1 00:00:05,600 --> 00:00:08,800 >>WELCOME TO THE DNA 2 00:00:08,800 --> 00:00:11,760 REPAIR INTEREST GROUP CONFERENCE 3 00:00:11,760 --> 00:00:12,120 SERIES. 4 00:00:12,120 --> 00:00:13,080 THIS IS THE LAST 1 OF THE SEASON 5 00:00:13,080 --> 00:00:17,200 AND WE WILL BE TAKING A BREAK IN 6 00:00:17,200 --> 00:00:24,840 JULIE AND --JULY AND AUGUST AND 7 00:00:24,840 --> 00:00:26,200 PICKING UP IN SEPTEMBER. 8 00:00:26,200 --> 00:00:30,840 THOSE OF YOU WHO WOULD LIKE TO 9 00:00:30,840 --> 00:00:32,520 BE UPDATED ON THE CONFERENCENESS 10 00:00:32,520 --> 00:00:35,360 AND ALSO INFORMATION ABOUT DNA 11 00:00:35,360 --> 00:00:39,000 REPAIR MEETINGS AND POST DOCS, 12 00:00:39,000 --> 00:00:44,320 PLEASE JOIN THE LISTSERV, THE 13 00:00:44,320 --> 00:00:47,320 DNA REPAIR INTEREST GROUP 14 00:00:47,320 --> 00:00:52,720 LISTSERV, AND SEND IT TO ME. 15 00:00:52,720 --> 00:00:58,360 WE'RE HAPPY TO HAVE 16 00:00:58,360 --> 00:01:00,920 DR. TOM KUNKEL SPEAK TO US, HE 17 00:01:00,920 --> 00:01:06,480 IS AT THE NIEHS WHERE HE'S BEEN 18 00:01:06,480 --> 00:01:12,520 FOR 40 YEARS IN 1982 BUT HE 19 00:01:12,520 --> 00:01:13,960 RECEIVED HIS EDUCATION AT THOMAS 20 00:01:13,960 --> 00:01:15,280 MOORE COLLEGE IN KENTUCKY. 21 00:01:15,280 --> 00:01:17,800 HE HAS A BACHELOR'S AND HE GREW 22 00:01:17,800 --> 00:01:22,320 UP ON A FARM IN KENTUCKY TO HELP 23 00:01:22,320 --> 00:01:24,080 US AND HE MOVED TO CINCINNATI 24 00:01:24,080 --> 00:01:25,600 UNIVERSITY 25 00:01:25,600 --> 00:01:28,800 WHERE HE GOT A MASTERS IN 26 00:01:28,800 --> 00:01:34,720 CELL BIOLOGY AND A Ph.D. IN 27 00:01:34,720 --> 00:01:36,800 DEVELOPMENTAL BIOLOGY AT THE 28 00:01:36,800 --> 00:01:38,960 UNIVERSITY OF CINCINNATI. 29 00:01:38,960 --> 00:01:42,240 HE THEN BEGAN POST DOCTORAL 30 00:01:42,240 --> 00:01:44,360 TRAINING WITH DR. LARRY LOWE WHO 31 00:01:44,360 --> 00:01:46,160 IS REALLY WELL KNOWN FOR HIS 32 00:01:46,160 --> 00:01:50,680 WORK IN MUTE O GENESIS, 33 00:01:50,680 --> 00:01:52,280 APPARENTLIA LARRY WAS INITIALLY 34 00:01:52,280 --> 00:01:54,480 IN PENNSYLVANIA BUT THEN WENT TO 35 00:01:54,480 --> 00:01:59,080 THE UNIVERSITY OF WASHINGTON TBH 36 00:01:59,080 --> 00:02:02,360 SEATTLE WHERE HE IS STILL. 37 00:02:02,360 --> 00:02:04,760 DR. KUNKEL STARTED WITH HIM IN 38 00:02:04,760 --> 00:02:06,120 1978 AND THEN STAYED WITH HIM 39 00:02:06,120 --> 00:02:11,600 UNTIL HE CAME TO NIH AND NIEHS 40 00:02:11,600 --> 00:02:13,280 AND RESEARCH TRIANGLE NORTH 41 00:02:13,280 --> 00:02:16,720 CAROLINA, OVER THE YEARS HE'S 42 00:02:16,720 --> 00:02:19,600 MOVED UP, HE WILL GRADUATE, HE 43 00:02:19,600 --> 00:02:21,600 BECAME THE CHIEF OF THE 44 00:02:21,600 --> 00:02:23,000 LABORATORY OF STRUCTURAL BIOLOGY 45 00:02:23,000 --> 00:02:25,840 AND ALSO DIRECTOR OF THE 46 00:02:25,840 --> 00:02:29,440 ENVIRONMENTAL BIOLOGY PROGRAM AT 47 00:02:29,440 --> 00:02:33,800 NIEHS, SINCE 2011 THOUGH, HE HAS 48 00:02:33,800 --> 00:02:38,400 A SPECIAL TITLE OF AN NIH 49 00:02:38,400 --> 00:02:39,160 DISTINGUISHED INVESTIGATOR. 50 00:02:39,160 --> 00:02:43,120 AND THOUGH HE IS VERY PROLIFIC 51 00:02:43,120 --> 00:02:45,760 IN RESEARCH HE'S RECEIVED MANY 52 00:02:45,760 --> 00:02:52,120 HONORS INCLUDING AN HONORARY 53 00:02:52,120 --> 00:02:54,120 DOCTOR, DOCTORAL DIPLOMA FROM 54 00:02:54,120 --> 00:02:57,360 [INDISCERNIBLE] UNIVERSITY IN 55 00:02:57,360 --> 00:03:00,160 SWEDEN. 56 00:03:00,160 --> 00:03:03,760 AND HE HAS BEEN ELECTED TO THE 57 00:03:03,760 --> 00:03:07,080 AMERICAN ACADEMY OF ARTS AND 58 00:03:07,080 --> 00:03:09,760 SCIENCES HAS BEEN ON NUMEROUS 59 00:03:09,760 --> 00:03:11,480 EDITORIAL BOARDS, GAVE MANY 60 00:03:11,480 --> 00:03:13,880 LECTURES AND KEY NOTE SPEECHES 61 00:03:13,880 --> 00:03:17,840 AND PUBLISHED MORE THAN 400 62 00:03:17,840 --> 00:03:19,680 PAPERS, RESEARCH PAPERS IN THE 63 00:03:19,680 --> 00:03:22,080 BEST JOURNAL IN THE COUNTRY, OR 64 00:03:22,080 --> 00:03:23,640 IN THE WORLD. 65 00:03:23,640 --> 00:03:27,160 DR. BOER HAS A LITTLE MORE TO 66 00:03:27,160 --> 00:03:27,480 SAY,. 67 00:03:27,480 --> 00:03:29,720 >> YEAH, JUST TO CHIME IN HERE, 68 00:03:29,720 --> 00:03:31,120 TOM, IT'S A PLEASURE TO HAVE YOU 69 00:03:31,120 --> 00:03:34,720 AS A TRUE LEADER IN THE FIELD OF 70 00:03:34,720 --> 00:03:36,080 DNA REPAIR, MUTOGENESIS AND HAS 71 00:03:36,080 --> 00:03:38,040 BEEN A LEADER FOR MANY YEARS, 72 00:03:38,040 --> 00:03:39,600 I'VE HEARD HIM ON MANY 73 00:03:39,600 --> 00:03:43,040 OCCASIONS, VERY OFTEN AS A KEY 74 00:03:43,040 --> 00:03:46,120 NOTE TALKING ABOUT MUTOGENESIS, 75 00:03:46,120 --> 00:03:47,760 FED ILLEGALSITY OF POLYMERASES, 76 00:03:47,760 --> 00:03:49,280 ROLE OF RNA AND IT'S REALLY 77 00:03:49,280 --> 00:03:52,200 GREAT TO HAVE YOU TOM AND I 78 00:03:52,200 --> 00:03:54,800 WANTED TO ALSO ADD THAT THIS 79 00:03:54,800 --> 00:03:57,480 SAYS YOU HAVE ACHIEVED NUMEROUS 80 00:03:57,480 --> 00:03:58,920 AWARDS, YOU'VE GONE THROUGH ALL 81 00:03:58,920 --> 00:04:05,680 LEVELS OF NIH AND NOW 82 00:04:05,680 --> 00:04:07,160 DISTINGUISHED INVESTIGATOR AND 83 00:04:07,160 --> 00:04:08,160 RECEIVED MANY AWARDS. 84 00:04:08,160 --> 00:04:11,560 I'M VERY IMPRESSED WITH THE 85 00:04:11,560 --> 00:04:12,360 PRINCESS TAKAMATSU, THAT WE 86 00:04:12,360 --> 00:04:13,680 TALKED ABOUT ONCE ON A TRIP WHEN 87 00:04:13,680 --> 00:04:15,680 WE WERE TOGETHER IN JAPAN, 88 00:04:15,680 --> 00:04:21,720 THAT'S 1 VERY PRESTIGIOUS AWAR. 89 00:04:21,720 --> 00:04:23,240 IT'S VERY GREAT TO HAVE YOU AND 90 00:04:23,240 --> 00:04:23,640 SEE YOU. 91 00:04:23,640 --> 00:04:27,280 >> TOM WILL BE SPEAKING ABOUT A 92 00:04:27,280 --> 00:04:32,600 NUMBER OF TOPICS, INITIALLY 93 00:04:32,600 --> 00:04:33,960 ADVERTISE WITH EXTRINSIC PROOF 94 00:04:33,960 --> 00:04:36,320 BREEDING OF THE DNA REPLICATION 95 00:04:36,320 --> 00:04:41,160 ERRORS BUT HE'S EXPANDED TO IN 96 00:04:41,160 --> 00:04:43,240 GENERAL EUKARYOTIC DNA 97 00:04:43,240 --> 00:04:45,560 REPLICATION FIDELITY BY THE BETA 98 00:04:45,560 --> 00:04:46,920 FAMILY OF DNA POLYMERASES. 99 00:04:46,920 --> 00:04:48,600 SO, IF YOU HAVE ANY QUESTIONS, 100 00:04:48,600 --> 00:04:54,240 PLEASE PUT IT IN THE CHAT BOX 101 00:04:54,240 --> 00:04:56,640 IT'LL BE DIRECTED TO ME AND 102 00:04:56,640 --> 00:04:57,960 DR. BOER AND WE WILL READ THE 103 00:04:57,960 --> 00:04:59,680 QUESTIONS AT THE END. 104 00:04:59,680 --> 00:05:00,720 THANK YOU. 105 00:05:00,720 --> 00:05:02,400 OKAY, TAKE IT AWAY, TOM. 106 00:05:02,400 --> 00:05:07,600 >> WELL, I WANT TO THANK KEN AND 107 00:05:07,600 --> 00:05:09,720 WILL FOR THE KIND INVITATION TO 108 00:05:09,720 --> 00:05:10,520 SPEAK HERE. 109 00:05:10,520 --> 00:05:18,720 THIS IS A REAL PLEASURE AND I'M 110 00:05:18,720 --> 00:05:20,600 LOOKING IF ORDER TO INTERACTING 111 00:05:20,600 --> 00:05:22,520 WITH ANY OF YOU IF YOU HAVE ANY 112 00:05:22,520 --> 00:05:22,800 QUESTIONS. 113 00:05:22,800 --> 00:05:23,960 FIRST THING I WANT TO SAY IS 114 00:05:23,960 --> 00:05:26,200 THIS TALK AND ALL MY TALKS FOR 115 00:05:26,200 --> 00:05:27,440 THE INDEFINITE FUTURE ARE GOING 116 00:05:27,440 --> 00:05:32,920 TO BE IN MEMORY OF SAM WILSON, 117 00:05:32,920 --> 00:05:35,080 SAM WAS A VERY CLOSE FRIEND OF 118 00:05:35,080 --> 00:05:35,960 MINE AND 1 OF THE BEST 119 00:05:35,960 --> 00:05:41,080 SCIENTISTS I KNOW AND HE DIED 120 00:05:41,080 --> 00:05:43,320 LAST YEAR AND WE ALL MISS HIM 121 00:05:43,320 --> 00:05:46,800 VERY MUCH, MANY OF THE PEOPLE IN 122 00:05:46,800 --> 00:05:47,480 THIS AUDIENCE, THAT'S PRETTY 123 00:05:47,480 --> 00:05:51,200 MUCH ALL I WANT TO SAY EXCEPT 124 00:05:51,200 --> 00:05:54,560 THAT HE REALLY WAS A FINE--HE 125 00:05:54,560 --> 00:05:57,800 AND I SHARED BIRTHDAYS ABOUT 10 126 00:05:57,800 --> 00:06:01,320 YEARS APART OR CLOSE TO SHARING 127 00:06:01,320 --> 00:06:04,760 BIRTHDAYS AND EVERY TIME I HAD A 128 00:06:04,760 --> 00:06:06,720 CAREER DECISION OF ANY SORT TO 129 00:06:06,720 --> 00:06:08,480 MAKE, I ALWAYS CONSULTED WITH 130 00:06:08,480 --> 00:06:11,320 SAM AND HE WAS ALWAYS VERY 131 00:06:11,320 --> 00:06:13,920 GENEROUS IN HIS MENTORING ADVICE 132 00:06:13,920 --> 00:06:17,000 IN ADDITION TO PROVIDING AN 133 00:06:17,000 --> 00:06:21,760 EXCELLENT EXAMPLE FOR ME OF HOW 134 00:06:21,760 --> 00:06:23,760 TO DO QUALITY SCIENCE SO I HAVE 135 00:06:23,760 --> 00:06:26,360 FOND MEMORIES OF SAM AND I HOPE 136 00:06:26,360 --> 00:06:27,160 YOU DO TOO. 137 00:06:27,160 --> 00:06:30,200 SO THE TITLE OF MY TALK IS 138 00:06:30,200 --> 00:06:31,160 EUKARYOTIC DNA REPLICATION 139 00:06:31,160 --> 00:06:35,400 FIDELITY BY THE B-FAMILY DNA 140 00:06:35,400 --> 00:06:35,680 POLYMERASES. 141 00:06:35,680 --> 00:06:38,480 THESE ARE THE PERSONS CURRENTLY 142 00:06:38,480 --> 00:06:39,560 IN MY GROUP. 143 00:06:39,560 --> 00:06:43,280 MY GROUP IS CURRENTLY AS SMALL 144 00:06:43,280 --> 00:06:45,880 AS IT'S BEEN FOR THE LAST 35 145 00:06:45,880 --> 00:06:49,120 YEARS, PARTLY DUE TO THE COVID 146 00:06:49,120 --> 00:06:49,440 PANDEMIC. 147 00:06:49,440 --> 00:06:50,720 BUT THE REAL POINT HERE IS IN 148 00:06:50,720 --> 00:06:52,240 ADDITION TO THANKING ALL OF THEM 149 00:06:52,240 --> 00:06:58,040 FOR THEIR FINE WORK IN MY GROUP. 150 00:06:58,040 --> 00:07:00,120 I WOULD LIKE YOU TO TAKE NOTE 151 00:07:00,120 --> 00:07:02,800 THAT I HAVE POST DOCTORAL 152 00:07:02,800 --> 00:07:03,800 POSITIONS CURRENTLY AVAILABLE IN 153 00:07:03,800 --> 00:07:05,000 MY LAB, IF YOU'RE INTERESTED, 154 00:07:05,000 --> 00:07:08,800 ACCEPTED ME AN E-MAIL AND WE CAN 155 00:07:08,800 --> 00:07:09,160 CHATS. 156 00:07:09,160 --> 00:07:11,640 THIS IS A TIMELINE FOR LIFE ON 157 00:07:11,640 --> 00:07:13,640 EARTH STARTING WITH FORMATION, 158 00:07:13,640 --> 00:07:14,840 THE MOON, THROUGH THE PRESENT 159 00:07:14,840 --> 00:07:22,840 DAY, AND ON THE UPPER RIGHT YOU 160 00:07:22,840 --> 00:07:24,920 CAN SEE VISIBLE LIFE, IT HAS 161 00:07:24,920 --> 00:07:26,960 ONLY BEEN AROUND FOR ABOUT A 162 00:07:26,960 --> 00:07:32,120 HALF OF A BILLION YEARS OF THE 163 00:07:32,120 --> 00:07:33,840 4.5 BILLION YEAR LIFE SPAN ON 164 00:07:33,840 --> 00:07:38,760 THIS OR SPAN ON THIS DIAGRAM AND 165 00:07:38,760 --> 00:07:41,680 HUMANS HAVE ONLY BEEN AROUND FOR 166 00:07:41,680 --> 00:07:46,640 ABOUT 200,000 YEARS, SO, 1 OF 167 00:07:46,640 --> 00:07:48,040 THE INTERESTING THINGS FROM MY 168 00:07:48,040 --> 00:07:51,760 PERSPECTIVE ABOUT THE WORK WE DO 169 00:07:51,760 --> 00:07:55,000 APPROXIMATE IS THE EVOLUTIONARY 170 00:07:55,000 --> 00:07:56,560 PERSPECTIVE AND 1 WAY TO SAY 171 00:07:56,560 --> 00:08:00,920 THAT IS JUST TO POSE A SIMPLE 172 00:08:00,920 --> 00:08:01,760 QUESTION. 