1 00:00:05,120 --> 00:00:10,320 >>WELCOME EVERYBODY TO 2 00:00:10,320 --> 00:00:14,720 THE DNA REPAIR INTEREST GROUP. 3 00:00:14,720 --> 00:00:21,040 WE ARE NOW IN OUR WEBEX FROM 4 00:00:21,040 --> 00:00:21,240 HOME. 5 00:00:21,240 --> 00:00:22,000 IN PRESENTATIONS AND WE'RE VERY 6 00:00:22,000 --> 00:00:25,520 GLAD YOU ARE ABLE TO JOIN US. 7 00:00:25,520 --> 00:00:29,640 ROB SOBOL IS OUR SPEAKER 8 00:00:29,640 --> 00:00:33,920 TODAY, HE WAS BORN IN NEW YORK, 9 00:00:33,920 --> 00:00:45,360 BUT THEN HE WENT TO COLLEGE AT 10 00:00:45,360 --> 00:00:49,880 ALGENEY COLLEGE AND THEN 11 00:00:49,880 --> 00:00:52,440 GOT HIS Ph.D. AND HE STARTED 12 00:00:52,440 --> 00:00:53,560 WORKING WITH SAM WILSON AT THAT 13 00:00:53,560 --> 00:00:56,520 TIME WHO WAS AT NIH AND AFTER HE 14 00:00:56,520 --> 00:00:57,960 GRADUATED HE WENT AROUND THE 15 00:00:57,960 --> 00:00:58,360 WORLD. 16 00:00:58,360 --> 00:01:00,560 HE WAS IN FRANCE AND THEN IN 17 00:01:00,560 --> 00:01:04,560 GERMANY AND THEN IN NORTH 18 00:01:04,560 --> 00:01:05,680 CAROLINA AT THE UNIVERSITY OF 19 00:01:05,680 --> 00:01:07,880 NORTH CAROLINA CHAPEL HILL AND 20 00:01:07,880 --> 00:01:10,960 HE WAS STAYING WITH SAM WILSON 21 00:01:10,960 --> 00:01:14,760 BETWEEN GALVESTON TEXAS AND THEN 22 00:01:14,760 --> 00:01:19,160 NIEHS UNTIL HE WENT UP TO 23 00:01:19,160 --> 00:01:20,080 PITTSBURGH FOR A WHILE. 24 00:01:20,080 --> 00:01:22,480 HE WAS THERE AND SINCE 2014 HE'S 25 00:01:22,480 --> 00:01:27,160 BEEN IN MOBILE ALABAMA. 26 00:01:27,160 --> 00:01:28,440 SX HEAT USA, THE UNIVERSITY OF 27 00:01:28,440 --> 00:01:33,640 SOUTH ALABAMA, HE WILL BE 28 00:01:33,640 --> 00:01:35,000 TALKING ABOUT THE MECHANISM OF 29 00:01:35,000 --> 00:01:37,920 BASE EXCISION REPAIR IN HUMAN 30 00:01:37,920 --> 00:01:40,720 CELLS AND WE WILL SEE IF 31 00:01:40,720 --> 00:01:42,520 DR. BORE HAS ANYTHING TO ADD. 32 00:01:42,520 --> 00:01:43,160 >> NOT REALLY. 33 00:01:43,160 --> 00:01:45,800 I JUST WELCOME YOU ROB, I'M 34 00:01:45,800 --> 00:01:50,120 LOOKING FORWARD TO THIS 35 00:01:50,120 --> 00:01:52,560 DISCUSSION, THE MECHANISM, AND 36 00:01:52,560 --> 00:01:53,720 EVERYTHING DONE WITH PAUL BETA 37 00:01:53,720 --> 00:01:54,720 AND PROTEIN AND [INDISCERNIBLE] 38 00:01:54,720 --> 00:01:57,600 AND THEN YOU HAVE ALSO 39 00:01:57,600 --> 00:02:01,160 CONTRIBUTED TO MANY ASPECTS OF 40 00:02:01,160 --> 00:02:06,600 BER INCLUDING AT THE NAD 41 00:02:06,600 --> 00:02:07,120 METABOLISM, MEETINGS AND 42 00:02:07,120 --> 00:02:10,000 DELIGHTED TO HAVE YOU SPEAK HERE 43 00:02:10,000 --> 00:02:14,600 FROM FROM OUR USA--FROM USA TO 44 00:02:14,600 --> 00:02:15,920 THE INTERNATIONAL FORUM. 45 00:02:15,920 --> 00:02:17,160 SO WELCOME, AND THANKS AND JUST 46 00:02:17,160 --> 00:02:18,960 1 MORE TIME WE HAVE A FEW PEOPLE 47 00:02:18,960 --> 00:02:20,560 HERE, WE HAVE ABOUT 5 OR 6 48 00:02:20,560 --> 00:02:23,280 PEOPLE WHO HAVE NOT MUTED THEIR 49 00:02:23,280 --> 00:02:24,760 MICROPHONE, IF YOU KINDLY WILL 50 00:02:24,760 --> 00:02:26,680 DO THAT, IT'S APPRECIATED 51 00:02:26,680 --> 00:02:29,360 BECAUSE WE--OTHERWISE WE GET 52 00:02:29,360 --> 00:02:31,640 BACKGROUND NOISE AND THEN, 53 00:02:31,640 --> 00:02:34,720 AGAIN, QUESTIONS BY CHAT, 54 00:02:34,720 --> 00:02:35,520 PLEASE. 55 00:02:35,520 --> 00:02:37,120 SO PLEASE ROB. 56 00:02:37,120 --> 00:02:39,040 >> OKAY, THANKS SO MUCH KEN AND 57 00:02:39,040 --> 00:02:42,320 WILL AND EVERYONE ATTENDING 58 00:02:42,320 --> 00:02:43,520 TODAY: WELCOME TO ALL OF YOU 59 00:02:43,520 --> 00:02:45,240 FROM HERE ON THE GULF COAST AND 60 00:02:45,240 --> 00:02:48,120 DISCUSS ONGOING WORK HERE ON 61 00:02:48,120 --> 00:02:49,680 MECHANISM OF BASE EXCISION 62 00:02:49,680 --> 00:02:51,080 REPAIR REGULATION AND THOSE OF 63 00:02:51,080 --> 00:02:52,560 YOU THAT GET TO VISIT US HERE 64 00:02:52,560 --> 00:02:54,160 THIS IS THE MITCHELL CANCER 65 00:02:54,160 --> 00:02:58,280 EN--STRATEGIESITUTE AS PART OF 66 00:02:58,280 --> 00:03:01,200 THE UNIVERSITY OF SOUTH ALABAMA 67 00:03:01,200 --> 00:03:03,000 SCHOOL OF MEDICINE AND MY LAB IS 68 00:03:03,000 --> 00:03:06,080 LOCATED HERE IN THE TOP AND 69 00:03:06,080 --> 00:03:07,880 WE'VE BEEN VERY HAPPY IN TERMS 70 00:03:07,880 --> 00:03:09,200 OF HOW WE'RE MOVING FORWARD 71 00:03:09,200 --> 00:03:09,400 HERE. 72 00:03:09,400 --> 00:03:11,200 I WILL GIVE YOU SOME BACKGROUND 73 00:03:11,200 --> 00:03:12,880 IN TERMS OF WHERE--HOW WE GOT TO 74 00:03:12,880 --> 00:03:15,240 WHERE WE ARE TODAY AND THEN I'LL 75 00:03:15,240 --> 00:03:18,200 SHARE SOME WORK THAT WE'RE JUST 76 00:03:18,200 --> 00:03:18,520 FINISHING UP. 77 00:03:18,520 --> 00:03:20,400 SO BECAUSE I DON'T WANT TO 78 00:03:20,400 --> 00:03:21,840 FORGET ANYTHING, THIS IS THE 79 00:03:21,840 --> 00:03:23,000 GROUP, SO THESE ARE THE FOLKS 80 00:03:23,000 --> 00:03:26,480 THAT ARE DOING ALL ACTUALLY ALL 81 00:03:26,480 --> 00:03:28,200 THE PIPETTING AND EXOTIC WORK 82 00:03:28,200 --> 00:03:29,800 THAT I'LL DESCRIBE TODAY AND I 83 00:03:29,800 --> 00:03:32,040 WILL BE REFERRING TO THE 84 00:03:32,040 --> 00:03:38,120 INDIVIDUALS THAT HAVE CONSIDERED 85 00:03:38,120 --> 00:03:38,440 THROUGHOUT. 86 00:03:38,440 --> 00:03:39,640 AND THEN OF COURSE I WANT TO 87 00:03:39,640 --> 00:03:41,560 THANK A FEW KEY FOLKS. 88 00:03:41,560 --> 00:03:45,240 THESE ARE MEMBERS OF 1 OF THE 89 00:03:45,240 --> 00:03:47,040 PROGRAMS AT OUR CENTERS SO 90 00:03:47,040 --> 00:03:49,960 NATALIE [INDISCERNIBLE] WHO HAS 91 00:03:49,960 --> 00:03:50,520 INTRODUCED US TO CONFOCAL 92 00:03:50,520 --> 00:03:51,840 MICROSCOPY AND HOW THAT CAN BE 93 00:03:51,840 --> 00:03:53,880 USED TO LOOK AT DNA REPAIR 94 00:03:53,880 --> 00:03:56,640 MECHANISM AT LEAST IN TERMS OF 95 00:03:56,640 --> 00:03:57,520 COMPLEX ASSEMBLY, SO WHICH IS 96 00:03:57,520 --> 00:03:59,520 WHAT I WILL FOCUS ON TODAY. 97 00:03:59,520 --> 00:04:00,640 MARIE [INDISCERNIBLE] WHO IS A 98 00:04:00,640 --> 00:04:08,120 PROFESSOR NOW IN THE DEPARTMENT 99 00:04:08,120 --> 00:04:13,120 HAS A HISTORY OF WORKING WITH 100 00:04:13,120 --> 00:04:15,240 VITAMINS AND N. A. D. MOLECULES 101 00:04:15,240 --> 00:04:16,880 AND [INDISCERNIBLE] WHO WE'VE 102 00:04:16,880 --> 00:04:19,720 WORKED ON SEVERAL PROJECTS AND 103 00:04:19,720 --> 00:04:21,840 THOSE OF YOU WHO KNOW SHE'S A 104 00:04:21,840 --> 00:04:22,800 STRUCTURAL BIOLOGIST AND WE'RE 105 00:04:22,800 --> 00:04:25,120 ALL HAPPY THEY ARE PART OF OUR 106 00:04:25,120 --> 00:04:27,240 CANCER CENTER AND OUR 107 00:04:27,240 --> 00:04:27,480 UNIVESITY. 108 00:04:27,480 --> 00:04:31,240 SO, AS WILL ALLUDED WE'VE BEEN 109 00:04:31,240 --> 00:04:32,520 WORKING ON BASIC CISION REPAIR 110 00:04:32,520 --> 00:04:34,320 AND FOR A LONG TIME AND IN 111 00:04:34,320 --> 00:04:35,040 PARTICULAR, WE'VE ALWAYS BEEN 112 00:04:35,040 --> 00:04:40,320 THINKING ABOUT IT FROM THE 113 00:04:40,320 --> 00:04:42,800 PERSPECTIVE OF PORP, AND TO GIVE 114 00:04:42,800 --> 00:04:46,040 YOU BACKGROUND, BUT JUST TO 115 00:04:46,040 --> 00:04:47,400 REORIENT EVERYONE, THESE PLAY A 116 00:04:47,400 --> 00:04:48,440 CRITICAL ROLE IN GENOME 117 00:04:48,440 --> 00:04:51,040 STABILITY AND THESE PROTEINS ARE 118 00:04:51,040 --> 00:04:53,320 ALL TO SOME EXTENT SOMEWHAT 119 00:04:53,320 --> 00:04:55,960 AFFECTED IN CANCER IN VARIOUS 120 00:04:55,960 --> 00:04:58,840 WAYS, AN INTERESTING ASPECT FOR 121 00:04:58,840 --> 00:05:00,760 EXAMPLE IS [INDISCERNIBLE] CROSS 122 00:05:00,760 --> 00:05:01,960 LINK REPAIR, WITH NEW FINDINGS 123 00:05:01,960 --> 00:05:05,160 AND THEN MANY OF THESE FACTORS 124 00:05:05,160 --> 00:05:08,640 AS I LIKE TO CALL THEM IN BASIC 125 00:05:08,640 --> 00:05:10,080 [INDISCERNIBLE] ARE CONSIDERED 126 00:05:10,080 --> 00:05:14,600 NOW TO BE NOVEL AND DRUGGABLE 127 00:05:14,600 --> 00:05:15,360 TARGETS FOR SYNTHETIC LETHALITY 128 00:05:15,360 --> 00:05:17,560 AND IT RAISES THE QUESTION OF 129 00:05:17,560 --> 00:05:19,440 WHAT OTHER FACTORS CAN WE THINK 130 00:05:19,440 --> 00:05:21,440 ABOUT AND WHAT I ALSO LIKE TO 131 00:05:21,440 --> 00:05:22,360 THINK ABOUT, WELL, 1 THING TO 132 00:05:22,360 --> 00:05:24,960 POINT OUT HERE IS OF COURSE THAT 133 00:05:24,960 --> 00:05:26,520 PART 1 AND ININCREASE IN 134 00:05:26,520 --> 00:05:28,440 BODYITORS ARE NOW CONSIDERED AS 135 00:05:28,440 --> 00:05:29,240 EFFECTIVE SMALL MOLECULES AND 136 00:05:29,240 --> 00:05:32,560 TREATMENT OF CANCER IN 137 00:05:32,560 --> 00:05:35,280 PARTICULAR FOR CANCERS WITH 138 00:05:35,280 --> 00:05:37,240 DEFECTS IN BRCCA 1 AND 2, WE ALL 139 00:05:37,240 --> 00:05:38,240 KNOW THESE FINDINGS BUT THIS 140 00:05:38,240 --> 00:05:40,840 SPEAKS TO WHAT WE'RE ENDEAVORING 141 00:05:40,840 --> 00:05:43,640 TOO IS UNCOVER THESE NOVEL 142 00:05:43,640 --> 00:05:45,320 EXCISION REPAIR OR SINGLE STRAND 143 00:05:45,320 --> 00:05:46,800 BREAK ARE REPAIR AND THOSE 144 00:05:46,800 --> 00:05:48,640 FACTORS THAT CAN BE REGULATED 145 00:05:48,640 --> 00:05:49,920 AND TARGETING SYSTEMYETED FOR 146 00:05:49,920 --> 00:05:51,240 EFFECTIVE RESPONSE IN CANCER, SO 147 00:05:51,240 --> 00:05:54,760 THAT'S THE BIG PICTURE OF OUR 148 00:05:54,760 --> 00:05:57,040 OVERALL THEME IN THE LAB. 149 00:05:57,040 --> 00:06:01,320 AGAIN GOING BACK IN TIME AS WE 150 00:06:01,320 --> 00:06:02,760 THINK ABOUT EXCISION REPAIR 151 00:06:02,760 --> 00:06:05,480 ORIGINALLY 1 THING ABOUT IT IS A 152 00:06:05,480 --> 00:06:06,720 VERY SIMPLE FORMAT, BASE DAMAGE 153 00:06:06,720 --> 00:06:09,760 IS INN DUCED BY A VARIETY OF 154 00:06:09,760 --> 00:06:12,480 SOURCES MOST OF THE CASES THAT 155 00:06:12,480 --> 00:06:14,840 DAMAGE IS REMOVED BY GLUE 156 00:06:14,840 --> 00:06:16,680 MARIOUS COSALATE AND BY BASIC 157 00:06:16,680 --> 00:06:18,680 SITES AND POLYMER ACE DATA 158 00:06:18,680 --> 00:06:20,400 CARRIES OUT 2 OF THESE TYPICAL 159 00:06:20,400 --> 00:06:22,800 FUNCTIONS IN THE CONTEXT OF THIS 160 00:06:22,800 --> 00:06:25,680 PATHWAY, BOTH TAYLORING THE GAP 161 00:06:25,680 --> 00:06:29,400 WITH THE PRIME DRP MICE FUNCTION 162 00:06:29,400 --> 00:06:30,800 AND THEN SYNTHESIZING DNA TO 163 00:06:30,800 --> 00:06:32,720 FILL THE GAP AND THEN 164 00:06:32,720 --> 00:06:34,840 SUBSEQUENTLY DO IT WITH THE 165 00:06:34,840 --> 00:06:35,200 LIGATION STEP. 166 00:06:35,200 --> 00:06:46,880 SO, WE KNOW OF COURSE THAT BER 167 00:06:46,880 --> 00:06:49,840 DATA IS RESPONSIBLE FOR A LOT OF 168 00:06:49,840 --> 00:06:50,560 DATA. 169 00:06:50,560 --> 00:06:52,960 SO BETA CAN COMPLEMENT THE KNOCK 170 00:06:52,960 --> 00:06:56,840 OUT TO PROVIDE SURVIVABILITY 171 00:06:56,840 --> 00:06:59,640 WHEREAS THE LYASE DEFICIENT 172 00:06:59,640 --> 00:07:01,440 MUTANT DOES NOT APPEAR TO 173 00:07:01,440 --> 00:07:03,160 PROVIDE THAT COMPLEMENTATION, SO 174 00:07:03,160 --> 00:07:05,560 THAT TELL US THAT THE FUNCTION 175 00:07:05,560 --> 00:07:07,160 IS IMPORTANT AND NOT JUST THE 176 00:07:07,160 --> 00:07:08,920 PROTEIN ITSELF OR AT LEAST IN 177 00:07:08,920 --> 00:07:11,040 THE CONOF EXPOSURE LIKE AN 178 00:07:11,040 --> 00:07:14,960 ALKALINE OR IN THIS CASE 179 00:07:14,960 --> 00:07:17,640 [INDISCERNIBLE], WHEN WE THINK 180 00:07:17,640 --> 00:07:18,840 ABOUT BASE EXCISION REPAIR 181 00:07:18,840 --> 00:07:19,960 OSINGLE BREAK REPAIR, AS I 182 00:07:19,960 --> 00:07:23,560 POINTED OUT WE HAVE TO REALIZE 183 00:07:23,560 --> 00:07:25,360 THAT IT PLAYS A CRITICAL ROLE 184 00:07:25,360 --> 00:07:26,440 AND FACTOR IN MAMMALIAN CELL 185 00:07:26,440 --> 00:07:29,440 THIS IS IS A COMPLICATED ENZYME 186 00:07:29,440 --> 00:07:31,360 WITH NUMEROUS DOMAINS AND THE 187 00:07:31,360 --> 00:07:35,000 GENERAL CONCEPT HERE IS THAT 188 00:07:35,000 --> 00:07:36,520 PARK RECOGNIZES THE BREAK AND IN 189 00:07:36,520 --> 00:07:39,040 THE PRESENCE OF N. A. D. 190 00:07:39,040 --> 00:07:40,840 GENERATES THESE POLYMERS OF 191 00:07:40,840 --> 00:07:42,240 RIBOSE THAT FUNCTION AS A 192 00:07:42,240 --> 00:07:44,240 LANDING PAD IF YOU WILL TO 193 00:07:44,240 --> 00:07:44,920 FACILITATE RECRUITMENT AND THEN 194 00:07:44,920 --> 00:07:46,920 OF COURSE WE HAVE TO REMEMBER 195 00:07:46,920 --> 00:07:49,520 THAT THERE ARE IN EFFECT 196 00:07:49,520 --> 00:07:51,720 REVERSAL REACTIONS, THE MAIN 197 00:07:51,720 --> 00:07:54,920 ENZYME [INDISCERNIBLE] WHICH 198 00:07:54,920 --> 00:07:59,160 REMOVES PRIMATES MARLY ALL THE 199 00:07:59,160 --> 00:08:01,080 RIBOSE MOIT ESTIMATE THAD AND 200 00:08:01,080 --> 00:08:04,240 THEN 3 WHICH TENDS TO BREAK DOWN 201 00:08:04,240 --> 00:08:06,520 THAT POLYMER AS ITS DEGRADING. 