1 00:00:06,760 --> 00:00:10,320 >>WELCOME TO THE DNA REPAIR 2 00:00:10,320 --> 00:00:10,840 INTEREST GROUP. 3 00:00:10,840 --> 00:00:13,280 I'M KEN KRAEMER. 4 00:00:13,280 --> 00:00:15,160 MY CO-HOST IS OFF WITH HIS 5 00:00:15,160 --> 00:00:15,440 FAMILY. 6 00:00:15,440 --> 00:00:18,840 YOU KNOW ABOUT THIS BECAUSE YOU 7 00:00:18,840 --> 00:00:25,440 SUBSCRIBE TO THE DNA REPAIR 8 00:00:25,440 --> 00:00:27,880 INTEREST GROUP IF YOU DON'T YOU 9 00:00:27,880 --> 00:00:33,520 SEND EMAIL KRAEMERK@MAIL.NIH.GOV 10 00:00:33,520 --> 00:00:36,040 AND ASK TO BE ON THE LIST IN 11 00:00:36,040 --> 00:00:44,320 JANUARY WE'RE GOING TO HAVE A 12 00:00:44,320 --> 00:00:47,920 TALK ON DOUBLE MECHANISM OF 13 00:00:47,920 --> 00:00:51,440 DOUBLE STRAND BREAKS AND IN 14 00:00:51,440 --> 00:00:52,680 FEBRUARY WE'LL SPEAK ABOUT THE 15 00:00:52,680 --> 00:01:00,840 DNA REPLICATION FOR DYNAMICS 16 00:01:00,840 --> 00:01:04,360 WITH CHEMOTHERAPY RESPONSE AND 17 00:01:04,360 --> 00:01:07,520 IN MARCH WE'LL HAVE MORE ON DNA 18 00:01:07,520 --> 00:01:11,760 RESPONSE ON ALL TELEMERES AND IN 19 00:01:11,760 --> 00:01:14,840 APRIL WE'LL HAVE A WRAP-UP OF 20 00:01:14,840 --> 00:01:19,720 DNA REPAIR MITOCHONDRIA IN THE 21 00:01:19,720 --> 00:01:22,280 NEURODEGENERATIN 22 00:01:22,280 --> 00:01:23,720 NEURODEGENERATION. 23 00:01:23,720 --> 00:01:27,680 WE'LL HAVE A TALK TODAY IS GOING 24 00:01:27,680 --> 00:01:30,800 TO GO FOR THE USUAL TIME BUT IF 25 00:01:30,800 --> 00:01:32,240 YOU FOLKS HAVE ANY QUESTIONS, 26 00:01:32,240 --> 00:01:34,480 PLEASE PUT IT INTO THE CHAT AND 27 00:01:34,480 --> 00:01:37,680 I WILL READ THE QUESTIONS AT THE 28 00:01:37,680 --> 00:01:38,800 END. 29 00:01:38,800 --> 00:01:41,760 AND TODAY'S SPEAKER, DR. KRISTIN 30 00:01:41,760 --> 00:01:46,840 ECKERT IS GOING TO SPEAK ABOUT 31 00:01:46,840 --> 00:01:49,760 DNA POLYMERASE NAVIGATION 32 00:01:49,760 --> 00:01:50,840 THROUGH DIFFICULT-TO-REPLACE 33 00:01:50,840 --> 00:01:52,760 SEQUENCE TO PREVENT GENOME 34 00:01:52,760 --> 00:01:54,320 INSTABILITY AND ASKED HER TO 35 00:01:54,320 --> 00:01:56,160 GIVE A SPECIAL INTRODUCTION 36 00:01:56,160 --> 00:01:58,280 ABOUT HER BACKGROUND AND WHAT 37 00:01:58,280 --> 00:02:03,400 IT'S LIKE TO BE ONE OF THE 38 00:02:03,400 --> 00:02:05,240 PIONEERING WOMEN IN SCIENCE THE 39 00:02:05,240 --> 00:02:06,080 PAST FEW YEARS. 40 00:02:06,080 --> 00:02:08,640 SO WHY DON'T WE TAKE IT AWAY, 41 00:02:08,640 --> 00:02:08,880 KRISTIN. 42 00:02:08,880 --> 00:02:13,240 >> THANK YOU, KEN FOR THAT 43 00:02:13,240 --> 00:02:14,640 INTRODUCTION AND THANK YOU TO 44 00:02:14,640 --> 00:02:17,760 EVERYONE FOR TAKING TIME AWAY 45 00:02:17,760 --> 00:02:19,320 FROM YOUR VERY FULL SCHEDULES TO 46 00:02:19,320 --> 00:02:20,440 JOIN MY PRESENTATION. 47 00:02:20,440 --> 00:02:24,560 TODAY I AM GOING TO TALK ABOUT A 48 00:02:24,560 --> 00:02:27,400 TOPIC NEAR AND DEAR TO MY HEART 49 00:02:27,400 --> 00:02:29,680 AND THAT IS THE TYPES OF GENETIC 50 00:02:29,680 --> 00:02:32,840 VARIATION THAT ARISE IN TUMORS. 51 00:02:32,840 --> 00:02:34,240 MY LAB VERY INTERESTED IN 52 00:02:34,240 --> 00:02:35,640 UNDERSTANDING THE MECHANISMS BY 53 00:02:35,640 --> 00:02:38,240 WHICH VARIOUS TYPES OF GENETIC 54 00:02:38,240 --> 00:02:41,760 VARIANTS ARE CREATED DURING THE 55 00:02:41,760 --> 00:02:45,840 IT PROCESS OF TUMORIGENESIS AND 56 00:02:45,840 --> 00:02:48,080 WE KNOW THEY'RE KEY FOR FUELING 57 00:02:48,080 --> 00:02:49,760 CANCER CELL EVOLUTION. 58 00:02:49,760 --> 00:02:53,480 THE TYPES OF GENETIC VARIATION 59 00:02:53,480 --> 00:02:55,760 IN OUR STUDIES RANGES FROM 60 00:02:55,760 --> 00:02:56,920 REPETITIVE SEQUENCES WITHIN THE 61 00:02:56,920 --> 00:03:00,320 GENOME TO MECHANISMS OF LARGE 62 00:03:00,320 --> 00:03:01,280 STRUCTURAL VARIATION. 63 00:03:01,280 --> 00:03:03,920 AND AS KEN MENTIONED, I PUT 64 00:03:03,920 --> 00:03:06,560 TOGETHER THE BEGINNING OF MY 65 00:03:06,560 --> 00:03:09,400 PRESENTATION IN A DIFFERENT WAY 66 00:03:09,400 --> 00:03:11,520 TO INTRODUCE YOU TO NOT ONLY 67 00:03:11,520 --> 00:03:15,800 THIS TOPIC BUT TO MYSELF. 68 00:03:15,800 --> 00:03:19,400 SO I ACTUALLY AM FROM A SMALL 69 00:03:19,400 --> 00:03:21,120 TOWN IN NORTHERN PENNSYLVANIA 70 00:03:21,120 --> 00:03:23,600 AND LIKE MANY STUDENTS I LOVED 71 00:03:23,600 --> 00:03:26,600 MY BIOLOGY AND CHEMISTRY CLASSES 72 00:03:26,600 --> 00:03:33,560 BUT WHEN IT WAS TIME TO GO TO 73 00:03:33,560 --> 00:03:37,720 SCHOOL IT WAS ONLY ONE CHOICE 74 00:03:37,720 --> 00:03:39,920 PENN STATE WHICH WAS VERY LITTLE 75 00:03:39,920 --> 00:03:42,800 TUITION AND THE STATE SCHOOL. 