173 00:08:01,760 --> 00:08:04,360 I LOVE SCIENCE FICTION MOVIES, I 174 00:08:04,360 --> 00:08:05,760 LOVE THE MIEWRY JUREACIC PARK 175 00:08:05,760 --> 00:08:08,280 AND WHEN I SAW THOSE DINOSAURS 176 00:08:08,280 --> 00:08:09,800 WALKING AROUND, I WONDERED HOW 177 00:08:09,800 --> 00:08:13,360 THEY WERE REPLICATING THEIR DNA 178 00:08:13,360 --> 00:08:15,520 AND THAT WAS MANY MILLIONS OF 179 00:08:15,520 --> 00:08:15,880 YEARS AGO. 180 00:08:15,880 --> 00:08:17,440 DO THEY HAVE THE SAME ENZYMES 181 00:08:17,440 --> 00:08:21,800 THAT I'M GOING TO TALK ABOUT IS 182 00:08:21,800 --> 00:08:25,440 INTERESTING TO ME. 183 00:08:25,440 --> 00:08:26,720 IN ADDITION, FROM THE STANDPOINT 184 00:08:26,720 --> 00:08:28,120 OF HOW WE'RE GOING TO 185 00:08:28,120 --> 00:08:29,800 INVESTIGATE THAT, HERE'S A SLIDE 186 00:08:29,800 --> 00:08:32,720 ON PROTEIN DYNAMICS AND 187 00:08:32,720 --> 00:08:34,960 MACROMOLECULAR MOTIONS THAT CAN 188 00:08:34,960 --> 00:08:38,080 OCCUR OVER TIME FRAMES OF 10 TO 189 00:08:38,080 --> 00:08:39,160 THE FOURTH SECONDS ON THE LEFT 190 00:08:39,160 --> 00:08:42,240 TO 10 TO THE MINUS 142ndS ON 191 00:08:42,240 --> 00:08:46,240 THE RIGHT HAND SIDE OF THE RED 192 00:08:46,240 --> 00:08:46,400 BAR. 193 00:08:46,400 --> 00:08:49,440 AND WHAT IS STUDIED ARE LISTED 194 00:08:49,440 --> 00:08:51,680 ABOUT THAT RED BAR AND HOW 195 00:08:51,680 --> 00:08:53,080 THEY'RE STUDIED IS LISTED BELOW 196 00:08:53,080 --> 00:08:57,400 THAT RED BAR AND THE POINT OF 197 00:08:57,400 --> 00:09:00,560 THESE 2 SLIDES IS TO SAY THAT 198 00:09:00,560 --> 00:09:03,520 OUR UNDERSTANDING OF DNA 199 00:09:03,520 --> 00:09:06,880 REPLICATION FIDELITY SPANS RANGE 200 00:09:06,880 --> 00:09:12,360 FROM 2 ANG STROMs, TO 2-METERS 201 00:09:12,360 --> 00:09:13,080 THE SIZE OF SOMEONE 202 00:09:13,080 --> 00:09:17,680 APPROXIMATELY AND 10 TO THE 14 203 00:09:17,680 --> 00:09:19,560 FRAMES MINUS TO 14-SECONDS 204 00:09:19,560 --> 00:09:22,080 GREATER THAN 14 YEARS, SO THAT'S 205 00:09:22,080 --> 00:09:24,240 A HUGE SPECTRUM OF SPACE AND 206 00:09:24,240 --> 00:09:27,360 TIME AND TO STUDY THAT THE 207 00:09:27,360 --> 00:09:29,040 TECHNOLOGIES THAT ARE CURRENTLY 208 00:09:29,040 --> 00:09:32,280 IN USE ARE STRUCTURAL BIOLOGY, 209 00:09:32,280 --> 00:09:34,560 BIOCHEMISTRY, GENETICS AND 210 00:09:34,560 --> 00:09:34,840 GENOMICS. 211 00:09:34,840 --> 00:09:37,320 AND I WILL ILLUSTRATE CERTAIN 212 00:09:37,320 --> 00:09:43,040 POINTS WITH EACH OF THOSE 4 213 00:09:43,040 --> 00:09:46,480 APPROACHES TO UNDERSTANDING DNA 214 00:09:46,480 --> 00:09:47,800 REPLICATION FIDELITY SO HERE ARE 215 00:09:47,800 --> 00:09:50,000 THE SUBUNITS OF THE DNA 216 00:09:50,000 --> 00:09:52,720 POLYMERASES IN 4 FAMILIES. 217 00:09:52,720 --> 00:09:56,080 THERE ARE 17 OF THEM THAT ARE 218 00:09:56,080 --> 00:09:57,240 NOW KNOWN, MOST OF THEM ARE 219 00:09:57,240 --> 00:09:58,600 SHOWN HERE AND WHAT I WANT TO 220 00:09:58,600 --> 00:10:01,200 FOCUS THIS TALK ON ARE DNA 221 00:10:01,200 --> 00:10:02,920 POLYMERASES OUT FOR DELTA AND 222 00:10:02,920 --> 00:10:07,560 EPSILON WHICH ARE IN THE 223 00:10:07,560 --> 00:10:07,960 B-FAMILY. 224 00:10:07,960 --> 00:10:09,920 HERE THEY ARE WITH A SUMMARY OF 225 00:10:09,920 --> 00:10:13,560 SOME OF THEIR FUNCTIONS AND 226 00:10:13,560 --> 00:10:15,360 PROPERTIES, THEY'RE ALL 227 00:10:15,360 --> 00:10:19,200 MULTISUBUNIT DNA POLYMERASES, 228 00:10:19,200 --> 00:10:21,720 THEY ARE HIGHLY CONSERVED FROM 229 00:10:21,720 --> 00:10:24,000 BUDDING YEAST, WHAT MY TALK WILL 230 00:10:24,000 --> 00:10:26,440 BE ABOUT THROUGH FISSION YEAST 231 00:10:26,440 --> 00:10:29,720 AND HUMANS AND THEY HAVE 232 00:10:29,720 --> 00:10:32,960 ACTIVITIES IN ADDITION TO THE 233 00:10:32,960 --> 00:10:40,120 DNA POLYMERASE ITSELF THAT ARE 234 00:10:40,120 --> 00:10:42,000 RELEVANT TO REPLICATION. 235 00:10:42,000 --> 00:10:43,840 THEY'RE FIDELITY FROM WORK THAT 236 00:10:43,840 --> 00:10:46,760 ME AND OTHER VS DONE FOR AT 10 237 00:10:46,760 --> 00:10:56,000 TO THE MINUS 4 AND BETTER FAN 238 00:10:56,000 --> 00:10:56,800 THAT PRACTICES PROEPSILON, AND 239 00:10:56,800 --> 00:11:01,680 DEDICATION IS WHAT I WILL TALK 240 00:11:01,680 --> 00:11:06,200 ABOUT HERE. 241 00:11:06,200 --> 00:11:08,000 SO NUCLEIIC DNA DEPENDS ON THE 242 00:11:08,000 --> 00:11:10,920 CONCENTRATIONS OF THE DNTs, 243 00:11:10,920 --> 00:11:13,120 AND THE RMTPs AND THAT 244 00:11:13,120 --> 00:11:13,800 INCLIEWRDS ABSOLUTE 245 00:11:13,800 --> 00:11:15,280 CONCENTRATIONS AND RELATIVE 246 00:11:15,280 --> 00:11:17,240 CONCENTRATIONS AND DEPENDS ON 247 00:11:17,240 --> 00:11:20,920 THE DNA POLYMERASES FOR CORRECT 248 00:11:20,920 --> 00:11:24,720 PROPERLY ALIGNED DTNP AND THE 249 00:11:24,720 --> 00:11:26,120 CORRECT SUGAR MOIETY, AND PROOF 250 00:11:26,120 --> 00:11:27,680 ON REPLICATION WHICH WE NOW 251 00:11:27,680 --> 00:11:30,360 BELIEVES OCCURS IN 2 FLAVORS, 1 252 00:11:30,360 --> 00:11:32,840 IS EXTRINSIC PROOF READING AND 253 00:11:32,840 --> 00:11:35,640 THE OTHER IS EXTRINSIC PROOF 254 00:11:35,640 --> 00:11:37,080 READERRING AND IT DEPENDS ON 255 00:11:37,080 --> 00:11:40,240 REPAIR OF ERRORS AFTER 256 00:11:40,240 --> 00:11:42,200 REPLICATION BY DNA MISMATCHED 257 00:11:42,200 --> 00:11:45,720 REPAIR AND NUCLEOTIDE EXCISION 258 00:11:45,720 --> 00:11:46,320 REPAIR. 259 00:11:46,320 --> 00:11:47,280 HERE ARE THE 4 PROCESSES THAT I 260 00:11:47,280 --> 00:11:53,640 WOULD LIKE TO CONSIDER IN THIS 261 00:11:53,640 --> 00:11:55,360 BRIEF TALK, 1 IS CHAIN 262 00:11:55,360 --> 00:11:56,800 ELONGATION ON A TEMPLATE THAT 263 00:11:56,800 --> 00:12:02,640 OCCURS ABOUT 6 BILLION TIMES FOR 264 00:12:02,640 --> 00:12:04,320 EVERY REPLICATION CYCLE, THAT'S 265 00:12:04,320 --> 00:12:08,960 HOW MANY NUCLEOTIDES ARE IN THE 266 00:12:08,960 --> 00:12:12,520 HUMAN GENOME. 267 00:12:12,520 --> 00:12:13,240 OKAZAKI FRAGMENT MATURATION 268 00:12:13,240 --> 00:12:15,480 WHICH I WILL MENTION BREFLY 269 00:12:15,480 --> 00:12:19,080 OCCURS 25 MILLION TIMES 270 00:12:19,080 --> 00:12:21,920 PERNUCLEAR GENOME. 271 00:12:21,920 --> 00:12:24,120 RIBONUCLEOTIDE EXCISION PAIRS, 272 00:12:24,120 --> 00:12:27,200 RIBONUCLEOTIDES INCORPORATED 273 00:12:27,200 --> 00:12:28,200 DURING REPLICATION OCCURS 274 00:12:28,200 --> 00:12:30,080 3 MILLION PER GENOME PER CYCLE 275 00:12:30,080 --> 00:12:31,960 AND MISMARCHED REPAIR OCCURS A 276 00:12:31,960 --> 00:12:35,240 HUNDRED OR MORE TIMES PER 277 00:12:35,240 --> 00:12:36,000 REPLICATION CYCLE. 278 00:12:36,000 --> 00:12:38,400 AND THE REASONS WHY I AM 279 00:12:38,400 --> 00:12:41,160 INTERESTED ARE UNDERSTANDING THE 280 00:12:41,160 --> 00:12:44,800 RESPONSIBLE MECHANISMS FOR THESE 281 00:12:44,800 --> 00:12:46,480 PROCESSES AND CONSISTENT WITH 282 00:12:46,480 --> 00:12:49,280 THE INTERESTS OF THE NATIONAL 283 00:12:49,280 --> 00:12:50,760 INSTITUTES OF HEALTH, THEIR 284 00:12:50,760 --> 00:12:52,080 CONNECTIONS TO HUMAN HEALTH AND 285 00:12:52,080 --> 00:12:54,680 AS I MENTIONED AT THE 286 00:12:54,680 --> 00:12:55,920 BEGENERATEDDING, THE 287 00:12:55,920 --> 00:12:57,800 EVOLUTIONARY CONSERVATION OF THE 288 00:12:57,800 --> 00:12:59,760 ENZYMES AND PROCESSES THAT OCCUR 289 00:12:59,760 --> 00:13:01,040 FOR EACH OF THESE 4 STEPS. 290 00:13:01,040 --> 00:13:04,920 SO WHAT I'M GOING TO DO NOW IS 291 00:13:04,920 --> 00:13:07,200 WALK THROUGH A TINY BIT OF 292 00:13:07,200 --> 00:13:08,440 INFORMATION THAT WE ACCUMULATED 293 00:13:08,440 --> 00:13:10,560 OVER 40 YEARS I'VE BEEN AT THE 294 00:13:10,560 --> 00:13:16,440 NIEHS, ON EACH OF THESE 4 295 00:13:16,440 --> 00:13:16,720 PROCESSES. 296 00:13:16,720 --> 00:13:20,600 ONE WAY TO DO THIS IS TO STUDY 297 00:13:20,600 --> 00:13:22,960 MUTATOR DNA POLYMERASES, SO 298 00:13:22,960 --> 00:13:28,440 HERE'S AN EXAMPLE OF THE POLE 299 00:13:28,440 --> 00:13:30,320 DELTA BINDING POCKET OF THIS 300 00:13:30,320 --> 00:13:31,080 CRYSTAL ENZYME STRUCTURE 301 00:13:31,080 --> 00:13:32,560 PUBLISHED BY THE CANNED WHAT LAB 302 00:13:32,560 --> 00:13:34,360 SEVERAL YEARS AGO AND IT SHOWS 303 00:13:34,360 --> 00:13:35,960 THE ACTIVEVIATE WITH A NEWLY 304 00:13:35,960 --> 00:13:39,400 FORMING BASE PAIR AND RESIDUES 305 00:13:39,400 --> 00:13:42,960 SURROUNDING THAT NEWLY FORMING 306 00:13:42,960 --> 00:13:47,480 BASE PAIR INCLUDING Y2613 NPOL 307 00:13:47,480 --> 00:13:50,200 DELTA WHICH IMMEDIATELY AT THE 308 00:13:50,200 --> 00:13:52,720 BOTTOM IS HIGHLY CONSERVED IN 309 00:13:52,720 --> 00:13:56,400 ALL 4 EUKARYOTIC DNA POLYMERASES 310 00:13:56,400 --> 00:13:57,440 AND IMMEDIATELY ADJAC TONIGHT 311 00:13:57,440 --> 00:13:58,720 THAT, THE RESIDUE THAT WE'VE 312 00:13:58,720 --> 00:14:02,320 CHANGED WHICH IS EITHER A LUCINE 313 00:14:02,320 --> 00:14:07,160 OR POLE ALPHA OR POLE DELTA OR A 314 00:14:07,160 --> 00:14:10,000 METRICS THININE, CONSERVED 315 00:14:10,000 --> 00:14:12,600 METHIONINE IN POLE EPSILONE. 316 00:14:12,600 --> 00:14:14,960 SO WE'VE CHANGED THOSE AMINO 317 00:14:14,960 --> 00:14:16,960 ACIDS TO OTHER AMINO ACIDS AND 318 00:14:16,960 --> 00:14:18,240 AMONG OTHER THINGS WE 319 00:14:18,240 --> 00:14:21,840 DEMONSTRATED THEY'RE ALL MUTATOR 320 00:14:21,840 --> 00:14:24,240 POLYMERASES AND HERE IS A WHOLE 321 00:14:24,240 --> 00:14:29,560 GENOME MUTATION RATE ANALYSIS OF 322 00:14:29,560 --> 00:14:32,680 8 DIP EMPLOYED BUDDING YEAST 323 00:14:32,680 --> 00:14:35,520 STRAIN, THEY ARE ALL CONTAINED 324 00:14:35,520 --> 00:14:39,280 EITHER THE WILD-TYPE AND/OR THE 325 00:14:39,280 --> 00:14:40,440 MUTATOR OF EPSILONE, ALPHA AND 326 00:14:40,440 --> 00:14:43,320 DELTA AND THEY CONTAIN 327 00:14:43,320 --> 00:14:45,760 MISMATCHED REPAIR EITHER 328 00:14:45,760 --> 00:14:48,640 PROEFFICIENT AND THIS IS A THIS 329 00:14:48,640 --> 00:14:52,400 IS A BRIEF SUMMARY OF WHAT WE 330 00:14:52,400 --> 00:14:54,240 LEARNED ABOUT PUBLICATION IN 331 00:14:54,240 --> 00:14:55,600 WHICH WE PERFORMED WHOLE GENOME 332 00:14:55,600 --> 00:14:58,040 SEQUENCING OF EACH OF THESE 8 333 00:14:58,040 --> 00:14:59,440 STRAINS, PASSAGE THROUGH ABOUT 334 00:14:59,440 --> 00:15:02,000 900 GENERATIONS OF GROWTH ON 335 00:15:02,000 --> 00:15:03,520 PETRI DISHES AND HERE'S SOME OF 336 00:15:03,520 --> 00:15:05,080 THE RESULTS, THIS IS THE NUMBER 337 00:15:05,080 --> 00:15:06,560 OF DISTRIBUTION OF SINGLE BASED 338 00:15:06,560 --> 00:15:11,120 CHANGES THAT WE DISCOVERED IN 339 00:15:11,120 --> 00:15:14,560 THESE 8 STRAINS, THE LEGEND IS 340 00:15:14,560 --> 00:15:16,880 ON THE UPPER LEFT AND EACH OF 341 00:15:16,880 --> 00:15:18,400 THESE COLORED MARKS THERE 342 00:15:18,400 --> 00:15:19,560 REPRESENT WHERE MUTATIONS WERE 343 00:15:19,560 --> 00:15:27,640 FOUND IN THE 16 CHROMOSOMES OF 344 00:15:27,640 --> 00:15:28,640 THESE YEAST STRAINS. 345 00:15:28,640 --> 00:15:33,160 AND THEN AFTER 9 GENERATIONS 346 00:15:33,160 --> 00:15:35,680 LOOKED AT EACH OF THESE IN BLUE 347 00:15:35,680 --> 00:15:43,440 AS WHOLE GENOMES THAT WE SEEK 348 00:15:43,440 --> 00:15:43,680 DATA. 349 00:15:43,680 --> 00:15:45,880 WE KNOW FROM THESE TYPES OF 350 00:15:45,880 --> 00:15:51,040 SAMPLES WE KNOW THAT LEUCINE 351 00:15:51,040 --> 00:15:54,520 L-THE IPADDA 9 AND DELTA 352 00:15:54,520 --> 00:15:58,600 INVITHOE AS CERTAIN SPECIFICITY 353 00:15:58,600 --> 00:16:00,720 INCLUDING THE G OPPOSITE T, 354 00:16:00,720 --> 00:16:02,440 MISPAIRS, 28 MORE FREQUENTLY 355 00:16:02,440 --> 00:16:03,640 THAN C OPPOSITE A AS DESCRIBED 356 00:16:03,640 --> 00:16:08,440 IN THE UPPER LEFT OF THIS SLIDE. 