202 00:08:06,520 --> 00:08:08,960 OF COURSE, PARK 1 IS HIGHLY 203 00:08:08,960 --> 00:08:10,000 REGULATED AS EVIDENCED BY A LOT 204 00:08:10,000 --> 00:08:12,840 OF THE WORK AND PEOPLE LISTENING 205 00:08:12,840 --> 00:08:16,360 TODAY AND THIS IS THROUGH POST 206 00:08:16,360 --> 00:08:17,680 TRANSLATIONAL MODIFICATION AMONG 207 00:08:17,680 --> 00:08:19,240 OTHER MECHANISMS AND WHAT WE'RE 208 00:08:19,240 --> 00:08:22,880 GOING TO TOUCH ON IN THE FINAL 209 00:08:22,880 --> 00:08:29,960 SECTION OF TODAY'S TALK IS SER6 210 00:08:29,960 --> 00:08:35,440 AND HOW IT MAY REGULATE PARK AND 211 00:08:35,440 --> 00:08:37,080 P A RP AND EXCISION REPAIR. 212 00:08:37,080 --> 00:08:38,800 SOPHISTICATED AS I'VE BEEN 213 00:08:38,800 --> 00:08:43,040 SAYING WE WE'VE BEEN LOOKING AT 214 00:08:43,040 --> 00:08:44,880 THE QUALITY RIBOSE, WHEN YOU 215 00:08:44,880 --> 00:08:47,040 HAVE A BER SUPPRESSED SITUATION 216 00:08:47,040 --> 00:08:52,040 FOR EXAMPLE, AND YOU EXPOSE AN 217 00:08:52,040 --> 00:08:53,560 ALKACCTINATOR AND YOU SEE THE 218 00:08:53,560 --> 00:08:55,720 ACCUMULATION BUT IF YOU HAVE AN 219 00:08:55,720 --> 00:08:58,840 ACTIVE OR HYPER ACT BASE 220 00:08:58,840 --> 00:09:00,840 EXCISION REPAIR SITUATION WITH 221 00:09:00,840 --> 00:09:04,520 LIMIT THE POLYMER ACE BET ASSUEY 222 00:09:04,520 --> 00:09:07,920 THE ACCUMULATION OF THE P A RP 223 00:09:07,920 --> 00:09:14,400 BETA IS FURTHER SUPPRESSED YOU 224 00:09:14,400 --> 00:09:18,640 SEE IT BRANDED ON THE EXPRESSION 225 00:09:18,640 --> 00:09:20,680 OF POLB, TO ELEMINATE THAT, 226 00:09:20,680 --> 00:09:22,120 KIEWM AWLINGS AND THEN 227 00:09:22,120 --> 00:09:24,600 INTERESTINGLY IF YOU EXPRESS THE 228 00:09:24,600 --> 00:09:27,640 MUTANT THAT CANNOT REPAIR THAT 229 00:09:27,640 --> 00:09:30,360 OR TRIM THE GAP, WHEN YOU SLIDE, 230 00:09:30,360 --> 00:09:32,600 THAT DOESN'T SEEM TO EFFECT THE 231 00:09:32,600 --> 00:09:34,320 P A RP ACTIVATION. 232 00:09:34,320 --> 00:09:40,480 SO THIS SUBJESTS THAT IN THAT 233 00:09:40,480 --> 00:09:42,240 MODEL THE BER LESION MIGHT BE 234 00:09:42,240 --> 00:09:45,640 VERY STRONG FOR ACTIVATING A 235 00:09:45,640 --> 00:09:45,840 LESION. 236 00:09:45,840 --> 00:09:48,000 SO WE'VE THOUGHT ABOUT THAT IN 237 00:09:48,000 --> 00:09:49,920 THAT MODEL FOR MANY YEARS AND 238 00:09:49,920 --> 00:09:51,880 THEN WHAT'S INTERESTING, TOO, IS 239 00:09:51,880 --> 00:09:53,720 THAT NOT ONLY IS P A RP A 240 00:09:53,720 --> 00:09:57,680 CRITICAL FACTOR BUT THEN THE NAD 241 00:09:57,680 --> 00:09:58,560 FACTORS INTO THIS. 242 00:09:58,560 --> 00:10:03,000 SO YOU CAN SUPPRESS THIS HYPER 243 00:10:03,000 --> 00:10:04,640 TENSATIVITY BECAUSE OF P A RP 244 00:10:04,640 --> 00:10:08,640 ACTIVATION SIMPLY BY ADDING NAD 245 00:10:08,640 --> 00:10:11,640 PRECURSORS, IN THIS CASE N MN 246 00:10:11,640 --> 00:10:14,280 THAT ACTS AS A PRECURSOR TO 247 00:10:14,280 --> 00:10:16,000 SYNTHESIZING N. A. B. AND NOW 248 00:10:16,000 --> 00:10:17,960 THIS IS OLDER WORK AND I WILL 249 00:10:17,960 --> 00:10:19,760 DISCUSS MORE DETAILED COMPONENTS 250 00:10:19,760 --> 00:10:21,520 THAT REALLY POINT AT N. A. D. 251 00:10:21,520 --> 00:10:24,640 BEING A CRITICAL FACTOR HERE. 252 00:10:24,640 --> 00:10:26,560 SO AGAIN, WE LOOK AT MAMMALIAN 253 00:10:26,560 --> 00:10:29,760 BASE REPAIR FROM THIS VIEW POINT 254 00:10:29,760 --> 00:10:31,360 AND AS WE'VE DISCUSSED AND 255 00:10:31,360 --> 00:10:34,240 REPORTED IN THE ABSENCE OF LET'S 256 00:10:34,240 --> 00:10:37,040 SAY COMPLETION OF REPAIR, YOU 257 00:10:37,040 --> 00:10:40,440 END UP WITH THIS RIBOS E 258 00:10:40,440 --> 00:10:42,440 SIMULATION AND THE RESULTING 259 00:10:42,440 --> 00:10:43,040 CELL DEATH. 260 00:10:43,040 --> 00:10:46,240 THIS PUTS P A RP UPSTREAM OF 261 00:10:46,240 --> 00:10:48,920 POLB AND NAD WOULD BE AN 262 00:10:48,920 --> 00:10:50,320 ESSENTIAL CO-FACTOR AND THIS HAS 263 00:10:50,320 --> 00:10:52,600 BEEN SUGGESTED AND SUPPORTED BY 264 00:10:52,600 --> 00:10:54,280 MANY OTHER STUDIES THAT WHEN 265 00:10:54,280 --> 00:10:57,240 DEPENDING ON NAD LEVELS YOU 266 00:10:57,240 --> 00:10:59,360 MIGHT SEE VARIOUS LEVELS OF CELL 267 00:10:59,360 --> 00:11:02,360 DEATH ASSOCIATED WITH THE BER 268 00:11:02,360 --> 00:11:02,680 ACCUMULATION. 269 00:11:02,680 --> 00:11:06,560 OF COURSE, NAD IS NOT SYNTHESIS 270 00:11:06,560 --> 00:11:09,440 IS NOT SPRAYED FORWARD, IT IS 271 00:11:09,440 --> 00:11:10,640 MANY DIFFERENT PATHWAYS, IT 272 00:11:10,640 --> 00:11:12,160 CONTRIBUTES TO THE PRODUCTION OF 273 00:11:12,160 --> 00:11:13,360 NIEE, AUDIENCE D AND THIS IS 274 00:11:13,360 --> 00:11:15,360 JUST SOME OF IT LAID OUT HERE 275 00:11:15,360 --> 00:11:18,160 FOR YOU ALL TO SEE AND OF COURSE 276 00:11:18,160 --> 00:11:21,360 THE NAD THAT'S SYNTHESIZED IS 277 00:11:21,360 --> 00:11:25,040 IMPORTANT FOR VARIOUS POST 278 00:11:25,040 --> 00:11:27,360 TRANSLATIONAL MODIFICATIONS 279 00:11:27,360 --> 00:11:29,320 INCLUDING B-ASSIMILATION AND 280 00:11:29,320 --> 00:11:32,000 MONOCLONAL ASSIMILATION AND 281 00:11:32,000 --> 00:11:33,520 POLYB ASSIMILATION AS WELL AS 282 00:11:33,520 --> 00:11:36,760 METABOLIC FUNCTIONS THAT N. A. 283 00:11:36,760 --> 00:11:39,080 D. CONTRIBUTES TO AND 1 CAN 284 00:11:39,080 --> 00:11:40,640 EFFECT THIS AND ASK QUESTIONS 285 00:11:40,640 --> 00:11:45,120 ABOUT NAD'S ROLE IN REPAIR VERY 286 00:11:45,120 --> 00:11:46,360 SIMPLY BY DEPLETING THIS WITH A 287 00:11:46,360 --> 00:11:52,840 NICE ININCREASE IN BODYITOR OF 288 00:11:52,840 --> 00:11:56,800 THIS NAM T ENZYME--NEVER MIND, 289 00:11:56,800 --> 00:11:59,240 I'M NOT POINTING--NAM T ENZYME 290 00:11:59,240 --> 00:12:01,480 YOU CAN INHIBIT EFFECTIVELY WITH 291 00:12:01,480 --> 00:12:04,880 FD6 ANDEE USE THIS A LOT TO 292 00:12:04,880 --> 00:12:06,240 EFFECT BASE CISION REPAIRS. 293 00:12:06,240 --> 00:12:08,600 THIS IS PATHWAY GIVES MOLECULE, 294 00:12:08,600 --> 00:12:11,080 VERY EFFECTIVE INHIBITOR WITHIN 295 00:12:11,080 --> 00:12:14,720 ABOUT 24 HOURS, GOING TO SEE 80% 296 00:12:14,720 --> 00:12:15,720 PROGRESSION OF NAD. 297 00:12:15,720 --> 00:12:16,760 SO WE ASKED THE SIMPLE QUESTION, 298 00:12:16,760 --> 00:12:19,600 IF WE TAKE A LOOK AT THE VARIETY 299 00:12:19,600 --> 00:12:20,960 OF [INDISCERNIBLE] AGENTS THAT 300 00:12:20,960 --> 00:12:23,040 ALL DAMAGE THE DNA IN VARIOUS 301 00:12:23,040 --> 00:12:24,280 DIFFERENT WAYS, AS CAN YOU SEE 302 00:12:24,280 --> 00:12:28,560 HERE BY MEASURED BY THE CONIC 303 00:12:28,560 --> 00:12:32,440 CHIP ASSAY, CAN YOU SEE ENHANCED 304 00:12:32,440 --> 00:12:34,560 DNA DAMAGE IN A VARIETY OF 305 00:12:34,560 --> 00:12:35,840 DIFFERENT AGENTS AND THE 306 00:12:35,840 --> 00:12:38,440 QUESTION SIMPLY IS, IF WE CHANGE 307 00:12:38,440 --> 00:12:40,840 THE NAD LEVELS DOES THAT AFFECT 308 00:12:40,840 --> 00:12:43,360 THE ACCUMULATION OR REPAIR OF 309 00:12:43,360 --> 00:12:44,760 THIS DNA DAMAGE, AND AS YOU 310 00:12:44,760 --> 00:12:47,840 MIGHT PREDICT, THIS IS NOT 311 00:12:47,840 --> 00:12:48,560 UNIVERSAL PHENOMENON. 312 00:12:48,560 --> 00:12:53,360 SO FOR EXAMPLE, A TOPOCID HAS 313 00:12:53,360 --> 00:12:55,800 NO--IS NOT AFFECTED BY LEVELS OF 314 00:12:55,800 --> 00:12:57,720 NAD, SO WE SUPPRESS THE NAD 315 00:12:57,720 --> 00:13:02,080 LEVELS TO ABOUT 10 OR 15% OF THE 316 00:13:02,080 --> 00:13:02,320 TOTAL. 317 00:13:02,320 --> 00:13:06,720 WE ASK IF A TOPOSIDE REPAIR, AND 318 00:13:06,720 --> 00:13:07,640 MEDIATED DNA DAMAGE IS THERE AND 319 00:13:07,640 --> 00:13:10,880 YOU CAN SEE THERE'S NO EFFECT, 320 00:13:10,880 --> 00:13:12,080 YET AGENTS LIKE MMS, WHICH 321 00:13:12,080 --> 00:13:15,000 HISTORICALLY IS THOUGHT TO GO 322 00:13:15,000 --> 00:13:16,440 THROUGH BASIC EXCISION REPAIR, 323 00:13:16,440 --> 00:13:20,280 YOU SEE THIS VERY ROBUST AND 324 00:13:20,280 --> 00:13:21,480 SIGNIFICANT EFFECT ON THE DAMAGE 325 00:13:21,480 --> 00:13:23,280 LEVELS AND THE REPAIR LEVELS SO 326 00:13:23,280 --> 00:13:26,600 AGAIN SUPPORTING A ROLE FOR NAD 327 00:13:26,600 --> 00:13:29,480 IS A CRITICAL BASE EXCISION 328 00:13:29,480 --> 00:13:29,840 REPAIR FACTOR. 329 00:13:29,840 --> 00:13:31,680 SO 1 OF THE QUESTIONS THAT WE 330 00:13:31,680 --> 00:13:36,640 HAD IN A RECENT REPORT IS CAN WE 331 00:13:36,640 --> 00:13:37,320 MODULATE REPAIR COMPLEX 332 00:13:37,320 --> 00:13:39,080 FORMATION WITH ALTERATIONS IN 333 00:13:39,080 --> 00:13:40,600 NAD LEVELS AND WE DID THIS IN 334 00:13:40,600 --> 00:13:42,200 THE CONTEXT OF A BIGGER STUDY 335 00:13:42,200 --> 00:13:44,720 AND THIS IS JUST SOME SUMMARIES 336 00:13:44,720 --> 00:13:49,560 HERE, SO WE LOOKED AT THE EFFECT 337 00:13:49,560 --> 00:13:54,360 OF EITHER SUPPRESSION OF NAD 338 00:13:54,360 --> 00:13:57,120 THROUGH FK H6, OR 339 00:13:57,120 --> 00:13:58,120 SUPPLEMENTATION OF PRECURSORS 6 340 00:13:58,120 --> 00:14:01,520 HOURS IN BOTH FETAL BOVINE SERUM 341 00:14:01,520 --> 00:14:03,320 AND IN HEATED ACTIVATED BOVINE 342 00:14:03,320 --> 00:14:05,840 SERUM AND IN SERUM FREE MEDIA 343 00:14:05,840 --> 00:14:08,240 AND REALLY WHAT WAS INTERESTING 344 00:14:08,240 --> 00:14:10,240 HERE WAS THAT WHEREAS WE SEE A 345 00:14:10,240 --> 00:14:13,120 ROBUST EFFECT IN THIS CONTEXT, 346 00:14:13,120 --> 00:14:21,640 EITHER IN SERUM FREE MEDIA AND 347 00:14:21,640 --> 00:14:25,720 IN--REALIZE THAT FETAL BOVINE 348 00:14:25,720 --> 00:14:35,040 SERUM DEGRADES THE COMPOUND AND 349 00:14:35,040 --> 00:14:36,880 IT DOES POINT AND REPAIR BECAUSE 350 00:14:36,880 --> 00:14:37,640 OF THAT DECREASED NIEE, AUDIENCE 351 00:14:37,640 --> 00:14:39,200 D LEVEL AND WE WILL GET INTO 352 00:14:39,200 --> 00:14:42,640 THIS A BIT MORE IN THE MEET OF 353 00:14:42,640 --> 00:14:43,560 THE STORY HERE. 354 00:14:43,560 --> 00:14:46,880 SO AGAIN, AS WE THINK ABOUT DNA 355 00:14:46,880 --> 00:14:49,200 REPAIR FROM A GLOBAL PERSPECTIVE 356 00:14:49,200 --> 00:14:50,200 IS OBVIOUSLY MANY, MANY 357 00:14:50,200 --> 00:14:53,120 DIFFERENT PATHWAYS AND PROTEINS 358 00:14:53,120 --> 00:14:53,400 INVOLVED. 