76 00:03:42,800 --> 00:03:46,400 DURING MY COURSES TO BE BECOME A 77 00:03:46,400 --> 00:03:49,680 MEDICAL TECHNOLOGIST WHICH WAS 78 00:03:49,680 --> 00:03:55,640 MY ORIGINAL TRACK THIS IS SCHWAB 79 00:03:55,640 --> 00:03:59,840 AUDITORIUM AND A GENETICS CLASS 80 00:03:59,840 --> 00:04:01,720 OF ABOUT 600 STUDENTS AND WHEN 81 00:04:01,720 --> 00:04:03,160 IT CAME TO THE TOPIC OF FRUIT 82 00:04:03,160 --> 00:04:04,480 FLIES AND MUTATIONS I FELL IN 83 00:04:04,480 --> 00:04:06,840 LOVE WITH THE CONCEPT THE GENOME 84 00:04:06,840 --> 00:04:08,720 COULD CHANGE AND GENETIC 85 00:04:08,720 --> 00:04:10,520 MUTATIONS HAPPEN AND THIS IS AN 86 00:04:10,520 --> 00:04:11,120 ACTIVE PROCESS. 87 00:04:11,120 --> 00:04:16,400 I HAD A VERY ASTUTE FACULTY 88 00:04:16,400 --> 00:04:17,520 MEMBER WHO INTERVIEWED ME AND 89 00:04:17,520 --> 00:04:21,200 TOLD ME YOU DON'T WANT TO BE A 90 00:04:21,200 --> 00:04:22,640 MEDICAL TECHNOLOGIST, YOU WANT 91 00:04:22,640 --> 00:04:24,760 GO TO RESEARCH TO WHICH I 92 00:04:24,760 --> 00:04:25,640 ANSWERED WHAT IS RESEARCH. 93 00:04:25,640 --> 00:04:27,600 AT THAT TIME I HAD NO CLUE. 94 00:04:27,600 --> 00:04:30,040 I DIDN'T KNOW WHAT GRADUATE 95 00:04:30,040 --> 00:04:31,600 SCHOOL WAS MUCH LESS GETTING A 96 00:04:31,600 --> 00:04:31,800 Ph.D.. 97 00:04:31,800 --> 00:04:36,000 SO HE DIRECTED ME TO A LAB WHERE 98 00:04:36,000 --> 00:04:37,840 I STUDIED BACTERIA DURING MY 99 00:04:37,840 --> 00:04:40,400 SENIOR YEAR IN COLLEGE AND 100 00:04:40,400 --> 00:04:45,960 ISOLATED MUTANT FORMS THAT COULD 101 00:04:45,960 --> 00:04:56,520 NO LONGER FIX NUTRIGEN AND I 102 00:04:56,520 --> 00:04:58,800 JOINED THE LAB FOR CANCER 103 00:04:58,800 --> 00:05:01,800 RESEARCH AT McCARDLE LAB AND 104 00:05:01,800 --> 00:05:04,640 HERE I WAS ABLE TO STUDY 105 00:05:04,640 --> 00:05:06,160 MUTATIONS IN THE CONTEXT OF A 106 00:05:06,160 --> 00:05:07,160 PERTINENT DISEASE OF CANCER. 107 00:05:07,160 --> 00:05:11,040 AT THE TIME I JOINED GRADUATE 108 00:05:11,040 --> 00:05:16,240 SCHOOL IN 1982 THE WAR ON CANCER 109 00:05:16,240 --> 00:05:17,840 WAS RELATIVELY YOUNG WE KNEW 110 00:05:17,840 --> 00:05:19,280 THERE WERE SPECIFIC THINGS IN 111 00:05:19,280 --> 00:05:20,600 OUR ENVIRONMENT THAT CAUSED 112 00:05:20,600 --> 00:05:25,360 CANCER AND I JOINED THE CHEMICAL 113 00:05:25,360 --> 00:05:26,840 CARCINOGENESIS GROUP TO STUDY 114 00:05:26,840 --> 00:05:29,240 HOW CARCINOGENS DAMAGE THE DNA 115 00:05:29,240 --> 00:05:31,200 AND THEN IMPORTANTLY HOW DOES 116 00:05:31,200 --> 00:05:34,440 THAT DAMAGED DNA ACTUALLY BECOME 117 00:05:34,440 --> 00:05:39,680 A MUTATION THAT CAN FUEL CANCER 118 00:05:39,680 --> 00:05:40,720 CELL EVOLUTION. 119 00:05:40,720 --> 00:05:42,840 SO FOR THOSE GRADUATE STUDENTS 120 00:05:42,840 --> 00:05:48,880 AND POST-DOCS AND EARLY CAREER 121 00:05:48,880 --> 00:05:50,680 TRAINEES GOING THROUGH GRADUATE 122 00:05:50,680 --> 00:05:52,240 SCHOOL YOU HAVE A SMALL 123 00:05:52,240 --> 00:05:55,560 CONTRIBUTION, MOST OF US, TO THE 124 00:05:55,560 --> 00:05:57,120 SCIENTIFIC FIELD. 125 00:05:57,120 --> 00:06:01,040 MY CONTRIBUTION WAS ACTUALLY 126 00:06:01,040 --> 00:06:04,040 FOCUSSED ON MUTO GENESIS. 127 00:06:04,040 --> 00:06:07,760 BACK IN THE 1980s WE STUDY 128 00:06:07,760 --> 00:06:10,840 SPECIFICITY WITH TARGET REPORTER 129 00:06:10,840 --> 00:06:18,040 ASSAY S THAT WE COULD LOOK AT 130 00:06:18,040 --> 00:06:20,400 SEQUENCING AND THIS WAS THE 131 00:06:20,400 --> 00:06:23,120 FORERUNNER OF WHAT WE NOW CALL 132 00:06:23,120 --> 00:06:24,800 MUTATIONAL SIGNATURE. 133 00:06:24,800 --> 00:06:27,800 STUDYING THIS IN HUMAN CELLS AND 134 00:06:27,800 --> 00:06:29,400 CULTURE I STUDIED THE 135 00:06:29,400 --> 00:06:31,240 SPECIFICITY I OBSERVED IN THE 136 00:06:31,240 --> 00:06:34,840 HUMAN CELLS WAS VERY MUCH AKIN 137 00:06:34,840 --> 00:06:36,880 TO WHAT YOU COULD OBSERVE IN 138 00:06:36,880 --> 00:06:40,040 TUMORS AFTER MICE WERE INJECTED 139 00:06:40,040 --> 00:06:50,880 WITH THE SAME ALKYLATING AGE 140 00:06:50,880 --> 00:06:53,200 AGAINSTS BUT IF YOU FIRST 141 00:06:53,200 --> 00:06:55,000 INDUCED THE IT SOS RESPONSE I 142 00:06:55,000 --> 00:06:56,840 SHIFT THE SPECIFICITY AND GOT A 143 00:06:56,840 --> 00:06:59,040 SPECTRUM AKIN TO WHAT I OBSERVED 144 00:06:59,040 --> 00:07:01,960 IN THE HUMAN CELLS. 145 00:07:01,960 --> 00:07:13,600 SO THIS REALLY FASCINATED ME. 146 00:07:13,600 --> 00:07:15,840 IT CHANGED MY WAY OF THINKING 147 00:07:15,840 --> 00:07:18,200 BECAUSE I THOUGHT HOW A DNA 148 00:07:18,200 --> 00:07:20,840 LESION INTERACTS WITH THE DNA 149 00:07:20,840 --> 00:07:22,840 POLYMERASE AND WHAT DO WE KNOW 150 00:07:22,840 --> 00:07:23,680 ABOUT THIS PROCESS. 151 00:07:23,680 --> 00:07:32,240 TO STUDY THIS FURTHER I JOINED 152 00:07:32,240 --> 00:07:37,760 TE LAB AND FOCUSSED ON HOW DNA 153 00:07:37,760 --> 00:07:39,200 POLYMERASES MAKE ERRORS EITHER 154 00:07:39,200 --> 00:07:40,920 WILD TYPE DNA REPLICATED OR 155 00:07:40,920 --> 00:07:42,840 DAMAGED DNA, HOW DO YOU GO FROM 156 00:07:42,840 --> 00:07:48,200 A LESION TO MUTATION? 