357 00:16:08,440 --> 00:16:09,960 AND IF YOU MAP THOSE ON THE 358 00:16:09,960 --> 00:16:11,400 CHROMOSOME, YOU CAN FIND THAT 359 00:16:11,400 --> 00:16:13,480 THE PREVENTIVE RENTERAL 360 00:16:13,480 --> 00:16:14,320 PERFORMANCE OF G-OPPOSITE T 361 00:16:14,320 --> 00:16:18,920 OCCURS JUST TO THE RIGHT OF 362 00:16:18,920 --> 00:16:19,640 THORIGEINSOT LEFT-HAND SIDE, AND 363 00:16:19,640 --> 00:16:22,440 JUST TO THE LEFT OF ORIGINS ON 364 00:16:22,440 --> 00:16:24,560 THE RIGHT HAND SIDE WHICH MEANS 365 00:16:24,560 --> 00:16:27,200 THAT THEY'RE PRIMARILY LAGS 366 00:16:27,200 --> 00:16:29,600 STRAND DNA REPLICATION ERRORS 367 00:16:29,600 --> 00:16:31,640 AND THEIR DISTRIBUTION ACROSS A 368 00:16:31,640 --> 00:16:33,640 CHROMOSOME IS IN RED AND BLUE 369 00:16:33,640 --> 00:16:35,040 X-DIAGRAM ON THE LOWER LEFT AND 370 00:16:35,040 --> 00:16:36,440 IF YOU CONSIDER THIS KIND OF 371 00:16:36,440 --> 00:16:38,320 INFORMATION FOR EACH OF THE 12 372 00:16:38,320 --> 00:16:41,080 MISPAIRS THAT CAN FORM, AND FOR 373 00:16:41,080 --> 00:16:44,080 EACH OF THE 3 MAJOR REPLICATIVE 374 00:16:44,080 --> 00:16:47,720 DNA POLYMERASES, ALPHA DELTA AND 375 00:16:47,720 --> 00:16:49,280 EPSILONE, WE COME UP WITH A 376 00:16:49,280 --> 00:16:50,600 MODEL THAT WE BELIEVE IS 377 00:16:50,600 --> 00:16:52,200 CURRENTLY GOING ON IN YEAST 378 00:16:52,200 --> 00:16:55,160 CELLS WHICH IS THAT POLE EPSILON 379 00:16:55,160 --> 00:16:59,440 IS THE MAJOR LEADING STRAND 380 00:16:59,440 --> 00:17:01,160 REPLICATION, AND THE POLE DELTA 381 00:17:01,160 --> 00:17:03,240 DOES THE BULK OF CHAIN 382 00:17:03,240 --> 00:17:05,120 ELONGATION OVER THE LAGGING 383 00:17:05,120 --> 00:17:05,720 STRAND. 384 00:17:05,720 --> 00:17:08,040 YOU CAN TEST THIS HYPOTHESIS 385 00:17:08,040 --> 00:17:11,720 FURTHER USING A PROCEDURE CALLED 386 00:17:11,720 --> 00:17:12,520 HYDROLYTIC DNA IN-SEQUENCING 387 00:17:12,520 --> 00:17:15,440 WHICH WE DEPRIVATIONED BACK IN 388 00:17:15,440 --> 00:17:17,480 PUBLISHED IN 2015, THAT'S THE 389 00:17:17,480 --> 00:17:18,920 PROCEDURE ITSELF, BUT THIS TELLS 390 00:17:18,920 --> 00:17:23,680 YOU WHERE EACH OF THE 3 391 00:17:23,680 --> 00:17:24,320 POLYMERASES INCORPORATE 392 00:17:24,320 --> 00:17:28,160 RIBONUCLEOTIDES INTO THE 393 00:17:28,160 --> 00:17:29,760 CHROMOSOMES IN NUCLEIC 394 00:17:29,760 --> 00:17:30,960 CHROMOSOMES IN BUDDING YEAST AND 395 00:17:30,960 --> 00:17:34,000 THIS IS 1 EXAMPLE OF FROM 2015 396 00:17:34,000 --> 00:17:35,960 WHERE IN THE MIDDLE OF THIS 397 00:17:35,960 --> 00:17:46,480 SLIDE, THE RED AND GREEN--WHERE 398 00:17:57,920 --> 00:17:59,200 THE RED AND BLUE SWITCH AND IF 399 00:17:59,200 --> 00:18:02,880 WE LOOK AT THE PATTERNS OF WHERE 400 00:18:02,880 --> 00:18:09,080 THESE 3 MUTANT DNA POLYMERASES 401 00:18:09,080 --> 00:18:09,800 INCORPORATE RIBONUCLEOTIDES AND 402 00:18:09,800 --> 00:18:12,600 WE GET A PICTURE THAT INDICATES 403 00:18:12,600 --> 00:18:16,440 THE ROLES OF REPLICATION AND 404 00:18:16,440 --> 00:18:17,960 IDENTIFIES REPLICATION ORIGINS, 405 00:18:17,960 --> 00:18:21,760 SO WHERE WE ARE RIGHT NOW, IS 406 00:18:21,760 --> 00:18:26,040 SHOWN IN THIS SLIDE, THE CURRENT 407 00:18:26,040 --> 00:18:31,880 MODEL IS SHOWN IN THE TOP PANEL 408 00:18:31,880 --> 00:18:35,000 A WHICH IS--SHOWS THAT POLE 409 00:18:35,000 --> 00:18:38,560 EPSILONE, IS THE MAJOR LAGGING 410 00:18:38,560 --> 00:18:40,480 STRAND REP LIAISON CASE, POLE 411 00:18:40,480 --> 00:18:44,440 DALTA IS THE MAJOR LAGGING 412 00:18:44,440 --> 00:18:46,880 STRAND, POLE EPSILONE IS THE 413 00:18:46,880 --> 00:18:50,120 MAJOR LEADING STRAND, SHOWS 414 00:18:50,120 --> 00:18:51,200 ORIGINS OF REPLICATION WHERE 415 00:18:51,200 --> 00:18:54,080 POLL ALPHA IS DOING MOST OF THE 416 00:18:54,080 --> 00:18:56,240 INITIATION OF OKSCRKS AKI 417 00:18:56,240 --> 00:18:57,680 FRAGMENTS ON THE LAST STRAND. 418 00:18:57,680 --> 00:19:01,080 HOWEVER, WE SINCE PUBLISHED THAT 419 00:19:01,080 --> 00:19:04,600 DURING INITIATION OR 420 00:19:04,600 --> 00:19:06,560 REPLICATION, POLL DELTA, 421 00:19:06,560 --> 00:19:09,160 SYNTHESIZES POLL ALPHA AND DELTA 422 00:19:09,160 --> 00:19:11,200 SIPGHTICIZES THESE ORIGINS AND 423 00:19:11,200 --> 00:19:13,080 REPLICATION AND THEN WHEN POLL 424 00:19:13,080 --> 00:19:17,400 DELTA BUMPS INTO THE POLL 425 00:19:17,400 --> 00:19:18,920 EPSILON, INACTIVE BUT NAILED IT, 426 00:19:18,920 --> 00:19:22,200 IT'S A COLLISION BETWEEN THOSE 2 427 00:19:22,200 --> 00:19:25,200 ENZYMES ALLOWING POLL EPSILONE, 428 00:19:25,200 --> 00:19:29,080 TO INITIATE LEADING STRAND 429 00:19:29,080 --> 00:19:29,880 REPLICATION, BUT WITHOUT DENYING 430 00:19:29,880 --> 00:19:32,920 THE FACT THAT THE WHOLE ALPHA 431 00:19:32,920 --> 00:19:33,800 AND DELTA SYNTHESIZED BOTH THE 432 00:19:33,800 --> 00:19:39,040 LEADING AND THE LAGGING STRAND 433 00:19:39,040 --> 00:19:41,800 WITH ORIGINS OF APPLICATIONS 434 00:19:41,800 --> 00:19:42,600 LIAISONPLICATION DURING 435 00:19:42,600 --> 00:19:44,040 APPLICATION AND THEN PANEL C 436 00:19:44,040 --> 00:19:45,560 ILLUSTRATES WHAT WE THINK IS 437 00:19:45,560 --> 00:19:47,840 GOING ON WHEN 2 REPLICATION 438 00:19:47,840 --> 00:19:49,720 FORKS COLLIDE WITH 1 ANOTHER 439 00:19:49,720 --> 00:19:53,920 FROM OPPOSITE ORIGINS AND THERE, 440 00:19:53,920 --> 00:19:56,440 EPSILONE, HANDINGS THE DNA BACK 441 00:19:56,440 --> 00:19:59,640 TO POLE DELTA WHICH DOES LEADING 442 00:19:59,640 --> 00:20:01,560 AND LAGGING, SO OUR OVERALL 443 00:20:01,560 --> 00:20:07,120 CURRENT WORKING HIPOLGT SIS IS 444 00:20:07,120 --> 00:20:09,560 THAT POLE EPSILONE IS THE 445 00:20:09,560 --> 00:20:11,000 LEADING DELTA REP LIAISON CASE 446 00:20:11,000 --> 00:20:14,880 BUT ALPHA AND DELTA MAY BE OF 447 00:20:14,880 --> 00:20:18,560 THE LEADING STRAND AT ORIGINS AT 448 00:20:18,560 --> 00:20:22,560 TERM NATIONS BEING A PREDOMINANT 449 00:20:22,560 --> 00:20:27,000 LEADING STRANDS OF THESE 450 00:20:27,000 --> 00:20:27,360 ENZYMES. 451 00:20:27,360 --> 00:20:28,640 SO REPLICATION IS COMPLICATED 452 00:20:28,640 --> 00:20:30,560 BUT FOR THE VAST MAJORITY OF 453 00:20:30,560 --> 00:20:34,240 REPLICATION, AT LEAST OF THE 454 00:20:34,240 --> 00:20:37,920 YEAST CHROMOSOMES WE BELIEVE 455 00:20:37,920 --> 00:20:39,280 POLE EPSILOINY IS THE LEADING 456 00:20:39,280 --> 00:20:44,040 CASE AND DELTA AND ALPHA ARE THE 457 00:20:44,040 --> 00:20:46,000 SYNTHESIZING, THESE FRAGMENTS. 458 00:20:46,000 --> 00:20:50,320 IN 1989 AND 1990 THERE WERE 22 459 00:20:50,320 --> 00:20:53,200 PUBLICATIONS, 1 BY KATHRYN AND 1 460 00:20:53,200 --> 00:20:56,400 BY FRED ANDULARLY, WHICH 461 00:20:56,400 --> 00:20:57,800 PROVIDED BIOCHEMICAL EVIDENCE 462 00:20:57,800 --> 00:21:00,640 SUGGESTING THAT AFTER A 463 00:21:00,640 --> 00:21:01,840 POLYMERASE GENERATES A MISMATCH, 464 00:21:01,840 --> 00:21:04,640 THAT MISMATCH CAN BE REMOVED BY 465 00:21:04,640 --> 00:21:07,920 AN EXONUCLEASE IN A SEPARATE 466 00:21:07,920 --> 00:21:08,520 PROTEIN. 467 00:21:08,520 --> 00:21:13,080 AND THAT'S WHAT WE NOW MY LAB 468 00:21:13,080 --> 00:21:15,800 REFERRED TO AS EXTRINSIC PROOF 469 00:21:15,800 --> 00:21:16,720 READING WHERE THE POLYMERASE 470 00:21:16,720 --> 00:21:18,400 THAT MAY HAVE BEEN THE MISTAKE 471 00:21:18,400 --> 00:21:20,360 IS NOT THE SAME PROTEIN THAT 472 00:21:20,360 --> 00:21:24,600 PROOF READS THIS THING. 473 00:21:24,600 --> 00:21:25,920 SO INTRINSIC PROOF READING OF 474 00:21:25,920 --> 00:21:29,040 MISMATCH IS MADE AND IS THEN 475 00:21:29,040 --> 00:21:30,240 MOVED WITHOUT INTERVENING 476 00:21:30,240 --> 00:21:32,160 DISASSOCIATION OR THE 477 00:21:32,160 --> 00:21:34,120 POLARIZEDDER MERACE, WHEREAS 478 00:21:34,120 --> 00:21:35,760 EXTRENSIC PROOF IF A MISMATCH IS 479 00:21:35,760 --> 00:21:39,240 MADE AND THAT DISASSOCIATES AND 480 00:21:39,240 --> 00:21:41,680 THEN SOME ENZYME THAT THEN BINDS 481 00:21:41,680 --> 00:21:43,560 TO THE MISMATCH IN THE 482 00:21:43,560 --> 00:21:45,880 EXONUCLEASE ACTIVE SITE AND 483 00:21:45,880 --> 00:21:46,360 REMOVES THE MISMATCH. 484 00:21:46,360 --> 00:21:48,080 SO WE WERE INTERESTED IN TESTING 485 00:21:48,080 --> 00:21:50,120 THIS HYPOTHESIS AND WE'VE DONE 486 00:21:50,120 --> 00:21:52,880 SO NOW, AND WE NOW HAVE EVIDENCE 487 00:21:52,880 --> 00:21:55,680 FOR EXTRINSIC PROOF READING OF 488 00:21:55,680 --> 00:21:58,360 REPLICATION ERRORS, BY POLL 489 00:21:58,360 --> 00:21:58,560 DELTA. 490 00:21:58,560 --> 00:22:02,160 ONE PUBLICATION WAS PUBLISHED IN 491 00:22:02,160 --> 00:22:06,680 2006 AND IT SHOWS A SYNERGISTIC 492 00:22:06,680 --> 00:22:08,000 INCREASE IN THE MUTATION RATE 493 00:22:08,000 --> 00:22:11,440 WHEN WE HAVE A MUTATOR 494 00:22:11,440 --> 00:22:14,680 DERIVATIVE OF POLL ALPHA ON THE 495 00:22:14,680 --> 00:22:16,760 LAGS STRAND INITIATING OKSAKI 496 00:22:16,760 --> 00:22:17,720 FRAGMENTS AND WE COMBINE THAT 497 00:22:17,720 --> 00:22:22,520 WITH A PROOF READING DEFICIENT 498 00:22:22,520 --> 00:22:25,040 DERIVATIVE OF POLE DELTA, 35 DB 499 00:22:25,040 --> 00:22:26,960 AND WHEN YOU COME BIEN THOSE 2, 500 00:22:26,960 --> 00:22:28,920 YOU GET A SYNERGISTIC INCREASE 501 00:22:28,920 --> 00:22:33,200 IN MUTATION RATE THAT'S 502 00:22:33,200 --> 00:22:35,840 ILLUSTRATED BY THAT HIGH GRADE 503 00:22:35,840 --> 00:22:44,520 BAR ON THE LEFT AND IN 2021, 504 00:22:44,520 --> 00:22:46,640 DR. ZHU LED A STUDY IN MY LAB 505 00:22:46,640 --> 00:22:49,280 WHICH SHOWS A SYNERGISTIC 506 00:22:49,280 --> 00:22:52,080 INCREASE ONLY PRIMARILY FOR POLE 507 00:22:52,080 --> 00:22:53,920 EPSILONE ERRORS SO THE DATA IN 508 00:22:53,920 --> 00:22:55,640 THIS ARE CONSISTENT WITH 509 00:22:55,640 --> 00:22:58,840 HYPOTHESIS THAT POLE DELTA CAN 510 00:22:58,840 --> 00:23:00,720 EXTRENSICICALLY DO THE 511 00:23:00,720 --> 00:23:01,560 REPLICATION ERRORS IN VIVO, AND 512 00:23:01,560 --> 00:23:06,360 AS YOU CAN SEE, SO FAR IN THE 513 00:23:06,360 --> 00:23:11,560 UPPER LEFT, POLL 2-4 514 00:23:11,560 --> 00:23:15,840 EXODEFICIENT ENZYME WHICH IS A 515 00:23:15,840 --> 00:23:17,840 PROEPSILONE POLYMERASE CANNOT 516 00:23:17,840 --> 00:23:22,840 EXTRINSICALLY PROOF READ ERRORS 517 00:23:22,840 --> 00:23:26,080 MADE BY POLE. 518 00:23:26,080 --> 00:23:28,440 BY POL-ALPHA SO SO FAR WE HAVE 519 00:23:28,440 --> 00:23:29,920 EXTRINSIC READING BY POL DELTA 520 00:23:29,920 --> 00:23:34,640 BUT NO EVIDENCE THAT POL E OR 521 00:23:34,640 --> 00:23:36,320 POL A CAN CONTRIBUTE TO THE SAME 522 00:23:36,320 --> 00:23:36,720 PROCESS. 523 00:23:36,720 --> 00:23:41,200 SO THIS IS A SUMMARY OF WHAT WE 524 00:23:41,200 --> 00:23:44,800 LEARNED SO FAR, EXTRINSIC POL 525 00:23:44,800 --> 00:23:45,880 READING, CORRECTS MISMATCHES 526 00:23:45,880 --> 00:23:48,080 MADE BY ALL 3 REP LIAISON CASES, 527 00:23:48,080 --> 00:23:49,640 IT'S EFFICIENT, IT'S INDEPENDENT 528 00:23:49,640 --> 00:23:51,840 OF POLYMERASE ACTIVITY OF POL 529 00:23:51,840 --> 00:23:54,520 DELTA, IT'S LARGELY INDEPENDENT 530 00:23:54,520 --> 00:23:56,760 OF DNA MISMATCH REPAIR, IT'S 531 00:23:56,760 --> 00:23:59,480 SPECIFICITY DIFFERS FROM 532 00:23:59,480 --> 00:24:00,800 INTRINSIC PROOF READING. 