359 00:14:53,400 --> 00:14:57,360 P A RP IS EMERGING TO PLAY A 360 00:14:57,360 --> 00:15:00,600 ROLE NOT ONLY IN BASE EXCISION 361 00:15:00,600 --> 00:15:02,160 REPAIR BUT NUCLEOTIDE EXCISION 362 00:15:02,160 --> 00:15:04,360 REPAIR AND VARIOUS COMPONENTS OF 363 00:15:04,360 --> 00:15:05,240 DOUBLE STRAND LAYER REPAIR AND 364 00:15:05,240 --> 00:15:08,040 IF YOU LAYER ON TOP OF THAT, THE 365 00:15:08,040 --> 00:15:09,960 NAD AND P A RP FORMATION THAT'S 366 00:15:09,960 --> 00:15:11,520 NECESSARY AND ALL OF THOSE CO 367 00:15:11,520 --> 00:15:13,040 FACTORS THAT ARE NECESSARY, YOU 368 00:15:13,040 --> 00:15:15,240 CAN SEE ENTIRE PATH, ALL THE 369 00:15:15,240 --> 00:15:17,320 PATHWAYS SEEM TO BE POST 370 00:15:17,320 --> 00:15:18,160 TRANSLATIONING MODIFIED IN SUCH 371 00:15:18,160 --> 00:15:20,760 A WAY THAT NAD IS PROBABLY A 372 00:15:20,760 --> 00:15:21,360 VERY CRITICAL FACTOR. 373 00:15:21,360 --> 00:15:23,840 THAT'S 1 OF THE COMPONENTS WE'VE 374 00:15:23,840 --> 00:15:26,840 BEEN INTERESTED IN PURSUING. 375 00:15:26,840 --> 00:15:29,120 WHAT WE'LL TALK ABOUT TODAY IS 376 00:15:29,120 --> 00:15:31,320 IN THE CONTEXT OF BASE CISION 377 00:15:31,320 --> 00:15:33,920 REPAIR SO AGAIN IF WE LOOK AT 378 00:15:33,920 --> 00:15:36,440 THIS MODEL, WE ALL KNOW THE 379 00:15:36,440 --> 00:15:38,920 COMPLEXITY HERE AS WE START TO 380 00:15:38,920 --> 00:15:42,200 THINK ABOUT IT, ABOUT 20 ENZYMES 381 00:15:42,200 --> 00:15:44,240 INVOLVED, BUT 1 OF THE THINGS 382 00:15:44,240 --> 00:15:46,520 THAT THIS OBVIOUSLY CHANGES IN 383 00:15:46,520 --> 00:15:48,440 THE CONTEXT OF [INDISCERNIBLE] 384 00:15:48,440 --> 00:15:52,840 WORRY ABOUT AND SO IN TERMS OF 385 00:15:52,840 --> 00:15:56,800 THE CHROMATIN MEDIATED REPAIR OR 386 00:15:56,800 --> 00:15:59,280 THE REPAIR A CHROMATIN P A RP 387 00:15:59,280 --> 00:16:00,720 BECOMES A VERY IMPORTANT PLAYER 388 00:16:00,720 --> 00:16:03,960 TO HELP UNWIND THE COMPACT 389 00:16:03,960 --> 00:16:06,240 SITUATION WHERE THE DNA IS 390 00:16:06,240 --> 00:16:11,440 EMBEDDED IN THESE HISTONES AND 391 00:16:11,440 --> 00:16:12,960 OTHER FACTORS AND THAT GIVES 392 00:16:12,960 --> 00:16:15,400 ACCESS TO THE DAMAGE DNA, THIS 393 00:16:15,400 --> 00:16:16,840 IS NOT A SURPRISE. 394 00:16:16,840 --> 00:16:19,840 P A RP IS WELL DOCUMENTED TO 395 00:16:19,840 --> 00:16:21,360 HAVE AIR ROLE IN CHROMATIN 396 00:16:21,360 --> 00:16:22,960 RELAXATION AND IF YOU SCOUR THE 397 00:16:22,960 --> 00:16:24,360 LITERATURE, CAN YOU SEE A HOST 398 00:16:24,360 --> 00:16:26,040 OF FACTORS THAT HAVE BEEN 399 00:16:26,040 --> 00:16:30,360 SUGGESTED TO BE P A RP DEPENDENT 400 00:16:30,360 --> 00:16:31,560 CHROMATIN RELAXATION OR 401 00:16:31,560 --> 00:16:34,160 NUCLEOSOME EVIKS FACTORS AND SO 402 00:16:34,160 --> 00:16:39,240 WE NOW MORE THINK ABOUT THE VER 403 00:16:39,240 --> 00:16:40,640 OR SINGLE STRAND BREAK REPAIR TO 404 00:16:40,640 --> 00:16:43,120 HAVE A STEP WHERE THERE'S AN 405 00:16:43,120 --> 00:16:46,760 ESSENTIAL COMPONENT HERE THAT 406 00:16:46,760 --> 00:16:47,760 FACILITATE THIS AND OUR GOAL IS 407 00:16:47,760 --> 00:16:50,440 TO UNDERSTAND IN THE CONTEXT OF 408 00:16:50,440 --> 00:16:53,320 BASE EXCISION REPAIR WHAT ARE 409 00:16:53,320 --> 00:16:55,240 THOSE FACTORS? 410 00:16:55,240 --> 00:16:59,120 AND OF COURSE P A RP IS NOT ONLY 411 00:16:59,120 --> 00:17:02,680 PLAYING A ROLE IN BASE EXCISION 412 00:17:02,680 --> 00:17:04,320 REPAIR OR SINGLE STRAND REPAIR, 413 00:17:04,320 --> 00:17:08,400 IT PLAYS AN PRNTD ROLE IN SINGLE 414 00:17:08,400 --> 00:17:10,720 REPAIR, AND OF LATE IT PLAYS AN 415 00:17:10,720 --> 00:17:12,400 IMPORTANT ROLE FOR THE 416 00:17:12,400 --> 00:17:13,720 REPLICATION SPORTS, MAYBE WITH 417 00:17:13,720 --> 00:17:17,720 REGARD TO THE [INDISCERNIBLE] 418 00:17:17,720 --> 00:17:19,480 FRAGMENT ACTIVATION AND THEN 419 00:17:19,480 --> 00:17:22,120 ULTIMATELY THE REGULATION OF THE 420 00:17:22,120 --> 00:17:23,840 REPLICATION FOR IT. 421 00:17:23,840 --> 00:17:25,640 SO IN OUR CURRENT WORK WE'RE 422 00:17:25,640 --> 00:17:29,600 INTERESTED NOT ONLY IN THE TEMP 423 00:17:29,600 --> 00:17:32,000 RETURN OF RESULTSLY MAPPING IF 424 00:17:32,000 --> 00:17:34,880 YOU WILL THE INTERACT OHM SO WE 425 00:17:34,880 --> 00:17:37,000 CAN DISCOVER THESE FACTORS THAT 426 00:17:37,000 --> 00:17:39,240 ARE ESSENTIAL FOR NOT ONLY 427 00:17:39,240 --> 00:17:41,200 SINGLE STRAND BREAK REPAIR AND 428 00:17:41,200 --> 00:17:42,880 BASE EXCISION REPAIR AND BUT 429 00:17:42,880 --> 00:17:46,080 ALSO REPLICATION FORKS AND I'M 430 00:17:46,080 --> 00:17:48,240 REALLY JUST TO FOCUS ON THE BER 431 00:17:48,240 --> 00:17:50,920 COMPONENT FOR TODAY'S TALK. 432 00:17:50,920 --> 00:17:52,200 SO THIS IS OUR GLOBAL 433 00:17:52,200 --> 00:17:54,080 PERSPECTIVE AND HOW TO MOVE 434 00:17:54,080 --> 00:17:56,480 FORWARD AND UNCOVERING THESE 435 00:17:56,480 --> 00:17:56,720 FACTORS. 436 00:17:56,720 --> 00:17:59,840 TEMP RETURN OF RESULTSLY, WE'LL 437 00:17:59,840 --> 00:18:02,840 FIRST START OFF WITH TEMPORALLY 438 00:18:02,840 --> 00:18:04,440 MASKING THESE FACTORS AND WE'VE 439 00:18:04,440 --> 00:18:07,760 EMBRACED A FEW OF THESE AND 440 00:18:07,760 --> 00:18:09,200 THESE CLASSICAL PROTEIN INTERACT 441 00:18:09,200 --> 00:18:10,240 OME ANALYSIS AND I WILL TALK ON 442 00:18:10,240 --> 00:18:12,080 THAT TODAY AS WELL. 443 00:18:12,080 --> 00:18:13,440 SO FOR THOSE WHO HAVE NOT USED 444 00:18:13,440 --> 00:18:15,840 THIS, I KNOW MANY OF YOU 445 00:18:15,840 --> 00:18:17,440 PROBABLY HAVE, BIOID IS KIND OF 446 00:18:17,440 --> 00:18:19,120 A UNIQUE SYSTEM, THERE'S MANY OF 447 00:18:19,120 --> 00:18:22,440 THESE SIMILAR SYSTEMS OUT THERE 448 00:18:22,440 --> 00:18:25,320 NOW, BIOID ALLOWS 1 TO USE YOUR 449 00:18:25,320 --> 00:18:28,240 PROTEIN WITH THIS E.COLI FACTOR 450 00:18:28,240 --> 00:18:29,840 BIRA, AND IT'S A MODIFIED FACTOR 451 00:18:29,840 --> 00:18:32,920 AND SO IN THE PRESENCE OF 452 00:18:32,920 --> 00:18:37,040 ELEVATED BOTTLE OF BIOTIN 1 THEN 453 00:18:37,040 --> 00:18:38,640 EFFECT BIOACCUMULATING THOSE 454 00:18:38,640 --> 00:18:40,520 PROTEIN IN ABOUT 10 NANO METERS 455 00:18:40,520 --> 00:18:43,520 SO WE THINK OF THIS AS A WAY OF 456 00:18:43,520 --> 00:18:45,720 TEMPORALLY MAPPING THE BER 457 00:18:45,720 --> 00:18:48,360 INTERACT OHM FOR EXAMPLE. 458 00:18:48,360 --> 00:18:50,040 SO THAT THAT END WE'VE TAKEN IT 459 00:18:50,040 --> 00:18:51,640 UPON OURSELVES OF LOOKING AT ALL 460 00:18:51,640 --> 00:18:53,440 OF THESE PROTEINS, I WILL TOUCH 461 00:18:53,440 --> 00:18:56,960 ON A FEW OF THEM TODAY SO FOR 462 00:18:56,960 --> 00:18:58,640 EXAMPLE, GLUE MARIOUS COSLACES, 463 00:18:58,640 --> 00:19:01,160 THE WAY WE,A PROACH THIS IS WE 464 00:19:01,160 --> 00:19:03,840 KNOCK OUT THE GLUE MARIOUS 465 00:19:03,840 --> 00:19:08,800 COSLACE AND WE REEXPRESS THIS 466 00:19:08,800 --> 00:19:09,560 CONSTITTATIVELY OR REGULATED AS 467 00:19:09,560 --> 00:19:13,040 A FUSION AND THEN THIS GIVES AN 468 00:19:13,040 --> 00:19:15,360 OPPORTUNITY TO IDENTIFY FOR 469 00:19:15,360 --> 00:19:17,120 EXAMPLE, DAMAGE INDUCED INTERACT 470 00:19:17,120 --> 00:19:19,040 OHM OR CONSTITTATIVE INTERACT 471 00:19:19,040 --> 00:19:27,560 OHM WHAT HAVE YOU. 472 00:19:27,560 --> 00:19:28,440 --MPG AND THE GLUE MARIOUS 473 00:19:28,440 --> 00:19:30,640 COSLACE AND THEN WE REEXPRESS IN 474 00:19:30,640 --> 00:19:33,360 THE NEXT SLIDE, I'LL JUST SHOW 475 00:19:33,360 --> 00:19:37,240 YOU WE REEXPRESSED THE 476 00:19:37,240 --> 00:19:38,640 [INDISCERNIBLE] AS A PREFUSION 477 00:19:38,640 --> 00:19:40,040 TO BIRA, AND OF COURSE WHAT 478 00:19:40,040 --> 00:19:42,360 WOULD WE EXPECT IF WE DO THIS, 479 00:19:42,360 --> 00:19:43,080 RIGHT? 480 00:19:43,080 --> 00:19:45,520 WE KNOW THAT THE METHYL GLUE 481 00:19:45,520 --> 00:19:47,080 MARIOUS COSLACED ARE ESSENTIAL 482 00:19:47,080 --> 00:19:50,440 FOR THE REPAIR FOR EXAMPLE OF 483 00:19:50,440 --> 00:19:52,120 THEN ALKAALATED BASIS AND 484 00:19:52,120 --> 00:19:53,160 CONTEXT OF BASIC EXCISION REPAIR 485 00:19:53,160 --> 00:19:54,600 AND WE WOULD EXPECT TO FIND 486 00:19:54,600 --> 00:19:56,280 FACTORS THAT ARE CRITICAL HERE 487 00:19:56,280 --> 00:19:58,800 AND 1 CAN'T FORGET THAT WHEN YOU 488 00:19:58,800 --> 00:20:02,480 EXTO THINGS LIKE MMS, YOU END UP 489 00:20:02,480 --> 00:20:04,680 ENGAGING OTHER PATHWAYS LIKE THE 490 00:20:04,680 --> 00:20:06,800 [INDISCERNIBLE] FAMILY THAT ARE 491 00:20:06,800 --> 00:20:07,400 ALSO RESPONSIBLE FOR REPAIRING 492 00:20:07,400 --> 00:20:09,000 THESE BASES AND WE'RE PROBABLY 493 00:20:09,000 --> 00:20:10,880 NOT SHOCKED TO BE ABLE TO FIND 494 00:20:10,880 --> 00:20:14,480 FACTORS BECAUSE IT'S BEEN 495 00:20:14,480 --> 00:20:17,600 SUGGESTED FOR EXAMPLE THAT THIS 496 00:20:17,600 --> 00:20:19,600 MPG GLUE MARIOUS COSLACE MAY 497 00:20:19,600 --> 00:20:21,240 INTERFERE WITH AND INTERACT WITH 498 00:20:21,240 --> 00:20:24,920 THE [INDISCERNIBLE] AT THE PH2, 499 00:20:24,920 --> 00:20:26,400 AND CONVERSELY IN BASE EXCISION 500 00:20:26,400 --> 00:20:30,680 REPAIR IN A RECENT REPORT IT WAS 501 00:20:30,680 --> 00:20:32,480 SUGGESTED THATOXIDATIVE DAMAGE 502 00:20:32,480 --> 00:20:35,200 MAY ACTUALLY INCORPORATE UVDB FR 503 00:20:35,200 --> 00:20:37,840 EXAMPLE AS A CENSUS, SO WE 504 00:20:37,840 --> 00:20:40,280 VISIONED THAT THERE'S PROBABLY 505 00:20:40,280 --> 00:20:44,160 FACTORS TO FACILITATE SOME OF 506 00:20:44,160 --> 00:20:45,760 THESE PATHWAYS FOR ITS PATHWAY 507 00:20:45,760 --> 00:20:46,760 COORDINATION SO WE'RE EXCITED TO 508 00:20:46,760 --> 00:20:48,080 CONTINUE TO PROCEED ON THIS, 509 00:20:48,080 --> 00:20:49,560 THIS IS GENERALLY DONE THE 510 00:20:49,560 --> 00:20:50,560 FOLLOWING WAY. 511 00:20:50,560 --> 00:20:55,160 WE DO MULTIPLE EXPOSURES AND 512 00:20:55,160 --> 00:20:57,120 THEN GLUE MARIOUS COSALATE ALL 513 00:20:57,120 --> 00:21:01,320 THESE PROTEINS, WE GENERALLY MAP 514 00:21:01,320 --> 00:21:03,440 THIS IN CONCERT WITH A 515 00:21:03,440 --> 00:21:03,720 CORRELATION. 