:28:17,791 IF YOU LOOK AT THE SINGLE STRAND 672 00:28:17,791 --> 00:28:21,528 DNA IT WAS REDUCED BY MDC1 673 00:28:21,528 --> 00:28:24,798 CONSISTENT MDC1 IS SUPPRESSING 674 00:28:24,798 --> 00:28:27,167 GAPS IN BRCA DEFICIENT CELLS. 675 00:28:27,167 --> 00:28:31,004 THEN JUST LIKE 53BP1 LOSS THIS 676 00:28:31,004 --> 00:28:34,942 PARP INHIBITOR RESISTANCE WAS 677 00:28:34,942 --> 00:28:40,214 DEPENDENT ON LIGASE3 AS WELL. 678 00:28:40,214 --> 00:28:47,221 IS MDC1 AT THE REPA ZONE. 679 00:28:47,221 --> 00:28:53,060 WE PERFORMED LIGATION ANALYSIS. 680 00:28:53,060 --> 00:28:55,896 53BP1 IN THE WILD CELLS AND SO 681 00:28:55,896 --> 00:28:57,164 THE MDC1. 682 00:28:57,164 --> 00:28:58,565 WE SEE IN THE CHASE PERIOD. 683 00:28:58,565 --> 00:29:05,239 NOW WHEN YOU LOOK AT THE B BRCA 684 00:29:05,239 --> 00:29:07,708 1 CELLS, IT IS HIGHER SORT OF 685 00:29:07,708 --> 00:29:09,776 MIRRORING THAT ACCUMULATION. 686 00:29:09,776 --> 00:29:13,213 SO WE THEN BEGAN TO THINK, YOU 687 00:29:13,213 --> 00:29:17,684 KNOW, HOW IS IT THIS REPIZOME 688 00:29:17,684 --> 00:29:22,389 ENRICHMENT COULD PLAY OUT TO 689 00:29:22,389 --> 00:29:23,590 CHROMATIN ENRICHMENT? 690 00:29:23,590 --> 00:29:25,559 HOW ARE BOTH PROTEINS ENRICHED 691 00:29:25,559 --> 00:29:28,061 IN THE CHROMATIN. 692 00:29:28,061 --> 00:29:30,564 WE THOUGHT ABOUT THIS PHENOMENON 693 00:29:30,564 --> 00:29:34,401 FROM SEVERAL YEARS AGO 53BP1 694 00:29:34,401 --> 00:29:35,569 NUCLEAR BODIES. 695 00:29:35,569 --> 00:29:38,739 THESE WERE IDENTIFIED TO BE 696 00:29:38,739 --> 00:29:43,010 PRESENCE IN UNPERTURBED CELLS 697 00:29:43,010 --> 00:29:46,213 AND PHENO COLON TREATMENT. 698 00:29:46,213 --> 00:29:50,784 THESE NUCLEAR BODIES INCLUDED 699 00:29:50,784 --> 00:29:55,022 BRCA 1 AND DEPEND ON -- IS BRCA 700 00:29:55,022 --> 00:30:00,394 1 DEFICIENCY SIMILAR TO TREATING 701 00:30:00,394 --> 00:30:02,229 CELLS WITH -- WILL IT CONTINUE 702 00:30:02,229 --> 00:30:05,666 THROUGHOUT THE CELL CYCLE IN THE 703 00:30:05,666 --> 00:30:09,069 CHROMAIN AND DEPEND ON H2AX. 704 00:30:09,069 --> 00:30:10,938 THOSE ARE OUR QUESTIONS. 705 00:30:10,938 --> 00:30:19,813 WE BEGAN TREATING CELLS WITH THE 706 00:30:19,813 --> 00:30:23,250 APHIDICOLIN, MDC1 WAS ENRICHED. 707 00:30:23,250 --> 00:30:27,087 AND COMPARABLE WITH 53BP1 CELLS. 708 00:30:27,087 --> 00:30:30,657 IT IS ENRICHED IN THE CHROMATIN 709 00:30:30,657 --> 00:30:31,558 THE BRCA DEFICIENT CELL. 710 00:30:31,558 --> 00:30:33,427 IF YOU WANT TO KNOW WHETHER THAT 711 00:30:33,427 --> 00:30:36,563 IS THROUGHOUT THE CELL CYCLE, WE 712 00:30:36,563 --> 00:30:39,266 PERFORMED THIS CYTOMETRY THAT 713 00:30:39,266 --> 00:30:42,135 ALLOWS US TO SEE IF WE EXTRACT 714 00:30:42,135 --> 00:30:44,771 OUR CELLS THAT WE'RE JUST 715 00:30:44,771 --> 00:30:50,777 LOOKING AT THE CHROMETIN, THE 716 00:30:50,777 --> 00:30:53,080 MDC1 ENRICHMENT IS NOT IN A 717 00:30:53,080 --> 00:30:54,381 PARTICULAR CELL CYCLE PHASE. 718 00:30:54,381 --> 00:30:57,517 IT DOES SEEM TO GO THROUGH THE 719 00:30:57,517 --> 00:30:59,353 CELL CYCLE. 720 00:30:59,353 --> 00:31:03,991 WHAT ABOUT H2AX, IS THAT DEPEND 721 00:31:03,991 --> 00:31:06,460 ON H2AX? 722 00:31:06,460 --> 00:31:11,031 WE DEPLETED IT AND LOOKED AT THE 723 00:31:11,031 --> 00:31:13,100 MDC1 ACCUMULATION AND SEE IT WAS 724 00:31:13,100 --> 00:31:14,167 CLEARLY REDUCED. 725 00:31:14,167 --> 00:31:22,109 IF YOU LOOK AT 53BP1 OR MDC1 OR 726 00:31:22,109 --> 00:31:23,744 A2HX DEPLETED AND IT IS REPORTED 727 00:31:23,744 --> 00:31:25,579 IN THE NUCLEAR BODIES. 728 00:31:25,579 --> 00:31:26,947 IF YOU LOOK BY WESTERN BLOT IT 729 00:31:26,947 --> 00:31:28,148 IS QUITE CLEAR. 730 00:31:28,148 --> 00:31:31,018 THE DEPLETION IS REDUCING MDC1 731 00:31:31,018 --> 00:31:35,322 AND REDUCING 50BP1CHROMATIN 732 00:31:35,322 --> 00:31:36,289 ASSOCIATION. 733 00:31:36,289 --> 00:31:42,663 AND SO WE NOW ASK IS H2AX IN THE 734 00:31:42,663 --> 00:31:44,264 REPLIZOME? 735 00:31:44,264 --> 00:31:45,832 THIS IS HAPPENING AT AND BEHIND 736 00:31:45,832 --> 00:31:47,467 THE FORK CELL. 737 00:31:47,467 --> 00:31:49,970 IN THE BRCA DEFICIENT CELL IT IS 738 00:31:49,970 --> 00:31:51,038 ACCUMULATING BEHIND THE 739 00:31:51,038 --> 00:31:53,740 REPLICATION FORK. 740 00:31:53,740 --> 00:31:59,846 MDC1, 53BP1, H2AX ARE ENHANCED 741 00:31:59,846 --> 00:32:01,748 BEHIND THE FORK WITH BRCA IS 742 00:32:01,748 --> 00:32:01,982 MISSING. 743 00:32:01,982 --> 00:32:04,418 IF YOU LOOK AT THE RESISTANCE OF 744 00:32:04,418 --> 00:32:07,020 PARP INHIBITOR, WE SEE IT AS 745 00:32:07,020 --> 00:32:10,457 WELL AS TELOS INHIBITOR. 746 00:32:10,457 --> 00:32:14,795 I WILL POINT OUT THIS WAS 747 00:32:14,795 --> 00:32:16,963 RECENTLY PUBLISHED. 748 00:32:16,963 --> 00:32:26,073 SO FINALLY, IT COMES TO MDC1 749 00:32:26,073 --> 00:32:32,779 RESTORE HOMOLGA CONFIRMATION? 750 00:32:32,779 --> 00:32:36,550 WE WANT TO SEE SOMETHING 751 00:32:36,550 --> 00:32:36,883 DIFFERENTIATE. 