533 00:24:00,800 --> 00:24:03,720 IT BALANCES LEADING AND LAGGING 534 00:24:03,720 --> 00:24:04,520 STRAND REPLICATION FIDELITY AND 535 00:24:04,520 --> 00:24:06,720 WE BELIEVE IT'S RELEVANT TO THE 536 00:24:06,720 --> 00:24:08,720 ORIGINS OF CANCER AND TO 537 00:24:08,720 --> 00:24:09,680 EVOLUTION AND IF YOU'RE 538 00:24:09,680 --> 00:24:15,600 INTERESTED IN THESE PROCESSES WE 539 00:24:15,600 --> 00:24:18,000 HAVE WRITTEN A LITTLE MINIREVIEW 540 00:24:18,000 --> 00:24:21,000 OF THIS PROCESS WHICH WILL BE 541 00:24:21,000 --> 00:24:22,840 PUBLISHED IN A FEW WEEKS IN THE 542 00:24:22,840 --> 00:24:24,800 JOURNAL OF DNA REPAIR. 543 00:24:24,800 --> 00:24:28,760 THIS IS THE SECOND SUBJECT OR 544 00:24:28,760 --> 00:24:30,360 OKSAKI FRAME MATURATION, THIS 545 00:24:30,360 --> 00:24:32,560 SLIDE IS SIMPLY PUT UP THERE TO 546 00:24:32,560 --> 00:24:37,200 TELL YOU THAT OKSAKI FRAGMENT 547 00:24:37,200 --> 00:24:39,720 MATURATION WHICH IS TD REMOVE 548 00:24:39,720 --> 00:24:41,440 WILL OF RIBOTIDES INCORPORATE 549 00:24:41,440 --> 00:24:43,280 BIDE POL ALPHA AND THEIR 550 00:24:43,280 --> 00:24:45,720 REPLACEMENT BY POL DELTA WITH 551 00:24:45,720 --> 00:24:47,920 DNA, FOLLOWED BY LIGATION TO 552 00:24:47,920 --> 00:24:51,120 FORM A CONTINUOUS LAGGING 553 00:24:51,120 --> 00:24:52,600 STRAND, THAT PROCESS IS VERY 554 00:24:52,600 --> 00:24:55,760 COMPLICATED AND CAN BE A SHORT 555 00:24:55,760 --> 00:24:57,600 PATCH PATHWAYOT LEFT OR A LONG 556 00:24:57,600 --> 00:25:00,240 PATCH PATHWAY ON THE RIGHT THAT 557 00:25:00,240 --> 00:25:09,320 CAN TAKE CARE OF THE LONG PATCH 558 00:25:09,320 --> 00:25:11,240 AND RETURN IT TO THE PATHWAY. 559 00:25:11,240 --> 00:25:12,880 THIS IS A SHORT PATCH PROCESS 560 00:25:12,880 --> 00:25:13,560 BUT ON THE LEFT-HAND SIDE YOU 561 00:25:13,560 --> 00:25:17,520 CAN SEE THAT WHEN WHEN A SHORT 562 00:25:17,520 --> 00:25:20,160 PATCH IS THERE, FLAP ENDOCLUE 563 00:25:20,160 --> 00:25:22,800 CLE ACE BINDS AND EXCISES THAT 564 00:25:22,800 --> 00:25:24,240 SHORT PATCH. 565 00:25:24,240 --> 00:25:27,080 AFTER SYNTHESIS BY POL DELTA TO 566 00:25:27,080 --> 00:25:29,440 CREATE THAT FLAP. 567 00:25:29,440 --> 00:25:33,440 AND THEN POL, THE FLAP 568 00:25:33,440 --> 00:25:34,920 ENDONUCLEACE DISASSOCIATES AND 569 00:25:34,920 --> 00:25:37,680 DNA LIGASE SEALS IT TO GIVE A 570 00:25:37,680 --> 00:25:39,320 CONTINUOUS LAGGING STRAND 571 00:25:39,320 --> 00:25:40,120 TEMPLATE. 572 00:25:40,120 --> 00:25:43,280 THAT'S THE PROCESS THAT COPIES 573 00:25:43,280 --> 00:25:48,720 THAT HAPPENS ROUGHLY 25 MILLION 574 00:25:48,720 --> 00:25:50,080 TIMES PER EUKARYOTIC NUCLEAR 575 00:25:50,080 --> 00:25:51,960 GENOME AND IT'S BEEN 576 00:25:51,960 --> 00:25:52,760 DISPLACEMENT SYNTHESIS AS YOU 577 00:25:52,760 --> 00:25:55,560 CAN SEE, SO WE'VE BEEN VERY 578 00:25:55,560 --> 00:25:57,040 INTERESTED IN ADDRESSING WHETHER 579 00:25:57,040 --> 00:25:59,040 THE FIDELITY OF THAT REACTION 580 00:25:59,040 --> 00:26:04,920 DIFFERS FROM THE FIDELITY OF 581 00:26:04,920 --> 00:26:06,160 MOST OF REPLICATION WHICH 582 00:26:06,160 --> 00:26:08,280 REPRESENTS MOST OF THIS ON AN 583 00:26:08,280 --> 00:26:08,640 OPEN TEMPLATE. 584 00:26:08,640 --> 00:26:10,320 WELL 1 WAY RECENTLY THAT WE'VE 585 00:26:10,320 --> 00:26:12,960 GOTTEN INVOLVED IN THAT, COMES 586 00:26:12,960 --> 00:26:15,240 FROM WORK DONE IMMEDIATELY NEXT 587 00:26:15,240 --> 00:26:18,920 DOOR TO US BY SCOTT WILLIAMS LAB 588 00:26:18,920 --> 00:26:20,760 WHO PUBLISHED THE HIGH 589 00:26:20,760 --> 00:26:22,800 RESOLUTION STRUCTURE OF DNA 590 00:26:22,800 --> 00:26:26,080 LIGASE 1, BOUND TO DNA AND WHAT 591 00:26:26,080 --> 00:26:29,880 THEY DISCOVER ON THE UPPER RIGHT 592 00:26:29,880 --> 00:26:31,680 OF THIS SLIDE IS THAT IN 593 00:26:31,680 --> 00:26:33,760 ADDITION TO THE CATALYTIC 594 00:26:33,760 --> 00:26:35,160 MAGNESIUM AT THE POLYMERASE OR 595 00:26:35,160 --> 00:26:37,360 AT THE LIGASE ACTIVE SITE, 596 00:26:37,360 --> 00:26:40,960 THERE'S A SECOND MAGNESIUM ION 597 00:26:40,960 --> 00:26:42,280 BOUND 3 NUCLEOTIDES UP STREAM OF 598 00:26:42,280 --> 00:26:44,320 WHERE THE LIGATION HAS TO OCCUR 599 00:26:44,320 --> 00:26:47,320 AND IF STRUCTURE IS SHOWN IN THE 600 00:26:47,320 --> 00:26:51,840 MIDDLE OF THIS SLIDE, THAT LED 601 00:26:51,840 --> 00:26:54,000 JESS WILLIAMS WHO IS SCOTT 602 00:26:54,000 --> 00:26:55,640 WILLIAMS WIFE BUT WORKS IN MY 603 00:26:55,640 --> 00:27:00,600 LAB TO ADDRESS THE QUESTION OR 604 00:27:00,600 --> 00:27:01,800 DNA LIGASE FIDELITY IS IMPORTANT 605 00:27:01,800 --> 00:27:02,720 FOR GENOME STABILITY AND THE 606 00:27:02,720 --> 00:27:06,000 APPROACH IS TO USE A BUDDING 607 00:27:06,000 --> 00:27:08,640 YEAST STRAIN AND TEST THE MUTE O 608 00:27:08,640 --> 00:27:11,200 GENIC CONSEQUENCES OF EXPRESSING 609 00:27:11,200 --> 00:27:12,600 A LIGASE MUTANT IN THAT STRAIN 610 00:27:12,600 --> 00:27:15,960 WHICH GETS RID OF THOSE HIGH 611 00:27:15,960 --> 00:27:17,680 FIDELITY MAGNESIUM BINDING SITE 612 00:27:17,680 --> 00:27:21,400 AND THIS IS WHAT JUST FOUND THAT 613 00:27:21,400 --> 00:27:24,480 THE SPECTRUM OF MUTANTS IN THAT 614 00:27:24,480 --> 00:27:27,920 LIGASE MUTANT ARE DOMINANTLY 615 00:27:27,920 --> 00:27:29,840 CHARACTERIZED BY 1 NUCLEOTIDE 616 00:27:29,840 --> 00:27:32,120 EDITIONS IN SHORT MONONUCLEOTIDE 617 00:27:32,120 --> 00:27:32,720 ROUNDS. 618 00:27:32,720 --> 00:27:36,720 THOSE ARE THOSE RED CLOSED 619 00:27:36,720 --> 00:27:39,000 TRIANGLES, YOU CAN SEE SEVERAL 620 00:27:39,000 --> 00:27:40,640 HOT SPOTS FOR THOSE EVENTS 621 00:27:40,640 --> 00:27:48,400 OCCURRING IN THE EURO 3 GENE AND 622 00:27:48,400 --> 00:27:52,520 WE BELIEVE THEY ARE REPLICATION 623 00:27:52,520 --> 00:27:54,880 ERRORS IN THE OGASAKI, LIGASE IN 624 00:27:54,880 --> 00:27:56,200 THE MUSEUM TABT AND WE GOT 625 00:27:56,200 --> 00:27:58,400 INTERESTED IN WHETHER THEY'RE 626 00:27:58,400 --> 00:27:59,760 SUBJECTED TO DNA MISMATCH REPAIR 627 00:27:59,760 --> 00:28:04,000 WHICH CAN BE MY MUTE S ALPHA OR 628 00:28:04,000 --> 00:28:06,040 MUTE S-BETA DEPENDENT REPAIR OR 629 00:28:06,040 --> 00:28:11,200 BY FLAP PROCESSING DURING 630 00:28:11,200 --> 00:28:14,960 OKASAKI, AND SO WE TESTED THIS 631 00:28:14,960 --> 00:28:17,840 IN FLAP ENDONUCLEACE AND BOTH OF 632 00:28:17,840 --> 00:28:22,160 THOSE PROCESSES DO AFFECT 633 00:28:22,160 --> 00:28:23,400 REPLICATION FIDELITY, AND THEY 634 00:28:23,400 --> 00:28:26,160 ARE SPECIFIC FOR CREATING A 635 00:28:26,160 --> 00:28:29,800 LARGE INCREASE IN MUTE O 636 00:28:29,800 --> 00:28:34,760 GENESIS, FOR PLUS 1 BASE FRAME 637 00:28:34,760 --> 00:28:36,320 SHIFTS IN SHORT HOMONUCLEOTIDE 638 00:28:36,320 --> 00:28:37,640 RUNS, SO HERE IS THE CURRENT 639 00:28:37,640 --> 00:28:41,200 WORKING MODEL IN THE LAB WHERE 640 00:28:41,200 --> 00:28:43,400 ON THE UPPER LEFT IS STRAND 641 00:28:43,400 --> 00:28:45,000 DISPLACEMENT SYNTHESIS TO CREATE 642 00:28:45,000 --> 00:28:47,440 THE FLAP, FOLLOWED BY A STRAND 643 00:28:47,440 --> 00:28:50,840 SLIPPAGE EVENT TO CREATE AN 644 00:28:50,840 --> 00:28:54,720 EXTRA HELICAL C-RESIDUE AND THEN 645 00:28:54,720 --> 00:28:58,480 FOLLOWING THAT UPON OR ONCE THAT 646 00:28:58,480 --> 00:29:00,800 OCCURS, YOU HAVE EXTRA C THAT 647 00:29:00,800 --> 00:29:04,560 CAN BE INCORPORATED AND THEN THE 648 00:29:04,560 --> 00:29:05,680 FLAP ENDONUCLEACE CLEAVES THE 649 00:29:05,680 --> 00:29:09,720 FLAP AND THEN LIGASE IS SUPPOSED 650 00:29:09,720 --> 00:29:11,680 TO LIGATE THIS INTERMEDIATE TO 651 00:29:11,680 --> 00:29:14,200 CONTAIN A CONTINUOUS LEADING 652 00:29:14,200 --> 00:29:16,000 STRAND BUT ON THE LOWER LEFT, 653 00:29:16,000 --> 00:29:18,880 YOU CAN SEE IF THE WILD-TYPE REP 654 00:29:18,880 --> 00:29:19,800 LIAISON CASE ENCOUNTERS THAT, 655 00:29:19,800 --> 00:29:25,360 CAN YOU HAVE A BOARD OF LIGATION 656 00:29:25,360 --> 00:29:29,320 AND END PROCESSING OR PROOF 657 00:29:29,320 --> 00:29:31,080 READING TO REMOVE THAT EXTRA 658 00:29:31,080 --> 00:29:32,600 NUCLEOTIDE BUT IF THE MUTANT 659 00:29:32,600 --> 00:29:36,480 FORM OF LIGASE IS THERE, THIS 660 00:29:36,480 --> 00:29:39,720 EE TO MUTE ANT EA, YOU GET 661 00:29:39,720 --> 00:29:41,560 INSERTIONS INSERTED BY INSERTING 662 00:29:41,560 --> 00:29:46,320 THE NEXT CORRECT C AND THEN MUTE 663 00:29:46,320 --> 00:29:46,840 O GENIC LIGATION. 664 00:29:46,840 --> 00:29:50,680 SO WE'RE VERY INTERESTED IN THE 665 00:29:50,680 --> 00:29:55,320 PROCESS, AS 1 SIGNATURE OF 666 00:29:55,320 --> 00:29:56,200 OKASAKI MATURATION AND WHAT I 667 00:29:56,200 --> 00:30:01,080 CAN SAY IS HAD --WHAT WE'RE DOIG 668 00:30:01,080 --> 00:30:02,600 NOW IS TO INVESTIGATE THAT WHOLE 669 00:30:02,600 --> 00:30:04,880 PROCESS BY WHOLE GENOME 670 00:30:04,880 --> 00:30:06,680 SEQUENCING TO EXAMINE THAT 671 00:30:06,680 --> 00:30:07,720 CHARACTERISTIC ERROR OR 672 00:30:07,720 --> 00:30:09,880 SIGNATURE PLUS 1 FRAME SHIFTS 673 00:30:09,880 --> 00:30:11,960 ACROSS THE ENTIRE NUCLEAR 674 00:30:11,960 --> 00:30:12,200 GENOME. 675 00:30:12,200 --> 00:30:17,440 I'LL LET YOU KNOW WHAT HAPPENS, 676 00:30:17,440 --> 00:30:18,040 SOMEDAY SOON. 677 00:30:18,040 --> 00:30:19,360 AND THEN HERE'S 1 OF THE MORE 678 00:30:19,360 --> 00:30:21,800 INTERESTING SLIDES, I WILL SHOW 679 00:30:21,800 --> 00:30:25,840 YOU REPRESENTS UNPUBLISHED 680 00:30:25,840 --> 00:30:27,240 OBSERVATIONS SO DON'T TAKE THIS 681 00:30:27,240 --> 00:30:28,520 TOO SERIOUSLY UNTIL WE HAVE TIME 682 00:30:28,520 --> 00:30:32,320 TO DO MORE WORK ON IT, BUT THIS 683 00:30:32,320 --> 00:30:41,240 IS THE EURO3 MUTANT SPECTRA FOR 684 00:30:41,240 --> 00:30:43,440 AN ACTIVE SITE MUTATION IN 685 00:30:43,440 --> 00:30:47,520 LIGASE 1, THAT'S THE MOST MIXED 686 00:30:47,520 --> 00:30:50,120 COMBINATION OF ERRORS CREATED 687 00:30:50,120 --> 00:30:51,320 DURING OKASAKI MATURATION THAT 688 00:30:51,320 --> 00:30:52,120 I'VE EVER SEEN. 689 00:30:52,120 --> 00:30:53,760 IT INCLUDES ALL KINDS OF 690 00:30:53,760 --> 00:30:58,160 MUTATIONS, PLUS 1S AND MINUS 1S, 691 00:30:58,160 --> 00:31:00,640 IN RUNS, NONE RUNS AND THEN THE 692 00:31:00,640 --> 00:31:02,800 BIG RED DRI ANGLES ARE INSERTION 693 00:31:02,800 --> 00:31:06,200 OF OF LARGER NUMBERS OF 694 00:31:06,200 --> 00:31:07,280 NUCLEOTIDES AND WHEN JESS AND I 695 00:31:07,280 --> 00:31:08,560 HAVE BEEN STUDYING THIS SPECTRUM 696 00:31:08,560 --> 00:31:11,240 FOR A COUPLE OF WEEKS NOW, AND 697 00:31:11,240 --> 00:31:15,760 WE BELIEVE IT INDICATES AT LEAST 698 00:31:15,760 --> 00:31:16,720 5 DIFFERENT MECHANISMS BY WHICH 699 00:31:16,720 --> 00:31:22,920 1 CAN GET A MUTATOR PHENOTYPE BY 700 00:31:22,920 --> 00:31:26,440 HAVING AN ACTIVE SITE POINT 701 00:31:26,440 --> 00:31:27,040 MUTATION IN LIGASE 1. 