516 00:21:03,720 --> 00:21:06,840 IN OTHER WORDS WE ENVISION IF WE 517 00:21:06,840 --> 00:21:08,960 EXPOSE THE CELLS DIRECTLY WE 518 00:21:08,960 --> 00:21:10,840 SHOULD SEE P A R ACCUMULATION 519 00:21:10,840 --> 00:21:12,000 AND THEN WE COORDINATE IT AND 520 00:21:12,000 --> 00:21:13,520 MAKE SURE ALL OUR SAMPLES LOOK 521 00:21:13,520 --> 00:21:14,080 LIKE THIS. 522 00:21:14,080 --> 00:21:19,880 AS YOU MIGHT EXPECT, THE MPG IS 523 00:21:19,880 --> 00:21:22,240 LABELED BY THE BIOTIN AS WELL AS 524 00:21:22,240 --> 00:21:25,600 THE NUCLEACE, WE ONLY SEE A 525 00:21:25,600 --> 00:21:27,480 SLIGHT DAMAGE-INDUCED INDUCTION 526 00:21:27,480 --> 00:21:30,400 OF THE BIOTIN AALATION OF THE 527 00:21:30,400 --> 00:21:31,800 CANNED WHAT. 528 00:21:31,800 --> 00:21:35,560 INTERESTINGLY WE SEE HISTONE H3 529 00:21:35,560 --> 00:21:36,200 AS THE LABELED THROUGHOUT 530 00:21:36,200 --> 00:21:38,080 SUGGESTING THAT THIS IS ALL 531 00:21:38,080 --> 00:21:43,880 OBVIOUSLY WITHIN THE CHROMATIN 532 00:21:43,880 --> 00:21:44,880 REGARDLESS OF THE AMOUNT OF 533 00:21:44,880 --> 00:21:46,240 DAMAGE WHICH IS AN INTERESTING 534 00:21:46,240 --> 00:21:47,840 FINDING IN AND OF ITSELF AND 535 00:21:47,840 --> 00:21:51,400 THEN WE PROCEED AND WE'RE REALLY 536 00:21:51,400 --> 00:21:56,440 FORTUNATE TO WORK WITH 537 00:21:56,440 --> 00:21:57,680 [INDISCERNIBLE] AND ZHANG AT THE 538 00:21:57,680 --> 00:22:01,640 UNIVERSITY OF PITTSBURGH AND WE 539 00:22:01,640 --> 00:22:03,840 GIVE THEM DOZENS AND DOZENS OF 540 00:22:03,840 --> 00:22:05,120 SAMPLES AND THIS GOES THROUGH 541 00:22:05,120 --> 00:22:07,640 STANDARD WORK FLOW AND NOT 542 00:22:07,640 --> 00:22:08,960 SURPRISINGLY, WE HAVE IDENTIFIED 543 00:22:08,960 --> 00:22:14,640 NOW OTHER FACTORS AS WELL AS 544 00:22:14,640 --> 00:22:19,520 PROTEINS INVOLVED IN B REPAIR 545 00:22:19,520 --> 00:22:21,240 ALTHOUGH JEN IS WORKING ON THIS 546 00:22:21,240 --> 00:22:22,440 AND I'M SURE SHE'S LISTENING AND 547 00:22:22,440 --> 00:22:24,440 SHE WOULD BE SHOOTING ME HERE IF 548 00:22:24,440 --> 00:22:27,040 WE REVEALED WHAT THEY ARE AT THE 549 00:22:27,040 --> 00:22:28,480 MOMENT BUT WE'RE--STAY TUNED, 550 00:22:28,480 --> 00:22:33,520 WE'RE CONTINUING TO WORK ON 551 00:22:33,520 --> 00:22:33,840 THAT. 552 00:22:33,840 --> 00:22:36,320 SO TD OTHER BIG COMPONENT THAT 553 00:22:36,320 --> 00:22:39,120 WE'RE PURSUING AT THE MOMENT IS 554 00:22:39,120 --> 00:22:40,720 ACTIVATION IN PARTICULAR WITH 555 00:22:40,720 --> 00:22:43,440 REGARDS TO SINGLE STRAND BREAK 556 00:22:43,440 --> 00:22:45,240 REPAIR AND BASIC EXCISION 557 00:22:45,240 --> 00:22:48,120 REPAIR, THIS IS ALSO QUITE 558 00:22:48,120 --> 00:22:50,040 PRODUCTIVE AND WE HAVE 2 GROUPS 559 00:22:50,040 --> 00:22:52,280 OF PROTEINS WE IDENTIFIED HERE, 560 00:22:52,280 --> 00:22:54,880 YOUR TYPICAL BASIC EXCISION 561 00:22:54,880 --> 00:22:56,960 REPAIR PROTEINS, PRIMARILY P A 562 00:22:56,960 --> 00:23:01,440 RPs AND CC1 AS FACTORS OF 563 00:23:01,440 --> 00:23:04,880 BIOACCUMULATED BY P A RP, BUT 564 00:23:04,880 --> 00:23:06,160 INTERESTINGLY ENOUGH WHERE 565 00:23:06,160 --> 00:23:10,240 REPLICATION FACTORS SUCH AS RFC1 566 00:23:10,240 --> 00:23:12,360 AND TCNA, WE THINK THESE FALL 567 00:23:12,360 --> 00:23:13,240 INTO 2 CATEGORIES, PROBABLY ON 568 00:23:13,240 --> 00:23:18,560 THE LEFT, THE ROLE OF P A RP IN 569 00:23:18,560 --> 00:23:20,640 BASE EXCISION REPAIR OR 570 00:23:20,640 --> 00:23:21,640 CLASSICAL SINGLE STRAND BREAK 571 00:23:21,640 --> 00:23:24,040 REPAIR AND PROBABLY ON THE RIGHT 572 00:23:24,040 --> 00:23:26,040 THE MECHANISM OF P A RP INDUCED 573 00:23:26,040 --> 00:23:29,160 OR P A RP MEDIATED REPLICATION 574 00:23:29,160 --> 00:23:31,640 STRESS AND SO FOR TODAY'S TALK, 575 00:23:31,640 --> 00:23:34,600 WE WILL JUST FOCUS ON BASE 576 00:23:34,600 --> 00:23:35,440 EXCISION REPAIR AND SINGLE 577 00:23:35,440 --> 00:23:37,040 STRAND BREAK REPAIR AS WE 578 00:23:37,040 --> 00:23:39,240 CLASSICALLY REFER TO IT. 579 00:23:39,240 --> 00:23:41,560 SO WHAT WE'RE INTERESTED HERE IS 580 00:23:41,560 --> 00:23:44,520 UNCOVERING THOSE PROTEIN AND 581 00:23:44,520 --> 00:23:46,040 HISTONE ACETYLATION AND CODE IF 582 00:23:46,040 --> 00:23:50,560 YOU WILL AND REGULATES CLASSICAL 583 00:23:50,560 --> 00:23:53,120 BASE EXCISION REPAIR. 584 00:23:53,120 --> 00:23:55,680 SO, AS A I REFERRED TO EARLIER, 585 00:23:55,680 --> 00:23:59,600 WE ENVISION THIS P A RP 1 586 00:23:59,600 --> 00:24:01,760 MEDIATED CHROMATIN UNWINDING AND 587 00:24:01,760 --> 00:24:03,640 SUBSEQUENT RECRUITMENT OF THE 588 00:24:03,640 --> 00:24:05,680 MODIFYING COMPLEXES AND WE 589 00:24:05,680 --> 00:24:06,680 CONSIDER THAT THERE'S A NUMBER 590 00:24:06,680 --> 00:24:09,600 OF THINGS TO THINK ABOUT, DNA 591 00:24:09,600 --> 00:24:12,200 DAMAGE RESPONSE FACTOR TO THE 592 00:24:12,200 --> 00:24:14,240 HISTONE CHAPERONS, CHROMATIN 593 00:24:14,240 --> 00:24:16,200 MODIFIERS AND EPIGENIC MODIFIERS 594 00:24:16,200 --> 00:24:17,560 AND OUR HYPOTHESIS HERE IS THAT 595 00:24:17,560 --> 00:24:19,840 THERE'S A DIN,A DAMAGE RESPONSE 596 00:24:19,840 --> 00:24:21,200 AND HISTONE MODIFICATION CODE 597 00:24:21,200 --> 00:24:26,960 THAT DICTATES AND REGULATES BASE 598 00:24:26,960 --> 00:24:30,960 EXCISION REPAIR OR SINGLE STRANT 599 00:24:30,960 --> 00:24:33,240 BREAK REPAIR,--SO HOW WE DO 600 00:24:33,240 --> 00:24:33,840 TRACK THIS? 601 00:24:33,840 --> 00:24:37,960 AGAIN WE'VE BEEN INTRODUCED TO 602 00:24:37,960 --> 00:24:39,840 CONFOCAL MICROSCOPY SO WE CAN 603 00:24:39,840 --> 00:24:41,840 TRACK COMPLEX ASSEMBLY AND 604 00:24:41,840 --> 00:24:43,440 DISASSEMBLY IN THE LIVE CELL. 605 00:24:43,440 --> 00:24:47,640 THE THIS INVOLVES THE LASER 606 00:24:47,640 --> 00:24:49,760 INDUCED DNA DAMAGE, AND 607 00:24:49,760 --> 00:24:54,320 PRIMARILY FOCUS ON EGFP TAG, POL 608 00:24:54,320 --> 00:24:58,920 B AND EGFP TAG ERCC1 AND TAGOT 609 00:24:58,920 --> 00:25:02,040 END TERM GNAWS AND TAG ON THE 610 00:25:02,040 --> 00:25:04,840 C-TERMINUS, AND THEN WE COMBINE 611 00:25:04,840 --> 00:25:07,280 THAT WITH INHIBITORS OR SELECT 2 612 00:25:07,280 --> 00:25:09,280 KNOCK OUTS SO WE CAN PROBE 613 00:25:09,280 --> 00:25:12,480 FUNCTION AND INVOLVEMENT IN 614 00:25:12,480 --> 00:25:13,080 COMPLEX ASSEMBLY. 615 00:25:13,080 --> 00:25:15,480 AND WE'VE BUILT THE SYSTEM 616 00:25:15,480 --> 00:25:17,520 PRIMARILY BUILT BY JILL ANDREWS 617 00:25:17,520 --> 00:25:20,320 WHO IS THE MANAGER OF OUR 618 00:25:20,320 --> 00:25:22,600 MICROSCOPY UNIT WHICH HE CALLS 619 00:25:22,600 --> 00:25:24,800 MIDAS AND IT GIVES US AN 620 00:25:24,800 --> 00:25:26,480 OPPORTUNITY TO HAVE A REALLY 621 00:25:26,480 --> 00:25:28,600 HANDS ON APPROACH TO GET THESE 622 00:25:28,600 --> 00:25:31,440 PLOTS HERE IF YOU WILL, SO FOR 623 00:25:31,440 --> 00:25:34,480 EXAMPLE, THE 405 LASER, 624 00:25:34,480 --> 00:25:36,200 PRIMARILY MAKES SINGLE AND 625 00:25:36,200 --> 00:25:37,400 DOUBLE STRAND BREAKS, OF COURSE 626 00:25:37,400 --> 00:25:38,640 THERE'S A SPECTRUM OF THINGS 627 00:25:38,640 --> 00:25:40,600 DEPENDING ON THE DOSE AND THEN 628 00:25:40,600 --> 00:25:44,280 WE ALSO USE THE 355 NANO METER 629 00:25:44,280 --> 00:25:46,200 LASER, THIS TENDS TO BE 630 00:25:46,200 --> 00:25:47,880 PRIMARILY MORE BASE CISION 631 00:25:47,880 --> 00:25:49,120 REPAIR AND SINGLE STRAND REPAIR 632 00:25:49,120 --> 00:25:51,080 LESIONS AND CAN YOU SEE THE 633 00:25:51,080 --> 00:25:52,280 KINETICS OF RECRUITMENT OF THE 634 00:25:52,280 --> 00:25:54,960 DOUBLE STRAND BREAK PROTEINS AND 635 00:25:54,960 --> 00:25:56,160 BASE PAIR PROTEINS ARE QUITE 636 00:25:56,160 --> 00:26:00,320 DIFFERENT AND WE USE THIS TO 637 00:26:00,320 --> 00:26:03,040 FOLLOW THE COMPLEX ASSEMBLY AND 638 00:26:03,040 --> 00:26:04,280 DISASSEMBLY AND WE REALLY 639 00:26:04,280 --> 00:26:06,640 DEVELOP 2 SYSTEMS, SOMETHING WE 640 00:26:06,640 --> 00:26:08,120 CALL MID AS [INDISCERNIBLE]. 641 00:26:08,120 --> 00:26:09,680 WHERE OF COURSE WE HAVE CONTROL 642 00:26:09,680 --> 00:26:11,120 CELLS THAT HAVE NO LASER INDUCED 643 00:26:11,120 --> 00:26:15,960 DAMAGE AND WE MONITOR THAT AS A 644 00:26:15,960 --> 00:26:18,400 CONTROL, THAT AREA OF INTEREST 645 00:26:18,400 --> 00:26:22,720 AND THEN 10 OTHER CELLS ARE 646 00:26:22,720 --> 00:26:25,840 MONITORED IN PARALLEL AND THEN 647 00:26:25,840 --> 00:26:26,720 THEY'RE CONTINUING OVER TIME TO 648 00:26:26,720 --> 00:26:28,840 ALLOW US TO GET THESE PLOTS, 649 00:26:28,840 --> 00:26:30,720 EPPED UP DOING THIS ABOUT 4 650 00:26:30,720 --> 00:26:33,400 TIMES OR MORE SO WE CAN GET AN 651 00:26:33,400 --> 00:26:34,640 AVERAGE OF 40 OR MORE CELLS TO 652 00:26:34,640 --> 00:26:37,480 GET A LOT OF RECRUITMENT FOR 653 00:26:37,480 --> 00:26:40,520 EXAMPLE AS SHOWN HERE FOR CC1 654 00:26:40,520 --> 00:26:43,040 AND THIS PARALLEL ANALYSIS AND 655 00:26:43,040 --> 00:26:44,080 PROVIDES QUANTIFICATION OF 656 00:26:44,080 --> 00:26:45,640 REPAIR FOR PROTEIN RECRUITMENT, 657 00:26:45,640 --> 00:26:47,400 WE DO 10 CELLS PER EXPERIENT, 658 00:26:47,400 --> 00:26:49,080 AT LEAST 40 FOR ANY OF THE PLOTS 659 00:26:49,080 --> 00:26:53,640 I WILL BE SHOWING AND THEN WE 660 00:26:53,640 --> 00:26:55,320 HAVE SOMETHING CALLED 661 00:26:55,320 --> 00:26:56,280 SERO-ANALYSIS WHERE WE LOOK AT 662 00:26:56,280 --> 00:26:57,400 THE SAME CELL OVER AND OVER 663 00:26:57,400 --> 00:26:58,720 AGAIN AND THIS GIVES US AN 664 00:26:58,720 --> 00:26:59,880 OPPORTUNITY TO MAKE SURE WE'RE 665 00:26:59,880 --> 00:27:06,800 NOT MISSING ANYTHING THE FIRST 666 00:27:06,800 --> 00:27:09,000 15-20 SECONDS IN THAT ANALYSIS. 667 00:27:09,000 --> 00:27:11,680 AND THIS PROVIDES RAPID 668 00:27:11,680 --> 00:27:12,560 SELF-QUANTIFICATION OF PROTEINS. 669 00:27:12,560 --> 00:27:15,600 NOW AGAIN WE'RE LOOKING AT THE 670 00:27:15,600 --> 00:27:17,520 STEPS OF BASE EXCISION REPAIR, 671 00:27:17,520 --> 00:27:19,760 THERE ARE 2 CRITICAL ENZYMATIC 672 00:27:19,760 --> 00:27:23,200 ROLES AND SO TO ADDRESS THIS 673 00:27:23,200 --> 00:27:24,000 WE'VE SIMPLY EXPRESSED DUE TO 674 00:27:24,000 --> 00:27:27,560 THE WILD TYPE OR THE 2 MUTANTS 675 00:27:27,560 --> 00:27:30,360 AS EFF FUSIONS AND YOU CAN SEE, 676 00:27:30,360 --> 00:27:31,160 BASICALLY WE DON'T SLEEP APNEA 677 00:27:31,160 --> 00:27:34,000 AND OBESITY ANY CHANGE 678 00:27:34,000 --> 00:27:36,240 SURPRISINGLY THAT IS THE PEAK 679 00:27:36,240 --> 00:27:37,560 RECRUITMENT TIME AND THE HALF 680 00:27:37,560 --> 00:27:40,400 LIFE IS ALL SIMILAR SUPG JESTING 681 00:27:40,400 --> 00:27:41,560 SORRY, SUGGESTING THAT 682 00:27:41,560 --> 00:27:44,960 RECRUITMENT IS NOT DEPENDENT ON 683 00:27:44,960 --> 00:27:47,040 FUNCTION AT LEAST IN THE THE 2 684 00:27:47,040 --> 00:27:49,040 OR 3 CELLS--EXCUSE ME--2 OR 3 685 00:27:49,040 --> 00:27:56,000 CELL TYPES WE'VE LOOKED AT. 686 00:27:56,000 --> 00:27:58,000 WE ASK ASK THE QUESTION, 687 00:27:58,000 --> 00:27:59,640 HOWEVER, BECAUSE WE'RE 688 00:27:59,640 --> 00:28:02,000 EXPRESSING THIS USUALLY ON LENTI 689 00:28:02,000 --> 00:28:03,000 VIRUS, IS THIS AFFECTED BY THE 690 00:28:03,000 --> 00:28:04,800 LEVEL OF THE ENDOGENOUS PROTEIN 691 00:28:04,800 --> 00:28:07,680 AND SO HERE WE'RE JUST COMPARING 692 00:28:07,680 --> 00:28:10,800 THE 3 PROTEINS OF INTEREST 693 00:28:10,800 --> 00:28:15,800 EXPRESSED AS A FUSION TO THE 694 00:28:15,800 --> 00:28:17,400 SAME SITUATION WHERE WE KNOCKED 695 00:28:17,400 --> 00:28:18,880 OUT ENDOGENOUS LEVEL AND THESE 696 00:28:18,880 --> 00:28:21,280 ARE PROTEINS ARE EXPRESSED AS 697 00:28:21,280 --> 00:28:23,080 CAS 9 RESISTANT FUSIONS AND YOU 698 00:28:23,080 --> 00:28:25,400 CAN SEE EFFECTIVELY NO CHANGE IN 699 00:28:25,400 --> 00:28:27,080 TIME OF PEAK AND NO CHANGE IN 700 00:28:27,080 --> 00:28:28,880 HALF LIFE SO REALLY THERE'S NO 701 00:28:28,880 --> 00:28:30,680 CHANGE, IN OTHER WORDS THE 702 00:28:30,680 --> 00:28:33,600 ENDOGENOUS PROTEIN IS NOT 703 00:28:33,600 --> 00:28:34,440 AFFECTING THAT. 704 00:28:34,440 --> 00:28:36,320 HOW ABOUT IF THE CHANGING LEVEL? 