752 00:32:36,883 --> 00:32:40,420 WE PERFORM THE STANDARD 753 00:32:40,420 --> 00:32:42,456 EXPERIMENT AND ADD -- YOU CAN 754 00:32:42,456 --> 00:32:49,362 SEE RAD51 FORMS, SHOWING THE 755 00:32:49,362 --> 00:32:51,865 SITES OF DNA DAMAGE NOT IN BRCA 756 00:32:51,865 --> 00:32:55,035 1 DEFICIENT CELLS, THAT HAS BEEN 757 00:32:55,035 --> 00:32:58,538 KNOWN SINCE THE MID 90s. 758 00:32:58,538 --> 00:33:02,342 THIS IS COMING BACK AS WE 759 00:33:02,342 --> 00:33:03,243 LEARNED QUITE CLEARLY FROM 760 00:33:03,243 --> 00:33:05,979 SEVERAL GROUPS WHEN YOU GET RID 761 00:33:05,979 --> 00:33:10,684 OF 53BP1 AND WE CAN SEE THE RAD 762 00:33:10,684 --> 00:33:13,153 FICATION IN THE RAD 51FOCI. 763 00:33:13,153 --> 00:33:15,455 IF YOU LOOK AT THE DEPLETION AND 764 00:33:15,455 --> 00:33:20,427 SCORE THE SAME RAD 51FOCI, WE DO 765 00:33:20,427 --> 00:33:27,000 NOT SEE RESTORATION OF THE RAD 766 00:33:27,000 --> 00:33:27,334 51NOCI. 767 00:33:27,334 --> 00:33:29,202 WE THINK THIS IS ULTIMATELY THE 768 00:33:29,202 --> 00:33:32,372 CORRECT OUTCOME HERE AND SO THIS 769 00:33:32,372 --> 00:33:35,876 THEN LEAVES THE POSSIBILITY THAT 770 00:33:35,876 --> 00:33:39,913 WE HAVE BRCA 1 AND MDC1 771 00:33:39,913 --> 00:33:42,516 DEFICIENT CELLS RESTORING GAP 772 00:33:42,516 --> 00:33:44,251 SUH PRESENTATION AND RESISTANCE 773 00:33:44,251 --> 00:33:45,952 AND WE CAN CONTINUE SO FAR 774 00:33:45,952 --> 00:33:47,754 WITHOUT BREAKING OUR MODEL, 775 00:33:47,754 --> 00:33:50,090 AGAIN, STILL TO BE DETERMINED 776 00:33:50,090 --> 00:33:51,992 WHAT IS GOING ON WITH THIS 777 00:33:51,992 --> 00:33:53,493 MUTANT HERE AS TO WHETHER 778 00:33:53,493 --> 00:34:00,100 THERE'S SOMETHING MORE TO TAKE 779 00:34:00,100 --> 00:34:03,136 APART HERE. 780 00:34:03,136 --> 00:34:07,741 THE INTERESTING TAKE HOME WORK 781 00:34:07,741 --> 00:34:10,877 IS RAD 51 DON'T RESIDE WITH -- 782 00:34:10,877 --> 00:34:15,415 WE DO NOT SEE RAD 51 IS FOCI AND 783 00:34:15,415 --> 00:34:17,918 WE DON'T SEE IN OTHER RESISTANT 784 00:34:17,918 --> 00:34:19,019 CELL LINES. 785 00:34:19,019 --> 00:34:22,689 RAD 51 FOCI IS BEING USED IN THE 786 00:34:22,689 --> 00:34:22,923 CLINIC. 787 00:34:22,923 --> 00:34:26,560 IT IS OBVIOUSLY, ONLY A PARTIAL 788 00:34:26,560 --> 00:34:27,561 BIOMARKER FOR WHAT RESPONSE WILL 789 00:34:27,561 --> 00:34:28,562 LOOK LIKE. 790 00:34:28,562 --> 00:34:31,832 AND SO WHAT WE HAVE TURNED TO 791 00:34:31,832 --> 00:34:32,465 THEN, OF COURSE, IS, YOU KNOW, 792 00:34:32,465 --> 00:34:35,435 THE IDEA THAT FOR ONE, A 793 00:34:35,435 --> 00:34:39,806 SNAPSHOT OF THE TUMOR YHROMATIN 794 00:34:39,806 --> 00:34:42,175 IS CLINICALLY INFORMATIVE. 795 00:34:42,175 --> 00:34:45,312 WE GRAB EVERYTHING IN THE 796 00:34:45,312 --> 00:34:46,813 CHROMATIN AND YOU ARE NOT ADDING 797 00:34:46,813 --> 00:34:48,448 ANY DNA DAMAGE. 798 00:34:48,448 --> 00:34:49,850 IT SUPPORTS THE IDEA THAT THERE 799 00:34:49,850 --> 00:34:52,519 IS ALREADY AN INHERIT GAP OR 800 00:34:52,519 --> 00:34:54,454 OTHERWISE OTHER PROBLEM IN THE 801 00:34:54,454 --> 00:34:55,889 BRCA DEFICIENT CELL FOR WHICH 802 00:34:55,889 --> 00:34:57,157 PROTEINS ARE BEGINNING TO 803 00:34:57,157 --> 00:34:59,025 ACCUMULATE AND TRYING TO DEAL 804 00:34:59,025 --> 00:35:01,728 WITH. 805 00:35:01,728 --> 00:35:03,864 MOREOVER, THE BIOMARKERS OF 806 00:35:03,864 --> 00:35:05,966 RESPONSE COULD MAYBE BE MORE 807 00:35:05,966 --> 00:35:08,068 LIKE THINGS THAT THE TUMOR HAS 808 00:35:08,068 --> 00:35:09,502 HIGH GAPS. 809 00:35:09,502 --> 00:35:11,371 WE CAN DEFINE GAP BIOMARKERS OF 810 00:35:11,371 --> 00:35:13,740 THE DNA OR THAT THE PROTEINS 811 00:35:13,740 --> 00:35:18,845 LIKE NBC1, GUAM-H1, WE THOUGHT 812 00:35:18,845 --> 00:35:21,414 OF DOUBLE STRAND BREAK REPAIR 813 00:35:21,414 --> 00:35:21,915 PROT 814 00:35:21,915 --> 00:35:23,550 PROTEINS, MAYBE THESE ARE MORE 815 00:35:23,550 --> 00:35:25,485 RELATED TO GAP INDUCTION. 816 00:35:25,485 --> 00:35:27,387 BECAUSE AS WE FIND, THEY 817 00:35:27,387 --> 00:35:29,656 ACTUALLY CAUSE THE GAPS IN THE 818 00:35:29,656 --> 00:35:31,224 BRCA DEFICIENT CELL AND PERSIST, 819 00:35:31,224 --> 00:35:34,794 WE THINK, IN THESE GAPS. 820 00:35:34,794 --> 00:35:36,263 MOREOVER, CHEMOTHERAPY WILL WORK 821 00:35:36,263 --> 00:35:37,697 IF WE ARE GOING TO MAXIMIZE GAPS 822 00:35:37,697 --> 00:35:45,672 WITH A PARP INHIBITOR, AN ATR 823 00:35:45,672 --> 00:35:45,939 INHIBITOR. 824 00:35:45,939 --> 00:35:48,441 WE NEED TO SUPPRESS WAYS CANCER 825 00:35:48,441 --> 00:35:52,646 ECELLS OVERCOME GAPS, EITHER BY 826 00:35:52,646 --> 00:35:54,047 EXPRESSING MORE RPA THAT 827 00:35:54,047 --> 00:35:57,484 PROTECTING THE GAP OR INHIBIT 828 00:35:57,484 --> 00:35:59,019 TLS USEFUL FOR PREVENTING GAPS 829 00:35:59,019 --> 00:36:01,821 IN THE FIRST PLACE AT THE 830 00:36:01,821 --> 00:36:06,126 REPLICATION FORK OR FILL IN GAPS 831 00:36:06,126 --> 00:36:09,162 POST REPUTABLY. 832 00:36:09,162 --> 00:36:12,532 I WANT TO THANK MY TEAM. 833 00:36:12,532 --> 00:36:14,034 MIN PENG HAS SPEARHEADED THIS 834 00:36:14,034 --> 00:36:16,536 WORK AND WORKED CLOSELY WITH 835 00:36:16,536 --> 00:36:17,871 SYLVIA ON THIS PROJECT. 836 00:36:17,871 --> 00:36:24,411 WE HAVE A GREAT GROUP 837 00:36:24,411 --> 00:36:33,286 CONTRIBUTING AS WELL. 838 00:36:33,286 --> 00:36:37,123 I NOW KNOW WE ARE AT DANA FARBER 839 00:36:37,123 --> 00:36:38,525 AS A POST DOC. 840 00:36:38,525 --> 00:36:40,160 I WANT TO TURN ALSO TO A LITTLE 841 00:36:40,160 --> 00:36:47,400 PLUG TO A MEETING THAT WE'RE 842 00:36:47,400 --> 00:36:50,870 GOING TO HAVE IF YOU ARE 843 00:36:50,870 --> 00:36:52,939 INTERESTED IN THIS AREA AND YOU 844 00:36:52,939 --> 00:36:54,374 HAVE A STORY TO PRESENT OR JUST 845 00:36:54,374 --> 00:36:56,309 WANT TO COME TO LISTEN, WE HAVE 846 00:36:56,309 --> 00:36:57,377 A GREAT LINEUP. 