702 00:31:27,040 --> 00:31:28,360 SO THIS IS WORK THAT WE'RE 703 00:31:28,360 --> 00:31:30,640 WORKING ON NOW TO BEGIN TO 704 00:31:30,640 --> 00:31:31,960 INVESTIGATE THOSE MECHANISMS AND 705 00:31:31,960 --> 00:31:34,520 WE WILL FOLLOW THIS SLIDE UP 706 00:31:34,520 --> 00:31:35,240 WITH PUBLICATIONS, SOMETIMES IN 707 00:31:35,240 --> 00:31:37,840 THE NEXT YEAR OR 2 INCLUDING 708 00:31:37,840 --> 00:31:39,840 WHOLE GENOME SEQUENCING TO SEE 709 00:31:39,840 --> 00:31:43,880 IF THIS KIND OF SPECIFICITY, 710 00:31:43,880 --> 00:31:46,280 BROAD SPECIFICITY IS ACROSS THE 711 00:31:46,280 --> 00:31:48,840 ENTIRE NUCLEAR GENOME, I SHOULD 712 00:31:48,840 --> 00:31:51,400 MENTION THE EURO-3 GENE IS ABOUT 713 00:31:51,400 --> 00:31:53,000 110,000th OF THE ENTIRE LENGTH 714 00:31:53,000 --> 00:31:55,000 OF THE 16 CHROMOSOMES IN YEAST, 715 00:31:55,000 --> 00:31:57,320 SO THERE'S A LOT OF 716 00:31:57,320 --> 00:31:58,520 OPPORTUNITIES TO INVESTIGATE, A 717 00:31:58,520 --> 00:32:00,920 LOT OF PROCESSES RNTION THAT 718 00:32:00,920 --> 00:32:04,200 COULD BE UNIQUE TO OKASAKI 719 00:32:04,200 --> 00:32:05,920 FRAGMENT MATURATION AND SINCE 720 00:32:05,920 --> 00:32:07,360 THAT INVOLVES STRAND 721 00:32:07,360 --> 00:32:10,560 DISPLACEMENT SYNTHESIS, WE ARE 722 00:32:10,560 --> 00:32:12,040 INTERESTED IN KNOWING IF PLUS 1 723 00:32:12,040 --> 00:32:14,640 ERRORS ARE COMMON TO STRAND 724 00:32:14,640 --> 00:32:15,280 DISPLACEMENT SYNTHESIS REACTIONS 725 00:32:15,280 --> 00:32:21,720 BY ANY OF THE OTHER 16 HUMAN DNA 726 00:32:21,720 --> 00:32:22,440 POLYMERASES BESIDES 727 00:32:22,440 --> 00:32:22,800 [INDISCERNIBLE]. 728 00:32:22,800 --> 00:32:24,400 IN FACT, WE HAVEN'T YET 729 00:32:24,400 --> 00:32:27,840 DEMONSTRATED THAT THOSE PLUS 1S 730 00:32:27,840 --> 00:32:30,760 ARE COMMON TO STRAND 731 00:32:30,760 --> 00:32:31,840 DISPLACEMENT BY POL DELTA PER 732 00:32:31,840 --> 00:32:33,360 SE, THAT'S JUST THE WORKING 733 00:32:33,360 --> 00:32:41,840 HYPOTHESIS AT THE MOMENT. 734 00:32:41,840 --> 00:32:43,880 HERE ARE RIBONUCLEOTIDES, ARE 735 00:32:43,880 --> 00:32:44,920 THEY INCORPORATED DURING 736 00:32:44,920 --> 00:32:45,240 REPLICATION? 737 00:32:45,240 --> 00:32:52,680 THIS IS FOR AN INCORRECT BASE. 738 00:32:52,680 --> 00:32:55,360 ON THE UPPER LEFT, IS THE SUGAR 739 00:32:55,360 --> 00:32:58,800 AND THAT CAN ATTACK THE SUGAR 740 00:32:58,800 --> 00:33:00,680 PHOSPHATE BACKBONE IN IT'S 741 00:33:00,680 --> 00:33:02,440 PRESENT IN DNA AND DO CLEAVE AND 742 00:33:02,440 --> 00:33:04,880 CREATE A NIK WITH UNLIGATABLE 743 00:33:04,880 --> 00:33:07,480 DNA ENDS WHICH HAVE TO BE 744 00:33:07,480 --> 00:33:08,960 PROCESSED. 745 00:33:08,960 --> 00:33:14,520 ON THE RIGHT IS HOW POLYMERASES, 746 00:33:14,520 --> 00:33:15,440 DNA POLYMERASES EXCLUDE 747 00:33:15,440 --> 00:33:19,000 RIBONUCLEOTIDES AND ON THE 748 00:33:19,000 --> 00:33:21,240 BOTTOM IS CONCENTRATIONS OF THE 749 00:33:21,240 --> 00:33:23,280 DNTPs AND THE RNAs IN YEAST 750 00:33:23,280 --> 00:33:26,800 MEASURED BY ANDRE CHAVEZ OUR 751 00:33:26,800 --> 00:33:27,480 COLLABORATOR IN THE UNIVERSITY 752 00:33:27,480 --> 00:33:29,240 AND YOU CAN SEE THAT THE 753 00:33:29,240 --> 00:33:30,960 CONCENTRATION OF THE 754 00:33:30,960 --> 00:33:34,760 DEOXYNUCLEOTIDE TRI POS FATES IS 755 00:33:34,760 --> 00:33:35,920 MUCH LOWER THAN THE 756 00:33:35,920 --> 00:33:40,200 CONCENTRATION OF THE RNTPs, BY 757 00:33:40,200 --> 00:33:42,600 DIFFERENCES OF 194 OR 36 FOLD, 758 00:33:42,600 --> 00:33:43,600 ET CETERA. 759 00:33:43,600 --> 00:33:46,960 SO THERE'S A LOT MORE RNTPs 760 00:33:46,960 --> 00:33:49,640 CONSIST WENT THEIR CRITICAL ROLE 761 00:33:49,640 --> 00:33:53,360 IN TRANSCRIPTION, BUT DOES THAT 762 00:33:53,360 --> 00:33:57,440 INCREASE ALSO DNA POLYMERASES TO 763 00:33:57,440 --> 00:33:58,960 INCORPORATE RIBONUCLEOTIDES 764 00:33:58,960 --> 00:34:02,360 BECAUSE IT'S--THEY'RE PRESENT IN 765 00:34:02,360 --> 00:34:06,320 EXCESS OVER DNTPs, THAT IS 766 00:34:06,320 --> 00:34:08,800 INCORPORATION ASSAY DONE INVITRO 767 00:34:08,800 --> 00:34:13,120 WITH POLs ALPHA, DELTA AND 768 00:34:13,120 --> 00:34:18,200 EPSILONE, AND THEY INDICATE 769 00:34:18,200 --> 00:34:20,880 RIBONUCLEOTIDES AS INDICATED BY 770 00:34:20,880 --> 00:34:22,320 THESE LINES WHERE THESE ARE 771 00:34:22,320 --> 00:34:24,840 INCORPORATED BY THE 3 DNA 772 00:34:24,840 --> 00:34:28,920 POLYMERASES AND IF 1 ASKS HOW 773 00:34:28,920 --> 00:34:31,480 CELLS THERE IS A CLEAR ESTIMATE 774 00:34:31,480 --> 00:34:36,760 OF A NUMBER OF RESIDUES 775 00:34:36,760 --> 00:34:38,200 GENERATED PERGENOME, NUCLEAR 776 00:34:38,200 --> 00:34:39,640 GENOME IN EUKARYOTES FOR A WHOLE 777 00:34:39,640 --> 00:34:41,840 VARIETY OF LESIONS THAT HAVE 778 00:34:41,840 --> 00:34:43,760 BEEN STUDIED FOR MORE THAN 50 779 00:34:43,760 --> 00:34:47,440 YEARS AND IF WE ASK HOW MANY 780 00:34:47,440 --> 00:34:48,120 RIBONUCLEOTIDES AREN'T 781 00:34:48,120 --> 00:34:49,320 INCORPORATED, WE CAN GIVE EACH 782 00:34:49,320 --> 00:34:51,720 OF THESE KINDS OF LESIONS 783 00:34:51,720 --> 00:34:54,400 STUDIES FROM THE LAST 50 YEARS, 784 00:34:54,400 --> 00:34:57,640 LITTLE CIRCLES WITH INCREASING 785 00:34:57,640 --> 00:34:58,240 SIZE REPRESENTING INCREASING 786 00:34:58,240 --> 00:35:01,200 NUMBER AND THEN WE CAN SHOW HOW 787 00:35:01,200 --> 00:35:02,360 MANY RIBONUCLEOTIDES ARE 788 00:35:02,360 --> 00:35:04,600 INCORPORATED AND YOU CAN SEE, 789 00:35:04,600 --> 00:35:11,840 RIBONUCLEOTIDES ARE THE MOST 790 00:35:11,840 --> 00:35:12,800 ABUNDANT NONNORMAL--THE 791 00:35:12,800 --> 00:35:14,040 NUCLEOTIDE TRI PHOSPHATE 792 00:35:14,040 --> 00:35:14,680 INCORPORATED DURING REPLICATION 793 00:35:14,680 --> 00:35:17,400 SO THIS GOT US VERY INTERESTED 794 00:35:17,400 --> 00:35:20,600 IN THIS PROCESS, AND THERE'S A 795 00:35:20,600 --> 00:35:22,040 SUMMARY OF WHAT JESS WILLIAMS 796 00:35:22,040 --> 00:35:24,320 AND I PUBLISHED IN THE ANNUAL 797 00:35:24,320 --> 00:35:25,480 REVIEWS OF BIOCHEMISTRY THEY 798 00:35:25,480 --> 00:35:28,520 THINK SHOULD BE OUT NOW, 799 00:35:28,520 --> 00:35:31,880 SUPPOSED TO COME OUT ONLINE IN 800 00:35:31,880 --> 00:35:35,960 MAY, AND IT HAS BOTH THE AMOUNT 801 00:35:35,960 --> 00:35:37,520 OF RIBONUCLEOTIDES INCORPORATED 802 00:35:37,520 --> 00:35:41,800 ON THE LEFT, THE DNA 803 00:35:41,800 --> 00:35:43,520 REPLICATIONS THAT DO THAT CAN 804 00:35:43,520 --> 00:35:48,040 PROOF READ, OR REMARE THOSE 805 00:35:48,040 --> 00:35:52,080 RIBONUCLEOTIDES BY RNA AGE TO 806 00:35:52,080 --> 00:35:52,640 DEPENDENT RIBONUCLEOTIDE 807 00:35:52,640 --> 00:35:57,240 EXCISION REPAIR OR BY 808 00:35:57,240 --> 00:35:58,320 TOPO-ISOMER ACE REMOVAL AND THEN 809 00:35:58,320 --> 00:36:02,480 THERE'S EVEN MORE THAT'S PUTS 810 00:36:02,480 --> 00:36:03,560 INNING OKAZAKI AND WE ARE 811 00:36:03,560 --> 00:36:04,720 BEGINNING TO STUDY THAT PROCESS 812 00:36:04,720 --> 00:36:06,640 AND ON THE RIGHT ARE THEINDS 813 00:36:06,640 --> 00:36:09,000 OF EEIVETS THAT THOSE 814 00:36:09,000 --> 00:36:11,680 RIBONUCLEOTIDES CAN HAVE IN 815 00:36:11,680 --> 00:36:13,720 CELLS INCLUDING REPLICATION, 816 00:36:13,720 --> 00:36:15,280 STRESS AND GENOME INSTABILITY 817 00:36:15,280 --> 00:36:16,000 WITH THE ACKNOWLEDGMENT THAT 818 00:36:16,000 --> 00:36:17,400 SOME OF THOSE EFFECTS COULD BE 819 00:36:17,400 --> 00:36:20,720 RELATED TO OTHER ROLES FOR RNA 820 00:36:20,720 --> 00:36:22,960 SAGE 2 FOR EXAMPLE IN RESOLVING 821 00:36:22,960 --> 00:36:24,720 ALL OF THOSE, SO IF YOU'RE 822 00:36:24,720 --> 00:36:28,080 INTERESTED IN THIS SUBJECT, YOU 823 00:36:28,080 --> 00:36:29,520 CAN READ THAT IN THE REVIEWS 824 00:36:29,520 --> 00:36:31,440 ARTICLE AS A WAY TO GET STARTED 825 00:36:31,440 --> 00:36:32,440 ON THAT. 826 00:36:32,440 --> 00:36:34,280 AND HERE IS EUKARYOTIC MISMATCH 827 00:36:34,280 --> 00:36:36,000 REPAIR, I DON'T HAVE A CLOCK IN 828 00:36:36,000 --> 00:36:40,440 FRONT OF ME, HOW AM I DOING ON 829 00:36:40,440 --> 00:36:40,840 TIME? 830 00:36:40,840 --> 00:36:43,000 >> YOU'RE DOING FINE, YOU STILL 831 00:36:43,000 --> 00:36:46,360 HAVE 15 MINUTES OR SO. 832 00:36:46,360 --> 00:36:48,840 >> OKAY, THAT'S PERFECT. 833 00:36:48,840 --> 00:36:50,880 THIS IS EUKARYOTIC MISMATCH DNA 834 00:36:50,880 --> 00:36:55,480 REPAIR AS WE INFER FROM A LOT OF 835 00:36:55,480 --> 00:36:58,120 LITERATURE, OCCURS IN YEAST OR 836 00:36:58,120 --> 00:36:59,800 HUMAN CELLS. 837 00:36:59,800 --> 00:37:02,280 IT'S RECOGNITION AT THE TOP OF A 838 00:37:02,280 --> 00:37:07,840 MISMATCH LEFT BEHIND BY THE 839 00:37:07,840 --> 00:37:10,040 REPLICATION FORK BY MUTASE ALPHA 840 00:37:10,040 --> 00:37:11,600 FOR EXAMPLE, AND THEN MUTATE 841 00:37:11,600 --> 00:37:14,480 ALPHA LEADS A SEPARATE 842 00:37:14,480 --> 00:37:17,080 HETERODIMER COMPRISED OF PMS 2 843 00:37:17,080 --> 00:37:25,160 EXPW MLH 1 AND THEN THERE'S THE 844 00:37:25,160 --> 00:37:35,600 INVOLVEMENT OF PC NA THAT 845 00:37:41,320 --> 00:37:43,400 ALLOWED THEM IN THE DNA STRAND 846 00:37:43,400 --> 00:37:44,800 AND THIS ALLOWS MISMATCH REMOVAL 847 00:37:44,800 --> 00:37:49,960 BY ANY OF SEVERAL PROCESSES, 848 00:37:49,960 --> 00:37:51,520 FOLLOWED BY DNA MISMATCHES ONCE 849 00:37:51,520 --> 00:37:54,080 IT'S GONE AND THEN LIGATION TO 850 00:37:54,080 --> 00:37:59,280 RESTORE GENOME INTEGRITY AND 851 00:37:59,280 --> 00:38:01,760 THAT'S JUST A VERY QUICK DIRTY 852 00:38:01,760 --> 00:38:03,360 REVIEW OF DNA MISMATCH REPAIR. 853 00:38:03,360 --> 00:38:07,440 WHAT WE'VE BEEN DOING IS 1 SMALL 854 00:38:07,440 --> 00:38:09,800 SUBSET OF THE KIND OF WORK THAT 855 00:38:09,800 --> 00:38:10,520 MANY INVESTIGATORS AROUND THE 856 00:38:10,520 --> 00:38:13,200 WORLD HAVE BEEN DOING, AND HERE 857 00:38:13,200 --> 00:38:17,120 IS 1 EXAMPLE OF 1 OF THE UTCOMES 858 00:38:17,120 --> 00:38:19,840 WE PUBLISHED LAST YEAR. 859 00:38:19,840 --> 00:38:25,000 SCOTT [ [INDISCERNIBLE] IN MY LB 860 00:38:25,000 --> 00:38:27,040 AND I LOOKED AT SEQUENCING A 861 00:38:27,040 --> 00:38:29,520 VARIETY OF OF SPECIES SHOWNOT 862 00:38:29,520 --> 00:38:30,120 LEFT-HAND SIDE AND ON THE 863 00:38:30,120 --> 00:38:32,680 EXTREME RIGHT IS THE EFFICIENCY 864 00:38:32,680 --> 00:38:35,480 OF MISMATCH REPAIR OF ERRORS 865 00:38:35,480 --> 00:38:37,400 MADE IN THOSE ORGANISMS BY THE 866 00:38:37,400 --> 00:38:39,160 REPLICATION MACHINERY AND YOU 867 00:38:39,160 --> 00:38:42,000 CAN SEE THAT THE MISMATCH REPAIR 868 00:38:42,000 --> 00:38:44,200 EFFICIENCY IS ESTIMATED TO BE 869 00:38:44,200 --> 00:38:46,000 ROUGHLY THE SAME IN ALL THESE 870 00:38:46,000 --> 00:38:49,560 ORGANISMS, WE FIND THAT 871 00:38:49,560 --> 00:38:50,680 INTERESTING RELATIVE TO 872 00:38:50,680 --> 00:38:52,960 UNDERSTANDING THE MECHANISMS OF 873 00:38:52,960 --> 00:38:55,560 DNA MISMATCH REPAIR, 1 OF THE 874 00:38:55,560 --> 00:38:58,640 WAYS THAT WE'RE INVESTIGATING 875 00:38:58,640 --> 00:39:02,240 THAT S&P MAY BE THAT MISMATCH IT 876 00:39:02,240 --> 00:39:04,400 ACTS IN THE DIRECTED MANNER 877 00:39:04,400 --> 00:39:05,600 POSSIBLY BECAUSE OF A SPECIAL 878 00:39:05,600 --> 00:39:08,720 RELATION TO THE REPLICATION 879 00:39:08,720 --> 00:39:10,800 COMPLEX THAT WAS FIRST PROPOSED 880 00:39:10,800 --> 00:39:13,800 BY WAGNER AND [INSCERNIBLE] IN 881 00:39:13,800 --> 00:39:17,400 1966 AND ON THE LOWER LEFT, CAN 882 00:39:17,400 --> 00:39:19,560 YOU SEE THERE MAY BE SOME 883 00:39:19,560 --> 00:39:21,040 SPECIAL RELATIONSHIP BETWEEN 884 00:39:21,040 --> 00:39:23,120 WHEN IT HOW REPLICATION ERRORS 885 00:39:23,120 --> 00:39:25,200 ARE MADE BY THE REPLICATIONS AND 886 00:39:25,200 --> 00:39:27,840 HOW THEY'RE REPAIRED BY THE 887 00:39:27,840 --> 00:39:28,600 MISMATCH REPAIR MACHINERY. 