705 00:28:36,320 --> 00:28:37,680 OF COURSE THOSE OF THAT YOU WORK 706 00:28:37,680 --> 00:28:40,160 WITH ANY KIND OF LENTI VIRUS, 707 00:28:40,160 --> 00:28:41,920 IT'S PROBABLY EXPRESSING THE 708 00:28:41,920 --> 00:28:44,040 PROTEIN AT 10 AND 20 OR 50 TIMES 709 00:28:44,040 --> 00:28:46,880 HIGHER THAN NORMAL AND SO, IN 710 00:28:46,880 --> 00:28:49,640 THE FIRST TASK WE ISOLATED BY 711 00:28:49,640 --> 00:28:53,080 FLOW, HIGH MEDIUM AND LOW 712 00:28:53,080 --> 00:28:56,040 EXPRESSERS, THESE HAVE NO CHANGE 713 00:28:56,040 --> 00:28:57,840 IN RECRUITMENT TIME OR PEAK 714 00:28:57,840 --> 00:28:59,440 LIFE, PEAK TIME OR HALF LIFE, SO 715 00:28:59,440 --> 00:29:03,640 IT DOESN'T SEEM TO BE A 716 00:29:03,640 --> 00:29:08,240 CONCENTRATION DEPENDENT BUT THAT 717 00:29:08,240 --> 00:29:09,240 STILL ALWAYS WORRIES US SO THE 718 00:29:09,240 --> 00:29:13,240 GOAL WAS TO TAG THE GENES WE'RE 719 00:29:13,240 --> 00:29:14,720 INTERESTED IN, AND THE END 720 00:29:14,720 --> 00:29:16,320 TERMINUS, AND THE C-TERMINUS 721 00:29:16,320 --> 00:29:22,040 ABOUT THIS DATA HERE IS THE END 722 00:29:22,040 --> 00:29:24,480 TEMPERATUREUS AND WE THINK WE'VE 723 00:29:24,480 --> 00:29:29,320 ONLY TAGGED 1 ALLELE, THIS GIVES 724 00:29:29,320 --> 00:29:35,280 A VERY NICE EXPRESS WITH 725 00:29:35,280 --> 00:29:37,080 PRIMARILY NUCLEAR WE'VE WE'VE 726 00:29:37,080 --> 00:29:38,400 SEEN THIS EXTREMELY 727 00:29:38,400 --> 00:29:38,760 [INDISCERNIBLE]. 728 00:29:38,760 --> 00:29:41,560 IN FACT WE SEE A HIGHER P-KITE 729 00:29:41,560 --> 00:29:46,960 AND THIS IS THE RESULT OF 730 00:29:46,960 --> 00:29:48,080 SIGNATURES SIGNAL TO NOISE 731 00:29:48,080 --> 00:29:50,040 LEVEL, AND ENDOGENOUS IN THE 732 00:29:50,040 --> 00:29:51,680 BETA BUT WE DON'T SEE ANY PEAK 733 00:29:51,680 --> 00:29:52,480 RECRUITMENT OR HALF LIFE. 734 00:29:52,480 --> 00:29:54,360 BUT THIS IS BECOMING 1 OF THE 735 00:29:54,360 --> 00:29:57,240 MORE POWERFUL WAYS WE WOULD LIKE 736 00:29:57,240 --> 00:29:59,680 TO MOVE FORWARD. 737 00:29:59,680 --> 00:30:02,160 WHAT ABOUT EXOCC1. 738 00:30:02,160 --> 00:30:06,160 WE THINK OF POLB AND EXOCC1 AS A 739 00:30:06,160 --> 00:30:07,120 HETEROGENEOUS ROW DIMER WHETHER 740 00:30:07,120 --> 00:30:09,160 THEY EXIST AS A HETEROGENEOUS 741 00:30:09,160 --> 00:30:10,200 ROW DIMER HERE, IT'S HARD TO 742 00:30:10,200 --> 00:30:15,440 KNOW BUT WE DO KNOW IF WE KNOCK 743 00:30:15,440 --> 00:30:20,160 OUT--OOPS SORRY--IF WE KNOCK OUT 744 00:30:20,160 --> 00:30:22,640 ECHO CC1, WE SEE LESS CHROMATIN 745 00:30:22,640 --> 00:30:25,720 AND NUCLEAR ACCUMULATION OF POL 746 00:30:25,720 --> 00:30:28,040 B POTENTIALLY BECAUSE OF ITS 747 00:30:28,040 --> 00:30:32,520 INTERACT BUT ALSO BECAUSE OF THE 748 00:30:32,520 --> 00:30:33,640 UBIQUITYINATION NEGOTIATED WITH 749 00:30:33,640 --> 00:30:35,040 SEPARATED INTO 2 PROTEINS AND 750 00:30:35,040 --> 00:30:38,920 THIS IS JUST PLOTTED RIGHT HERE. 751 00:30:38,920 --> 00:30:40,320 BUT WE CAN ASK QUESTIONS WHETHER 752 00:30:40,320 --> 00:30:47,240 OR NOT THE RECRUITMENT IS 753 00:30:47,240 --> 00:30:48,400 DEPENDENT ON EXOCC1. 754 00:30:48,400 --> 00:30:49,800 SO WE SIMPLY KNOCKED IT OUT AS 755 00:30:49,800 --> 00:30:53,280 SHOWN HERE AND YOU CAN SEE AS 756 00:30:53,280 --> 00:30:55,760 YOU MIGHT PREDICT IN THE ABSENCE 757 00:30:55,760 --> 00:30:58,320 OF XRCC1 THERE'S NO RECRUITMENT 758 00:30:58,320 --> 00:31:00,640 OF POLB TO THE SITE OF DAMAGE. 759 00:31:00,640 --> 00:31:03,440 IT'S NOT ONLY THE EXPRESSION OF 760 00:31:03,440 --> 00:31:05,440 XRCC1 THAT IS NEEDED, IT'S 761 00:31:05,440 --> 00:31:08,160 ACTUALLY THAT INTERACTION, SO 762 00:31:08,160 --> 00:31:12,360 YOU CAN MUTATE POLB SO IT CANNOT 763 00:31:12,360 --> 00:31:14,240 INTERACT WITH XRCC1, AND YOU SEE 764 00:31:14,240 --> 00:31:15,760 NO RECRUITMENT ON THESE 765 00:31:15,760 --> 00:31:17,440 CONDITIONS, SO THE RECRUITMENT 766 00:31:17,440 --> 00:31:18,960 THEN REQUIRES BOTH THE PRESENE 767 00:31:18,960 --> 00:31:23,160 OF THE PROTEIN AND INTERACTION 768 00:31:23,160 --> 00:31:26,720 BETWEEN THE 2 AND AGAIN, THIS 769 00:31:26,720 --> 00:31:28,920 TRIP, THIS TM-MUTANT IS A MUTANT 770 00:31:28,920 --> 00:31:30,160 THAT'S A SEPARATION OF FUNCTION 771 00:31:30,160 --> 00:31:34,120 AND IT BLOCKS THAT INTERACTION. 772 00:31:34,120 --> 00:31:37,040 SO TO BE HONEST, I STILL DON'T 773 00:31:37,040 --> 00:31:38,920 KNOW IF THESE PROTEINS ARE 774 00:31:38,920 --> 00:31:39,920 RECRUITED AS A HETEROGENEOUS ROW 775 00:31:39,920 --> 00:31:41,440 DIMER AND THAT'S AN ONGOING 776 00:31:41,440 --> 00:31:43,120 QUESTION THAT WIEWR INTERESTED 777 00:31:43,120 --> 00:31:43,840 IN. 778 00:31:43,840 --> 00:31:44,520 --WE'RE INTERESTED IN. 779 00:31:44,520 --> 00:31:49,680 IN OTHER WORDS IS POLB THERE AND 780 00:31:49,680 --> 00:31:50,920 XRCC1 COME TO THE SIDE? 781 00:31:50,920 --> 00:31:52,640 AND WE DON'T KNOW THAT 782 00:31:52,640 --> 00:31:54,800 SPECIFICALLY AT THIS POINT. 783 00:31:54,800 --> 00:31:58,680 SO WE KNOW HOWEVER THAT BOTH 784 00:31:58,680 --> 00:32:00,960 POLB AND XRCC1 IRPT ACTION AND 785 00:32:00,960 --> 00:32:05,680 THE EXPRESSION IS ESSENTIAL 786 00:32:05,680 --> 00:32:08,400 COMPLEX [INDISCERNIBLE]--WHAT 787 00:32:08,400 --> 00:32:08,840 ABOUT DISASSEMBLY? 788 00:32:08,840 --> 00:32:11,760 SO AS I SHOWED YOU HERE, WE GET 789 00:32:11,760 --> 00:32:14,920 VERY NICE TREATMENT OF XRCC1? 790 00:32:14,920 --> 00:32:17,960 WE DO THIS NOW IN THE ABSENCE OF 791 00:32:17,960 --> 00:32:20,960 POL B, IT'S DOWN STREAM MIGHT 792 00:32:20,960 --> 00:32:22,640 NOT EXPECT ANY EFFECT BUT IN 793 00:32:22,640 --> 00:32:26,800 FACT, IN THE ABSENCE OF POLB, WE 794 00:32:26,800 --> 00:32:31,840 SEE NO RECRUITMENT EFFECT BUT 795 00:32:31,840 --> 00:32:35,840 LOSS OF POLB SHOWS US HERE THAT 796 00:32:35,840 --> 00:32:36,560 XRCC1 DEMONSTRATES SLOWER 797 00:32:36,560 --> 00:32:37,640 DISASSEMBLY FROM THE REPAIR 798 00:32:37,640 --> 00:32:40,640 COMPLEX IN THE ABSENCE OF POLB. 799 00:32:40,640 --> 00:32:44,440 SO THERE'S SORT OF A--2 SIDES OF 800 00:32:44,440 --> 00:32:45,360 THIS COIN. 801 00:32:45,360 --> 00:32:47,400 YOU NEED XRCC1 FOR POLB TO COME 802 00:32:47,400 --> 00:32:49,160 THERE AND THEN YOU NEED POLB FOR 803 00:32:49,160 --> 00:32:51,720 IT TO BE THERE FOR THE 804 00:32:51,720 --> 00:32:52,640 DISASSEMBLY COME IS KIND OF 805 00:32:52,640 --> 00:32:53,960 INTERESTING AND THEN WE BUILT 806 00:32:53,960 --> 00:32:56,800 ANOTHER VECTOR SYSTEM WHERE WE 807 00:32:56,800 --> 00:32:59,000 EXPRESS THE EGFP TAG, XRCC1 808 00:32:59,000 --> 00:33:02,000 EITHER IN THE ABSENCE OF AN 809 00:33:02,000 --> 00:33:05,480 EMPTY SECOND CASSETTE, OR 810 00:33:05,480 --> 00:33:07,600 THE--OR THE POLB THAT'S 811 00:33:07,600 --> 00:33:09,400 RESISTANT TO THE GUIDE RNA AND 812 00:33:09,400 --> 00:33:11,880 WHEN YOU EXPRESS THE EMPTY 813 00:33:11,880 --> 00:33:14,280 SECOND CASSETTE, AGAIN, YOU SEE 814 00:33:14,280 --> 00:33:17,280 THE SAME PERSISTENT FOCI OF 815 00:33:17,280 --> 00:33:19,640 XRCC1 BUT THEN THE PRESENCE OF 816 00:33:19,640 --> 00:33:21,600 POLB IN THAT KNOCK OUT REVERSES 817 00:33:21,600 --> 00:33:24,280 THAT PHENOTYPE AS WE SEE HERE. 818 00:33:24,280 --> 00:33:25,600 SO THIS SUGGESTS TO US THAT 819 00:33:25,600 --> 00:33:30,080 AGAIN, YOU HAVE TO HAVE POLB 820 00:33:30,080 --> 00:33:32,640 THERE TO AFFECT THAT DISASSEMBLY 821 00:33:32,640 --> 00:33:34,480 WHICH IS A NOVEL FINDING AND 822 00:33:34,480 --> 00:33:38,040 THIS S&P JUST THAT SERO-ANALYSIS 823 00:33:38,040 --> 00:33:41,440 AND WE ARE NOT MISSING ANY TIME 824 00:33:41,440 --> 00:33:43,640 POINTS OF XRCC1 RECRUITMENT. 825 00:33:43,640 --> 00:33:46,560 SO POLB AND XRCC1 ASSEMBLY 826 00:33:46,560 --> 00:33:49,640 SHOULD BE REGULATED BY POLB 827 00:33:49,640 --> 00:33:51,040 RIBOSE OF COURSE, THIS IS THE 828 00:33:51,040 --> 00:33:52,040 MODEL WE'VE BEEN THINKING ABOUT 829 00:33:52,040 --> 00:33:53,960 WHICH IS CONSIST WENT THIS UPPER 830 00:33:53,960 --> 00:33:55,640 MODEL HERE AND SO AT FIRST WE 831 00:33:55,640 --> 00:33:58,240 WENT AHEAD AND JUST DID SOME, 832 00:33:58,240 --> 00:33:59,720 YOU KNOW IMMUNE O FLUORESCENCE 833 00:33:59,720 --> 00:34:01,240 AND YOU CAN FIX YOURSELF AFTER A 834 00:34:01,240 --> 00:34:04,520 MINUTE AND SEE THAT NICE POLB 835 00:34:04,520 --> 00:34:09,240 FOCI, AND THEN YOU CAN STAIN 836 00:34:09,240 --> 00:34:11,960 WITH AN ANTIP A R-ANTIBODY IS 837 00:34:11,960 --> 00:34:13,240 SIMILAR TO WHAT EVERYONE USES 838 00:34:13,240 --> 00:34:14,840 AND IT'S RIGHT ON TOP OF EACH 839 00:34:14,840 --> 00:34:15,040 OTHER. 840 00:34:15,040 --> 00:34:16,960 IF YOU HAVE DONE THIS, YOU 841 00:34:16,960 --> 00:34:19,920 REALIZE THIS IS PAINFUL BUT BUT 842 00:34:19,920 --> 00:34:21,240 WE CAN CONSISTENTLY DEMONSTRATE 843 00:34:21,240 --> 00:34:24,040 THAT THE IMPORTANCE OF P A RP IN 844 00:34:24,040 --> 00:34:26,920 THIS CONTEXT, IF WE KNOCK OUT P 845 00:34:26,920 --> 00:34:29,640 A RP, WE SEE A SUPPRESSION OF 846 00:34:29,640 --> 00:34:30,520 THAT RECRUITMENT AND CONSISTENT 847 00:34:30,520 --> 00:34:34,480 WITH THE REQUIREMENT FOR THAT 848 00:34:34,480 --> 00:34:36,760 POLB RIBOSE, AND SIMILARLY IF WE 849 00:34:36,760 --> 00:34:45,040 INHIBIT P A RP WE SEE A COMPLETE 850 00:34:45,040 --> 00:34:46,520 ATTENUATION OF THE POLYMER WITH 851 00:34:46,520 --> 00:34:49,280 THE P A RP INHIBITOR, YOU SEE 852 00:34:49,280 --> 00:34:51,400 THAT POL BETA STICKS AROUND AND 853 00:34:51,400 --> 00:34:52,400 DOESN'T COME OFF THE SITE. 854 00:34:52,400 --> 00:34:57,040 WE SEE THE SAME THING FOR XRCC1 855 00:34:57,040 --> 00:34:59,480 FOR EXAMPLE. 856 00:34:59,480 --> 00:35:00,160 INTERESTINGLY, [INDISCERNIBLE] 857 00:35:00,160 --> 00:35:02,440 WHICH IS THE ENSIEM THAT REMOVES 858 00:35:02,440 --> 00:35:06,760 THE VERY LAST ADP RIBOSE, AND 859 00:35:06,760 --> 00:35:09,880 NOT SURPRISING BECAUSE IT'S 1 860 00:35:09,880 --> 00:35:12,240 ADP RIBOSE UNIT. 861 00:35:12,240 --> 00:35:14,520 SO AGAIN, THE MODEL THAT WE'RE 862 00:35:14,520 --> 00:35:17,320 THINKING ABOUT HERE, WE USE 863 00:35:17,320 --> 00:35:19,520 THESE 2 PROTEINS TO UNDERSTAND 864 00:35:19,520 --> 00:35:21,840 THE COMPLEX ASSEMBLY AND 865 00:35:21,840 --> 00:35:25,920 DISASSEMBLY STEP, AND WE ENHANCE 866 00:35:25,920 --> 00:35:29,040 OUR MECHANISTIC ANALYSIS BY MORE 867 00:35:29,040 --> 00:35:31,720 EFFECTIVELY TRACKING POLYB 868 00:35:31,720 --> 00:35:32,840 RIBOSE, SO THAT IMMUNE O 869 00:35:32,840 --> 00:35:34,440 FLUORESCENCE IS NOT FOWN TO DO 870 00:35:34,440 --> 00:35:36,840 AND NOT REALTIME AND NOT 871 00:35:36,840 --> 00:35:40,440 QUANTITATIVE AND WE ASKED THE 872 00:35:40,440 --> 00:35:41,680 QUESTION--CAN WE MONITOR THE 873 00:35:41,680 --> 00:35:46,040 ACCUMULATION OF P A R FROM THESE 874 00:35:46,040 --> 00:35:47,400 REVIEWS EXPERIMENTS, YOU KNOW TO 875 00:35:47,400 --> 00:35:49,440 GET AWAY FROM THIS VERY PAINFUL 876 00:35:49,440 --> 00:35:52,400 APPROACH AND TO DO THAT, WE 877 00:35:52,400 --> 00:35:53,760 DEVELOPED SOMETHING WHICH WE 878 00:35:53,760 --> 00:35:58,360 CALL REAL P A R WHICH IS A P A 879 00:35:58,360 --> 00:35:59,960 R BINDING DOMAIN FUSED EGFP AND 880 00:35:59,960 --> 00:36:02,400 THIS ALLOWS US TO MEASURE P A R, 881 00:36:02,400 --> 00:36:05,160 FORMATION AND ACCUMULATION IN 882 00:36:05,160 --> 00:36:05,440 REALTIME. 