847 00:36:57,377 --> 00:37:00,213 SO PLEASE CHECK OUT THAT. 848 00:37:00,213 --> 00:37:04,150 AND CONSIDER THAT IN YOUR 849 00:37:04,150 --> 00:37:04,384 FUTURE. 850 00:37:04,384 --> 00:37:07,821 ALSO AS MENTIONED EARLIER, BY 851 00:37:07,821 --> 00:37:11,024 KAREN, WE STARTED A GENOME 852 00:37:11,024 --> 00:37:11,858 INTEGRITY PROGRAM UMASS AND 853 00:37:11,858 --> 00:37:13,126 INITIATE HIRING THIS FALL. 854 00:37:13,126 --> 00:37:14,294 SO PLEASE CONSIDER THAT IF YOU 855 00:37:14,294 --> 00:37:16,363 ARE LOOKING FOR A JOB IN THE 856 00:37:16,363 --> 00:37:16,863 NEAR FUTURE. 857 00:37:16,863 --> 00:37:19,099 AND SO FINALLY, I JUST WANT TO 858 00:37:19,099 --> 00:37:20,200 LEAVE, YOU KNOW, WITH THIS SORT 859 00:37:20,200 --> 00:37:23,103 OF IDEA THAT IF WE HAD COME TO 860 00:37:23,103 --> 00:37:25,271 THE STUDY OF THE BRCA PROTEINS 861 00:37:25,271 --> 00:37:28,575 AT THIS TIME, WE MIGHT HAVE SORT 862 00:37:28,575 --> 00:37:30,643 OF STARTED WITH THINKING, OH, 863 00:37:30,643 --> 00:37:32,846 THESE PROTEINS THEY REPAIR GAPS 864 00:37:32,846 --> 00:37:35,482 AND THEY PREVENT GAPS AND AGAIN, 865 00:37:35,482 --> 00:37:37,851 IS THIS THE FUNDAMENTAL ROLE FOR 866 00:37:37,851 --> 00:37:41,087 THESE PROTEINS POTENTIALLY IN 867 00:37:41,087 --> 00:37:43,957 THE REPLICATION FOR AN UNPER 868 00:37:43,957 --> 00:37:47,660 UNPERTURBED CONDITIONS. 869 00:37:47,660 --> 00:37:49,396 OTHER FUNCTIONS WERE 870 00:37:49,396 --> 00:37:49,696 HIGHLIGHTED. 871 00:37:49,696 --> 00:37:54,968 THEY ARE EASIER TO DESCRIBE IN 872 00:37:54,968 --> 00:37:55,468 PHENOTYPES THAT REQUIRE 873 00:37:55,468 --> 00:37:57,137 DIFFERENT ASSAYS THAT ARE JUST 874 00:37:57,137 --> 00:37:58,471 COMING ONLINE NOW. 875 00:37:58,471 --> 00:38:00,273 I GUESS I WILL STOP THERE AND 876 00:38:00,273 --> 00:38:02,642 JUST WANT TO POINT OUT MY 877 00:38:02,642 --> 00:38:03,943 ATTEMPTS TO TRY TO GET THE FIELD 878 00:38:03,943 --> 00:38:08,348 TO THINK ABOUT THESE MODELS AND 879 00:38:08,348 --> 00:38:13,253 WAS VERY HAPPY TO SEE THE -- 880 00:38:13,253 --> 00:38:15,488 INHIBITOR FIELD REIMAGINING 881 00:38:15,488 --> 00:38:17,323 RATHER THAN A DOUBLE STRAND 882 00:38:17,323 --> 00:38:19,859 BREAK BEING THE CAUSE OF 883 00:38:19,859 --> 00:38:28,735 SYNTHETIC LETHALITY, IT WOULD BE 884 00:38:28,735 --> 00:38:28,935 BRICK. 885 00:38:28,935 --> 00:38:31,971 AND -- WHY INHIBTORS ARE 886 00:38:31,971 --> 00:38:33,073 SENSITIVE TO BRCA DEFICIENT 887 00:38:33,073 --> 00:38:33,273 CELLS. 888 00:38:33,273 --> 00:38:35,442 SO I THINK I WILL STOP THERE AND 889 00:38:35,442 --> 00:38:36,276 HAPPY TO TAKE ANY QUESTIONS. 890 00:38:36,276 --> 00:38:44,684 THANK YOU. 891 00:38:44,684 --> 00:38:47,353 >> CAN I JUST REMIND EVERYONE, 892 00:38:47,353 --> 00:38:49,422 TO PLEASE PUT ANY QUESTIONS YOU 893 00:38:49,422 --> 00:38:51,858 MIGHT HAVE FOR SHARON IN THE 894 00:38:51,858 --> 00:38:52,292 CHAT. 895 00:38:52,292 --> 00:38:53,159 THANK YOU, SHARON. 896 00:38:53,159 --> 00:38:55,261 THAT WAS A REALLY, REALLY 897 00:38:55,261 --> 00:38:56,930 INTERESTING TALK. 898 00:38:56,930 --> 00:38:57,897 >> THANK YOU. 899 00:38:57,897 --> 00:39:00,266 >> SO, YEAH, SO PLEASE TYPE IN 900 00:39:00,266 --> 00:39:02,168 YOUR QUESTION IN THE CHAT BOX. 901 00:39:02,168 --> 00:39:05,939 I SEE THERE IS A QUESTION FROM 902 00:39:05,939 --> 00:39:08,441 PATTY OPRESKO, SHE MENTIONED A 903 00:39:08,441 --> 00:39:10,009 GREAT TALK, SHARON. 904 00:39:10,009 --> 00:39:11,611 DO YOU HAVE AN IDEA HOW LARGE 905 00:39:11,611 --> 00:39:12,812 THESE GAPS ARE? 906 00:39:12,812 --> 00:39:15,014 DO YOU KNOW IF TEMPLATE 907 00:39:15,014 --> 00:39:17,150 SWITCHING HAS ANY ROLE IN 908 00:39:17,150 --> 00:39:19,552 PROCESSING OR FILLING THESE GAPS 909 00:39:19,552 --> 00:39:21,521 AND PARP INHIBITOR RESISTANCE? 910 00:39:21,521 --> 00:39:22,122 >> OKAY. 911 00:39:22,122 --> 00:39:22,856 YEAH. 912 00:39:22,856 --> 00:39:23,656 PATTY, GREAT QUESTION. 913 00:39:23,656 --> 00:39:25,125 WE DON'T HAVE ANY IDEA HOW BIG 914 00:39:25,125 --> 00:39:26,392 THESE GAPS ARE. 915 00:39:26,392 --> 00:39:30,797 WE ARE WORKING VERY HARD WITH 916 00:39:30,797 --> 00:39:31,931 NANAPOR SEQUENCING TO TRY TO GET 917 00:39:31,931 --> 00:39:33,366 A HANDLE ON THAT. 918 00:39:33,366 --> 00:39:34,968 SO WE THINK THAT WILL BE REALLY 919 00:39:34,968 --> 00:39:37,704 TELLING TO SEE HOW BIG THEY ARE 920 00:39:37,704 --> 00:39:38,805 AND HOW MANY THEY ARE AND HOW 921 00:39:38,805 --> 00:39:40,573 FAR APART THEY ARE. 922 00:39:40,573 --> 00:39:45,879 SO WITH TEMPLATE SWITCHING, ITS 923 00:39:45,879 --> 00:39:51,451 ROLE IN THE BRCA AND 53BP1 924 00:39:51,451 --> 00:39:53,486 DEFICIENT SELLS WAS HIGHLIGHTED 925 00:39:53,486 --> 00:39:56,222 BY WORK WHERE YOU COULD SEE A 926 00:39:56,222 --> 00:39:58,558 MUTATION PATTERN THAT WAS MORE 927 00:39:58,558 --> 00:39:59,826 CONSISTENT WITH A TEMPLATE 928 00:39:59,826 --> 00:40:03,563 SWITCH PATTERN THAT CHANGED FROM 929 00:40:03,563 --> 00:40:06,299 THE BRCA DEFICIENT CELL WITH 930 00:40:06,299 --> 00:40:07,667 TRANSLESION SYNTHESIS MUTATION 931 00:40:07,667 --> 00:40:08,001 PATTERN. 932 00:40:08,001 --> 00:40:10,970 THERE IS EVIDENCE FOR TEMPLATE 933 00:40:10,970 --> 00:40:12,572 SWITCH AND I THINK THE REAL 934 00:40:12,572 --> 00:40:14,841 STRONG EVIDENCE FOR THE 935 00:40:14,841 --> 00:40:16,309 MECHANISMS REGULATING THAT STILL 936 00:40:16,309 --> 00:40:16,709 REMAIN. 