157 00:07:48,200 --> 00:07:51,240 AND HOW WE KNOW DNA POLYMERASE 158 00:07:51,240 --> 00:07:52,920 ERRORS ARE KEY DETERMINATES OF 159 00:07:52,920 --> 00:07:54,280 THE ERRORS THAT OCCUR DURING 160 00:07:54,280 --> 00:08:00,440 TUMOR CELL EF -- EVOLUTION AND 161 00:08:00,440 --> 00:08:05,800 THE DNA THAT DO THIS ARE THE DNA 162 00:08:05,800 --> 00:08:08,240 POLYMERASE AND WE'RE AT AN 163 00:08:08,240 --> 00:08:11,440 EXCITING TIME IN THE LATE 1980s 164 00:08:11,440 --> 00:08:14,200 AND WE HAD EXTRA CRYSTAL 165 00:08:14,200 --> 00:08:16,200 STRUCTURES WHERE THE FIELD WAS 166 00:08:16,200 --> 00:08:17,040 ADVANCING AND HAD DNA 167 00:08:17,040 --> 00:08:20,840 POLYMERASES AND THIS IS A 168 00:08:20,840 --> 00:08:22,800 STRUCTURE OF ONE OF THE FIRST 169 00:08:22,800 --> 00:08:24,320 POLYMERASES CRYSTALLIZED WITH 170 00:08:24,320 --> 00:08:28,640 THE DNA AND SUBSTRATES AT HIGH 171 00:08:28,640 --> 00:08:29,840 RESOLUTION AND WE ALSO HAD 172 00:08:29,840 --> 00:08:36,640 CRYSTAL STRUCTURES OF THE DNA 173 00:08:36,640 --> 00:08:38,360 AND OLGA KINARD PUBLISHED AND 174 00:08:38,360 --> 00:08:41,600 STUDIED HOW THE STRUCTURE OF DNA 175 00:08:41,600 --> 00:08:43,720 MISPAIRS WAS DIFFERENT THAN THE 176 00:08:43,720 --> 00:08:49,240 STRUCTURE OF WHAT WE KNOW IS THE 177 00:08:49,240 --> 00:08:49,920 DIFFERENT PAIRS IN THE 178 00:08:49,920 --> 00:08:54,040 LABORATORY I WAS ABLE TO STUDY 179 00:08:54,040 --> 00:08:56,720 DNA POLYMERASE AND BECOME A BIO 180 00:08:56,720 --> 00:09:02,040 CHEMIST AND I LAEARNED IT'S A 181 00:09:02,040 --> 00:09:05,680 FAMILY OF ENZYMES AND WAS ABLE 182 00:09:05,680 --> 00:09:10,880 TO WORK FROM HIV REVERSE 183 00:09:10,880 --> 00:09:12,800 TRANSCRIPTASE AND LEARNED TIME 184 00:09:12,800 --> 00:09:14,920 MANAGEMENT SKILLS BECAUSE AT THE 185 00:09:14,920 --> 00:09:18,840 TIME AS THE POST-DOC MY THIRD 186 00:09:18,840 --> 00:09:22,840 CHILD WAS BORN AND MY SONS ISAAC 187 00:09:22,840 --> 00:09:28,040 AND IAN I HAD AS A GRADUATE 188 00:09:28,040 --> 00:09:30,680 STUDENT BUT THE REAL CHALLENGE 189 00:09:30,680 --> 00:09:35,480 FOR ME FOR HOW DO YOU DO 190 00:09:35,480 --> 00:09:38,240 WORK-LIFE BALANCE CAME DURING MY 191 00:09:38,240 --> 00:09:39,480 POST-DOC YEARS WHERE PERFORMING 192 00:09:39,480 --> 00:09:41,800 THE EXPERIMENTS, WRITING THE 193 00:09:41,800 --> 00:09:43,200 MANUSCRIPTS AND DOING THE 194 00:09:43,200 --> 00:09:44,320 THINKING. 195 00:09:44,320 --> 00:09:49,600 THIS WAS A FORMATIVE TIME FOR ME 196 00:09:49,600 --> 00:09:50,840 AS A POST-DOCTORAL FELLOW. 197 00:09:50,840 --> 00:09:52,240 ARMED WITH ALL THIS NEW 198 00:09:52,240 --> 00:09:54,680 KNOWLEDGE THEN I WAS LOOKING FOR 199 00:09:54,680 --> 00:09:59,720 FACULTY POSITIONS AND I ACTUALLY 200 00:09:59,720 --> 00:10:08,360 JOINED THE FACULTY AT PENN STATE 201 00:10:08,360 --> 00:10:11,280 AND JOINED THE CAMPUS OF 202 00:10:11,280 --> 00:10:13,760 MEDICINE AND IN THE DEPARTMENT 203 00:10:13,760 --> 00:10:15,600 OF PATHOLOGY AND THE DEPARTMENT 204 00:10:15,600 --> 00:10:17,840 WAS GROWING AND A NEW DIVISION 205 00:10:17,840 --> 00:10:20,960 WAS BEING EXPANDED FOR RESEARCH 206 00:10:20,960 --> 00:10:23,040 AND SPECIFICALLY LOOKING FOR 207 00:10:23,040 --> 00:10:25,240 PEOPLE STUDYING CANCER RESEARCH. 208 00:10:25,240 --> 00:10:27,560 SO PUTTING EVERYTHING TOGETHER 209 00:10:27,560 --> 00:10:29,840 FOR MY GRADUATE SCHOOL TRAINING 210 00:10:29,840 --> 00:10:34,800 AND MY POST-DOCTORAL TRAINING I 211 00:10:34,800 --> 00:10:37,920 WENT ON TO STUDY ONE OF THE 212 00:10:37,920 --> 00:10:39,880 BIOCHEMICAL MECHANISM UNDER LIES 213 00:10:39,880 --> 00:10:43,680 THE IT MUTATIONS DURING CANCER 214 00:10:43,680 --> 00:10:44,000 DEVELOPMENT. 215 00:10:44,000 --> 00:10:46,840 SO MY LABORATORY EXCUSE ME 216 00:10:46,840 --> 00:10:48,000 PHONE. 217 00:10:48,000 --> 00:10:49,200 SORRY ABOUT THAT. 218 00:10:49,200 --> 00:10:51,480 MY LABORATORY HAS SEVERAL 219 00:10:51,480 --> 00:10:52,640 EXPERIMENTAL APPROACHES THAT WE 220 00:10:52,640 --> 00:11:00,400 USE TO STUDY THE MECHANISMS OF 221 00:11:00,400 --> 00:11:02,800 MUTATION IN HUMAN CELLS DURING 222 00:11:02,800 --> 00:11:04,960 CANCER DEVELOPMENT RANGE FROM 223 00:11:04,960 --> 00:11:05,960 BIOCHEMISTRY THE TOPIC OF WHAT 224 00:11:05,960 --> 00:11:07,120 I'LL TELL YOU ABOUT TODAY, 225 00:11:07,120 --> 00:11:09,640 LOOKING AT THE RESPONSES OF 226 00:11:09,640 --> 00:11:12,120 CELLS IN CULTURE TO VARIOUS 227 00:11:12,120 --> 00:11:14,600 TYPES OF STRESSES AND HOW THAT 228 00:11:14,600 --> 00:11:18,440 CHANGES THE PROCESS OF MUTO 229 00:11:18,440 --> 00:11:21,280 GENESIS AND NOW RECENTLY USING 230 00:11:21,280 --> 00:11:25,800 GENOMICS TO EXPLORE WHAT'S 231 00:11:25,800 --> 00:11:50,480 HAPPENING IN HUMAN TISSUES. 232 00:11:50,480 --> 00:11:52,920 THE DNA HAS TO BE COMPLETELY 233 00:11:52,920 --> 00:11:57,440 DUPLICATED AND FINISHED BEFORE 234 00:11:57,440 --> 00:12:00,840 CELLS DIVIDE IN CYTOKINESIS. 235 00:12:00,840 --> 00:12:03,840 SO MY LAB HAS STUDIED OVER THE 236 00:12:03,840 --> 00:12:06,240 YEARS POLYMERASES FROM VARIOUS 237 00:12:06,240 --> 00:12:06,680 FAMILIES. 