883 00:36:05,440 --> 00:36:08,360 AND IT TURNED OUT TO BE NOT SO 884 00:36:08,360 --> 00:36:12,160 TRIVIAL IN THAT THERE ARE 10 885 00:36:12,160 --> 00:36:14,680 MORE BINDING DOMAINS, WE CREATED 886 00:36:14,680 --> 00:36:17,240 THIS REAGENT FOR ALL OF THEM. 887 00:36:17,240 --> 00:36:18,440 THEY ALL EXPRESS REASONABLY 888 00:36:18,440 --> 00:36:20,240 WELL, SOME OF THEM HAVE UNIQUE 889 00:36:20,240 --> 00:36:21,040 EXPRESSION PROFILES AND WE 890 00:36:21,040 --> 00:36:23,840 TESTED THEM ALL FOR 4 OR 5 AND 891 00:36:23,840 --> 00:36:25,200 355 NANO METER RECRUITMENT AND 892 00:36:25,200 --> 00:36:28,240 ONLY 1 OF THEM RECRUITED THE 893 00:36:28,240 --> 00:36:30,240 LASER INDUCED DNA DAMAGE, 894 00:36:30,240 --> 00:36:31,240 FORTUNATELY AT LEAST, AND THIS 895 00:36:31,240 --> 00:36:33,520 IS SOME OF THAT DATA HERE, 896 00:36:33,520 --> 00:36:36,360 HERE'S AN EXAMPLE OF THAT 897 00:36:36,360 --> 00:36:37,840 RECRUITMENT, WE GET A VERY NICE 898 00:36:37,840 --> 00:36:40,760 RECRUITMENT AND AGAIN IT'S A P A 899 00:36:40,760 --> 00:36:42,720 R-BINDING DOMAIN LINKED TO GFP 900 00:36:42,720 --> 00:36:45,040 AND SO THE CARTOON OF HOW WE 901 00:36:45,040 --> 00:36:47,240 ENVISION IT WORKING AND IT'S 902 00:36:47,240 --> 00:36:48,920 QUITE SPECIFIC. 903 00:36:48,920 --> 00:36:52,800 SO WE GET VERY NICE RECRUITMENT 904 00:36:52,800 --> 00:36:54,240 KINETICS AND XRCC1 TO SOME 905 00:36:54,240 --> 00:36:57,720 EXTENT BUT YET IF WE MUTATE THE 906 00:36:57,720 --> 00:37:00,360 RESIDUES KNOWN TO BLOCK P A R 907 00:37:00,360 --> 00:37:02,840 BNDING YOU ELIMINATE THAT 908 00:37:02,840 --> 00:37:03,720 RECRUITMENT SO WE'RE KIND OF 909 00:37:03,720 --> 00:37:05,320 EXCITED ABOUT THAT AND IN 910 00:37:05,320 --> 00:37:07,920 ADDITION, SIMILAR TO WHAT WE SAW 911 00:37:07,920 --> 00:37:10,000 FOR POLB AND XRCC1, IF YOU 912 00:37:10,000 --> 00:37:12,440 INHIBIT P A RP, YOU INHIBIT ANY 913 00:37:12,440 --> 00:37:15,200 RECRUITMENT AND IF YOU INHIBIT P 914 00:37:15,200 --> 00:37:17,520 A R, THE RECRUITMENT LEVEL 915 00:37:17,520 --> 00:37:19,320 PERSISTS OF THIS REAL P A R 916 00:37:19,320 --> 00:37:19,760 REAGENT. 917 00:37:19,760 --> 00:37:23,320 SO WE'RE USING THIS NOW IN ALL 918 00:37:23,320 --> 00:37:23,840 OF OUR STUDIES. 919 00:37:23,840 --> 00:37:30,280 NOW WE CAN USE THIS TO PROBE P A 920 00:37:30,280 --> 00:37:32,320 RP FORMATION KINETICS ALONG WITH 921 00:37:32,320 --> 00:37:32,840 ASSEMBLY AND DISASSEMBLY. 922 00:37:32,840 --> 00:37:34,600 SO 1 OF THE QUESTIONS WE ASKED 923 00:37:34,600 --> 00:37:37,880 IS LOSS OF POLB IMPACT INDUCED P 924 00:37:37,880 --> 00:37:40,040 A R FORMATION, TO REMIND YOU IN 925 00:37:40,040 --> 00:37:43,000 THIS CONTEXT WE SAW THAT WHEN WE 926 00:37:43,000 --> 00:37:48,080 EXPOSED THIS HIGH LEVELS OF 927 00:37:48,080 --> 00:37:51,160 [INDISCERNIBLE], WE GET 928 00:37:51,160 --> 00:37:55,000 ACCUMULATION OF P A RP, AND THIS 929 00:37:55,000 --> 00:37:58,040 CONTAINS XRCC1 OR REAL P A R AND 930 00:37:58,040 --> 00:38:02,440 THIS IS THE REAL PAR DATA. 931 00:38:02,440 --> 00:38:04,840 SURPRISINGLY WE SEE NO CHANGE IN 932 00:38:04,840 --> 00:38:07,760 THE ACCUMULATION OR APPEARANCE 933 00:38:07,760 --> 00:38:09,640 OF THE PAR SIGNAL AS EVIDENCED 934 00:38:09,640 --> 00:38:12,360 BY THE REAL PAR REAGENT. 935 00:38:12,360 --> 00:38:14,840 AND THIS SUGGESTS POTENTIALLY 936 00:38:14,840 --> 00:38:16,560 THERE AT THESE ADMITTEDLY VERY 937 00:38:16,560 --> 00:38:18,960 LOW LEVELS OF DNA DAMAGE, IS A 938 00:38:18,960 --> 00:38:21,960 BACK UP ENZYME THAT'S 939 00:38:21,960 --> 00:38:25,840 FACILITATING REPAIR OF THE P A 940 00:38:25,840 --> 00:38:28,640 R ACTIVATION LESION SUCH AS 941 00:38:28,640 --> 00:38:31,160 POLQ, POLI, OR OTHERS, SO IS 942 00:38:31,160 --> 00:38:33,160 PAR JUST SO PROBUST THAT WE 943 00:38:33,160 --> 00:38:35,240 ACTUALLY DON'T SEE IT 944 00:38:35,240 --> 00:38:37,360 ACCUMULATED VERY MUCH AND THAT'S 945 00:38:37,360 --> 00:38:39,120 SOMETHING UNDER STUDY NOW. 946 00:38:39,120 --> 00:38:42,320 AGAIN WE'RE INTERESTED IN THESE 947 00:38:42,320 --> 00:38:42,560 FACTORS. 948 00:38:42,560 --> 00:38:44,640 WE'VE LOOKEDDA THE THESE ON 2 949 00:38:44,640 --> 00:38:46,040 PROTEINS HERE, WE LOOKED AT REAL 950 00:38:46,040 --> 00:38:48,000 P A R AND THEN WHAT ABOUT 951 00:38:48,000 --> 00:38:48,600 NIEE, AUDIENCE D, DOES THAT 952 00:38:48,600 --> 00:38:50,240 CONTRIBUTE IN THE SAME CONTEXT 953 00:38:50,240 --> 00:38:56,080 IN TERMS OF REGULATING COMPLEX 954 00:38:56,080 --> 00:38:58,440 ASSEMBLY, AND AS WE REFERRED TO 955 00:38:58,440 --> 00:39:00,160 EARLIER, YES, IN VERY 956 00:39:00,160 --> 00:39:02,880 EFFECTIVELY, IF YOU SUPPRESS 957 00:39:02,880 --> 00:39:04,920 LEVELS OF CELLULAR NAD AND THEN 958 00:39:04,920 --> 00:39:06,640 ASK CAN THEE PROTEINS RECRUIT, 959 00:39:06,640 --> 00:39:08,360 YOU BASICALLY DON'T SEE ANY 960 00:39:08,360 --> 00:39:11,400 RECRUITMENT AND THAT IS LIKELY 961 00:39:11,400 --> 00:39:12,960 BECAUSE YOU CANNOT SEE A 962 00:39:12,960 --> 00:39:17,880 MEASURABLE LEVEL OF P A RP 963 00:39:17,880 --> 00:39:19,840 ACTIVATION BUT WHAT ABOUT 964 00:39:19,840 --> 00:39:21,240 ALTERATIONS IN NAD VIABILITY, IS 965 00:39:21,240 --> 00:39:22,760 THAT SOMETHING WE REALLY HAVE TO 966 00:39:22,760 --> 00:39:23,440 WORRY ABOUT? 967 00:39:23,440 --> 00:39:24,760 WELL, AGAIN WE TRIED TO MAKE 968 00:39:24,760 --> 00:39:26,040 WILL ARGUMENT HERE THAT WE 969 00:39:26,040 --> 00:39:29,640 SHOULD CONSIDER FACTORS SUCH AS 970 00:39:29,640 --> 00:39:32,760 NAD AS DER REGULATORY FACTORS, 971 00:39:32,760 --> 00:39:35,160 IN FACT THE BIOSYNTHESIS OF NAD 972 00:39:35,160 --> 00:39:40,640 IS WHICH IS A SUBSTRATE FOR BOTH 973 00:39:40,640 --> 00:39:42,240 IS COMPARTMENTALIZED AND HIGHLY 974 00:39:42,240 --> 00:39:42,960 REGULATED, THESE HAVE BEEN 975 00:39:42,960 --> 00:39:52,160 LINKED TO THE AGING PROCESS AND 976 00:39:52,160 --> 00:39:53,840 CANCER RELATED BIOSYNTHESIS IN 977 00:39:53,840 --> 00:39:57,200 DIFFERENT TISSUES SO IT'S BECOME 978 00:39:57,200 --> 00:39:59,520 VERY COMMON THESE DAYS THAT 979 00:39:59,520 --> 00:40:00,880 INCREASING NAD VIABILITY MAY 980 00:40:00,880 --> 00:40:02,320 PROVIDE AN OPPORTUNITY TO 981 00:40:02,320 --> 00:40:03,880 INCREASE CELLULAR DNA REPAIR 982 00:40:03,880 --> 00:40:05,040 CAPACITY, I WILL POINT OUT 983 00:40:05,040 --> 00:40:06,440 HOWEVER, IT REMAINS TO BE 984 00:40:06,440 --> 00:40:08,480 DETERMINED IF TOO MUCH OF A GOOD 985 00:40:08,480 --> 00:40:10,480 THING CAN ALSO BE DETRIMENTAL, 986 00:40:10,480 --> 00:40:13,480 AND THAT'S SOMETHING WE ARE ALSO 987 00:40:13,480 --> 00:40:13,800 CONSIDERING. 988 00:40:13,800 --> 00:40:15,280 SO IT BECAME REALLY INTERESTING 989 00:40:15,280 --> 00:40:17,240 THEN, WE WERE THEN WORKING WITH 990 00:40:17,240 --> 00:40:17,880 MARIE [INDISCERNIBLE], WE WERE 991 00:40:17,880 --> 00:40:23,800 ABLE TO GET A NICE SOURCE OF 992 00:40:23,800 --> 00:40:25,040 [INDISCERNIBLE] OR DISCIPLINARY 993 00:40:25,040 --> 00:40:27,880 HYDRONIKO TIN O ROBOSIDE, IT'S A 994 00:40:27,880 --> 00:40:31,400 PRECURSOR TO NAD, READILY GETS 995 00:40:31,400 --> 00:40:34,200 SYNTHESIZED INTO NAD AND IN FACT 996 00:40:34,200 --> 00:40:35,560 UNDER NONSTRESSED CONDITIONS, IT 997 00:40:35,560 --> 00:40:38,040 CAN RAISE THE LEVEL FOR A FEW 998 00:40:38,040 --> 00:40:40,400 HOURS AND OVER 10-15 FOLD HIGHER 999 00:40:40,400 --> 00:40:42,000 THAN FORMAL, THIS BECOMES 1000 00:40:42,000 --> 00:40:43,760 INTERESTING BECAUSE IN SOME CELL 1001 00:40:43,760 --> 00:40:48,880 TYPES WE FIND THAT P A RP MUST 1002 00:40:48,880 --> 00:40:52,680 BE IF YOU WILL--IT DOES NOT HAVE 1003 00:40:52,680 --> 00:40:53,480 ENOUGH NAD AVAILABLE. 1004 00:40:53,480 --> 00:40:55,040 SO HERE'S AN EXAMPLE OF A CELL 1005 00:40:55,040 --> 00:40:58,720 TYPE WHERE IF WE ECPOSE THE 1006 00:40:58,720 --> 00:41:01,440 HYDROGEN PEROXIDE AND WE DROVE 1007 00:41:01,440 --> 00:41:02,320 FOR PAR ACCUMULATION, IT'S 1008 00:41:02,320 --> 00:41:04,320 PRETTY LOW BUT WHEN WE TREAT THE 1009 00:41:04,320 --> 00:41:06,520 SOLES WITH NRA WE SEE THIS 1010 00:41:06,520 --> 00:41:09,160 EXTREMELY ROBUST AND P A RP 1011 00:41:09,160 --> 00:41:10,160 ACTIVATES AND SUGGESTING THAT 1012 00:41:10,160 --> 00:41:12,920 IT'S MISSING A LOT OF SUBSTRATE 1013 00:41:12,920 --> 00:41:14,480 AVAILABILITY IN THIS CONTEXT SO 1014 00:41:14,480 --> 00:41:18,600 WE ASK SIMPLY IF WE TREAT WITH 1015 00:41:18,600 --> 00:41:19,720 NRH DOES THAT AFFECT BASE 1016 00:41:19,720 --> 00:41:20,640 EXCISION REPAIR AND OF COURSE 1017 00:41:20,640 --> 00:41:26,640 IT'S WHAT WE SEE AND WE SEE 1018 00:41:26,640 --> 00:41:27,880 INCREASED RECRUITMENT, INCREASED 1019 00:41:27,880 --> 00:41:30,360 XRCC1 RECRUITMENT AND OF COURSE 1020 00:41:30,360 --> 00:41:30,960 INCREASED PAR-ACCUMULATION. 1021 00:41:30,960 --> 00:41:32,280 SO AGAIN WE THINK THAT NAD HAS 1022 00:41:32,280 --> 00:41:34,040 TO BE AN IMPORTANT FACTOR WHEN 1023 00:41:34,040 --> 00:41:35,280 CONSIDERING ALL THESE THINGS. 1024 00:41:35,280 --> 00:41:39,120 BUT THESE ARE NOT THE ONLY--P A 1025 00:41:39,120 --> 00:41:41,080 RP IS NOT THE ONLY PLAYER IN THE 1026 00:41:41,080 --> 00:41:42,440 FIELD, WHAT ABOUT MODIFIERS AND 1027 00:41:42,440 --> 00:41:46,960 WHEN WE'RE THINKING ABOUT NAD, 1028 00:41:46,960 --> 00:41:49,080 OBVIOUSLY THAT BRINGS US TO THE 1029 00:41:49,080 --> 00:41:51,480 [INDISCERNIBLE], AND WE FOCUSED 1030 00:41:51,480 --> 00:41:55,600 A LITTLE TIME ON CERT 6, NOW WHY 1031 00:41:55,600 --> 00:41:57,240 IS CERT 6 IMPORTANT FOR BER? 1032 00:41:57,240 --> 00:41:59,040 I WILL GET TO THAT IN THE NEXT 1033 00:41:59,040 --> 00:41:59,840 SLIDE. 1034 00:41:59,840 --> 00:42:01,320 WE'VE DONE THAT BY KNOCKING CERT 1035 00:42:01,320 --> 00:42:03,640 6 OUT IN ALL OF THESE CELLS AND 1036 00:42:03,640 --> 00:42:05,160 OF COURSE CERT 6 WAS SUGGESTED 1037 00:42:05,160 --> 00:42:08,640 THAT THE ROLLING BASIS REPAIR BY 1038 00:42:08,640 --> 00:42:09,520 AN UNKNOWN MECHANISM SOMETIME 1039 00:42:09,520 --> 00:42:11,240 AGO, WE KNOW AT LEAST FROM 1040 00:42:11,240 --> 00:42:14,160 REPORTS THAT IT CAN MODIFY BER 1041 00:42:14,160 --> 00:42:15,560 PROTEINS SO WE HAVEN'T--WE 1042 00:42:15,560 --> 00:42:18,040 HAVEN'T ASKED IF ANY OF OUR 1043 00:42:18,040 --> 00:42:21,240 PROTEINS ARE MODIFIED BY SERT 6 1044 00:42:21,240 --> 00:42:23,960 YET, THAT'S UNDERSTUDIED, WE DO 1045 00:42:23,960 --> 00:42:27,560 KNOW HOWEVER THAT CERT 6 AND 1046 00:42:27,560 --> 00:42:28,320 KNOWN TORACETALATE HISTONS THAT 1047 00:42:28,320 --> 00:42:30,920 CERTAINLY CAN BE PART OF AN 1048 00:42:30,920 --> 00:42:32,840 INTERESTING COMPONENT WITH 1049 00:42:32,840 --> 00:42:36,440 REGARD TO CELLULAR BASE EXCISION 1050 00:42:36,440 --> 00:42:36,640 REPAIR. 