937 00:40:16,709 --> 00:40:18,578 YOU CAN IMAGINE YOU WOULD NEED 938 00:40:18,578 --> 00:40:22,315 TO RESECT THE GAP TO GET IT A 939 00:40:22,315 --> 00:40:24,484 CERTAIN SIZE FOR PROMOTING 940 00:40:24,484 --> 00:40:25,351 TEMPLATE SWITCH. 941 00:40:25,351 --> 00:40:26,853 UNDERSTANDING HOW THAT IS 942 00:40:26,853 --> 00:40:34,260 REGULATED STILL IS OUTSTANDING. 943 00:40:34,260 --> 00:40:37,630 >> JOHN HAS A QUESTION, ARE GAPS 944 00:40:37,630 --> 00:40:44,037 HARD TO REPAIR WHEN LIGASE IS 945 00:40:44,037 --> 00:40:47,774 NOT DEPLETED? 946 00:40:47,774 --> 00:40:48,374 >> OKAY. 947 00:40:48,374 --> 00:40:50,810 SO MEANING IF YOU HAVE THE 948 00:40:50,810 --> 00:40:52,879 LIGASE, YOU SHOULD BE ABLE TO 949 00:40:52,879 --> 00:40:59,619 REPAIR GAPS, CORRECT? 950 00:40:59,619 --> 00:41:00,420 YES. 951 00:41:00,420 --> 00:41:00,620 OKAY. 952 00:41:00,620 --> 00:41:03,122 SO I THINK IT COMES DOWN TO 953 00:41:03,122 --> 00:41:05,658 LAGGING STRAND SYNTHESIS IS 954 00:41:05,658 --> 00:41:07,727 COORDINATED WHEN ONE OKAZAKY IS 955 00:41:07,727 --> 00:41:10,330 COMING IN AND THE OTHER IS READY 956 00:41:10,330 --> 00:41:13,099 TO BE PROCESSED AND THE LIGATION 957 00:41:13,099 --> 00:41:15,935 WOULD BE READILY ACHIEVED. 958 00:41:15,935 --> 00:41:18,805 IF YOU HAVE AN IMPEDIMENT WITH 959 00:41:18,805 --> 00:41:22,141 PROCESSING OF THE FLAPS OR THE 960 00:41:22,141 --> 00:41:24,577 FLAPS GET HIGHLY OVERLY 961 00:41:24,577 --> 00:41:26,846 RESECTION, TOO MUCH OR TOO 962 00:41:26,846 --> 00:41:28,414 LITTLE RESECTION COULD IMPACT 963 00:41:28,414 --> 00:41:30,483 THE ABILITY TO PUT THE TWO FLAPS 964 00:41:30,483 --> 00:41:31,484 TOGETHER AND SEAL THE DEAL. 965 00:41:31,484 --> 00:41:34,120 SO, YES, CERTAINLY, YOU KNOW, IN 966 00:41:34,120 --> 00:41:36,789 THE ABSENCE OF THE LIGASE YOU 967 00:41:36,789 --> 00:41:37,957 HAVE A LOT OF PROBLEMS. 968 00:41:37,957 --> 00:41:43,029 IF YOU HAVE A LIGASE AND NOT 969 00:41:43,029 --> 00:41:45,965 RESECTING OR OVERSECTING 970 00:41:45,965 --> 00:41:47,567 OKAZAKY, YOU ARE GOING TO HAVE 971 00:41:47,567 --> 00:41:47,834 TROUBLE. 972 00:41:47,834 --> 00:41:50,870 >> ELISE IS ASKING, ARE THERE 973 00:41:50,870 --> 00:41:52,739 GENOMIC REGIONS MORE SENSITIVE 974 00:41:52,739 --> 00:41:53,940 TO GAP FORMATION THAN OTHERS IS 975 00:41:53,940 --> 00:41:54,707 >> YEAH. 976 00:41:54,707 --> 00:41:56,342 THAT IS SUCH A GOOD QUESTION. 977 00:41:56,342 --> 00:41:57,677 I WOULD THINK THE ANSWER WOULD 978 00:41:57,677 --> 00:41:59,412 BE YES AND THAT IS WHAT WE HOPE 979 00:41:59,412 --> 00:42:04,150 THE NANA4 SEQUENCING WILL SHOW. 980 00:42:04,150 --> 00:42:06,819 FOR ONE, THE RELATIONSHIP 981 00:42:06,819 --> 00:42:08,288 BETWEEN GAPS AND SECONDARY 982 00:42:08,288 --> 00:42:10,023 STRUCTURE AND I HAD A SLIDE FOR 983 00:42:10,023 --> 00:42:14,127 THAT IS HERE, THAT WE ARE 984 00:42:14,127 --> 00:42:17,764 LEARNING FROM A MODEL FROM 985 00:42:17,764 --> 00:42:19,432 ANOTHER GROUP, IF YOU HAVE A GAP 986 00:42:19,432 --> 00:42:21,534 SUBSTRATE AND YOU HAVE THE 987 00:42:21,534 --> 00:42:22,902 ABILITY TO FORM SECONDARY 988 00:42:22,902 --> 00:42:24,737 STRUCTURES, WELL, THAT REALLY 989 00:42:24,737 --> 00:42:28,174 INTERFERES WITH GAP REPAIR. 990 00:42:28,174 --> 00:42:30,209 AND IF YOU CAN RAPIDLY RESECT 991 00:42:30,209 --> 00:42:35,982 AND GET THESE SORT OF RAD 51 992 00:42:35,982 --> 00:42:38,051 STRANDS BINDING THAT CAN 993 00:42:38,051 --> 00:42:38,818 FACILITATE REPAIR. 994 00:42:38,818 --> 00:42:41,154 GOING THE OTHER WAY, IF YOU HAVE 995 00:42:41,154 --> 00:42:43,389 SECONDARY STRUCTURES, YOU KNOW, 996 00:42:43,389 --> 00:42:46,059 THEY HAVE THE OPPORTUNITY TO 997 00:42:46,059 --> 00:42:50,596 FORM IN A GAP OBVIOUSLY, THAT IS 998 00:42:50,596 --> 00:42:52,231 GOING TO BE GAP REPAIR. 999 00:42:52,231 --> 00:42:54,300 YOU WOULD EXPECT TO FIND 1000 00:42:54,300 --> 00:42:55,868 SECONDARY STRUCTURES IN REGIONS 1001 00:42:55,868 --> 00:42:57,570 OF UNREPAIRED GAPS. 1002 00:42:57,570 --> 00:42:58,705 SO THE ANSWER I GUESS IS YES. 1003 00:42:58,705 --> 00:43:01,140 THERE SHOULD BE A ROLE FOR 1004 00:43:01,140 --> 00:43:07,146 SECONDARY STRUCTURES INTERFERING 1005 00:43:07,146 --> 00:43:08,147 WITH GAP RESOLUTION. 1006 00:43:08,147 --> 00:43:11,384 >> SO HERE WE HAVE A QUESTION 1007 00:43:11,384 --> 00:43:12,952 FROM DAVID. 1008 00:43:12,952 --> 00:43:13,720 GREAT TALK. 1009 00:43:13,720 --> 00:43:17,457 CAN YOU DRIP HOW A T R AND THE 1010 00:43:17,457 --> 00:43:22,261 REPLICATION CHECKPOINTS GAP 1011 00:43:22,261 --> 00:43:25,431 FORMATION HOW DO YOU USE APR 1012 00:43:25,431 --> 00:43:25,765 INHIBITOR -- 1013 00:43:25,765 --> 00:43:28,835 >> THE MECHANISM GAPS ARE 1014 00:43:28,835 --> 00:43:30,536 RESENTED FOR REPAIR AS OPPOSED 1015 00:43:30,536 --> 00:43:33,206 TO INTERFERING WITH A REPAIR, A 1016 00:43:33,206 --> 00:43:35,608 T R WILL ULTIMATELY REGULATE 1017 00:43:35,608 --> 00:43:35,808 THAT. 1018 00:43:35,808 --> 00:43:43,449 AND SO BECAUSE ATR HAS SO MANY 1019 00:43:43,449 --> 00:43:45,952 TARGETS IT WILL TAKE WORK TO 1020 00:43:45,952 --> 00:43:47,320 EXTRAPOLATE THE CRITICAL 1021 00:43:47,320 --> 00:43:50,390 PATHWAYS FOR THAT. 1022 00:43:50,390 --> 00:43:52,925 ATR INHIBITOR IS LEADING TO A 1023 00:43:52,925 --> 00:43:53,426 LOT OF GAPS. 1024 00:43:53,426 --> 00:43:55,027 THE MECHANISM BY WHICH THOSE 1025 00:43:55,027 --> 00:43:57,597 GAPS ARE FORMING WE DON'T QUITE 1026 00:43:57,597 --> 00:43:59,499 FULLY UNDERSTAND BEYOND THE MOST 1027 00:43:59,499 --> 00:44:01,100 OBVIOUS WHICH IS REPLICATION IS 1028 00:44:01,100 --> 00:44:03,202 NOT SLOWING PROPERLY AND NOT 1029 00:44:03,202 --> 00:44:05,304 BEING REGULATED BUT IN THE GAPS 1030 00:44:05,304 --> 00:44:06,606 ALREADY FORMED DOES IT HAVE A 1031 00:44:06,606 --> 00:44:06,906 ROLE? 1032 00:44:06,906 --> 00:44:08,608 I THINK THIS IS WHAT PEOPLE ARE 1033 00:44:08,608 --> 00:44:10,877 TAKING APART AND CERTAINLY 1034 00:44:10,877 --> 00:44:12,445 SEEING A ROLE FOR CHECKPOINT 1035 00:44:12,445 --> 00:44:16,749 SIGNALLING AND GAP PROCESSING. 