238 00:12:06,680 --> 00:12:09,640 I HAVE ACCORDING TO POSTULATED 239 00:12:09,640 --> 00:12:10,800 FUNCTIONS HERE. 240 00:12:10,800 --> 00:12:16,480 WE'LL BE TALKING ABOUT THE 241 00:12:16,480 --> 00:12:18,480 REPLICATIVE POLYMERASES AND 242 00:12:18,480 --> 00:12:19,640 WE'VE DONE A LITTLE BIT OF WORK 243 00:12:19,640 --> 00:12:22,120 WITH DNA REPAIR ENZYMES BUT MOST 244 00:12:22,120 --> 00:12:26,040 THE WORK HAS BEEN ON WHAT I 245 00:12:26,040 --> 00:12:27,680 GROUP TOGETHER AND CALL 246 00:12:27,680 --> 00:12:33,040 SPECIALIZED POLYMERASES. 247 00:12:33,040 --> 00:12:40,480 ENZYMES THAT HAVE AND CREATE 248 00:12:40,480 --> 00:12:54,840 GENOME REPLICATION. 249 00:12:54,840 --> 00:12:59,040 AND WE WANTED TO STUDY THE HUMAN 250 00:12:59,040 --> 00:12:59,440 GENO 251 00:12:59,440 --> 00:12:59,680 GENOME. 252 00:12:59,680 --> 00:13:00,440 AS SOON AS YOU ASK THE QUESTION 253 00:13:00,440 --> 00:13:02,600 WHAT IS THE SEQUENCE OF HUMAN 254 00:13:02,600 --> 00:13:04,000 GENOME YOU ARE UP TOO THE 255 00:13:04,000 --> 00:13:06,560 REALIZATION THAT 60% OF THE 256 00:13:06,560 --> 00:13:08,720 HUMAN GENOME IS COMPRISED OF 257 00:13:08,720 --> 00:13:10,000 REPETITIVE DNA SEQUENCES OF 258 00:13:10,000 --> 00:13:10,840 VARIOUS TYPES. 259 00:13:10,840 --> 00:13:14,240 MANY ARE FAMILIAR WITH THE 260 00:13:14,240 --> 00:13:26,840 MICROSATELLITES FOUND AT 261 00:13:26,840 --> 00:13:30,320 TELEMERE SEQUENCES AND FOUND 262 00:13:30,320 --> 00:13:35,760 WITHIN COMMON FRAGILE TYPES AND 263 00:13:35,760 --> 00:13:46,240 OTHERS CONSEQUENCES AND 264 00:13:46,240 --> 00:13:47,680 IMPORTANTLY REPETITIVE SEQUENCES 265 00:13:47,680 --> 00:13:50,640 ARE FOUND THROUGHOUT THE GENOME 266 00:13:50,640 --> 00:13:57,200 AND CAN BE SITES OF RECURRENT 267 00:13:57,200 --> 00:13:57,840 STRUCTURAL VARIATION IN CANCER 268 00:13:57,840 --> 00:13:58,040 CELLS. 269 00:13:58,040 --> 00:14:01,120 WHEN YOU THINK OF REPETITIVE 270 00:14:01,120 --> 00:14:02,600 SEQUENCES YOU HAVE TO THINK 271 00:14:02,600 --> 00:14:03,640 ABOUT THE POLYMERASES. 272 00:14:03,640 --> 00:14:05,120 THEY HAVE TWO SUBSTRATES. 273 00:14:05,120 --> 00:14:07,680 ONE IS THE DNA AND ONE IS THE 274 00:14:07,680 --> 00:14:11,400 INCOMING DNTP. 275 00:14:11,400 --> 00:14:14,840 DNA COMES IN MANY SECONDARY 276 00:14:14,840 --> 00:14:23,960 STRUCTURES AS A BIO PHYSICIST 277 00:14:23,960 --> 00:14:28,720 CAN BE RANDOM AND WHEN YOU BEGIN 278 00:14:28,720 --> 00:14:29,600 THE SEQUENCE OF DNA IT WILL FOLD 279 00:14:29,600 --> 00:14:35,840 INTO A SECONDARY STRUCTURE. 280 00:14:35,840 --> 00:14:37,720 AND THEY'RE CALLED IN THE FIELD 281 00:14:37,720 --> 00:14:42,840 AS NON BDNA. 282 00:14:42,840 --> 00:14:42,960 DEF 283 00:14:51,520 --> 00:14:58,800 AND THEY MAKE A 284 00:14:58,800 --> 00:14:59,440 SOMATOMUTOGENESIS AND DISCUSSED 285 00:14:59,440 --> 00:15:00,120 IN THE LITERATURE AS DIFFICULT 286 00:15:00,120 --> 00:15:01,040 TO REPLICATION. 287 00:15:01,040 --> 00:15:04,880 TODAY WE'LL TAKE A DEEP DIVE TOO 288 00:15:04,880 --> 00:15:08,680 THE DIFFICULT TO REPLICATE 289 00:15:08,680 --> 00:15:13,680 SECOND-CHANCES I'LL CALL DITORS. 290 00:15:13,680 --> 00:15:15,720 IMAGINE YOU'RE A CAR AND THE DNA 291 00:15:15,720 --> 00:15:17,680 POLYMERASE IS THE DRIVER AND YOU 292 00:15:17,680 --> 00:15:20,960 NEED TO NAVIGATE THE TRACK THE 293 00:15:20,960 --> 00:15:22,240 HUMAN GENOME. 294 00:15:22,240 --> 00:15:25,040 AT TIMES YOU NEED TO DRIVE NICE 295 00:15:25,040 --> 00:15:25,920 SMOOTH ROADS AND OTHER TIMES 296 00:15:25,920 --> 00:15:30,040 YOU'LL ENCOUNTER OTHER OBSTACLES 297 00:15:30,040 --> 00:15:34,840 SUCH AS HAIRPINS, BUMPS IN THE 298 00:15:34,840 --> 00:15:46,440 ROAD WILL YOU NAVIGATE TO THE 299 00:15:46,440 --> 00:15:48,160 END ON TIME AND NOT MAKE MISTAKE 300 00:15:48,160 --> 00:15:49,720 TO FLIP YOUR CAR OVER AND THE 301 00:15:49,720 --> 00:15:53,160 OUTCOMES OF INHIBITION AND 302 00:15:53,160 --> 00:15:55,080 ERRORS ARE ACTUALLY TWO EVENTS 303 00:15:55,080 --> 00:15:57,680 IMPACTFUL FOR GENOME STABILITY 304 00:15:57,680 --> 00:15:59,440 AND CAN LEAD TO CHROMOSOME 305 00:15:59,440 --> 00:16:03,640 BREAKAGE AND MUTATION. 306 00:16:03,640 --> 00:16:12,440 TODAY LIKE I SAID WE'LL FOCUS ON 307 00:16:12,440 --> 00:16:17,920 THE DETOURS AND THE DIFFICULT TO 308 00:16:17,920 --> 00:16:19,040 REPLACE AND WE'LL SEE WHAT 309 00:16:19,040 --> 00:16:21,840 CONSTITUTES A DETOUR AND WHAT 310 00:16:21,840 --> 00:16:25,080 MAKES A SEQUENCE DIFFICULT TO 311 00:16:25,080 --> 00:16:25,320 REPLACE. 312 00:16:25,320 --> 00:16:28,520 OUR FOCUS WILL BE ON DNA 313 00:16:28,520 --> 00:16:30,680 POLYMERASE AND SHOW YOU HOW WE 314 00:16:30,680 --> 00:16:32,640 USE BIOCHEMISTRY TO GET TO THE 315 00:16:32,640 --> 00:16:33,000 DETOUR. 