888 00:39:28,600 --> 00:39:33,640 AND SOME OF THE PROCESSES THAT 889 00:39:33,640 --> 00:39:36,560 WE'RE INVESTIGATING INCLUDE 890 00:39:36,560 --> 00:39:37,320 MISMATCHED REPAIR EFFICIENCY 891 00:39:37,320 --> 00:39:39,640 POLYMERASE THAT MAKES THE ERROR, 892 00:39:39,640 --> 00:39:44,520 THE MISMATCH REPAIR SUBWAY THAT 893 00:39:44,520 --> 00:39:47,080 OPERATING, IS MUTE S ALPHA OR 894 00:39:47,080 --> 00:39:49,480 MUTE S BETA, 2 DIFFERENT 895 00:39:49,480 --> 00:39:50,000 HETERODIMERS, MISMATCHED 896 00:39:50,000 --> 00:39:51,000 COMPOSITION, WHETHER THE ERROR 897 00:39:51,000 --> 00:39:53,600 IS A LEADING STRAND OR LAGGING 898 00:39:53,600 --> 00:39:57,680 STRAND REPLICATION ERROR, THE 899 00:39:57,680 --> 00:39:59,240 SEQUENCE CONTEXT REPLICATION 900 00:39:59,240 --> 00:40:00,640 TIMING, CHROMOSOMAL LOCATION AND 901 00:40:00,640 --> 00:40:02,840 CHROMATIN STAFF, SO I WILL SHOW 902 00:40:02,840 --> 00:40:05,840 YOU JUST VERY QUICKLY A COUPLE 903 00:40:05,840 --> 00:40:08,240 OF UNPUBLISHED OBSERVATIONS THAT 904 00:40:08,240 --> 00:40:11,760 WERE CURRENTLY WORKING HARD ON 905 00:40:11,760 --> 00:40:12,120 IT. 906 00:40:12,120 --> 00:40:13,960 ONE IS WE KNOW FROM LITERATURE 907 00:40:13,960 --> 00:40:16,360 THAT'S BEEN PUBLISHED INCLUDING 908 00:40:16,360 --> 00:40:18,560 EVIDENCE BY PAUL MODRICH WHO WON 909 00:40:18,560 --> 00:40:21,560 THE NOBEL PRIZE IN 2015 FOR HIS 910 00:40:21,560 --> 00:40:22,800 WORK ON THIS MISMATCH REPAIR, 911 00:40:22,800 --> 00:40:25,040 THAT THERE IS A REQUIREMENT FOR 912 00:40:25,040 --> 00:40:28,240 PC NA IN DNA MISMATCH REPAIR, 913 00:40:28,240 --> 00:40:34,480 AND WE PUBLISHED IN 1996 THAT 914 00:40:34,480 --> 00:40:37,160 THAT REQUIREMENT IS AT A STEP 915 00:40:37,160 --> 00:40:38,040 PRECEDING DNA RESENTH SIS. 916 00:40:38,040 --> 00:40:42,040 SO THERE ON THE LEFT IS A LITTLE 917 00:40:42,040 --> 00:40:44,120 CARTOON OF MISMATCH REPAIR 918 00:40:44,120 --> 00:40:51,760 PROTEINS AND ON THE EXTREME LEFT 919 00:40:51,760 --> 00:40:53,480 OF MSH3 THE END TERMINUS AND YOU 920 00:40:53,480 --> 00:40:57,600 CAN FIND WHAT'S CALLED A PC NA 921 00:40:57,600 --> 00:41:00,280 BINDING MOTIF, AND IT IS 922 00:41:00,280 --> 00:41:04,200 CONSERVED IN A VARIETY OF 923 00:41:04,200 --> 00:41:07,040 DIFFERENT PROTEINS INCLUDING 924 00:41:07,040 --> 00:41:09,080 MSH 6 AND MSH 3, WHICH ARE HALF 925 00:41:09,080 --> 00:41:12,880 OF THE COMPONENT ALONG WITH MSH2 926 00:41:12,880 --> 00:41:17,120 OF EITHER MUTE S ALPHA OR MUTE S 927 00:41:17,120 --> 00:41:18,840 DELTA AND ON THE RIGHT HAND SIDE 928 00:41:18,840 --> 00:41:20,240 IS SHOWING THAT INVESTIGATING 929 00:41:20,240 --> 00:41:23,960 THE ROLE OF PC NA AND MISMATCH 930 00:41:23,960 --> 00:41:26,160 REPAIR AND COMPLICATED BY THE 931 00:41:26,160 --> 00:41:27,920 FACT THAT AND I PULLED THIS OUT 932 00:41:27,920 --> 00:41:30,000 OF THE LITERATURE, THIS IS THE 933 00:41:30,000 --> 00:41:32,800 SEQUENCE ALIGNMENT OF THE PC NA 934 00:41:32,800 --> 00:41:34,360 BINDING MOTIFS FOR HUMAN BINDING 935 00:41:34,360 --> 00:41:44,560 PROTEINS AND THE LIST IS VERY 936 00:41:44,560 --> 00:41:54,680 LONG. 937 00:41:56,840 --> 00:42:00,160 DEFECTS IN BINDING, AND NOTED PC 938 00:42:00,160 --> 00:42:10,440 NA, BECAUSE PC NA, BUT RATHER PC 939 00:42:10,440 --> 00:42:13,640 NA BINDING MOTIF AND MSH3 AND 940 00:42:13,640 --> 00:42:16,800 MSH6 AND ASK WHAT EFFECT IT HAS 941 00:42:16,800 --> 00:42:17,880 ON REPLICATION FIDELITY. 942 00:42:17,880 --> 00:42:19,280 SO WE'RE VIGOROUSLY INVOLVE 943 00:42:19,280 --> 00:42:21,680 INDEED THAT PROCESS, AND WE 944 00:42:21,680 --> 00:42:27,880 SHOULD HAVE SOME ANSWERS SOON TO 945 00:42:27,880 --> 00:42:31,760 COMPLEMENT THE WORK IN MSH-6 AND 946 00:42:31,760 --> 00:42:33,360 3 THAT'S BEEN DONE NOW FOR 947 00:42:33,360 --> 00:42:35,840 SEVERAL YEARS, 1 OF THOSE IS 948 00:42:35,840 --> 00:42:36,800 SECOND UNPUBLISHED OBSERVATION 949 00:42:36,800 --> 00:42:39,800 THAT WE HAVE, THAT IF YOU TACK A 950 00:42:39,800 --> 00:42:43,320 MUTATOR VARIANT OF POL DELTA, 951 00:42:43,320 --> 00:42:45,200 THE L612 M-MUTANT AND YOU KNOCK 952 00:42:45,200 --> 00:42:46,600 OUT MISMATCH REPAIR, BUT YOU 953 00:42:46,600 --> 00:42:50,840 ONLY KNOCK OUT THE MS-3, H3 954 00:42:50,840 --> 00:42:53,160 COMPONENT OF MUTE S BETA WHAT 955 00:42:53,160 --> 00:42:55,720 YOU GET IS THERE'S A STRONG 956 00:42:55,720 --> 00:42:58,160 MUTATOR PHENOTYPE THAT'S SORT OF 957 00:42:58,160 --> 00:43:01,320 SELECTED FOR THE LOSS OF GC BASE 958 00:43:01,320 --> 00:43:04,600 PAIRS IN HOMONUCLEOTIDE RUNS. 959 00:43:04,600 --> 00:43:06,720 SO THIS IS 1 WAY OF SHOWING THAT 960 00:43:06,720 --> 00:43:09,400 SO FAR WE BELIEVE THAT MSH3, 961 00:43:09,400 --> 00:43:13,800 MEASURED BY MUTE O GENESIS AT 962 00:43:13,800 --> 00:43:16,840 THE ERO 3 GENE IS A COMPONENT OF 963 00:43:16,840 --> 00:43:22,720 MUTE S BETA THAT WHEN INACTIVE 964 00:43:22,720 --> 00:43:25,240 LEADS TO MUTATIONS IN MINUS 1 965 00:43:25,240 --> 00:43:27,440 BASE FRAME SHIFTS FROM GC RUNS 966 00:43:27,440 --> 00:43:31,720 AT LEAST IN THE URO3 GENE AND 967 00:43:31,720 --> 00:43:34,360 WE'RE CURRENTLY INVESTIGATING 968 00:43:34,360 --> 00:43:35,880 THIS HYPOTHESIS USING A WHOLE 969 00:43:35,880 --> 00:43:37,960 GENOME SEQUENCING TO SEE WHAT 970 00:43:37,960 --> 00:43:40,040 THE SPECIFICITY ACROSS THE WHOLE 971 00:43:40,040 --> 00:43:40,400 GENOME IS. 972 00:43:40,400 --> 00:43:46,520 AND HERE IS AN EXAMPLE OF THAT. 973 00:43:46,520 --> 00:43:51,200 IF YOU ENACTIVATE MUTE S ALPHA 974 00:43:51,200 --> 00:43:56,240 BY KNOCKING OUT MSH-26, YOU GET 975 00:43:56,240 --> 00:43:57,720 A MUTATOR PHENOTYPE THAT WAS 976 00:43:57,720 --> 00:44:01,480 BASICALLY FOR ALL KINDS OF 977 00:44:01,480 --> 00:44:03,400 ERRORS INCLUDING SUBSTITUTIONS, 978 00:44:03,400 --> 00:44:05,160 1 BASE DELETIONS, 2 BASE 979 00:44:05,160 --> 00:44:09,400 DELETIONS, 3 BASE DELETIONS, AND 980 00:44:09,400 --> 00:44:12,360 IT'S SPECIFICITY IS UNUSUAL, IN 981 00:44:12,360 --> 00:44:17,920 THAT AS THE DELETION SIZE GET 982 00:44:17,920 --> 00:44:21,720 LARGER, THE RATIO OF ERRORS IN 983 00:44:21,720 --> 00:44:24,840 MUTE S ALPHA VERSUS MUTE S BETA 984 00:44:24,840 --> 00:44:26,320 CHANGES. 985 00:44:26,320 --> 00:44:29,480 SO FOR SUBSTITUTIONS ONLY MUTSA 986 00:44:29,480 --> 00:44:34,000 APPEARS TO WORK. 987 00:44:34,000 --> 00:44:36,200 BUT FOR--ON THE LEFT-HAND SIDE 988 00:44:36,200 --> 00:44:39,320 FOR LARGER DELETIONS, ONLY MUTSB 989 00:44:39,320 --> 00:44:42,640 SEEMS TO WORK. 990 00:44:42,640 --> 00:44:45,360 SO FOR EXAMPLE, FOR INSERTION 991 00:44:45,360 --> 00:44:46,600 DELETION ERRORS ACROSS THE WHOLE 992 00:44:46,600 --> 00:44:49,680 GENOME ARE GREATER THAN 1 BASE 993 00:44:49,680 --> 00:44:51,560 PAIR, KNOCKING OUT MUTE S ALPHA 994 00:44:51,560 --> 00:44:56,360 GIVES A 9 FOLD MUTATED PHENOTYPE 995 00:44:56,360 --> 00:44:58,440 BUT KNOCKING OUT MUTE S BETA 996 00:44:58,440 --> 00:45:00,080 GIVES 117 TOLD MUTATOR 997 00:45:00,080 --> 00:45:00,920 PHENOTYPE. 998 00:45:00,920 --> 00:45:05,480 SO WE'RE CURRENTLY CONTINUING TO 999 00:45:05,480 --> 00:45:07,960 INVESTIGATE THIS TO CONFIRM OR 1000 00:45:07,960 --> 00:45:08,840 DENY THIS HYPOTHESIS, WHAT I CAN 1001 00:45:08,840 --> 00:45:13,040 TELL YOU IS NOW WE HAVE BETWEEN 1002 00:45:13,040 --> 00:45:15,440 50 AND 100,000 INDEL MUTATIONS 1003 00:45:15,440 --> 00:45:18,400 ARRIVING FROM THE WHOLE GENOME 1004 00:45:18,400 --> 00:45:19,360 SEQUENCING MACHINE WHICH WE CAN 1005 00:45:19,360 --> 00:45:21,120 LOOK AT THIS IN MUCH GREATER 1006 00:45:21,120 --> 00:45:27,080 DEPTH AND WE WILL BE PUBLISHING 1007 00:45:27,080 --> 00:45:28,840 ON THIS IN PROBABLY SOMETIME 1008 00:45:28,840 --> 00:45:30,840 2023. 1009 00:45:30,840 --> 00:45:34,000 THIS IS A SUMMARY OF WHAT I JUST 1010 00:45:34,000 --> 00:45:34,440 TOLD YOU. 1011 00:45:34,440 --> 00:45:39,920 I WILL LET YOU STARE AT THAT IF 1012 00:45:39,920 --> 00:45:41,160 YOU WOULD LIKE AND I WOULD BE 1013 00:45:41,160 --> 00:45:42,720 HAPPY TO ANSWER ANY QUESTIONS. 1014 00:45:42,720 --> 00:45:53,080 THANK YOU VERY MUCH. 1015 00:45:58,600 --> 00:46:00,800 >> THANK YOU VERY MUCH, TOM. 1016 00:46:00,800 --> 00:46:02,840 WE HAVE A COUPLE QUESTIONS HERE, 1017 00:46:02,840 --> 00:46:06,440 I WILL READ THE FIRST IF 1018 00:46:06,440 --> 00:46:08,760 DR. SOBEL, IS THERE ANY EVIDENCE 1019 00:46:08,760 --> 00:46:09,960 OF UNSYNTHESIZED DNA ORIGIN 1020 00:46:09,960 --> 00:46:15,440 FIRING SUCH AS THAT DUE TO ATR 1021 00:46:15,440 --> 00:46:20,520 INHIBITION AT ALL FOR FIDELITY 1022 00:46:20,520 --> 00:46:21,120 APPROVE READING CAPACITY. 1023 00:46:21,120 --> 00:46:23,360 >> TO MY KNOWLEDGE THE ANSWER IS 1024 00:46:23,360 --> 00:46:23,600 NO. 1025 00:46:23,600 --> 00:46:26,720 SO FAR OUR ABILITY TO ANSWER 1026 00:46:26,720 --> 00:46:30,200 THAT QUESTION COMES FROM WHOLE 1027 00:46:30,200 --> 00:46:31,800 GENOME SEQUENCING AND THERE WE 1028 00:46:31,800 --> 00:46:34,080 LOOK AT--WE CAN LOOK AT FOR 1029 00:46:34,080 --> 00:46:36,200 EXAMPLE, AN EARLY FIRING ORIGIN 1030 00:46:36,200 --> 00:46:37,600 AND SEE DIFFERENCES IN MUTE O 1031 00:46:37,600 --> 00:46:41,240 GENESIS FROM THE LATE FIRING 1032 00:46:41,240 --> 00:46:41,480 ORIGIN. 1033 00:46:41,480 --> 00:46:44,280 OR SEE DIFFERENCES IN 1034 00:46:44,280 --> 00:46:44,880 RIBONUCLEOTIDE CORPORATION BUT 1035 00:46:44,880 --> 00:46:49,520 WE DON'T HAVE THE RESOLUTION YET 1036 00:46:49,520 --> 00:46:52,520 TO LOOK AT INDIVIDUAL ORIGINS AT 1037 00:46:52,520 --> 00:46:53,680 THE KIND OF DEPTH THAT WOULD BE 1038 00:46:53,680 --> 00:47:02,400 NEEDED TO ANSWER THAT QUESTION. 