1036 00:44:16,749 --> 00:44:20,453 >> OKAY. 1037 00:44:20,453 --> 00:44:24,724 THERE IS A COMMENT FROM YOUNG 1038 00:44:24,724 --> 00:44:24,891 KIM. 1039 00:44:24,891 --> 00:44:29,562 HOW IS IT ASSOCIATED WITH GAMMA 1040 00:44:29,562 --> 00:44:31,964 H2AX AND HOW MUCH WOULD YOU 1041 00:44:31,964 --> 00:44:42,208 EXPECT TO SEE AT UNPERTURBED 1042 00:44:42,208 --> 00:44:43,009 FORKS? 1043 00:44:43,009 --> 00:44:44,277 THE SECOND PART? 1044 00:44:44,277 --> 00:44:47,346 >> THROUGH GAMMA H2AX. 1045 00:44:47,346 --> 00:44:49,549 >> DOING THESE LIGATION ASSAYS 1046 00:44:49,549 --> 00:44:51,951 IN THE ABSENCE OR PRESENCE OF 1047 00:44:51,951 --> 00:44:53,352 ONE OR THE OTHER GENES IS 1048 00:44:53,352 --> 00:44:54,287 SOMETHING ONGOING. 1049 00:44:54,287 --> 00:44:58,024 IF YOU LOOK AT THE CHROMATIN IN 1050 00:44:58,024 --> 00:45:07,533 A CELL WHERE YOU DEPLETE H2AX, 1051 00:45:07,533 --> 00:45:09,902 BDC1 AND DROPS OUT. 1052 00:45:09,902 --> 00:45:11,204 IT IS THE SAME RESULT WITH 1053 00:45:11,204 --> 00:45:14,140 DOUBLE STRAND BREAKS AND NUCLEAR 1054 00:45:14,140 --> 00:45:14,373 BODIES. 1055 00:45:14,373 --> 00:45:16,809 I WOULD GUESS THE ABILITY FOR 1056 00:45:16,809 --> 00:45:20,379 MDC1 TO BE MAINTAINED IN THE 1057 00:45:20,379 --> 00:45:23,416 REPRIZOME IS HIGHLY DEPENDENT ON 1058 00:45:23,416 --> 00:45:24,851 H2AX. 1059 00:45:24,851 --> 00:45:28,054 IT IS PROPOSED TO GET TO THE 1060 00:45:28,054 --> 00:45:30,556 FLAP THROUGH ITS RANNE BINDING 1061 00:45:30,556 --> 00:45:31,023 DOMAIN. 1062 00:45:31,023 --> 00:45:32,859 IT COULD BE IT IS GETTING THERE 1063 00:45:32,859 --> 00:45:40,099 ON ITS OWN AND H2X MDC1 IS 1064 00:45:40,099 --> 00:45:42,902 COMING THROUGH, THE ORDER IS NOT 1065 00:45:42,902 --> 00:45:46,706 CLEARED, BUT IT IS PREDICTED FOR 1066 00:45:46,706 --> 00:45:47,006 MAINTENANCE. 1067 00:45:47,006 --> 00:45:48,574 TO ANSWER YOUR QUESTION, WE 1068 00:45:48,574 --> 00:45:51,978 HAVEN'T FULLY WORKED IT OUT. 1069 00:45:51,978 --> 00:45:57,483 HOW MANY MDC1 AS THE TOTAL? 1070 00:45:57,483 --> 00:45:59,151 I DON'T HAVE THE ANSWER. 1071 00:45:59,151 --> 00:46:02,088 >> HOW LARGE DO YOU THINK THESE 1072 00:46:02,088 --> 00:46:05,491 GAPS ARE FOR BRCA 1 DEFECTS? 1073 00:46:05,491 --> 00:46:06,325 >> OKAY. 1074 00:46:06,325 --> 00:46:08,794 SO IN ABSENCE OF BRCA 1, WE'VE 1075 00:46:08,794 --> 00:46:09,595 GOT GAPS. 1076 00:46:09,595 --> 00:46:11,831 WE DON'T KNOW HOW BIG THEY ARE. 1077 00:46:11,831 --> 00:46:14,634 PEOPLE WHO DO EM HAVE LOOKED AND 1078 00:46:14,634 --> 00:46:15,301 THEY CAN SEE THEM. 1079 00:46:15,301 --> 00:46:17,470 SO THAT MEANS THEY ARE BIGGER 1080 00:46:17,470 --> 00:46:20,806 THAN THE SIZE, YOU KNOW, 1081 00:46:20,806 --> 00:46:21,908 REQUIRED TO SEE THINGS. 1082 00:46:21,908 --> 00:46:24,410 I BELIEVE THAT, I DON'T KNOW, 50 1083 00:46:24,410 --> 00:46:35,087 TO 200 OR SOMETHING NUCLEOTIDES. 1084 00:46:35,087 --> 00:46:37,456 THE REASON THEY ARE SO TOXIC, IF 1085 00:46:37,456 --> 00:46:40,259 YOU RESECT THEM WITH OTHER 1086 00:46:40,259 --> 00:46:41,494 NUCLEASES YOU GET IN TROUBLE AND 1087 00:46:41,494 --> 00:46:42,929 NOT ABLE TO FILL THEM. 1088 00:46:42,929 --> 00:46:46,165 MY GUESS IS THEY ARE EXPANDING 1089 00:46:46,165 --> 00:46:47,466 AND SO HOW MUCH THEY ARE 1090 00:46:47,466 --> 00:46:49,201 EXPANDING AND WHAT IS THE 1091 00:46:49,201 --> 00:46:51,070 THRESHOLD THAT IS GOING TO 1092 00:46:51,070 --> 00:46:51,904 ULTIMATELY POTENTIALLY KILL 1093 00:46:51,904 --> 00:46:55,074 THESE CELLS REMAINS TO BE 1094 00:46:55,074 --> 00:46:58,811 DETERMINED. 1095 00:46:58,811 --> 00:47:00,313 >> OKAY. 1096 00:47:00,313 --> 00:47:01,280 WE HAVE ANOTHER QUESTION. 1097 00:47:01,280 --> 00:47:02,848 GREAT TALK. 1098 00:47:02,848 --> 00:47:04,917 THANK YOU. 1099 00:47:04,917 --> 00:47:07,987 DO YOU THINK PARP INHIBITOR 1100 00:47:07,987 --> 00:47:11,324 WOULD ASSERT THEIR EFFECTS OTHER 1101 00:47:11,324 --> 00:47:12,525 THAN -- 1102 00:47:12,525 --> 00:47:12,992 >> SO. 1103 00:47:12,992 --> 00:47:15,595 YEAH, THIS IS A PAPER WE 1104 00:47:15,595 --> 00:47:16,729 PUBLISHED, NATURE COMMUNICATIONS 1105 00:47:16,729 --> 00:47:17,930 WHERE WE ASKED THE QUESTION IS 1106 00:47:17,930 --> 00:47:21,901 PARP INHIBITOR WORKING IN A BRCA 1107 00:47:21,901 --> 00:47:23,035 1 DIVISION BACKGROUND? 1108 00:47:23,035 --> 00:47:26,872 BECAUSE IT IS TRAPPING OR IS IT 1109 00:47:26,872 --> 00:47:29,875 THIS BASIC REPLICATION ACTIVITY 1110 00:47:29,875 --> 00:47:31,210 THAT IS THE THING THAT IS SO 1111 00:47:31,210 --> 00:47:32,778 IMPORTANT OR BOTH? 1112 00:47:32,778 --> 00:47:35,448 IS IT GETTING TRAPPED IN 1113 00:47:35,448 --> 00:47:36,849 REPLICATION GAPS FROM FAILURE OF 1114 00:47:36,849 --> 00:47:40,186 PARP ACTIVITY AND PARP ALSO 1115 00:47:40,186 --> 00:47:40,953 GETTING TRAPPED. 