1051 00:42:36,640 --> 00:42:39,080 AND THEN IT'S ALSO KNOWN THAT 1052 00:42:39,080 --> 00:42:40,200 CERT 6 CAN EFFECT CHROMATIN 1053 00:42:40,200 --> 00:42:42,200 RELAXATION AND THIS IS THE 1054 00:42:42,200 --> 00:42:45,240 MEASUREMENT OF THE CONTEXT OF 1055 00:42:45,240 --> 00:42:46,800 DOUBLE STRANDED BREAK FORMATION, 1056 00:42:46,800 --> 00:42:48,440 WE'RE NOT SURE IF THAT'S THE 1057 00:42:48,440 --> 00:42:49,840 CONTEXT OF BASE EXCISION REPAIR 1058 00:42:49,840 --> 00:42:51,320 AND MAYBE MORE RELEVANT IS THAT 1059 00:42:51,320 --> 00:42:52,800 CERT 6 HAS BEEN SHOWN TO 1060 00:42:52,800 --> 00:42:56,320 ACTIVATE P A RP 1 IN RESPONSE TO 1061 00:42:56,320 --> 00:42:58,000 DOUBLE STRANDED BREAKS BUT NOT 1062 00:42:58,000 --> 00:43:00,680 BY DEASSIMILATION BUT IN FACT BY 1063 00:43:00,680 --> 00:43:01,680 THE ROBUST ACETYLATION SO WE 1064 00:43:01,680 --> 00:43:03,040 THOUGHT ABOUT THAT AND WONDERED 1065 00:43:03,040 --> 00:43:05,920 CAN THAT BE RESPONSIBLE FOR WHAT 1066 00:43:05,920 --> 00:43:09,120 YOU KNOW--CAN THAT BE RELATED TO 1067 00:43:09,120 --> 00:43:10,680 WHY CERT 6 WILL EFFECT BASE 1068 00:43:10,680 --> 00:43:11,800 EXCISION REPAIR IN THE EARLIER 1069 00:43:11,800 --> 00:43:14,160 STUDIES SO WE TOOK A LOOK AT 1070 00:43:14,160 --> 00:43:16,480 THAT AND AGAIN USING THOSE KNOCK 1071 00:43:16,480 --> 00:43:20,080 OUTS I REFERRED TO AND SHOWED, 1072 00:43:20,080 --> 00:43:22,760 SURPRISINGLY WE GET ALMOST A 1073 00:43:22,760 --> 00:43:24,960 COMPLETE ATTENUATION OF POLB 1074 00:43:24,960 --> 00:43:26,160 RECRUITMENT AND REDUCED DNA 1075 00:43:26,160 --> 00:43:28,240 DAMAGE WHEN WE KNOCK OUT CERT 6, 1076 00:43:28,240 --> 00:43:34,360 AND SIMILARLY ALMOST A COMPLETE 1077 00:43:34,360 --> 00:43:36,440 ATTENUATION OF XRCC1 IN THE 1078 00:43:36,440 --> 00:43:38,040 ROBBERIA SENSE OF CERT 6, BUT P 1079 00:43:38,040 --> 00:43:40,920 A RP ACTIVATION IS NOT AFFECTED 1080 00:43:40,920 --> 00:43:43,040 IN FACT IT'S ALMOST THE REVERSE, 1081 00:43:43,040 --> 00:43:44,640 WE ALMOST SEE MORE P A RP 1082 00:43:44,640 --> 00:43:46,440 ACTIVATION IN THE ABSENCE OF 1083 00:43:46,440 --> 00:43:49,040 THIS, SO THIS CERT 6 THEN 1084 00:43:49,040 --> 00:43:50,480 EFFECTS THE RECRUITMENT BY 1085 00:43:50,480 --> 00:43:53,520 MODULATING P A RP THE CC1 1086 00:43:53,520 --> 00:43:55,360 COMPLEX OR EVEN THE CHROMATIN 1087 00:43:55,360 --> 00:43:58,480 STATE ANY TYPE OF RECRUITMENT 1088 00:43:58,480 --> 00:43:59,880 BUT DOES NOT IMPACT LASER 1089 00:43:59,880 --> 00:44:02,200 INDUCED P A RP FORMATIONS, SO 1090 00:44:02,200 --> 00:44:03,480 IT'S DIFFERENT THAN WHAT'S SEEN 1091 00:44:03,480 --> 00:44:04,680 FOR DOUBLE STRAND BREAK REPAIR 1092 00:44:04,680 --> 00:44:07,400 AND WE THINK THIS IS INTERESTING 1093 00:44:07,400 --> 00:44:09,680 AND SO THE FACTORS THEN THAT ARE 1094 00:44:09,680 --> 00:44:11,600 AFFECTING THIS COMPLEX ASSEMBLY 1095 00:44:11,600 --> 00:44:14,920 ARE SHOWN HERE, BUT OF COURSE WE 1096 00:44:14,920 --> 00:44:16,760 HAVE TO THINK--SO NOW WE'RE 1097 00:44:16,760 --> 00:44:17,800 PURSUING THIS IN TERMS OF ALL 1098 00:44:17,800 --> 00:44:19,120 THE OTHER FACTORS THAT ARE 1099 00:44:19,120 --> 00:44:22,720 NECESSARY, AS WELL AS THE OTHERS 1100 00:44:22,720 --> 00:44:23,960 BUT OF COURSE WE ONLY LOOKEDDA 1101 00:44:23,960 --> 00:44:26,080 THE A FEW OF THESE PROTEINS, 1102 00:44:26,080 --> 00:44:30,240 THEY'RE OBVIOUSLY MORE, WE HAVE 1103 00:44:30,240 --> 00:44:32,240 AN ONGOING PROJECT WITH 1104 00:44:32,240 --> 00:44:33,720 [INDISCERNIBLE] AND TREY 1105 00:44:33,720 --> 00:44:35,960 [INDISCERNIBLE] AND THEY'VE 1106 00:44:35,960 --> 00:44:37,240 BUILT NOW SOMETHING THEY CALL 1107 00:44:37,240 --> 00:44:40,480 HIGHER UP MODEL OF DNA REPAIR 1108 00:44:40,480 --> 00:44:43,080 WHICH IS FROM OMICS SCALE 1109 00:44:43,080 --> 00:44:44,400 INTERACTION DATA SO BASICALLY GO 1110 00:44:44,400 --> 00:44:47,240 GOES OUT AND PULLS TOGETHER ALL 1111 00:44:47,240 --> 00:44:49,080 KNOWN PHYSICAL INTERACTIONS, IN, 1112 00:44:49,080 --> 00:44:51,200 RNA, CO EXPRESSIONS, PROTEIN CO 1113 00:44:51,200 --> 00:44:56,760 EXPRESSIONS, GENETIC CO 1114 00:44:56,760 --> 00:44:57,560 EXPRESSIONS GENETIC LEARNING 1115 00:44:57,560 --> 00:44:59,720 TYPE OF MODEL FOR DNA REPAIR, I 1116 00:44:59,720 --> 00:45:01,160 WILL NOT GO THROUGH ALL THE 1117 00:45:01,160 --> 00:45:04,520 DETAILS, THIS IS HOPEFULLY 1118 00:45:04,520 --> 00:45:09,040 COMING OUT SOON, BUT AS YOU 1119 00:45:09,040 --> 00:45:11,160 DRILL DOWN TO DNAREPAIR 1120 00:45:11,160 --> 00:45:12,440 COMMENNENTS THERE ARE 4 MODELS 1121 00:45:12,440 --> 00:45:15,040 THAT CAN COME OUT OF THIS DATA 1122 00:45:15,040 --> 00:45:16,440 AND OF COURSE WE WANTED TO ASK 1123 00:45:16,440 --> 00:45:19,240 THE QUESTION FWE TAKE A LOOK AT 1124 00:45:19,240 --> 00:45:21,040 THE SHORT BATCH FACTORS NOT ONLY 1125 00:45:21,040 --> 00:45:24,240 DO WE HAVE TO CONSIDER DNR CC1 1126 00:45:24,240 --> 00:45:26,560 BUT WE HAVE TO FACTOR THOSE IN 1127 00:45:26,560 --> 00:45:27,840 AS WELL AND WE LOOKEDDA THE 1128 00:45:27,840 --> 00:45:30,840 THOSE IN ADDITION TO OUR WORK 1129 00:45:30,840 --> 00:45:34,240 WITH POLB AND XRCC1 AND ASKING 1130 00:45:34,240 --> 00:45:38,040 IF ARE THEY ALL POLB DEPENDENT 1131 00:45:38,040 --> 00:45:41,840 AND THEY HAVE A STRONG P A RP 1132 00:45:41,840 --> 00:45:42,920 DEPENDENCE. 1133 00:45:42,920 --> 00:45:48,920 INTERESTINGLY SO THAT LIGASE 3, 1134 00:45:48,920 --> 00:45:50,720 AND MULTIPLE--ALMOST ENTIRELY AS 1135 00:45:50,720 --> 00:45:52,720 WELL AS APDX, AND IT HAS A 1136 00:45:52,720 --> 00:45:55,640 PARTIAL P A RP DEPENDENCE OF 1137 00:45:55,640 --> 00:45:57,200 COURSE, REAL P A R, YOU DON'T 1138 00:45:57,200 --> 00:45:59,320 SEE ANYTHING IN THE ABSENCE OF P 1139 00:45:59,320 --> 00:46:00,600 A RP ACTIVATION AND THEN IF YOU 1140 00:46:00,600 --> 00:46:04,400 LOOK AT THOSE KINDS OF PEAKS 1141 00:46:04,400 --> 00:46:05,960 THERE'S INTERESTING DATA SHOWING 1142 00:46:05,960 --> 00:46:07,400 UP HERE AND THEY'RE CORRELATED 1143 00:46:07,400 --> 00:46:09,680 AND THEN HAVE YOU HALF LIFE OF 1144 00:46:09,680 --> 00:46:10,640 RECRUITMENT IS ALSO AN 1145 00:46:10,640 --> 00:46:12,440 INTERESTING DATA SET TO LOOK AT. 1146 00:46:12,440 --> 00:46:14,960 WHAT IT WILL ITS US HERE AS WE 1147 00:46:14,960 --> 00:46:16,640 LAY THIS ON TOP OF EACH OTHER 1148 00:46:16,640 --> 00:46:22,160 THAT SOME OF THESE PROTEIN VS 1149 00:46:22,160 --> 00:46:24,160 VERY SIMILAR KINETICS AND THIS 1150 00:46:24,160 --> 00:46:30,000 IS POLB AND APTX AND THEN EXRCC1 1151 00:46:30,000 --> 00:46:30,960 AND OTHERS ARE CO LOCALIZING AND 1152 00:46:30,960 --> 00:46:39,240 THEN OF COURSE THE REAL P A RP 1153 00:46:39,240 --> 00:46:40,000 PERSISTS SO WE'RE ERPSYCHESSED 1154 00:46:40,000 --> 00:46:42,760 TO THINK ABOUT HOW WE REORGANIZE 1155 00:46:42,760 --> 00:46:45,560 THIS, BUT IF WE SEE THE 1156 00:46:45,560 --> 00:46:47,360 RECRUITMENT THEN THE XRCC1 1157 00:46:47,360 --> 00:46:49,240 RECRUITMENT TENDS TO BE ALL ON 1158 00:46:49,240 --> 00:46:51,160 ITS OWN, PROBABLY COORDINATING 1159 00:46:51,160 --> 00:46:52,640 THESE 3 FACTORS HERE, AND THEN 1160 00:46:52,640 --> 00:46:54,240 THAT CHANGES WHAT WE LOOK AT 1161 00:46:54,240 --> 00:46:55,760 HERE TO MAYBE SOMETHING MORE 1162 00:46:55,760 --> 00:46:57,640 LIKE THIS AND OF COURSE, THEY'RE 1163 00:46:57,640 --> 00:46:58,840 PROBABLY NOT ALL COMING HERE 1164 00:46:58,840 --> 00:47:01,240 TOGETHER, WE'RE PROBABLY LOOKING 1165 00:47:01,240 --> 00:47:04,840 AT MULTIPLE EVENTS, IN A SINGLE 1166 00:47:04,840 --> 00:47:05,920 LASER INDUCED SHOT, BUT AT LEAST 1167 00:47:05,920 --> 00:47:08,840 THIS IS 1 WAY TO THINK ABOUT IT 1168 00:47:08,840 --> 00:47:09,880 AS WE MOVE FORWARD. 1169 00:47:09,880 --> 00:47:11,720 SO FOR ALL OF THESE FACTORS 1170 00:47:11,720 --> 00:47:14,040 THOUGH, HOW DID THE CERTS, P A 1171 00:47:14,040 --> 00:47:15,920 RP AND CHROMATIN MODIFIERS 1172 00:47:15,920 --> 00:47:17,520 IMPACT THIS ASSEMBLY AND THERE 1173 00:47:17,520 --> 00:47:18,400 ARE RULES FOR EACH? 1174 00:47:18,400 --> 00:47:21,320 AND THIS IS SORT OF AN ONGOING 1175 00:47:21,320 --> 00:47:21,520 STORY. 1176 00:47:21,520 --> 00:47:24,040 AND SO, I'M GOING TO END WITH 1177 00:47:24,040 --> 00:47:25,120 THAT MODEL THERE, AND I'LL BE 1178 00:47:25,120 --> 00:47:28,240 HAPPY TO TAKE QUESTIONS MOVING 1179 00:47:28,240 --> 00:47:28,880 FORWARD. 1180 00:47:28,880 --> 00:47:39,320 THANKS VERY MUCH. 1181 00:47:39,320 --> 00:47:40,920 >> THANK YOU FOR THE VERY NICE 1182 00:47:40,920 --> 00:47:41,240 TALK THERE. 1183 00:47:41,240 --> 00:47:42,640 WE HAVE A NUMBER OF QUESTIONS 1184 00:47:42,640 --> 00:47:43,840 SUBMITTED IN THE CHAT. 1185 00:47:43,840 --> 00:47:45,280 WILL, DO YOU WANT TO START? 1186 00:47:45,280 --> 00:47:50,040 >> I WILL START, CAN YOU MUTE 1187 00:47:50,040 --> 00:47:51,080 PLEASE PEOPLE MUTE. 1188 00:47:51,080 --> 00:47:53,400 WHAT NAD LEVELS AND CANCER 1189 00:47:53,400 --> 00:48:00,840 CELLS, ARE THEY ELEVATED? 1190 00:48:00,840 --> 00:48:06,840 IT'S A COMPLEX QUESTION, YOU CAN 1191 00:48:06,840 --> 00:48:07,440 ADDRESS IT. 1192 00:48:07,440 --> 00:48:11,040 BOB, YOU'RE MUTED. 1193 00:48:11,040 --> 00:48:12,240 >> OKAY, HOW'S THAT, BETTER? 1194 00:48:12,240 --> 00:48:14,160 SO THANK YOU FOR MUCH FOR THAT 1195 00:48:14,160 --> 00:48:16,600 QUESTION, IT'S A COMPLEX 1196 00:48:16,600 --> 00:48:18,040 QUESTION AND WE'VE NOT DIRECTLY 1197 00:48:18,040 --> 00:48:21,440 COMPARED CANCER CELLS TO 1198 00:48:21,440 --> 00:48:23,000 THEIR--LET'S SAY NORMAL BECAUSE 1199 00:48:23,000 --> 00:48:25,720 HOW CAN YOU DEFINE THAT? 1200 00:48:25,720 --> 00:48:28,640 SO WE DON'T KNOW THE ANSWERS, 1201 00:48:28,640 --> 00:48:31,040 SPECIFICALLY, I WOULD SAY IT'S 1202 00:48:31,040 --> 00:48:32,840 REALLY HARD TO TELL THAT 1203 00:48:32,840 --> 00:48:33,840 QUANTITATIVELY BECAUSE WHAT ARE 1204 00:48:33,840 --> 00:48:38,200 YOU COMPARING IT TO? 1205 00:48:38,200 --> 00:48:38,720 >> SOME CANCER CELLS ARE 1206 00:48:38,720 --> 00:48:41,120 DIRECTLY AFFECTED IN THE NAD 1207 00:48:41,120 --> 00:48:44,000 METABOLISM AND THEREFORE CLEARLY 1208 00:48:44,000 --> 00:48:47,320 HAVE VERYIANT LEVELS OF NAD. 1209 00:48:47,320 --> 00:48:49,640 >> YEAH, WE'VE SEEN A LOT OF 1210 00:48:49,640 --> 00:48:52,720 DIFFERENT PHENOTYPES, WE'VE SEEN 1211 00:48:52,720 --> 00:48:54,800 SITUATIONS WHERE LET'S SAY 1212 00:48:54,800 --> 00:48:56,240 PRECURSOR SUPPLEMENTATION DOES 1213 00:48:56,240 --> 00:48:59,280 NOT GIVE RISE TO MORE NAD 1214 00:48:59,280 --> 00:49:03,280 PRODUCED AND OTHERS WHERE 1215 00:49:03,280 --> 00:49:04,080 PRECURSOR SUPPLEMENTATION DOES 1216 00:49:04,080 --> 00:49:05,800 GIVE RISE TO NAD SO THAT'S ALL 1217 00:49:05,800 --> 00:49:09,440 THE FACTORS THAT ARE NECESSARY 1218 00:49:09,440 --> 00:49:12,000 FOR BIOSYNTHESIS AND HOW THEY'RE 1219 00:49:12,000 --> 00:49:12,440 AFFECTED. 1220 00:49:12,440 --> 00:49:14,400 SO I DON'T THINK IT'S AN EASY 1221 00:49:14,400 --> 00:49:17,200 ANSWER BUT I THINK IT'S A GREAT 1222 00:49:17,200 --> 00:49:17,440 QUESTION. 1223 00:49:17,440 --> 00:49:20,400 >> I'M GOING TO GO TO THE NEXT 1224 00:49:20,400 --> 00:49:21,800 PERSON HERE, BEVERLY, DO YOU 1225 00:49:21,800 --> 00:49:24,760 HAVE ANY THOUGHTS ABOUT POLB 1226 00:49:24,760 --> 00:49:26,080 RECRUITMENT, SITES EVER DAMAGED 1227 00:49:26,080 --> 00:49:29,520 IN THE MITOCHONDRIA HA AND 1228 00:49:29,520 --> 00:49:32,240 WHETHER THEY'RE P A RP 1229 00:49:32,240 --> 00:49:32,520 DEPENDENT? 1230 00:49:32,520 --> 00:49:33,560 >> IT'S I GOOD QUESTION. 1231 00:49:33,560 --> 00:49:34,760 I THINK IT'S WELL ESTABLISH THAD 1232 00:49:34,760 --> 00:49:37,600 P A RP IS NONAPOPTOTIC NOT IN 1233 00:49:37,600 --> 00:49:38,920 THE MITOCHONDRIA SO THAT MAY NOT 1234 00:49:38,920 --> 00:49:39,560 BE THE CASE. 1235 00:49:39,560 --> 00:49:43,360 WE HAVEN'T LOOKED AT ALL AT POLB 1236 00:49:43,360 --> 00:49:45,440 RECRUITMENT IN TERMS OF LATE 1237 00:49:45,440 --> 00:49:46,760 DAMAGE INDUCED RECRUITMENT 1238 00:49:46,760 --> 00:49:48,560 BECAUSE THAT'S A WHOLE OTHER SET 1239 00:49:48,560 --> 00:49:51,040 OF EXPERIMENTS, BUT I WOULD SAY, 1240 00:49:51,040 --> 00:49:53,440 IT'S LIKELY NOT P A RP INDUCED. 