1039 00:47:02,400 --> 00:47:03,360 >> THANK YOU. 1040 00:47:03,360 --> 00:47:06,720 STEVE LLOYD ASKS, THANKS FOR A 1041 00:47:06,720 --> 00:47:08,000 FANTASTIC OVERVIEW, MIX WIDE NEW 1042 00:47:08,000 --> 00:47:13,440 DATA, ARE THE FREQUENCIES OF 1043 00:47:13,440 --> 00:47:15,840 RIBONUCLEOTIDE INCORPORATION AND 1044 00:47:15,840 --> 00:47:18,000 REPAIR COMPARABLE BETWEEN 1045 00:47:18,000 --> 00:47:20,320 MITOCHONDRIAL DNA VARIOUSUS THAT 1046 00:47:20,320 --> 00:47:21,840 OBSERVED IN NUCLEAR DNA. 1047 00:47:21,840 --> 00:47:26,360 >> SO THAT'S A GREAT QUESTION, I 1048 00:47:26,360 --> 00:47:28,840 DIDN'T TALK ABOUT THE WORK IN 1049 00:47:28,840 --> 00:47:33,120 MITOCHONDRIA BECAUSE IT'S NOT MY 1050 00:47:33,120 --> 00:47:35,480 OWN AND ANDREW CLOSSON IN MY 1051 00:47:35,480 --> 00:47:38,200 LAB, LEFT THE LAB IN TWEBT 16, I 1052 00:47:38,200 --> 00:47:43,560 THINK IT WAS, MIGHT BE 2017 AND 1053 00:47:43,560 --> 00:47:44,440 JOINED UNIVERSITY OF 1054 00:47:44,440 --> 00:47:47,840 [INDISCERNIBLE] WHERE HE IS 1055 00:47:47,840 --> 00:47:51,800 CURRENTLY ASKING QUESTIONS ABOUT 1056 00:47:51,800 --> 00:47:53,960 RIBOYOU NUCLEOTIDE INCORPORATION 1057 00:47:53,960 --> 00:47:57,400 INTO THE MITOCHONDRIA GENOME IN 1058 00:47:57,400 --> 00:48:01,680 COLLABORATION WITH COLLEAGUES OF 1059 00:48:01,680 --> 00:48:04,680 HIS OWN AND WHEN WE LEFT HERE HE 1060 00:48:04,680 --> 00:48:06,800 AGREED THAT HE WOULD TAKE THAT 1061 00:48:06,800 --> 00:48:07,840 MITOCHONDRIAL FOCUS WITH HIM AND 1062 00:48:07,840 --> 00:48:10,280 THAT I WOULD NOT WORK ON IT. 1063 00:48:10,280 --> 00:48:14,480 BUT WHAT I WOULD SUGGEST IS 1064 00:48:14,480 --> 00:48:15,480 SINCE MITOCHONDRIAL RIBOYOU THIS 1065 00:48:15,480 --> 00:48:17,920 CLE O TIDE INCORPORATION OCCURS 1066 00:48:17,920 --> 00:48:23,040 VERY FREQUENTLY, BEEN SHOWN A 1067 00:48:23,040 --> 00:48:24,680 LONG TIME AGO, WITH THE 1068 00:48:24,680 --> 00:48:28,560 SPECIFICITY OF THAT KIND OF 1069 00:48:28,560 --> 00:48:29,560 RIBONUCLEOTIDE INCORPORATION 1070 00:48:29,560 --> 00:48:33,040 PRESUMABLY BY DNA POLYMERASE 1071 00:48:33,040 --> 00:48:33,920 GAMMA THE MITOCHONDRIAL REP 1072 00:48:33,920 --> 00:48:35,080 LIAISON CASE, IF YOU'RE 1073 00:48:35,080 --> 00:48:35,800 INTERESTED IN WHAT'S BECOME 1074 00:48:35,800 --> 00:48:38,560 FOUND OUT ABOUT THAT, I WOULD 1075 00:48:38,560 --> 00:48:39,400 HIGHLY RECOMMEND READING THE 1076 00:48:39,400 --> 00:48:44,360 ARTICLES THAT HAVE BEEN 1077 00:48:44,360 --> 00:48:46,400 PUBLISHED BY ANDERS CLOSSON OVER 1078 00:48:46,400 --> 00:48:47,880 THE LAST FEW YEARS, VERY 1079 00:48:47,880 --> 00:48:49,440 INTERESTING WORK BUT NOT OURS. 1080 00:48:49,440 --> 00:48:51,080 >> CAN I JUST FOLLOW UP ON THAT 1081 00:48:51,080 --> 00:48:54,760 QUESTION, AND ASK YOU, SO, IT'S 1082 00:48:54,760 --> 00:48:57,560 AMAZING HOW COMMON THESE RIPE O 1083 00:48:57,560 --> 00:48:58,160 NUCLEOTIDE INCORPORATIONS ARE 1084 00:48:58,160 --> 00:49:02,160 AND YOU SAY THAT THEY WILL LEAD 1085 00:49:02,160 --> 00:49:04,560 TO VARIOUS CONDITIONS AND 1086 00:49:04,560 --> 00:49:09,080 DISEASES AND CONCERNS, ARE THERE 1087 00:49:09,080 --> 00:49:12,400 ANY KNOWN ADDITIONS SORE 1088 00:49:12,400 --> 00:49:13,960 DISEASES FOR EXAMPLE, WHERE 1089 00:49:13,960 --> 00:49:17,600 THERE IS A PARTICULARLY HIGH 1090 00:49:17,600 --> 00:49:18,840 INCORPORATION OF RIBONUCLEOTIDES 1091 00:49:18,840 --> 00:49:19,640 ECISIONS WHERE THEY'RE 1092 00:49:19,640 --> 00:49:21,600 PARTICULARLY HIGH NUMBER OF 1093 00:49:21,600 --> 00:49:26,120 RIBONUCLEOTIDES THAT HAVE BEEN 1094 00:49:26,120 --> 00:49:26,440 INCORPORATED? 1095 00:49:26,440 --> 00:49:30,600 >> SO, I CAN GIVE YOU A VERY 1096 00:49:30,600 --> 00:49:33,520 GOOD EXAMPLE OF RIBONUCLEOTIDES 1097 00:49:33,520 --> 00:49:35,720 ARE INCORPORATE BIDE THE REP 1098 00:49:35,720 --> 00:49:39,120 LIAISON CASES, AND THEY'RE 1099 00:49:39,120 --> 00:49:41,320 NORMALLY REPAIRED BY 1100 00:49:41,320 --> 00:49:43,480 RIBONUCLEOTIDE EXCISION REPAIR 1101 00:49:43,480 --> 00:49:47,160 AND IF THAT KIND OF REPAIR IS 1102 00:49:47,160 --> 00:49:48,360 DISABLED, THAT RIBONUCLEOTIDE 1103 00:49:48,360 --> 00:49:49,560 EXCISION REPAIR AND THEY'RE LEFT 1104 00:49:49,560 --> 00:49:53,200 IN THE GENOME, THEY ARE SUBJECT 1105 00:49:53,200 --> 00:49:57,720 TO CLEAVAGE BY TOPOISOMERACE, 1106 00:49:57,720 --> 00:49:59,480 WHICH IS AN ENZYME THAT RELIEVES 1107 00:49:59,480 --> 00:50:01,760 SUPER COILS IN DNA AS PART OF 1108 00:50:01,760 --> 00:50:05,880 THE NORMAL REPLICATION PROCESS, 1109 00:50:05,880 --> 00:50:10,400 MOST OF THAT INCISION, 1110 00:50:10,400 --> 00:50:13,440 RIBONUCLEOTIDES BY RNA--WELL BY 1111 00:50:13,440 --> 00:50:21,240 TOPOISOMMER ACE 1 IS PRETTY 1112 00:50:21,240 --> 00:50:21,600 ACCURATE. 1113 00:50:21,600 --> 00:50:23,080 BHU IFLET RIBONUCLEOTIDE IS 1114 00:50:23,080 --> 00:50:25,560 REPETITIVE IN THE ELEMENT THEN 1115 00:50:25,560 --> 00:50:27,640 ISOMER ACE CAN CLEAVE THE DNA 1116 00:50:27,640 --> 00:50:31,040 AND RESEAL IT AFTER A SECOND 1117 00:50:31,040 --> 00:50:35,280 CLEAVAGE WHICH LEADS TO DELETION 1118 00:50:35,280 --> 00:50:38,640 OF 2 TO 5 NUCLEOTIDES IN 1119 00:50:38,640 --> 00:50:42,960 REPETITIVE DNA SEQUENCES. 1120 00:50:42,960 --> 00:50:44,800 AND THERE IS A PULL LITERATURE 1121 00:50:44,800 --> 00:50:49,200 ON THE CAUSES OF CANCER, THE 1122 00:50:49,200 --> 00:50:52,760 MUTATIONAL CAUSES OF CANCER SO 1123 00:50:52,760 --> 00:50:54,400 PEOPLE ARE DOING RESEQUENCING OF 1124 00:50:54,400 --> 00:50:55,960 TUMOR DNA PRACTICES LOTS OF 1125 00:50:55,960 --> 00:50:59,480 DIFFERENT KINDS OF CANCER AND 1126 00:50:59,480 --> 00:51:00,680 DEFINING THE KINDS OF MUTATIONS 1127 00:51:00,680 --> 00:51:03,040 THAT ARE LIKELY TO BE 1128 00:51:03,040 --> 00:51:07,560 RESPONSIBLE FOR INDIVIDUAL KINDS 1129 00:51:07,560 --> 00:51:11,000 OF CANCER AND THEY PRESENT ERROR 1130 00:51:11,000 --> 00:51:12,800 SIGNATURES, VARIOUS KINDS OF 1131 00:51:12,800 --> 00:51:13,840 BASE SUBSTITUTIONS OR DELETIONS 1132 00:51:13,840 --> 00:51:18,600 AND 1 OF THOSE SIGNATURES IS THE 1133 00:51:18,600 --> 00:51:20,840 FREQUENT PRODUCTION OF 2-5 PAIR 1134 00:51:20,840 --> 00:51:23,000 BASE DELETIONS AND THE 1135 00:51:23,000 --> 00:51:26,360 IMPLICATION IS THAT THEY MAY BE 1136 00:51:26,360 --> 00:51:30,160 CAUSED BY CLEAVAGE OR 1137 00:51:30,160 --> 00:51:31,200 RIBONUCLEOTIDES BY TOPOISOMMER 1138 00:51:31,200 --> 00:51:35,440 ACE 1 AS PART OF THE NORMAL 1139 00:51:35,440 --> 00:51:41,960 PROCESS OF DNA REPLICATION 1140 00:51:41,960 --> 00:51:42,320 REPAIR. 1141 00:51:42,320 --> 00:51:44,480 THERE ARE OTHER INDICATORS THAT 1142 00:51:44,480 --> 00:51:54,120 RIBONUCLEOTIDES MAY BE IMPORTANT 1143 00:51:54,120 --> 00:51:54,920 IN CERTAIN IMMUNOLOGICAL 1144 00:51:54,920 --> 00:51:55,720 DISEASES, I DON'T FOLLOW THAT 1145 00:51:55,720 --> 00:51:57,800 CLOSE LOW BUT THE ANSWER TO YOUR 1146 00:51:57,800 --> 00:51:59,360 QUESTION IS DEFINITELY A YES AS 1147 00:51:59,360 --> 00:52:02,760 SUGGESTED BY THE MUTATIONAL 1148 00:52:02,760 --> 00:52:03,280 SIGNATURES. 1149 00:52:03,280 --> 00:52:04,720 >> OKAY, HERE'S ANOTHER 1150 00:52:04,720 --> 00:52:05,000 QUESTION. 1151 00:52:05,000 --> 00:52:07,400 >> QUESTION FROM MICHAEL 1152 00:52:07,400 --> 00:52:08,760 [INDISCERNIBLE]: IN LIGHT OF 1153 00:52:08,760 --> 00:52:09,680 YOUR INTEREST IN EVOLUTION, WHY 1154 00:52:09,680 --> 00:52:11,440 DO YOU THINK THAT THERE ARE 1155 00:52:11,440 --> 00:52:16,360 SEPARATE LEADING AND LAGGING 1156 00:52:16,360 --> 00:52:16,960 POLYMERASES APPROXIMATE POLD 1157 00:52:16,960 --> 00:52:19,080 SEEMS TO BE ABLE TO DO BOTH 1158 00:52:19,080 --> 00:52:26,840 LEADINGINGS AND LAGGING, GREAT 1159 00:52:26,840 --> 00:52:27,240 TALK. 1160 00:52:27,240 --> 00:52:30,240 >> YEAH, SO IT DEPENDS ON THE 1161 00:52:30,240 --> 00:52:33,800 ORGANISM, FOR INSTANCE THE MAJOR 1162 00:52:33,800 --> 00:52:39,560 AND LEADING BACTERIA E.COLI IS 1 1163 00:52:39,560 --> 00:52:42,640 ENZYME DNA POLYMERASE, 3 SO IT 1164 00:52:42,640 --> 00:52:44,160 DOESN'T JUST FROM COMPARING THAT 1165 00:52:44,160 --> 00:52:46,720 TO WORK DONE IN EUKARYOTIC 1166 00:52:46,720 --> 00:52:48,480 NUCLEOTIDES CLER DNA 1167 00:52:48,480 --> 00:52:52,000 REPLICATION, THE CONSERVATION IS 1168 00:52:52,000 --> 00:52:55,520 NOT PERFECT. 1169 00:52:55,520 --> 00:52:57,240 WE--WHY THERE IS A LEADING 1170 00:52:57,240 --> 00:52:59,280 STRAND REP LIAISON CASE IN 1171 00:52:59,280 --> 00:53:03,560 UCARIOTS THAT DIFFERS FROM THE 1172 00:53:03,560 --> 00:53:04,680 LAGGING STRAND PROBABLY REFLECTS 1173 00:53:04,680 --> 00:53:08,520 THE NATURE OF THE REPLICATION 1174 00:53:08,520 --> 00:53:11,280 THAT OCCURS SO THE LEADING 1175 00:53:11,280 --> 00:53:16,040 STRAND, 60 SIS BY POLE IS 1176 00:53:16,040 --> 00:53:20,440 PRIMARILY THOUGHT TO BE LARGELY 1177 00:53:20,440 --> 00:53:23,280 CONTINUOUS WITHOUT INTERVENING 1178 00:53:23,280 --> 00:53:26,640 DISASSOCIATION POL E GRABS ON AT 1179 00:53:26,640 --> 00:53:29,080 ORIGIN AND SYNTH CISES THOUSANDS 1180 00:53:29,080 --> 00:53:30,920 OF NUCLEOTIDES WITHOUT EVERY SIS 1181 00:53:30,920 --> 00:53:32,480 ASSOCIATED UNDER NATURAL DNA 1182 00:53:32,480 --> 00:53:33,680 CONDITIONS WHEREAS THE LAGS 1183 00:53:33,680 --> 00:53:40,800 STRAND HAS TO BE REPLICATED BY 1184 00:53:40,800 --> 00:53:51,320 CONTINUOUS INITIATION BY POLA 1185 00:53:51,720 --> 00:53:53,840 FOLLOWED BY LESS HIGHLY 1186 00:53:53,840 --> 00:53:55,800 POSSESSIVE BUT NOT NEARLY AS 1187 00:53:55,800 --> 00:53:59,280 POSSESSIVE OF SYNTHESIS BY POL 1188 00:53:59,280 --> 00:54:03,320 DA WHICH HAS TO TO OKZAKI THAT 1189 00:54:03,320 --> 00:54:05,440 SEAL THOSE REGIONS OF DNA BUT 1190 00:54:05,440 --> 00:54:05,600 IN. 1191 00:54:05,600 --> 00:54:10,240 WHY THAT HAS TO BE DIFFERENT ON 1192 00:54:10,240 --> 00:54:15,200 AN AFLIEWGZARY TIME FRAME IS 1193 00:54:15,200 --> 00:54:15,880 CERTAINLY AN INTERESTING 1194 00:54:15,880 --> 00:54:17,560 QUESTION THAT WE ALREADY KNOW 1195 00:54:17,560 --> 00:54:21,920 EXCEPTIONS AND MIGHT NOT BE 1196 00:54:21,920 --> 00:54:24,000 COMPLETELY TRUE BUT IT IS 1197 00:54:24,000 --> 00:54:27,440 INTERESTING TO THINK HOW FEW 1198 00:54:27,440 --> 00:54:29,080 ORGANISMS WE ACTUALLY KNOW WHAT 1199 00:54:29,080 --> 00:54:35,080 THE MECHANISM OF DNA REPLICATION 1200 00:54:35,080 --> 00:54:39,720 SO THE--WE'RE USING YEAST AS A 1201 00:54:39,720 --> 00:54:40,320 EUKARYOTIC NUCLEAR REP LIAISON 1202 00:54:40,320 --> 00:54:42,120 CASE AND WE THINK THERE ARE A 1203 00:54:42,120 --> 00:54:42,920 LOT OF SIMILARITIES BETWEEN 1204 00:54:42,920 --> 00:54:46,160 YEAST AND HUMANS AND MICE AND 1205 00:54:46,160 --> 00:54:49,920 OTHERS EUKARYOTIC NUCLEAR 1206 00:54:49,920 --> 00:54:52,920 SYSTEMS BUT WHETHER THAT'SA 1207 00:54:52,920 --> 00:54:53,960 COMPLETELY CONSERVED 1208 00:54:53,960 --> 00:54:55,080 EVOLUTIONARILY IS SIMPLY NOT 1209 00:54:55,080 --> 00:54:59,600 KNOWN YET BUT IT'S OUR WORKING 1210 00:54:59,600 --> 00:55:01,920 HYPOTHESIS AS TO--MIKE ASKED A 1211 00:55:01,920 --> 00:55:05,480 GREAT QUESTION, WHY, WHY ARE 1212 00:55:05,480 --> 00:55:06,040 THEY DIFFERENT? 1213 00:55:06,040 --> 00:55:11,120 FOR THOSE THAT ARE DRNT, I DON'T 1214 00:55:11,120 --> 00:55:11,400 KNOW WHY. 1215 00:55:11,400 --> 00:55:11,760 >> THANK YOU. 1216 00:55:11,760 --> 00:55:14,960 AND THEN WE HAVE A QUESTION FROM 1217 00:55:14,960 --> 00:55:17,080 STEPHEN LLOYD, RELATIVE TO MAP 1218 00:55:17,080 --> 00:55:18,400 ORIGINS OF REPLICATION, ARE 1219 00:55:18,400 --> 00:55:21,560 THERE DISTRIBUTIONS EVER 1220 00:55:21,560 --> 00:55:23,000 POTENTIAL G-QUADROUGH ATOM PLEX 1221 00:55:23,000 --> 00:55:28,000 SEQUENCES RANDOM OR CLUSTERED? 1222 00:55:28,000 --> 00:55:33,640 ARE THESE DISTRIBUTIONS? 1223 00:55:33,640 --> 00:55:34,640 >> YEAH, SO ... THE ANSWER TO 1224 00:55:34,640 --> 00:55:36,280 THAT MUST BE NO BUT I'M NOT 1225 00:55:36,280 --> 00:55:38,480 ENOUGH OF AN EXPERT ON 1226 00:55:38,480 --> 00:55:42,440 G-QUADROUGH ATOM PLEXS TO KNOW 1227 00:55:42,440 --> 00:55:44,240 ABOUT HOW RANDOM OR NONRANDOM 1228 00:55:44,240 --> 00:55:47,520 THEIR DISTRIBUTION IS IN THE 1229 00:55:47,520 --> 00:55:48,400 GENOME. 1230 00:55:48,400 --> 00:55:53,000 THAT WOULD TAKE ASKING THAT 1231 00:55:53,000 --> 00:55:55,560 QUESTION OF THE EXPERTS ON THAT 1232 00:55:55,560 --> 00:55:55,920 SUBJECT. 