1116 00:47:40,953 --> 00:47:44,390 TOGETHER OUR DATA DON'T PROVIDE 1117 00:47:44,390 --> 00:47:47,093 ANY EVIDENCE FOR TRAPPING BEING 1118 00:47:47,093 --> 00:47:50,296 AS IMPORTANT AS THE PARP 1119 00:47:50,296 --> 00:47:50,596 ACTIVITY. 1120 00:47:50,596 --> 00:47:54,934 AGAIN, IF YOU REMOVE THE -- HELO 1121 00:47:54,934 --> 00:47:58,537 CASE WE SEE PARP ACTIVITY GOES 1122 00:47:58,537 --> 00:48:00,640 DRAMATICALLY DOWN. 1123 00:48:00,640 --> 00:48:03,042 PARP BECOMES SEQUESTERED. 1124 00:48:03,042 --> 00:48:08,014 IF YOU COMBINE WITH BRCA LOSS. 1125 00:48:08,014 --> 00:48:09,382 THAT IS SIMILAR TO KNOCKOUT 1126 00:48:09,382 --> 00:48:11,484 CELLS WHERE THERE IS NO 1127 00:48:11,484 --> 00:48:11,784 TRAPPING. 1128 00:48:11,784 --> 00:48:14,520 PARP KNOCKOUT IS HIGHLY TOXIC. 1129 00:48:14,520 --> 00:48:16,789 THAT WAS THE ORIGINAL 1130 00:48:16,789 --> 00:48:18,691 OBSERVATION IN 2005 NATURE 1131 00:48:18,691 --> 00:48:18,924 PAPERS. 1132 00:48:18,924 --> 00:48:22,795 LOSS OF PARP ALONE, NO HIB HIB 1133 00:48:22,795 --> 00:48:25,364 TOR, IS GOING TO BE TOXIC WITH 1134 00:48:25,364 --> 00:48:26,165 PARP BRCA. 1135 00:48:26,165 --> 00:48:29,135 WE KNOW PARP TRAPPING CAN BE 1136 00:48:29,135 --> 00:48:31,170 BAD, WHEN THE BRCA 1 DEFICIENT 1137 00:48:31,170 --> 00:48:32,705 BACKGROUND WE DON'T THINK THAT 1138 00:48:32,705 --> 00:48:34,040 TRAPPING IS A MAJOR CONTRIBUTOR 1139 00:48:34,040 --> 00:48:37,276 TO THE DEATH, BUT, AGAIN, WE 1140 00:48:37,276 --> 00:48:40,913 KNOW XRCC1 LOSS TRAPPING IS A 1141 00:48:40,913 --> 00:48:41,447 PROBLEM. 1142 00:48:41,447 --> 00:48:43,716 TO GET RID OF PARP IS BETTER. 1143 00:48:43,716 --> 00:48:45,951 TRAPPING CAN BE BAD, BUT IT IS 1144 00:48:45,951 --> 00:48:47,019 CONTEXT DEPENDENT. 1145 00:48:47,019 --> 00:48:51,357 THANK YOU. 1146 00:48:51,357 --> 00:48:52,792 >> I HAVE JUST ONE QUESTION 1147 00:48:52,792 --> 00:48:53,459 MYSELF. 1148 00:48:53,459 --> 00:48:53,959 >> OKAY. 1149 00:48:53,959 --> 00:48:56,228 >> SHARON, COULD YOU SPECULATE 1150 00:48:56,228 --> 00:48:58,998 ON WHETHER THE MDC IS SERVE AS A 1151 00:48:58,998 --> 00:49:00,032 BIOMARKER FOR THERAPY RESPONSE? 1152 00:49:00,032 --> 00:49:01,701 >> OH, YEAH. 1153 00:49:01,701 --> 00:49:04,203 YOU KNOW, WE ARE LOOKING AT THAT 1154 00:49:04,203 --> 00:49:14,246 NOW. 1155 00:49:14,246 --> 00:49:14,847 WE LOOKED ACROSS A SERIES OF 1156 00:49:14,847 --> 00:49:15,481 BREAST AND OVARIAN CANCER CELL 1157 00:49:15,481 --> 00:49:15,681 LINES. 1158 00:49:15,681 --> 00:49:16,315 WHEN BRCA 1 OR BRCA 2 WAS LOW, 1159 00:49:16,315 --> 00:49:20,886 MDC1, # 53BP1 TREND HIGHER. 1160 00:49:20,886 --> 00:49:30,696 SO THE QUESTION IS WHETHER THAT 1161 00:49:30,696 --> 00:49:31,230 WILL HOLD UP ACROSS TUMOR 1162 00:49:31,230 --> 00:49:31,464 SAMPLES. 1163 00:49:31,464 --> 00:49:32,098 WE ARE GOING IN THAT DIRECTION 1164 00:49:32,098 --> 00:49:32,264 NOW. 1165 00:49:32,264 --> 00:49:32,765 THAT IS WHERE WE ARE AT. 1166 00:49:32,765 --> 00:49:33,399 IT COULD PROVIDE A BIOMARKER FOR 1167 00:49:33,399 --> 00:49:34,100 GAPS AND, AGAIN, HOW MUCH SIGNAL 1168 00:49:34,100 --> 00:49:35,334 DO YOU NEED TO HAVE TO KNOW THEY 1169 00:49:35,334 --> 00:49:36,869 ARE GOING TO BE SENSITIVE IS THE 1170 00:49:36,869 --> 00:49:37,803 CRITICAL QUESTION. 1171 00:49:37,803 --> 00:49:39,939 WHAT IS THE LOAD THAT YOU NEED? 1172 00:49:39,939 --> 00:49:42,575 SO WE ARE HOPING WE CAN USE 1173 00:49:42,575 --> 00:49:46,378 THAT, COMPARE IT TO RAD 51 FOCI 1174 00:49:46,378 --> 00:49:48,681 AND SEE IF THIS IS EQUALLY OR 1175 00:49:48,681 --> 00:49:50,015 BETTER AS A MARKER. 1176 00:49:50,015 --> 00:49:50,850 >> THANK YOU. 1177 00:49:50,850 --> 00:49:53,452 >> YES. 1178 00:49:53,452 --> 00:49:57,656 >> WE HAVE AGES AGO, WE WERE 1179 00:49:57,656 --> 00:50:00,559 PUZZLED BY THE COMPARISON OF 1180 00:50:00,559 --> 00:50:01,994 SINGLE STRAND BREAKS AND DOUBLE 1181 00:50:01,994 --> 00:50:03,462 STRAND BREAKS AND WONDERED WHY 1182 00:50:03,462 --> 00:50:06,232 THEY WERE SO, IF SINGLE STRAND 1183 00:50:06,232 --> 00:50:10,035 BREAKS WERE NOT TOXIC, WHY WERE 1184 00:50:10,035 --> 00:50:11,604 CELLS DYING? 1185 00:50:11,604 --> 00:50:12,037 >> YEAH. 1186 00:50:12,037 --> 00:50:14,874 >> THE ANSWER IS EVENTUALLY YOU 1187 00:50:14,874 --> 00:50:17,810 LINE UP A SINGLE STRAND BREAK 1188 00:50:17,810 --> 00:50:18,911 OPPOSITE ANOTHER AND WOULD LEAD 1189 00:50:18,911 --> 00:50:19,979 TO A DOUBLE STRAND BREAK. 1190 00:50:19,979 --> 00:50:20,846 >> SURE. 1191 00:50:20,846 --> 00:50:22,615 >> IS THAT A CONCEPT STILL 1192 00:50:22,615 --> 00:50:23,015 VIABLE? 1193 00:50:23,015 --> 00:50:25,317 >> THIS IS VERY MUCH A CONCEPT. 1194 00:50:25,317 --> 00:50:28,420 SO PEOPLE GENERALLY THINK OF 1195 00:50:28,420 --> 00:50:31,490 SINGLE STRAND BREAKS OR PARP 1196 00:50:31,490 --> 00:50:37,329 COMPLEXES ARE COLLAPSING THE 1197 00:50:37,329 --> 00:50:37,830 FORK. 1198 00:50:37,830 --> 00:50:39,765 IT IS INEVITABLE A SINGLE STRAND 1199 00:50:39,765 --> 00:50:43,102 WOULD HIT A REPLICATION FORK AND 1200 00:50:43,102 --> 00:50:45,104 AT SOME CONFIRMATION WOULD LEAD 1201 00:50:45,104 --> 00:50:47,039 TO A DOUBLE STRAND BREAK. 