316 00:16:33,000 --> 00:16:35,040 NEXT I WANT TO FOCUS ON OUR MORE 317 00:16:35,040 --> 00:16:36,880 RECENT WORK ASKING THE QUESTION 318 00:16:36,880 --> 00:16:38,480 OF WHAT IS THE COMPLETE 319 00:16:38,480 --> 00:16:42,000 LANDSCAPE OF DETOURS WITHIN THE 320 00:16:42,000 --> 00:16:43,800 HUMAN GENOME AND FINALLY I'M 321 00:16:43,800 --> 00:16:46,640 JUST GOING TO TOUCH ON A 322 00:16:46,640 --> 00:16:48,760 QUESTION THAT IS A SEPARATE 323 00:16:48,760 --> 00:16:50,640 PROJECT IN MY LABORATORY AND 324 00:16:50,640 --> 00:16:54,000 THAT IS WHEN DO THESE DETOURS 325 00:16:54,000 --> 00:16:55,440 ACTUALLY BECOME THREATS TO 326 00:16:55,440 --> 00:16:56,120 GENOME STABILITY DURING CANCER 327 00:16:56,120 --> 00:16:59,400 CELL EVOLUTION. 328 00:16:59,400 --> 00:17:03,920 SO TO DEFINE DETOURS, WE SET OUT 329 00:17:03,920 --> 00:17:06,880 MANY YEARS AGO TO TRY TO SET UP 330 00:17:06,880 --> 00:17:09,760 A BIOCHEMICAL SYSTEM TO ASK WHAT 331 00:17:09,760 --> 00:17:12,320 MAKES A SEQUENCE DIFFICULT TO 332 00:17:12,320 --> 00:17:16,280 REPLICATION. 333 00:17:16,280 --> 00:17:22,280 WE'RE FOCUSSED ON THE LOCUS TO 334 00:17:22,280 --> 00:17:22,480 STUDY. 335 00:17:22,480 --> 00:17:27,280 FRA 16 IS A FRAGILE SITE WITH 336 00:17:27,280 --> 00:17:30,080 COMMON BREAKS FREQUENTLY SEEN IN 337 00:17:30,080 --> 00:17:32,080 TUMOR CELLS AND USED THE GENOME 338 00:17:32,080 --> 00:17:34,440 BROWSER TO IDENTIFY DIFFERENT 339 00:17:34,440 --> 00:17:40,440 WINDOWS WITHIN THE FRA 16 LOCUS 340 00:17:40,440 --> 00:17:43,760 THAT HARBOR VARIOUS TYPES OF 341 00:17:43,760 --> 00:17:49,440 REPETITIVE SEQUENCES SUCH AS 342 00:17:49,440 --> 00:17:49,840 QUASI-PALINDROMES. 343 00:17:49,840 --> 00:17:51,480 WE NEXT CLONED THE SEQUENCES 344 00:17:51,480 --> 00:17:54,880 INTO THE FACTOR SO ISOLATE 345 00:17:54,880 --> 00:17:57,280 SINGLE STRAND DNA FORMS OF THE 346 00:17:57,280 --> 00:17:58,560 VARIOUS DITORS. 347 00:17:58,560 --> 00:18:01,120 ALL THE SEQUENCES ARE CLONED IN 348 00:18:01,120 --> 00:18:03,520 THE SAME LOCATION AND HAVE A 349 00:18:03,520 --> 00:18:05,640 CONSTANT UPSTREAM AND DOWN 350 00:18:05,640 --> 00:18:06,480 STREAM REGION SO THE POLYMERASE 351 00:18:06,480 --> 00:18:09,920 WILL TAKE A RUNNING START DOWN 352 00:18:09,920 --> 00:18:11,240 THE HIGHWAY AND ENCOUNTER THE 353 00:18:11,240 --> 00:18:12,040 VARIOUS DETOURS. 354 00:18:12,040 --> 00:18:15,760 AS A CONTROL, WE ACTUALLY CLONED 355 00:18:15,760 --> 00:18:18,840 IN A SEQUENCE FROM FRA 16D THAT 356 00:18:18,840 --> 00:18:22,160 HAS AN EQUIVALENT AT CONTENT OF 357 00:18:22,160 --> 00:18:25,840 76% BUT NO REPETITIVE ELEMENTS. 358 00:18:25,840 --> 00:18:26,840 SO OUR ASSAY IS FAIRLY 359 00:18:26,840 --> 00:18:27,240 STRAIGHTFORWARD. 360 00:18:27,240 --> 00:18:33,640 IT'S A PRIMER EXTENSION EASY 361 00:18:33,640 --> 00:18:35,640 WITH QUANTIFICATION AT THE 362 00:18:35,640 --> 00:18:42,840 NUCLEAR STRAND LEVEL AND ALLOW 363 00:18:42,840 --> 00:18:45,440 THE POLYMERASES TO SYNTHESIZE 364 00:18:45,440 --> 00:18:48,040 THROUGH AND SEPARATE ALONG SIDE 365 00:18:48,040 --> 00:18:49,640 THE DNA SEQUENCING LADDER TO SEE 366 00:18:49,640 --> 00:18:50,960 EXACTLY AT WHAT POINT THE 367 00:18:50,960 --> 00:18:54,880 PRODUCTS ARE ACCUMULATED. 368 00:18:54,880 --> 00:18:58,840 WE THEN CAN QUANTITATE USING 369 00:18:58,840 --> 00:18:59,400 QUANTITATION AND HOW MANY 370 00:18:59,400 --> 00:19:02,280 PRODUCTS ARE ACCUMULATED WITHIN 371 00:19:02,280 --> 00:19:07,800 THE DITORS SEQUENCE VERSUS HOW 372 00:19:07,800 --> 00:19:09,280 MANY ARE ABLE TO SYNTHESIZE 373 00:19:09,280 --> 00:19:12,480 THROUGH THE DITORS INTO THE DOWN 374 00:19:12,480 --> 00:19:12,840 STREAM REGION. 375 00:19:12,840 --> 00:19:14,480 THIS A QUICK SNAPSHOT OF THE 376 00:19:14,480 --> 00:19:15,040 FIRST ASSAY. 377 00:19:15,040 --> 00:19:18,840 THIS IS LOOKING AT THE R1 TO R5 378 00:19:18,840 --> 00:19:21,280 WHICH ARE DIFFERENT REGIONS, THE 379 00:19:21,280 --> 00:19:22,840 DIFFERENT WINDOWS ALONG THE FRA 380 00:19:22,840 --> 00:19:25,280 16 LOCUS AND WHAT YOU CAN SEE IS 381 00:19:25,280 --> 00:19:28,720 THAT THERE ARE SEVERAL REGIONS 382 00:19:28,720 --> 00:19:30,840 WHERE DNA POLYMERASES ARE FAIRLY 383 00:19:30,840 --> 00:19:32,240 HAPPY SYNTHESIZING THROUGH. 384 00:19:32,240 --> 00:19:34,400 THIS DIFFERENT COLORED BARS ARE 385 00:19:34,400 --> 00:19:35,960 INCREASING TIME OF SYNTHESIS. 386 00:19:35,960 --> 00:19:38,480 HOWEVER YOU CAN SEE THAT FOR THE 387 00:19:38,480 --> 00:19:41,920 TWO STRANDS THESE ARE 388 00:19:41,920 --> 00:19:42,560 COMPLIMENTARY STRANDS A AND B 389 00:19:42,560 --> 00:19:45,320 THERE ARE SLOW ZONES AND THE 390 00:19:45,320 --> 00:19:48,640 SLOW ZONE IS WITHIN A SPECIFIC 391 00:19:48,640 --> 00:19:54,840 CHROMOSOMAL REGION OF FRA 16D 392 00:19:54,840 --> 00:19:57,680 AND FOR INSTANCE HARBOR TWO 393 00:19:57,680 --> 00:20:00,240 REPETITIVE SEQUENCE ONE SAY LONG 394 00:20:00,240 --> 00:20:02,520 A28 MICROSATELLITE AND THE OTHER 395 00:20:02,520 --> 00:20:07,480 IS A QUASI-PALINDROME OF 36 396 00:20:07,480 --> 00:20:07,760 SPACES. 