1241 00:49:53,440 --> 00:49:57,920 >> LET ME JUST SAY THAT MANY 1242 00:49:57,920 --> 00:50:04,040 PEOPLE INCLUDING OURSELVES FIND 1243 00:50:04,040 --> 00:50:06,040 THEM--THAT MAY BE VARYING IN 1244 00:50:06,040 --> 00:50:08,200 TYPES OF CELL TYPES AND I KNOW 1245 00:50:08,200 --> 00:50:11,120 IN SOME OF THE P A RP MEETINGS, 1246 00:50:11,120 --> 00:50:12,400 THAT'S A BIG CONTROVERSY BUT 1247 00:50:12,400 --> 00:50:15,240 WE'VE NOT MEASURED IT SO IT'S 1248 00:50:15,240 --> 00:50:16,880 HARD TO KNOW. 1249 00:50:16,880 --> 00:50:24,640 >> WE HAVE 1 FROM AMANDA 1250 00:50:24,640 --> 00:50:26,320 [INDISCERNIBLE], DO YOU SEE THE 1251 00:50:26,320 --> 00:50:28,360 RECRUITMENT IS FROM OTHER DNA 1252 00:50:28,360 --> 00:50:29,880 DAMAGE RESPONSE P A RPs, 1253 00:50:29,880 --> 00:50:33,600 WHETHER IT'S 2 OR 3, SEEN NO 1254 00:50:33,600 --> 00:50:35,240 RECRUITMENT OF POL B IN THOSE 1255 00:50:35,240 --> 00:50:36,800 CASES HERE? 1256 00:50:36,800 --> 00:50:37,800 >> RIGHT, WE HAVEN'T--THAT'S A 1257 00:50:37,800 --> 00:50:39,480 GREAT QUESTION AND I'M GLAD YOU 1258 00:50:39,480 --> 00:50:41,640 SAW THAT. 1259 00:50:41,640 --> 00:50:42,520 YES THERE'S DEFINITELY A 1260 00:50:42,520 --> 00:50:44,880 DIFFERENCE IN P A RP 1 AND KNOCK 1261 00:50:44,880 --> 00:50:51,680 OUT INHIBITION SINCE AT LEAST 1262 00:50:51,680 --> 00:50:53,000 INHIBITORS WILL P A RP 2, AND 1263 00:50:53,000 --> 00:50:54,840 THEY ARE PLAYING A ROLE AND WE 1264 00:50:54,840 --> 00:50:56,320 HAVEN'T SPECIFICALLY ASKED THAT 1265 00:50:56,320 --> 00:51:04,160 QUESTION, BUT THAT'S A GREAT 1266 00:51:04,160 --> 00:51:04,960 QUESTION. 1267 00:51:04,960 --> 00:51:07,040 >> SO THEN SARAH ASKED IF YOU 1268 00:51:07,040 --> 00:51:08,040 HAVE TRIED SUPPLEMENTATION OF 1269 00:51:08,040 --> 00:51:10,280 OUR CELLS WITH NAR TO INCREASE 1270 00:51:10,280 --> 00:51:12,040 NAD LEVELS AND YOU DID THAT BUT 1271 00:51:12,040 --> 00:51:13,760 MAYBE YOU COULD EXTEND ON THAT? 1272 00:51:13,760 --> 00:51:15,440 >> YEAH, SOPHISTICATEDY WE TRIED 1273 00:51:15,440 --> 00:51:18,040 A WHOLE BUNCH OF AGENTS AND NAR 1274 00:51:18,040 --> 00:51:21,200 WORKS REALLY WELL IN STRESSED 1275 00:51:21,200 --> 00:51:24,920 CELLS, WE TEND NOT TO SEE BASAL 1276 00:51:24,920 --> 00:51:27,640 INDUCTION OF NAD LEVELS BY NR 1277 00:51:27,640 --> 00:51:29,160 ALONE AT LEAST IN THE CELL TYPES 1278 00:51:29,160 --> 00:51:30,240 WE'VE LOOKED AT. 1279 00:51:30,240 --> 00:51:35,240 IT DOES WORK REALLY WELL TO 1280 00:51:35,240 --> 00:51:37,440 COMPLEMENT, LET'S SAY FK86 1281 00:51:37,440 --> 00:51:38,440 INDUCED NAD SUPPLEMENTATION AND 1282 00:51:38,440 --> 00:51:40,840 NR WILL BRING IT BACK TO NORMAL. 1283 00:51:40,840 --> 00:51:41,800 THAT'S BEEN REPORTED. 1284 00:51:41,800 --> 00:51:42,920 WE'VE REPORTED ON IT AND WILL 1285 00:51:42,920 --> 00:51:45,600 AND OTHER VS REPORTED ON THAT, 1286 00:51:45,600 --> 00:51:48,880 BUT THE NRH TENDS TO BE A MUCH 1287 00:51:48,880 --> 00:51:50,240 MORE--BECAUSE WE DON'T ACTUALLY 1288 00:51:50,240 --> 00:51:54,480 KNOW HOW NRH IS CONVERTED IN NAD 1289 00:51:54,480 --> 00:51:55,840 WE DON'T KNOW THE FACTORS THAT 1290 00:51:55,840 --> 00:51:57,840 ARE NECESSARY AND IT SEEMS TO BE 1291 00:51:57,840 --> 00:52:02,040 MUCH MORE ARE BUST MECHANISM OF 1292 00:52:02,040 --> 00:52:06,640 NAD BIOSYNTHESIS IF YOU WILL 1293 00:52:06,640 --> 00:52:07,000 STIMULATION. 1294 00:52:07,000 --> 00:52:08,960 >> FROM KYLE MILLER HERE TO 1295 00:52:08,960 --> 00:52:10,760 EVERYONE, DOES NICOTINE O MID 1296 00:52:10,760 --> 00:52:14,000 WHICH IS A SEROTONIN INHIBITOR 1297 00:52:14,000 --> 00:52:16,560 SHE SAYS TREATMENT GIVE YOU THE 1298 00:52:16,560 --> 00:52:19,240 SAME RESULT AS 6 KNOCK OUT OR 1299 00:52:19,240 --> 00:52:22,880 BER FACTOR RECRUITMENT WITH DNA 1300 00:52:22,880 --> 00:52:23,120 DAMAGE? 1301 00:52:23,120 --> 00:52:27,240 >> GREAT QUESTION. 1302 00:52:27,240 --> 00:52:27,840 >> --DEACETYLATION DEPENDENT? 1303 00:52:27,840 --> 00:52:28,840 >> THAT'S A GREAT QUESTION, WE 1304 00:52:28,840 --> 00:52:29,760 DON'T KNOW THE ANSWER. 1305 00:52:29,760 --> 00:52:32,160 >> CAN I JUST THROW IN A 1306 00:52:32,160 --> 00:52:33,880 QUESTION HERE, ROB, SO YOU 1307 00:52:33,880 --> 00:52:35,360 MENTIONED THAT SOMETIMES THAT 1308 00:52:35,360 --> 00:52:38,440 THE RECRUITMENT THAT IS 1309 00:52:38,440 --> 00:52:39,640 INDEPENDENT UPON ACTIVITY OF THE 1310 00:52:39,640 --> 00:52:41,320 PROTEIN OR SOMETHING, SO WE TALK 1311 00:52:41,320 --> 00:52:43,640 A LOT ABOUT PHENOMENON CALLED 1312 00:52:43,640 --> 00:52:44,920 PIGGYBACK OR SOMETHING ELSE, 1313 00:52:44,920 --> 00:52:47,040 THAT VERY OFTEN AND WE'VE DONE A 1314 00:52:47,040 --> 00:52:48,240 LOT OF RECRUITMENT EXPERIMENTS, 1315 00:52:48,240 --> 00:52:51,240 YOU SEE OTHER THINGS BEING 1316 00:52:51,240 --> 00:52:53,320 RECRUITED THAT MAY OR MAY NOT GO 1317 00:52:53,320 --> 00:52:55,080 THERE IN ITS OWN FUNCTION BUT 1318 00:52:55,080 --> 00:52:57,480 BEING CARRIED ALONG WITH THE 1319 00:52:57,480 --> 00:52:59,080 PROTEIN THAT BIND TO AND SINCE 1320 00:52:59,080 --> 00:53:02,280 WITH ALL THESE MEASURES, YOU DO, 1321 00:53:02,280 --> 00:53:03,800 DO YOU HAVE ANY FEEL FOR THAT 1322 00:53:03,800 --> 00:53:07,080 AND OF COURSE, IT'S HARD TO 1323 00:53:07,080 --> 00:53:07,800 DIFFERENTIATE THESE THINGS BUT 1324 00:53:07,800 --> 00:53:10,800 HOW DO YOU SEE THIS? 1325 00:53:10,800 --> 00:53:11,880 IS RECRUITMENT ALWAYS AN 1326 00:53:11,880 --> 00:53:13,680 INDICATOR OF AN ACTIVE FUNCTION 1327 00:53:13,680 --> 00:53:15,920 OF THE PROTEIN AT THE SITE OF 1328 00:53:15,920 --> 00:53:16,120 THE-- 1329 00:53:16,120 --> 00:53:17,080 >> THATYA A GREAT QUESTION. 1330 00:53:17,080 --> 00:53:19,040 THAT'S WHY I RAISED THAT 1 1331 00:53:19,040 --> 00:53:20,880 STATEMENT THERE THAT WHAT WE 1332 00:53:20,880 --> 00:53:24,400 STILL DON'T KNOW IS THE--LET'S 1333 00:53:24,400 --> 00:53:29,440 SAY THIS CONTEXT POLB AND XRCC1 1334 00:53:29,440 --> 00:53:30,400 ARE THE ONLY HETEROGENEOUS ROW 1335 00:53:30,400 --> 00:53:31,680 DIMER AT SITES THAT ARE DAMAGED 1336 00:53:31,680 --> 00:53:33,880 AND SOME OF THE BIOLOGY WOULD 1337 00:53:33,880 --> 00:53:36,040 ARGUE THAT THEY HAVE INDEPENDENT 1338 00:53:36,040 --> 00:53:37,760 FUNCTIONS SO THEY'RE LIKELY NOT 1339 00:53:37,760 --> 00:53:39,080 ALWAYS A HETEROGENEOUS ROW DIMER 1340 00:53:39,080 --> 00:53:41,080 BUT YOU KNOW WE'RE TRYING TO 1341 00:53:41,080 --> 00:53:47,760 BUILD THE FLUORESCENT TOOLS AND 1342 00:53:47,760 --> 00:53:49,560 OTHER AND THE ANSWER OF THAT 1343 00:53:49,560 --> 00:53:51,240 QUESTION WE DON'T KNOW THE 1344 00:53:51,240 --> 00:53:52,840 ANSWER, IT'S JUST BEING BROUGHT 1345 00:53:52,840 --> 00:53:54,200 IN BECAUSE IT'S BOUND AS AN 1346 00:53:54,200 --> 00:53:57,000 EXAMPLE BUT WE DON'T KNOW THE 1347 00:53:57,000 --> 00:53:57,240 ANSWER. 1348 00:53:57,240 --> 00:54:01,320 >> YOU HAVE A QUESTION KEN? 1349 00:54:01,320 --> 00:54:08,080 >> YES, THERE'S 1 FROM--I HAVE A 1350 00:54:08,080 --> 00:54:08,360 QUESTION. 1351 00:54:08,360 --> 00:54:12,320 >> WHENEVER I HAVE PATIENTS WITH 1352 00:54:12,320 --> 00:54:15,040 SERIES POINTSA DOSA PIGMENTOSA, 1353 00:54:15,040 --> 00:54:19,320 THEY HAVE UDDB2, MUTATIONS THEY 1354 00:54:19,320 --> 00:54:21,920 CLINICALLY GET CANCER, HAVE YOU 1355 00:54:21,920 --> 00:54:26,040 STUDIED CELLS THAT ARE DEFICIENT 1356 00:54:26,040 --> 00:54:27,320 IN UDD2? 1357 00:54:27,320 --> 00:54:30,440 NA'S A PA, RP--WE'VE NOT, THE 1358 00:54:30,440 --> 00:54:32,000 PAPER WAS PRETTY EXCITING TO 1359 00:54:32,000 --> 00:54:33,320 DEMONSTRATE THAT IT HAD A ROLE 1360 00:54:33,320 --> 00:54:37,680 OR LIKELY AS A ROLE IN THE 1361 00:54:37,680 --> 00:54:39,480 LESION RECOGNITION INOXIDATIVE 1362 00:54:39,480 --> 00:54:40,800 DAMAGE, WE DID NOT ANSWER THAT 1363 00:54:40,800 --> 00:54:42,840 QUESTION, I THINK WE KNOCKED IT 1364 00:54:42,840 --> 00:54:44,400 OUT IN SOMEBODY WAS LOOKING AT 1365 00:54:44,400 --> 00:54:49,920 THAT YOU BUT I DON'T KNOW THE ANSWER 1366 00:54:49,920 --> 00:54:50,600 TO THE QUESTION,. 1367 00:54:50,600 --> 00:54:52,800 >> ANOTHER QUESTION CAME IN, DO 1368 00:54:52,800 --> 00:54:57,040 YOU FIND RAD 51 IN YOUR OGG1 1369 00:54:57,040 --> 00:54:58,160 BIOID EXPERIMENTS, I KNOW I 1370 00:54:58,160 --> 00:55:01,240 SHOULDN'T ASK BUT I DO ANYWAY 1371 00:55:01,240 --> 00:55:01,560 SINCE-- 1372 00:55:01,560 --> 00:55:03,160 >> YEAH, WE DO, WE ARE JUST 1373 00:55:03,160 --> 00:55:11,160 ANALYZING THE DATA SO WE DON'T 1374 00:55:11,160 --> 00:55:11,560 KNOW IT. 1375 00:55:11,560 --> 00:55:13,640 >> IS THE EFFECT ON RECRUITMENT 1376 00:55:13,640 --> 00:55:15,120 WITH THE CERT 6 KNOCK OUT 1377 00:55:15,120 --> 00:55:22,280 ANYTHING TO DO WITH THE HISTONE 1378 00:55:22,280 --> 00:55:23,760 ACETYLATION OR CORRELATION? 1379 00:55:23,760 --> 00:55:26,040 MAYBE THE RIBOSOLLATION THAT IS 1380 00:55:26,040 --> 00:55:27,640 ACTIVATED BY DNA DAMAGE? 1381 00:55:27,640 --> 00:55:29,360 >> THAT'S THE QUESTION, THAT'S 1382 00:55:29,360 --> 00:55:35,720 WHAT WE'RE TESTING NOW. 1383 00:55:35,720 --> 00:55:38,040 WE'VE ACTUALLY KNOCKED OUT ALL 1384 00:55:38,040 --> 00:55:39,720 THE [INDISCERNIBLE] AND ASKING 1385 00:55:39,720 --> 00:55:41,040 GLOBALLY HOW THEY AFFECT THIS 1386 00:55:41,040 --> 00:55:42,440 BUT RIGHT NOW WE'RE FOCUSING 1387 00:55:42,440 --> 00:55:44,240 THIS STUDY ON CERT 6 AND THEN 1388 00:55:44,240 --> 00:55:47,520 THE QUESTION BECOMES IS THE HOST 1389 00:55:47,520 --> 00:55:50,800 OF ACETYLATION POTENTIAL SITES 1390 00:55:50,800 --> 00:55:51,640 AND WE'RE ADDRESSING THOSE 1391 00:55:51,640 --> 00:55:56,560 QUESTIONS AS WE SPEAK. 1392 00:55:56,560 --> 00:55:59,080 >> I THINK THAT'S WHAT WE HAVE 1393 00:55:59,080 --> 00:56:00,280 FOR QUESTIONS. 1394 00:56:00,280 --> 00:56:03,760 >> THERE'S A COMMENT, THERE'S A 1395 00:56:03,760 --> 00:56:05,240 COMMENT FROM SARAH 1396 00:56:05,240 --> 00:56:06,040 [INDISCERNIBLE], ROB, I CAN'T 1397 00:56:06,040 --> 00:56:08,680 WAIT FOR THIS PAPER TO COME OUT! 1398 00:56:08,680 --> 00:56:09,840 GREAT STORY! 1399 00:56:09,840 --> 00:56:11,960 >> ALL RIGHT, WELL, MAKE SURE 1400 00:56:11,960 --> 00:56:14,000 I--I WILL SEND IT TO HER AS SOON 1401 00:56:14,000 --> 00:56:17,120 AS IT COMES. 1402 00:56:17,120 --> 00:56:19,360 [LAUGHTER] 1403 00:56:19,360 --> 00:56:19,640 >> OKAY. 1404 00:56:19,640 --> 00:56:22,680 >> WELL, THANKS AGAIN ROB. 1405 00:56:22,680 --> 00:56:23,440 >> THANKS, EVERYBODY. 1406 00:56:23,440 --> 00:56:25,240 >> NICE SLIDE LECTURE AND THANKS 1407 00:56:25,240 --> 00:56:28,240 EVERYBODY FOR ATTENDING. 1408 00:56:28,240 --> 00:56:30,240 >> YES AND THANK YOU VERY MUCH 1409 00:56:30,240 --> 00:56:32,640 FOR THE FINE TALK AND FOR THE 1410 00:56:32,640 --> 00:56:36,960 VIEW OF WHAT IT'S LIKE DOWN IN 1411 00:56:36,960 --> 00:56:38,760 THE SOUTHERN USA. 1412 00:56:38,760 --> 00:56:40,160 >> ALL RIGHT, THANKS SO MUCH AND 1413 00:56:40,160 --> 00:56:41,000 YOU ALL BE SAFE AND HAVE A GOOD 1414 00:56:41,000 --> 00:58:57,920 DAY