1202 00:50:47,039 --> 00:50:50,543 IT COMES DOWN TO IS THAT HOW THE 1203 00:50:50,543 --> 00:50:51,911 CELLS DIE THROUGH THE DOUBLE 1204 00:50:51,911 --> 00:50:54,146 STRAND BREAK OR CAN A SINGLE 1205 00:50:54,146 --> 00:50:57,216 STRAND OF DNA BREAK EXPAND, 1206 00:50:57,216 --> 00:50:59,819 RIGHT, AND HAVE ITS OWN LETHAL 1207 00:50:59,819 --> 00:51:01,187 CONSEQUENCES OUTSIDE OF HAVING 1208 00:51:01,187 --> 00:51:04,924 TO HIT THE REPLICATION FORK. 1209 00:51:04,924 --> 00:51:07,026 SO THAT'S REALLY IMPORTANT 1210 00:51:07,026 --> 00:51:08,394 INTERESTING AREA THAT I THINK 1211 00:51:08,394 --> 00:51:10,963 NEEDS TO BE FIGURED OUT BECAUSE 1212 00:51:10,963 --> 00:51:12,731 OTHERWISE WE ARE GOING TO KEEP 1213 00:51:12,731 --> 00:51:13,866 INVOKING A DOUBLE STRAND BREAK, 1214 00:51:13,866 --> 00:51:16,001 WHICH MIGHT BE RIGHT, BUT IF IT 1215 00:51:16,001 --> 00:51:17,670 IS NOT RIGHT, MAYBE THERE ARE 1216 00:51:17,670 --> 00:51:19,004 OTHER THERAPIES WE CAN USE THAT 1217 00:51:19,004 --> 00:51:24,710 DON'T REQUIRE THAT SITUATION. 1218 00:51:24,710 --> 00:51:25,044 >> OKAY. 1219 00:51:25,044 --> 00:51:26,245 GO AHEAD. 1220 00:51:26,245 --> 00:51:27,913 THERE'S MORE QUESTIONS IN THE 1221 00:51:27,913 --> 00:51:28,113 CHAT. 1222 00:51:28,113 --> 00:51:29,248 >> WE HAVE A QUESTION. 1223 00:51:29,248 --> 00:51:30,382 GREAT TALK. 1224 00:51:30,382 --> 00:51:32,852 IT LOOKS LIKE A THE RX IS RICH 1225 00:51:32,852 --> 00:51:35,554 AT THE REPLICATING DNA ABSENCE 1226 00:51:35,554 --> 00:51:37,723 OF BRCA 1. 1227 00:51:37,723 --> 00:51:39,425 IS THERE ANY FUNCTIONAL FOR A 1228 00:51:39,425 --> 00:51:41,427 THE RX TO BE THERE? 1229 00:51:41,427 --> 00:51:41,794 THANKS? 1230 00:51:41,794 --> 00:51:44,797 >> SO THE PROTEIN IS A THE RX? 1231 00:51:44,797 --> 00:51:45,631 >> YES. 1232 00:51:45,631 --> 00:51:52,371 A THE RX. 1233 00:51:52,371 --> 00:51:52,571 ATRX. 1234 00:51:52,571 --> 00:51:53,806 AT THE REPLICATION FORK? 1235 00:51:53,806 --> 00:51:55,040 I DON'T THINK I HAVE A GOOD 1236 00:51:55,040 --> 00:51:56,508 ANSWER FOR THIS. 1237 00:51:56,508 --> 00:51:57,610 I DON'T THINK I KNOW TOO MUCH 1238 00:51:57,610 --> 00:52:00,613 ABOUT THAT. 1239 00:52:00,613 --> 00:52:01,080 SORRY. 1240 00:52:01,080 --> 00:52:01,513 YEAH. 1241 00:52:01,513 --> 00:52:02,581 >> OKAY. 1242 00:52:02,581 --> 00:52:05,784 THERE IS ANOTHER QUESTION FROM 1243 00:52:05,784 --> 00:52:06,318 JOHN. 1244 00:52:06,318 --> 00:52:08,854 ASKING DO COMMON SAS SAYS REVEAL 1245 00:52:08,854 --> 00:52:10,556 THESE GAP AND TELL YOU TIME FOR 1246 00:52:10,556 --> 00:52:11,457 REPAIR OR RESISTANCE? 1247 00:52:11,457 --> 00:52:11,924 >> YEAH. 1248 00:52:11,924 --> 00:52:16,228 I THINK, I MEAN, WE HAVE STAYED 1249 00:52:16,228 --> 00:52:18,030 AWAY FROM COMMON ASSAYS BECAUSE 1250 00:52:18,030 --> 00:52:19,932 THE EXPERTS I TALKED TO SAY THEY 1251 00:52:19,932 --> 00:52:22,801 DON'T FULLY ONLY REPORT ON 1252 00:52:22,801 --> 00:52:25,137 SINGLE STRANDED DNA BREAKS 1253 00:52:25,137 --> 00:52:27,039 VERSUS DOUBLE STRANDED DNA 1254 00:52:27,039 --> 00:52:27,506 BREAKS. 1255 00:52:27,506 --> 00:52:30,309 SO I THINK IF, YOU KNOW, THE 1256 00:52:30,309 --> 00:52:32,111 DRUG OF INTEREST WAS CLEARLY 1257 00:52:32,111 --> 00:52:34,380 ONLY MAKING ONE KIND OF LESION 1258 00:52:34,380 --> 00:52:36,615 YOU COULD USE IT TO ASK ABOUT 1259 00:52:36,615 --> 00:52:37,816 RESOLUTION AND WHATEVER. 1260 00:52:37,816 --> 00:52:39,451 BUT IF YOU DON'T KNOW AND YOU 1261 00:52:39,451 --> 00:52:41,520 ARE MIXING IN CHEMOTHERAPY, YOU 1262 00:52:41,520 --> 00:52:42,922 DON'T KNOW HOW IT WORKS AND 1263 00:52:42,922 --> 00:52:45,024 LOOKING AT THAT, YOU ARE CLEARLY 1264 00:52:45,024 --> 00:52:47,226 NOT ABLE TO DISCRIMINATE. 1265 00:52:47,226 --> 00:52:50,329 IT JUST ADDS COMPLEXITY TO 1266 00:52:50,329 --> 00:52:50,629 COMPLEXITY. 1267 00:52:50,629 --> 00:52:52,898 THAT IS WHY WE STAYED AWAY FROM 1268 00:52:52,898 --> 00:52:53,065 IT. 1269 00:52:53,065 --> 00:52:54,934 WE ARE TRYING OTHER APPROACHES 1270 00:52:54,934 --> 00:52:57,202 TO MEASURE GAPS IN DNA THAT 1271 00:52:57,202 --> 00:53:02,841 HOPEFULLY WILL BE ABLE TO 1272 00:53:02,841 --> 00:53:08,781 RESOLVE THESE ISSUES. 1273 00:53:08,781 --> 00:53:11,884 >> THERE ARE STUDIES DONE BY ONE 1274 00:53:11,884 --> 00:53:14,853 OF THE PIONEERS IN CELL CULTURE 1275 00:53:14,853 --> 00:53:17,022 AT THE CANCER INSTITUTE WAS 1276 00:53:17,022 --> 00:53:18,891 CATHERINE SANFORD. 1277 00:53:18,891 --> 00:53:23,028 AND SHE WAS DOING G2 CHROMOSOME 1278 00:53:23,028 --> 00:53:24,930 HYPERSENSITIVITY AT RADIATION. 1279 00:53:24,930 --> 00:53:29,735 AND SHE WOULD FIND SINGLE 1280 00:53:29,735 --> 00:53:34,740 STRAND -- LOOKING AT TERIATYPES 1281 00:53:34,740 --> 00:53:38,444 AND CHROMOSOME GAPS OR BREAKS 1282 00:53:38,444 --> 00:53:39,345 REPRESENTING SINGLE STRAND OR 1283 00:53:39,345 --> 00:53:40,546 DOUBLE STRAND DNA. 1284 00:53:40,546 --> 00:53:44,616 SHE WAS ABLE TO CHARACTERIZE 1285 00:53:44,616 --> 00:53:47,019 CELL LINES FROM CANCER PRONE 1286 00:53:47,019 --> 00:53:47,987 DISEASES TO NOT CANCER PRONE 1287 00:53:47,987 --> 00:53:48,687 DISEASES ON THIS LEVEL. 1288 00:53:48,687 --> 00:53:50,089 >> THAT IS INTERESTING. 1289 00:53:50,089 --> 00:53:52,324 >> SHE DID A SERIES OF STUDIES 1290 00:53:52,324 --> 00:53:56,862 ON THOSE. 1291 00:53:56,862 --> 00:53:59,898 VERY PRECISE USING THE RIGHT 1292 00:53:59,898 --> 00:54:01,266 GLASSWARE, THE RIGHT CULTURE 1293 00:54:01,266 --> 00:54:01,967 CONDITIONS, LIGHTS AND 1294 00:54:01,967 --> 00:54:02,267 EVERYTHING. 1295 00:54:02,267 --> 00:54:03,302 >> OH, THAT IS GREAT. 