397 00:20:07,760 --> 00:20:10,040 LOOKING UP CLOSE AT THE 398 00:20:10,040 --> 00:20:14,320 NUCLEOTIDE RESOLUTION, WE ARE 399 00:20:14,320 --> 00:20:21,160 NOW LOOKING AT THE DNA 400 00:20:21,160 --> 00:20:23,840 POLYMERASE DELTA REGION AND YOU 401 00:20:23,840 --> 00:20:25,840 CAN SEE THE POLYMERASE STARTS 402 00:20:25,840 --> 00:20:28,160 OUT AND REALLY HAS DIFFICULTY 403 00:20:28,160 --> 00:20:30,120 SYNTHESIZING THROUGH THIS SUCH 404 00:20:30,120 --> 00:20:31,840 THAT FAIRLY LITTLE PRODUCT IS 405 00:20:31,840 --> 00:20:34,320 FORMED DOWNSTREAM AND THAT IS 406 00:20:34,320 --> 00:20:35,880 QUANTITATED HERE ON THE RIGHT 407 00:20:35,880 --> 00:20:36,480 FOR YOU. 408 00:20:36,480 --> 00:20:42,800 WE BELIEVE THIS IS DUE TO DNA 409 00:20:42,800 --> 00:20:44,560 BENDING AS IT SYNTHESIZES THE 410 00:20:44,560 --> 00:20:46,840 SECOND STRAND. 411 00:20:46,840 --> 00:20:56,280 THE TRIMER DUPLEX STEM BECOMES 412 00:20:56,280 --> 00:21:08,800 BENT AND EVEN IN THE PRESENCE OF 413 00:21:08,800 --> 00:21:10,960 PCNA AND RCNA CONTINUALLY 414 00:21:10,960 --> 00:21:12,680 LOADING THE CLAMP YOU LOOK AT 415 00:21:12,680 --> 00:21:16,040 THE LAST LANE PLUS THE 416 00:21:16,040 --> 00:21:17,440 EXPERIMENT THIS IS PLUS EXCESS 417 00:21:17,440 --> 00:21:19,840 DA DNA TRAP AND ALL THE PRODUCTS 418 00:21:19,840 --> 00:21:25,120 THEN ARE WITHIN THIS DETOUR IN 419 00:21:25,120 --> 00:21:26,440 THIS FRA 16 REGION WITH NO 420 00:21:26,440 --> 00:21:28,400 PRODUCTS EXTENDED DOWN STREAM. 421 00:21:28,400 --> 00:21:31,840 THIS IS ANOTHER EXAMPLE IN THE 422 00:21:31,840 --> 00:21:35,360 DATA WERE PUBLISHED IN 423 00:21:35,360 --> 00:21:38,800 COLLABORATION WITH WORK ON FLEX 424 00:21:38,800 --> 00:21:41,320 1 WITHIN THE FRA 1 LOCUS. 425 00:21:41,320 --> 00:21:45,160 I CHOSE THIS BECAUSE YOU CAN SEE 426 00:21:45,160 --> 00:21:49,360 HERE THIS IS THE ENZYME AND YOU 427 00:21:49,360 --> 00:21:54,840 CAN SEE THE LIGHT DEPENDENT 428 00:21:54,840 --> 00:21:58,160 INCREASE IN DETERMINATION AND 429 00:21:58,160 --> 00:21:59,280 THE POLYMERASE DELTA IS 430 00:21:59,280 --> 00:22:05,960 STRUGGLING TO GET THROUGH THE 431 00:22:05,960 --> 00:22:11,080 LONG REPEAT AND TO SUMMARIZE THE 432 00:22:11,080 --> 00:22:15,320 DATA WE CAN SEE DITORS CAN BE 433 00:22:15,320 --> 00:22:17,520 DEFINED BY THEIR LENGTH 434 00:22:17,520 --> 00:22:21,360 DEPENDENT INHIBITION OF HUMAN 435 00:22:21,360 --> 00:22:22,840 POLYMERASE DELTA. 436 00:22:22,840 --> 00:22:25,280 SHORT REPEATS ARE ARE NOT A 437 00:22:25,280 --> 00:22:27,840 PROBLEM, INTERRUPTED REPEATS BUT 438 00:22:27,840 --> 00:22:29,440 IT'S THE LONG MACRO SATELLITES 439 00:22:29,440 --> 00:22:35,360 OR THE QUASI-P PA-PALINDROMES THAT 440 00:22:35,360 --> 00:22:37,000 ARE QUITE STABLE AND IDENTIFIED 441 00:22:37,000 --> 00:22:41,920 A NEW ONE IN COLLABORATION 442 00:22:41,920 --> 00:22:44,880 IDENTIFYING THE HEXONUCLEOTIDE 443 00:22:44,880 --> 00:22:48,400 REPEAT IN A RECENT STUDY. 444 00:22:48,400 --> 00:22:52,480 SO FORTUNATELY FOR THE HUMAN DNA 445 00:22:52,480 --> 00:22:54,200 THERE'S HELP FOR REPLICATIVE 446 00:22:54,200 --> 00:22:56,600 POLYMERASES AND OUR LAB HAS BEEN 447 00:22:56,600 --> 00:23:01,640 STUDYING WHAT ENZYMES CAN HELP 448 00:23:01,640 --> 00:23:09,840 AND LOOKED AT ENZYMES BAY -- 449 00:23:09,840 --> 00:23:12,000 BETA AND KAPPA AND WHAT ASSISTS 450 00:23:12,000 --> 00:23:15,840 IN REPLICATION AND SHOW YOU NEW 451 00:23:15,840 --> 00:23:19,320 DATA TODAY THAT WE'VE JUST 452 00:23:19,320 --> 00:23:22,840 GOTTEN THE PAST YEAR LOOKING AT 453 00:23:22,840 --> 00:23:27,280 THE EFFECTS OF RPA. 454 00:23:27,280 --> 00:23:29,440 THIS IS A SNAPSHOT TAKEAWAY 455 00:23:29,440 --> 00:23:30,600 PICTURE LOOKING AT THE 456 00:23:30,600 --> 00:23:31,720 DIFFERENCE BETWEEN THE 457 00:23:31,720 --> 00:23:36,960 REPLICATIVE ENZYMES AND THE 458 00:23:36,960 --> 00:23:37,840 SPECIAL POLYMERASE. 459 00:23:37,840 --> 00:23:40,120 THEY'RE INHIBITED WITHIN THE DO 460 00:23:40,120 --> 00:23:40,560 TOUR. 461 00:23:40,560 --> 00:23:44,240 IT'S A QUASIPALINDROME WHICH I 462 00:23:44,240 --> 00:23:46,280 MENTIONED HAS A LONG STEM. 463 00:23:46,280 --> 00:23:49,880 ALL THREE ARE INHIBITED AT THE 464 00:23:49,880 --> 00:23:55,320 BASE OF THE STEM WHEREAS 465 00:23:55,320 --> 00:23:59,680 POLYMERASES BETA AND CAN 466 00:23:59,680 --> 00:24:01,920 SYNTHESIZE READILY INTO THE DOWN 467 00:24:01,920 --> 00:24:02,880 STREAM REGION. 468 00:24:02,880 --> 00:24:08,600 WE PUBLISHED SEVERAL PAPERS 469 00:24:08,600 --> 00:24:10,440 SHOWING THEY CAN DISPLAY THROUGH 470 00:24:10,440 --> 00:24:16,440 TE DITORS THAT INHIBIT DELTA AND 471 00:24:16,440 --> 00:24:19,880 WANTED TO SHOW YOU DATA SHOWING 472 00:24:19,880 --> 00:24:24,240 WE BELIEVE THAT THEY CAN 473 00:24:24,240 --> 00:24:26,000 INTERACT AND CAN ACTUALLY BE 474 00:24:26,000 --> 00:24:31,280 ENGAGED AT THE FORK. 475 00:24:31,280 --> 00:25:39,120 THIS IS AND IF WE GIVE EITHER 476 00:25:39,120 --> 00:25:41,240 THEY'RE VERY EFFICIENT AT TAKING 477 00:25:41,240 --> 00:25:44,840 OVER THOSE TERMINATED PRODUCTS 478 00:25:44,840 --> 00:25:46,920 AND COMPLETING SYNTHESIS THROUGH 479 00:25:46,920 --> 00:25:50,920 THIS REGION. 