1233 00:55:55,920 --> 00:55:59,200 I DO KNOW THEY ARE HIGHLY 1234 00:55:59,200 --> 00:55:59,760 SPECIALIZED SEQUENCES, 1235 00:55:59,760 --> 00:56:02,480 REPLICATION, SO WHEN WE DO WHOLE 1236 00:56:02,480 --> 00:56:03,200 GENOME SEQUENCING EVENTUALLY WE 1237 00:56:03,200 --> 00:56:09,120 ARE GOING TO LACK AT FIDELITY 1238 00:56:09,120 --> 00:56:11,160 ISSUES RELATED TO GQUADROUGH 1239 00:56:11,160 --> 00:56:13,000 ATOM PLEXS VERSUS NORMAL 1240 00:56:13,000 --> 00:56:14,880 NONQUADROUGH ATOM PLEX 1241 00:56:14,880 --> 00:56:16,760 CONTAINING DNA, BUT RIGHT NOW, 1242 00:56:16,760 --> 00:56:18,760 OUR WHOLE GENOME SEQUENCING DATA 1243 00:56:18,760 --> 00:56:23,160 NEED TO BE INCREASED IN REPORTER 1244 00:56:23,160 --> 00:56:27,440 DENSITY IN ORDER TO HAVE ENOUGH 1245 00:56:27,440 --> 00:56:33,000 MUTATIONS IN THE GENOME TO START 1246 00:56:33,000 --> 00:56:33,800 INVESTIGATING THOSE VERY 1247 00:56:33,800 --> 00:56:35,880 IMPORTANT QUESTIONS SO I 1248 00:56:35,880 --> 00:56:38,040 HONESTLY DON'T KNOW BUT THE 1249 00:56:38,040 --> 00:56:39,920 PEOPLE WHO STUDY G-QUADROUGH 1250 00:56:39,920 --> 00:56:41,560 ATOM PLEXES AND SHOW SOME OF IT 1251 00:56:41,560 --> 00:56:43,520 ARE IN THIS AUDIENCE WOULD BE 1252 00:56:43,520 --> 00:56:47,920 BETTER TO ASK THAN ME. 1253 00:56:47,920 --> 00:56:50,760 I HAVE A QUESTION OF MY OWN, YOU 1254 00:56:50,760 --> 00:56:53,960 ARE USING SEQUENCING TECHNIQUES, 1255 00:56:53,960 --> 00:56:55,880 WHOLE GENOME SEQUENCING WHICH 1256 00:56:55,880 --> 00:56:59,960 USES POLYMERASES ITSELF, TO 1257 00:56:59,960 --> 00:57:02,200 MEASURE AREAS IN POLYMERASES AND 1258 00:57:02,200 --> 00:57:04,360 ALGORITHMS HAVE BEEN DEVELOPED 1259 00:57:04,360 --> 00:57:06,240 WHO OVERLOOK THE ERRORS THAT 1260 00:57:06,240 --> 00:57:08,480 INTRODUCED BY THE POLYMERASES IN 1261 00:57:08,480 --> 00:57:12,320 THE SEQUENCES, HOW DO YOU DEAL 1262 00:57:12,320 --> 00:57:12,880 WITH THAT? 1263 00:57:12,880 --> 00:57:17,080 >> THE MAIN WAY WE DEAL WITH IT 1264 00:57:17,080 --> 00:57:19,320 IS WHAT WE'RE DOING IS LOOKING 1265 00:57:19,320 --> 00:57:21,240 AT MUSEUM TAIGDZ RATES OVER 1266 00:57:21,240 --> 00:57:27,120 MULTIPLE PASSAGES ON A PETRI 1267 00:57:27,120 --> 00:57:28,400 DISH AND THEN ASKING WHAT ARE 1268 00:57:28,400 --> 00:57:29,760 THOSE, WHAT ARE THE SAME--WHAT 1269 00:57:29,760 --> 00:57:32,880 ARE THE RATES FOR THE SAME KIND 1270 00:57:32,880 --> 00:57:38,200 OF ERRORS WHEN THAT STRAIN WAS 1271 00:57:38,200 --> 00:57:41,520 NOT PASSAGE THROUGH SAY 900 1272 00:57:41,520 --> 00:57:47,520 REPLICATION CYCLES SO WE ALWAYS 1273 00:57:47,520 --> 00:57:49,080 INTERPRET OUR DATA AS BEING THE 1274 00:57:49,080 --> 00:57:50,640 CONSEQUENCE OF THOSE REPLICATION 1275 00:57:50,640 --> 00:57:57,680 ERRORS BECAUSE WHAT WE SUBTRACT 1276 00:57:57,680 --> 00:57:59,880 OUT OF THAT IS THE BACKGROUND 1277 00:57:59,880 --> 00:58:00,960 MUTATIONS THAT WERE THERE AT THE 1278 00:58:00,960 --> 00:58:02,040 START AND THOSE BACKGROUND 1279 00:58:02,040 --> 00:58:05,040 MUTATIONS THAT WERE THERE BEFORE 1280 00:58:05,040 --> 00:58:12,480 THOSE 900 GENERATIONS ENCOMPASS 1281 00:58:12,480 --> 00:58:14,000 THINGS LIKE DNA SEQUENCING 1282 00:58:14,000 --> 00:58:15,720 ERRORS IN THE PROCEDURES ITSELF. 1283 00:58:15,720 --> 00:58:18,480 >> WE HAVE A QUESTION FROM SEAN 1284 00:58:18,480 --> 00:58:19,280 [INDISCERNIBLE] HERE, DEAR 1285 00:58:19,280 --> 00:58:21,320 THOMAS REALLY GREAT TALK, THIS 1286 00:58:21,320 --> 00:58:31,840 IS THE LIGASE 1 MEDIATED OKAY 1287 00:58:37,920 --> 00:58:39,640 LIGATION KOWP ILLEGALS OF 1288 00:58:39,640 --> 00:58:40,280 VARIATIONS. 1289 00:58:40,280 --> 00:58:41,440 >> COULD YOU REPEAT THAT. 1290 00:58:41,440 --> 00:58:44,840 >> YEAH, REALLY GREAT TALK, 1291 00:58:44,840 --> 00:58:48,480 LIGASE 1 MEDIATED OKAZAKI 1292 00:58:48,480 --> 00:58:51,600 LIGATION FOR THE PC R TO START 1293 00:58:51,600 --> 00:59:02,080 ANOTHER FRAGMENT SYNTHESIS ON 1294 00:59:02,840 --> 00:59:03,520 THE LAGS STRAND? 1295 00:59:03,520 --> 00:59:07,920 I DON'T KNOW WE'VE NEVER LOOKED, 1296 00:59:07,920 --> 00:59:09,840 THE OKAZAKI FRAGMENT INVOLVES 1297 00:59:09,840 --> 00:59:11,560 THE PIECE THAT WAS SYNTHESIZED 1298 00:59:11,560 --> 00:59:12,960 BE THE PRIME END OF THAT 1299 00:59:12,960 --> 00:59:13,680 PRACTICING MENTORSHIP SKILL, 1300 00:59:13,680 --> 00:59:15,360 BEING LIGATED TO THE 1 THAT'S 1301 00:59:15,360 --> 00:59:19,480 COMPLETED SO IN TERMS OF PCA, PC 1302 00:59:19,480 --> 00:59:23,480 NA BEING LIMITED FOR INITIATION 1303 00:59:23,480 --> 00:59:24,520 OF ADDITIONAL OKAZAKI FRAGMENTS 1304 00:59:24,520 --> 00:59:30,480 I DON'T THINK THE DATA WE HAVE 1305 00:59:30,480 --> 00:59:31,200 ADDRESS THAT QUESTION. 1306 00:59:31,200 --> 00:59:33,760 COULD VERY WELL BE BUT I DON'T 1307 00:59:33,760 --> 00:59:34,000 KNOW. 1308 00:59:34,000 --> 00:59:35,800 >> HERE'S ANOTHER QUESTION BY 1309 00:59:35,800 --> 00:59:37,200 DR. ROTH: HAVE YOU LOOKED AT 1310 00:59:37,200 --> 00:59:38,800 THE KIND OF MUTATIONS FOR THE 1311 00:59:38,800 --> 00:59:41,280 LIGASE 1 ON THE OTHER SITE 1312 00:59:41,280 --> 00:59:45,360 MUTANT IN THE CONTEXT OF A FEN1 1313 00:59:45,360 --> 00:59:45,760 NULL BACKGROUND? 1314 00:59:45,760 --> 00:59:50,880 >> WE ARE DOING THAT AS WE 1315 00:59:50,880 --> 00:59:51,200 SPEAK. 1316 00:59:51,200 --> 00:59:55,520 BUT THE WHOLE GENOME SEQUENCING 1317 00:59:55,520 --> 00:59:56,480 EXPERIMENTS WHICH WE THINK WOULD 1318 00:59:56,480 --> 01:00:00,000 BE THE FIRST WAY TO ADDRESS THAT 1319 01:00:00,000 --> 01:00:00,480 QUESTION. 1320 01:00:00,480 --> 01:00:02,440 THEY TAKE QUITE A WHILE SO YOU 1321 01:00:02,440 --> 01:00:06,480 HAVE TO SPEND SOME TIME DOING 1322 01:00:06,480 --> 01:00:07,680 THE STRAIN CONSTRUCTIONS, THEN 1323 01:00:07,680 --> 01:00:09,160 YOU HAVE TO PASSAGE THEM THROUGH 1324 01:00:09,160 --> 01:00:11,720 A LARGE ENOUGH GENERATION OF 1325 01:00:11,720 --> 01:00:14,520 REPLICATION CYCLES TO GET ENOUGH 1326 01:00:14,520 --> 01:00:15,640 MUTANTS TO HAVE SOMETHING TO 1327 01:00:15,640 --> 01:00:17,640 SAY, THEN YOU HAVE TO SEQUENCE 1328 01:00:17,640 --> 01:00:21,800 THEM, THEN YOU HAVE TO DO THE 1329 01:00:21,800 --> 01:00:22,920 BIOINFORMATIC ANALYSIS AND THEN 1330 01:00:22,920 --> 01:00:23,720 WE'LL KNOW. 1331 01:00:23,720 --> 01:00:25,400 FOR EXAMPLE, SOME OF THE 1332 01:00:25,400 --> 01:00:28,200 EXPERIMENTS THAT WE JUST 1333 01:00:28,200 --> 01:00:29,240 INITIATED FOR EXAMPLE, I 1334 01:00:29,240 --> 01:00:36,080 MENTIONED 1 OF THE NEW POST DOCS 1335 01:00:36,080 --> 01:00:37,560 HERE, MAHINA, SHE IS CURRENTLY 1336 01:00:37,560 --> 01:00:39,760 DOING A WHOLE GENOME SEQUENCING 1337 01:00:39,760 --> 01:00:41,560 EXPERIMENT THAT WILL TAKE 1338 01:00:41,560 --> 01:00:43,040 PROBABLY BETWEEN 6 MONTHS AND A 1339 01:00:43,040 --> 01:00:52,720 YEAR OF PASSAGING CELLS ON A PET 1340 01:00:52,720 --> 01:00:53,920 -PETRI DISH FOR WE SEQUENCE 1341 01:00:53,920 --> 01:00:55,560 THE WHOLE NUMBER OF MUTANTS AND 1342 01:00:55,560 --> 01:00:57,520 GET A REQUESTY TO THE QUESTION 1343 01:00:57,520 --> 01:00:58,120 SHE'S ASKING. 1344 01:00:58,120 --> 01:00:59,400 SO THAT WAS A GREAT QUESTION BUT 1345 01:00:59,400 --> 01:01:02,800 I DON'T KNOW THE ANSWER YET. 1346 01:01:02,800 --> 01:01:08,240 >> OKAY, AND 1 LAST QUESTION 1347 01:01:08,240 --> 01:01:10,080 HERE, FROM ANITA 1348 01:01:10,080 --> 01:01:12,680 [INDISCERNIBLE], CDC 9 LIGASE 1349 01:01:12,680 --> 01:01:13,920 FUNCTIONS ALL IN MITOCHONDRIA, 1350 01:01:13,920 --> 01:01:17,320 WHAT ARE THE EFFECTS OF THE 1351 01:01:17,320 --> 01:01:19,720 LIGASE DEFICIENCIES YOU STUDY ON 1352 01:01:19,720 --> 01:01:26,200 THE MAINTENANCE OF MTDNA IN 1353 01:01:26,200 --> 01:01:27,960 YEAST CELLS, I GUESS YOU ALREADY 1354 01:01:27,960 --> 01:01:29,400 SAID SOMETHING ABOUT T. 1355 01:01:29,400 --> 01:01:31,400 >> SO I HONESTLY DON'T KNOW 1356 01:01:31,400 --> 01:01:41,560 BECAUSE AS I MENTIONED BEFORE, 1357 01:01:41,560 --> 01:01:44,680 WE'RE LEAVING THE THE 1358 01:01:44,680 --> 01:01:45,440 MITOCHONDRIAL ANALYSIS WHO 1359 01:01:45,440 --> 01:01:47,040 DECIDED IT WOULD BE A POST DOC 1360 01:01:47,040 --> 01:01:48,880 OF HIS INFORMATION SO WE'VE NOT 1361 01:01:48,880 --> 01:01:54,760 ASKED THAT QUESTION 1362 01:01:54,760 --> 01:01:55,840 EXPERIMENTALLY PERHAPS EXPPERS 1363 01:01:55,840 --> 01:01:56,000 HAS. 1364 01:01:56,000 --> 01:01:58,960 THE ONLY THING WE'RE DOING ON 1365 01:01:58,960 --> 01:02:00,400 MITOCHONDRIA IS COLLABORATION 1366 01:02:00,400 --> 01:02:02,480 WITH BILL COPELAND DOWN THE 1367 01:02:02,480 --> 01:02:02,680 HALL. 1368 01:02:02,680 --> 01:02:06,920 HE'S 1 OF THE WORLD'S EXPERTS ON 1369 01:02:06,920 --> 01:02:07,600 MITOCHONDRIAL DNA REPLICATION 1370 01:02:07,600 --> 01:02:11,200 AND OUR WORK WITH BILL HAS TO DO 1371 01:02:11,200 --> 01:02:15,320 WITH THE ORIGIN OF MUTATIONS IN 1372 01:02:15,320 --> 01:02:19,200 MITOCHONDRIAL DNA. 1373 01:02:19,200 --> 01:02:19,600 NOT RIBONUCLEOTIDE 1374 01:02:19,600 --> 01:02:20,240 INCORPORATION. 1375 01:02:20,240 --> 01:02:23,120 SO I'M SORRY, IICISM DEE DON'T 1376 01:02:23,120 --> 01:02:24,520 HAVE THE ANSWER. 1377 01:02:24,520 --> 01:02:31,880 >> OKAY, WELL, THANK YOU SO MUCH 1378 01:02:31,880 --> 01:02:32,080 AGAIN. 1379 01:02:32,080 --> 01:02:33,960 I THINK WE HAVE TO STOP HERE. 1380 01:02:33,960 --> 01:02:37,560 AND THAT WAS A WONDERFUL LECTURE 1381 01:02:37,560 --> 01:02:37,720 TOM. 1382 01:02:37,720 --> 01:02:38,280 >> THANK YOU. 1383 01:02:38,280 --> 01:02:40,280 >> YES, THANK YOU TOM, IT WAS AN 1384 01:02:40,280 --> 01:02:41,440 EXCELLENT REVIEW, WE'RE HOPING 1385 01:02:41,440 --> 01:02:43,600 WE WILL BE ABLE TO POST A NUMBER 1386 01:02:43,600 --> 01:02:45,040 OF THESE SLIDES WITH THESE 1387 01:02:45,040 --> 01:02:48,560 SUMMARIES ON IT AND PEOPLE CAN 1388 01:02:48,560 --> 01:02:50,320 GO AND REVIEW THEM AT THEIR 1389 01:02:50,320 --> 01:02:52,240 LEISURE, YOU WERE PRESENTING AN 1390 01:02:52,240 --> 01:02:54,440 INCREDIBLE AMOUNT OF DATA THAT 1391 01:02:54,440 --> 01:02:56,120 YOU'VE ACCUMULATED OVER 40 YEARS 1392 01:02:56,120 --> 01:02:58,640 AND YOU'RE VERY HUMBLY INDICATE 1393 01:02:58,640 --> 01:03:02,560 THAT THAT'S ONLY A SMALL PORTION 1394 01:03:02,560 --> 01:03:04,760 OF WHAT WE SEE NOW, SO WE SEE 1395 01:03:04,760 --> 01:03:05,760 WHY YOU'RE STILL EXCITED ABOUT 1396 01:03:05,760 --> 01:03:08,360 THIS AND STILL WORKING VERY 1397 01:03:08,360 --> 01:03:09,440 DILLIENTLY IN THE LAB AND 1398 01:03:09,440 --> 01:03:11,880 PLANNING EXPERIMENTS THAT WILL 1399 01:03:11,880 --> 01:03:16,400 TAKE SEVERAL YEARS TO COMPLETE. 1400 01:03:16,400 --> 01:03:20,640 SO--THAT REMINDS ME TO REMIND 1401 01:03:20,640 --> 01:03:22,160 STUDENTS IN THE AUDIENCE, IF 1402 01:03:22,160 --> 01:03:25,280 ANYBODY'S LOOKING FOR A POST DOC 1403 01:03:25,280 --> 01:03:26,480 AND YOU'RE INTERESTED WHAT I 1404 01:03:26,480 --> 01:03:27,880 JUST TALKED ABOUT YOU'RE MORE 1405 01:03:27,880 --> 01:03:30,320 THAN WELCOME TO CONTACT ME. 1406 01:03:30,320 --> 01:03:31,200 >> OKAY. 1407 01:03:31,200 --> 01:03:31,480 >> OKAY. 1408 01:03:31,480 --> 01:03:32,920 THANK YOU. 1409 01:03:32,920 --> 01:03:33,360 >> YOU'RE WELCOME. 1410 01:03:33,360 --> 01:03:34,000 >> GREAT TO SEE YOU. 1411 01:03:34,000 --> 00:00:00,000 >> ABSOLUTELY THANK YOU GUYS.