1296 00:54:03,302 --> 00:54:05,004 >> THERE IS A WHOLE SERIES OF 1297 00:54:05,004 --> 00:54:06,739 PAPERS ABOUT THAT AND THAT SEEMS 1298 00:54:06,739 --> 00:54:08,807 TO HAVE BEEN LOST TO HISTORY. 1299 00:54:08,807 --> 00:54:11,777 >> I'M GOING TO GO LOOK THAT UP. 1300 00:54:11,777 --> 00:54:15,948 HER NAME IS CATHERINE STANFORD? 1301 00:54:15,948 --> 00:54:23,989 >> SANFORD. 1302 00:54:23,989 --> 00:54:24,323 S-A-N-F-O-R-D. 1303 00:54:24,323 --> 00:54:28,193 >> WHY AREN'T LONG GAPS 1304 00:54:28,193 --> 00:54:28,627 ATTACKED -- 1305 00:54:28,627 --> 00:54:31,930 >> IS THAT THE WORD, LONG? 1306 00:54:31,930 --> 00:54:33,132 >> YEAH. 1307 00:54:33,132 --> 00:54:34,099 LONG GAPS. 1308 00:54:34,099 --> 00:54:36,068 >> I'M NOT SAYING THEY ARE NOT. 1309 00:54:36,068 --> 00:54:37,503 THAT IS SOMETHING ONE CAN LOOK 1310 00:54:37,503 --> 00:54:38,871 AT. 1311 00:54:38,871 --> 00:54:40,472 HOW GAPS KILL IS NOT CLEAR, 1312 00:54:40,472 --> 00:54:40,939 RIGHT. 1313 00:54:40,939 --> 00:54:42,274 CERTAINLY THEY COULD CONVERT TO 1314 00:54:42,274 --> 00:54:43,675 DOUBLE STRAND BREAKS. 1315 00:54:43,675 --> 00:54:45,677 WE KNOW DOUBLE STRAND BREAKS ARE 1316 00:54:45,677 --> 00:54:47,146 VERY TOXIC AND COULD BE 1317 00:54:47,146 --> 00:54:48,881 ULTIMATELY HOW THEY KILL. 1318 00:54:48,881 --> 00:54:50,849 I'M JUST ARGUING THE 1319 00:54:50,849 --> 00:54:51,817 VULNERABILITY IS THE GAP THAT 1320 00:54:51,817 --> 00:54:54,219 BRCA 1 PROTECTS FROM. 1321 00:54:54,219 --> 00:54:55,888 THAT IS WHAT I'M REALLY ARGUING. 1322 00:54:55,888 --> 00:54:58,090 IF THEY ULTIMATELY CONVERT TO 1323 00:54:58,090 --> 00:54:59,391 DOUBLE STRAND BREAK AND THAT IS 1324 00:54:59,391 --> 00:55:02,828 HOW THE CELL DIES IS UNCLEAR. 1325 00:55:02,828 --> 00:55:05,297 OR WHETHER A GAP, YOU KNOW, WILL 1326 00:55:05,297 --> 00:55:08,333 TANGLE UP INTO A SECONDARY 1327 00:55:08,333 --> 00:55:09,468 STRUCTURE, WHICH, YOU KNOW, IS 1328 00:55:09,468 --> 00:55:13,105 GOING TO ITSELF BE BLOCKING 1329 00:55:13,105 --> 00:55:14,807 REPLICATION TRANSCRIPTION, 1330 00:55:14,807 --> 00:55:19,011 RECRUITING NUCLEASES LEADING TO 1331 00:55:19,011 --> 00:55:20,846 GENOMIC INSTABILITY AND 1332 00:55:20,846 --> 00:55:22,414 POTENTIALLY ELIMINATING CRITICAL 1333 00:55:22,414 --> 00:55:22,648 GENES. 1334 00:55:22,648 --> 00:55:24,483 THERE ARE SO MANY WAYS THIS 1335 00:55:24,483 --> 00:55:25,951 COULD UNFOLD AND THIS IS WHAT WE 1336 00:55:25,951 --> 00:55:27,286 ARE TRYING TO TAKE APART. 1337 00:55:27,286 --> 00:55:28,620 YOUR QUESTION IS A GOOD ONE. 1338 00:55:28,620 --> 00:55:30,923 IT IS POSSIBLE THAT THERE'S A 1339 00:55:30,923 --> 00:55:32,791 ROLE HERE THAT COULD FURTHER BE 1340 00:55:32,791 --> 00:55:35,194 INVOLVED IN THE MECHANISM. 1341 00:55:35,194 --> 00:55:41,533 ABSOLUTELY. 1342 00:55:41,533 --> 00:55:44,870 >> OKAY. 1343 00:55:44,870 --> 00:55:50,409 THERE IS ANOTHER QUESTION -- 1344 00:55:50,409 --> 00:55:53,645 SORRY, I CANNOT PRONOUNCE RIGHT. 1345 00:55:53,645 --> 00:55:55,047 GREAT TALK. 1346 00:55:55,047 --> 00:55:57,316 DO YOU THINK LABELING LONG DNA 1347 00:55:57,316 --> 00:56:03,021 IS TO DETECT SS DNA FOCI? 1348 00:56:03,021 --> 00:56:07,025 >> SO NONDENATURING, WHEN YOU 1349 00:56:07,025 --> 00:56:09,428 LOAD UP THE CELL WITH AN ANALOG, 1350 00:56:09,428 --> 00:56:11,497 IF THERE IS A SPOT, IT WILL 1351 00:56:11,497 --> 00:56:12,231 RECOGNIZE IT. 1352 00:56:12,231 --> 00:56:14,233 I THINK IT IS VERY GOOD. 1353 00:56:14,233 --> 00:56:15,167 IT WORKS QUITE WELL. 1354 00:56:15,167 --> 00:56:17,769 AGAIN, YOU DON'T KNOW IN THE GAP 1355 00:56:17,769 --> 00:56:21,006 IS STARTING IN REPLICATION OR 1356 00:56:21,006 --> 00:56:22,774 NOT UNLESS YOU CAN MARK 1357 00:56:22,774 --> 00:56:23,075 REPLICATION. 1358 00:56:23,075 --> 00:56:24,710 BUT I THINK IT IS QUITE GOOD. 1359 00:56:24,710 --> 00:56:25,477 IT DOESN'T TELL YOU ANYTHING 1360 00:56:25,477 --> 00:56:27,613 ABOUT THE SIZE OF THE GAP, 1361 00:56:27,613 --> 00:56:27,813 RIGHT. 1362 00:56:27,813 --> 00:56:29,982 IT JUST SAYS YOU HAVE GAPS. 1363 00:56:29,982 --> 00:56:31,283 AGAIN, IT IS NOT GOING TO 1364 00:56:31,283 --> 00:56:33,018 DISCRIMINATE ABOUT NUMBERS AND 1365 00:56:33,018 --> 00:56:35,020 SIZE AND, BUT, YEAH, IF YOU WANT 1366 00:56:35,020 --> 00:56:36,321 TO KNOW IF YOUR DRUG OF ACTION 1367 00:56:36,321 --> 00:56:38,957 IS MAKING GAPS, YOU CAN DO THAT. 1368 00:56:38,957 --> 00:56:44,296 FOR SURE. 1369 00:56:44,296 --> 00:56:45,197 >> GREAT. 1370 00:56:45,197 --> 00:56:46,665 I DON'T SEE ANY ADDITIONAL 1371 00:56:46,665 --> 00:56:46,932 QUESTIONS. 1372 00:56:46,932 --> 00:56:48,433 >> OKAY. 1373 00:56:48,433 --> 00:56:48,634 GREAT. 1374 00:56:48,634 --> 00:56:49,501 THANK YOU. 1375 00:56:49,501 --> 00:56:50,869 >> PLEASE JOIN ME IN THANKING 1376 00:56:50,869 --> 00:56:53,505 DR. CANTOR FOR THE WONDERFUL 1377 00:56:53,505 --> 00:56:54,339 TALK. 1378 00:56:54,339 --> 00:56:56,842 AND THANK YOU SO MUCH FOR 1379 00:56:56,842 --> 00:56:57,075 TALKING. 1380 00:56:57,075 --> 00:56:59,044 WE'RE LOOKING FORWARD FOR MORE 1381 00:56:59,044 --> 00:56:59,878 FINDINGS FROM YOUR GROUP. 1382 00:56:59,878 --> 00:57:00,913 >> OKAY. 1383 00:57:00,913 --> 00:57:02,781 WELL, THANK YOU SO MUCH. 1384 00:57:02,781 --> 00:57:03,815 APPRECIATE YOU GUYS. 1385 00:57:03,815 --> 00:57:05,450 HAVE A GOOD DAY. 1386 00:57:05,450 --> 00:57:06,451 >> KEEP IN TOO MUCH. 1387 00:57:06,451 --> 00:57:07,019 BYE-BYE. 1388 00:57:07,019 --> 00:57:16,762 >> BYE.