480 00:25:50,920 --> 00:25:53,480 WE ALSO SHOWED IN THE PAPER 481 00:25:53,480 --> 00:26:08,840 THEY'RE RESISTANT TO ACETYL 482 00:26:08,840 --> 00:26:10,880 CHOLINE AND WHAT WE SHOWED IS 483 00:26:10,880 --> 00:26:18,880 THAT IF WE DO THE SAME REACTION 484 00:26:18,880 --> 00:26:26,840 IN THE PRESENCE APHIDICOLIN IS 485 00:26:26,840 --> 00:26:36,040 DEPENDENT ON BETA AND KAPPA. 486 00:26:36,040 --> 00:26:38,080 I WANTED TO SHOW NEW DATA ON 487 00:26:38,080 --> 00:26:39,640 DITORS REPLICATION. 488 00:26:39,640 --> 00:26:43,640 I GET ASKED WHAT IS THE IMPACT 489 00:26:43,640 --> 00:26:46,440 OF A SINGLE STRAND BINDING 490 00:26:46,440 --> 00:26:49,960 PROTEIN ON RPA SYNTHESIS. 491 00:26:49,960 --> 00:26:53,040 IT'S EXPECTED TO MELT DOWN 492 00:26:53,040 --> 00:26:53,600 SECONDARY STRUCTURES AND 493 00:26:53,600 --> 00:27:04,440 STIMULATE SYNTHESIS. 494 00:27:04,440 --> 00:27:09,360 I HAVE A COLLABORATION WITH PENN 495 00:27:09,360 --> 00:27:13,040 LAB WITH A GIFTED BIOCHEMIST AND 496 00:27:13,040 --> 00:27:14,840 CHEMIST AND HE AND HIS GRADUATE 497 00:27:14,840 --> 00:27:19,640 STUDENT HAVE NOW BEEN ABLE TO 498 00:27:19,640 --> 00:27:22,880 RECAPITULATE A SYSTEM 499 00:27:22,880 --> 00:27:24,440 BIOCHEMICALLY WHERE WE CAN 500 00:27:24,440 --> 00:27:28,480 MEASURE RPA SUCCESS AND THIS 501 00:27:28,480 --> 00:27:30,400 SLIDE SHOWS THEIR SYSTEM WHERE 502 00:27:30,400 --> 00:27:33,680 WE CAN ACTUALLY CONFIRM THAT RPA 503 00:27:33,680 --> 00:27:36,520 IS ACTIVE IN IT'S UNWINDING 504 00:27:36,520 --> 00:27:38,480 ABILITY. 505 00:27:38,480 --> 00:27:42,440 SO IN THIS ASSAY WHAT THEY DID 506 00:27:42,440 --> 00:27:44,240 WAS TO SYNTHESIZE A DITORS 507 00:27:44,240 --> 00:27:46,400 CONTAINING TEMPLATE. 508 00:27:46,400 --> 00:27:55,520 THIS IS AN AT25 REPEAT THAT IS 509 00:27:55,520 --> 00:28:01,800 FOUND IN FRA 3B AND SUCH THAT 510 00:28:01,800 --> 00:28:04,440 WHEN THE HAIRPIN IS FORMED YOU 511 00:28:04,440 --> 00:28:05,840 HAVE HIGH THREAT ACTIVITY AND 512 00:28:05,840 --> 00:28:08,680 YOU CAN SEE THAT HERE 513 00:28:08,680 --> 00:28:09,040 QUANTITATIVELY. 514 00:28:09,040 --> 00:28:12,520 WHEN RPA IS ADDED TO THE 515 00:28:12,520 --> 00:28:14,880 REACTION THE SIGNAL GOES AWAY 516 00:28:14,880 --> 00:28:19,480 VERY QUICKLY AS RPA ASKS TO BIND 517 00:28:19,480 --> 00:28:25,960 THE DNA AND UNWIND THE HAIRPIN 518 00:28:25,960 --> 00:28:30,840 AND THIS SUBSTRATE IS ADDED IT 519 00:28:30,840 --> 00:28:34,240 JUMPS OVER AND WE GET OUR FRET 520 00:28:34,240 --> 00:28:34,680 ACTIVITY BACK. 521 00:28:34,680 --> 00:28:38,080 THIS SYSTEM THEY WERE ABLE TO 522 00:28:38,080 --> 00:28:45,440 SHOW RPA IS ACTIVE AND WE CAN 523 00:28:45,440 --> 00:28:48,240 LOSE PCNA ON TO THE REACTIONS. 524 00:28:48,240 --> 00:28:52,000 SO THIS DATA NOW ARE LOOKING AT 525 00:28:52,000 --> 00:28:56,040 WHAT HAPPENS WHEN RPA AND PCNA 526 00:28:56,040 --> 00:29:02,000 ARE LOADED ON TO A TEMPLATE. 527 00:29:02,000 --> 00:29:06,640 AND THERE'S CONDITIONS THEN TO 528 00:29:06,640 --> 00:29:08,720 LOOK AT SYNTHESIS OF PAL DELTA 529 00:29:08,720 --> 00:29:10,560 THROUGH THE HAIRPIN REGION. 530 00:29:10,560 --> 00:29:14,840 YOU CAN SEE HERE THE GEL SHOWING 531 00:29:14,840 --> 00:29:19,960 POL DELTA SYNTHESIZES AND HAS A 532 00:29:19,960 --> 00:29:23,640 BRIEF PAUSE AT THE HAIRPIN AND 533 00:29:23,640 --> 00:29:24,480 REPLICATES THROUGH THE END 534 00:29:24,480 --> 00:29:25,960 GIVING FULL LENGTH PRODUCT AND 535 00:29:25,960 --> 00:29:28,040 THE DATA IS QUANTITATED HERE. 536 00:29:28,040 --> 00:29:31,960 UNDER THE CONDITIONS OF EXCESS 537 00:29:31,960 --> 00:29:37,040 RPA, THIS PARTICULAR DITORS IS 538 00:29:37,040 --> 00:29:39,680 NOT A SIGNIFICANT BLOCK TO POL 539 00:29:39,680 --> 00:29:42,040 DELTA AND WE WANT TO LOOK AT THE 540 00:29:42,040 --> 00:29:45,120 EFFECTS OF RPA ON OTHER DITORS 541 00:29:45,120 --> 00:29:47,840 OF OTHER SEQUENCES TO DETERMINE 542 00:29:47,840 --> 00:29:52,040 WHICH DITORS RPA IS MOST ACTIVE 543 00:29:52,040 --> 00:29:56,320 IN HELPING POL DELTA SYNTHESIS. 544 00:29:56,320 --> 00:29:57,720 SYNTHESIZE. 545 00:29:57,720 --> 00:30:00,560 FOR THE SECOND HALF OF MY TALK I 546 00:30:00,560 --> 00:30:02,280 WANTED TO SHIFT GEARS TO 547 00:30:02,280 --> 00:30:03,560 UNPUBLISHED DATA AND WHAT WE'VE 548 00:30:03,560 --> 00:30:04,560 BEEN WORKING ON OVER THE LAST 549 00:30:04,560 --> 00:30:05,840 TWO YEARS AND THAT IS TO ANSWER 550 00:30:05,840 --> 00:30:07,960 THE QUESTION OF WHAT IS THE 551 00:30:07,960 --> 00:30:09,440 COMPLETE LANDSCAPE OF DITORS IN 552 00:30:09,440 --> 00:30:11,560 THE HUMAN GENOME AND TO ASK 553 00:30:11,560 --> 00:30:16,040 WHETHER ANY OF THESE SEQUENCES 554 00:30:16,040 --> 00:30:18,080 CAN ACT AS COMPLETE ROAD BLOCKS 555 00:30:18,080 --> 00:30:21,760 OR OBSTRUCTIONS TO DNA 556 00:30:21,760 --> 00:30:22,160 POLYMERASES. 557 00:30:22,160 --> 00:30:25,920 THIS PROJECT BEGAN AS AN 558 00:30:25,920 --> 00:30:30,840 INTEGRATIVE APPROACH WITH TWO 559 00:30:30,840 --> 00:30:34,560 OTHER COLLEAGUES AT PENN STATE 560 00:30:34,560 --> 00:30:37,920 UNIVERSITY AND ONE WHO IS I 561 00:30:37,920 --> 00:30:39,280 STATISTICAL GENOMICS GURU AND 562 00:30:39,280 --> 00:30:42,040 TOGETHER THE THREE OF US HAVE 563 00:30:42,040 --> 00:30:43,960 ACTUALLY BEEN CROSS-TRAINING 564 00:30:43,960 --> 00:30:45,800 MANY GRADUATE STUDENTS AND 565 00:30:45,800 --> 00:30:49,240 POST-DOCS TO ASK THE QUESTION OF 566 00:30:49,240 --> 00:30:53,800 HOW REPETITIVE SEQUENCES ARE 567 00:30:53,800 --> 00:30:55,640 MAINTAINED IN PRIMATE GENOMES 568 00:30:55,640 --> 00:30:58,200 AND IN THE HUMAN GENOME. 569 00:30:58,200 --> 00:31:01,960 SO WE TOOK THIS CROSS-TRAINING 570 00:31:01,960 --> 00:31:03,280 COLLABORATIVE APPROACH AND 571 00:31:03,280 --> 00:31:04,600 APPLIED IT TO ANSWERING THE 572 00:31:04,600 --> 00:31:09,160 QUESTION OF WHAT ARE THE ALL 573 00:31:09,160 --> 00:31:10,680 DITORS WITHIN THE HUMAN GENOME. 574 00:31:10,680 --> 00:31:12,960 THIS WORK WAS PUBLISHED A FEW 575 00:31:12,960 --> 00:31:14,400 YEARS AGO AND IT IS THE BRAIN 576 00:31:14,400 --> 00:31:22,840 CHILE OF A FORMER GRADUATE 577 00:31:22,840 --> 00:31:30,600 STUDENT AND A POST-DOC AND HE 578 00:31:30,600 --> 00:31:37,840 HAD AN IDEA I DIDN'T THINK WOULD 579 00:31:37,840 --> 00:31:40,400 WORK AND READ THERE WAS A VALUE 580 00:31:40,400 --> 00:31:41,920 IT GIVES EVERY TIME IT 581 00:31:41,920 --> 00:31:44,040 INCORPORATES A BASE AND HIS IDEA 582 00:31:44,040 --> 00:31:47,400 IS ISN'T THIS A KINETIC VALUE 583 00:31:47,400 --> 00:31:48,680 FOR A DNA POLYMERASE GOING 584 00:31:48,680 --> 00:31:51,280 THROUGH THE DIFFERENT VALUES OF 585 00:31:51,280 --> 00:31:54,880 THE GENOME AND I SAID YES, THIS 586 00:31:54,880 --> 00:31:56,840 WOULD BE A K POL VALUE IF YOU 587 00:31:56,840 --> 00:31:59,640 WANTED TO EXTRAPOLATE IT SO HE 588 00:31:59,640 --> 00:32:05,920 TOOK THE DATA AND THEN ANNOTATED 589 00:32:05,920 --> 00:32:10,120 ALL THE NON-BDNA SEQUENCES AND 590 00:32:10,120 --> 00:32:15,240 SHE APPLIED THE TESTING TO 591 00:32:15,240 --> 00:32:17,840 COMPUTE HE'S DISTRIBUTION TO 592 00:32:17,840 --> 00:32:19,400 ANALYZE STATISTICAL 593 00:32:19,400 --> 00:32:21,800 DISTRIBUTIONS AND THEIR 594 00:32:21,800 --> 00:32:23,520 DIFFERENCES AND THEY FOUND THE 595 00:32:23,520 --> 00:32:25,680 SHAPES DO DIFFER DEPENDING ON 596 00:32:25,680 --> 00:32:28,800 THE SEQUENCE BEING EXAMINED AND 597 00:32:28,800 --> 00:32:31,480 ALL THE G4 MOTIFS THEY EXAMINED 598 00:32:31,480 --> 00:32:36,200 HAD SIGNIFICANTLY HIGHER IPDs 599 00:32:36,200 --> 00:32:38,160 COMPARED TO CONTROLLED WINDOWS 600 00:32:38,160 --> 00:32:42,000 AND IDENTIFY ANOTHER NON-B 601 00:32:42,000 --> 00:32:44,840 MOTIFS WITH ALTERED 602 00:32:44,840 --> 00:32:47,440 POLYMERIZATION KINETICS 603 00:32:47,440 --> 00:32:49,000 INCLUDING THOSE IDENTIFIED IN 604 00:32:49,000 --> 00:32:52,080 THE STUDY SUCH AS A-PHASE 605 00:32:52,080 --> 00:32:52,400 REPEAT. 606 00:32:52,400 --> 00:32:55,280 THEY ALSO SHOWED THE SEQUENCING 607 00:32:55,280 --> 00:32:57,520 ERROR RATES DURING THE SYNTHESIS 608 00:32:57,520 --> 00:33:01,480 ARE ARE ACTUALLY INCREASED FOR 609 00:33:01,480 --> 00:33:02,400 G4 REGIONS VERSUS MOTIF FREE 610 00:33:02,400 --> 00:33:20,000 WINDOWS. 611 00:33:20,000 --> 00:33:20,440 THEN THEY LOOKED AT THE 612 00:33:24,080 --> 00:33:34,040 G4LOCI AND FOUND THE SNIPS 613 00:33:34,040 --> 00:33:37,960 COMPARED TO THE CONTROL WINDOWS 614 00:33:37,960 --> 00:33:44,120 OF THE GENOME. 615 00:33:44,120 --> 00:33:46,480 I WANT YOU TO REMEMBER THE 616 00:33:46,480 --> 00:33:48,600 CONCEPT OF STABLE G4s. 617 00:33:48,600 --> 00:33:51,640 THE QUADRANT ANALYSIS IS 618 00:33:51,640 --> 00:33:54,720 ACTUALLY BUILT ON -- IT TAKES 619 00:33:54,720 --> 00:33:59,240 INTO ACCOUNT G4 SEQ DATA AS WELL 620 00:33:59,240 --> 00:34:02,040 AS PARTICULAR MOTIF. 621 00:34:02,040 --> 00:34:03,520 THE QUADRANT SCORES ARE GIVEN 622 00:34:03,520 --> 00:34:05,440 DIFFERENT VALUES AND A SCORE 623 00:34:05,440 --> 00:34:10,520 GREATER THAN 19 WAS CONSIDERED 624 00:34:10,520 --> 00:34:16,720 BY THE WRITERS OF THE ALGORITHM 625 00:34:16,720 --> 00:34:19,280 TO BE STABLE TO THE GENOME. 626 00:34:19,280 --> 00:34:21,520 IF YOU LOOKED AT STABLE G4s 627 00:34:21,520 --> 00:34:24,520 THERE'S A HIGHER DIFFERENTIAL 628 00:34:24,520 --> 00:34:25,080 BETWEEN SNIP FREQUENCY AND 629 00:34:25,080 --> 00:34:27,240 CONTROL WINDOWS. 630 00:34:27,240 --> 00:34:29,480 SO MY LAB NOW HAS STEPPED INTO 631 00:34:29,480 --> 00:34:32,920 THIS PROJECT AND TAKEN OVER TO 632 00:34:32,920 --> 00:34:39,360 ASK WHETHER G4s CAN BE DITORS 633 00:34:39,360 --> 00:34:42,040 AND WHETHER THEY CAN AFFECT 634 00:34:42,040 --> 00:34:43,600 FIDELITY AND ARE ALSO ASKING 635 00:34:43,600 --> 00:34:46,880 WHETHER G4 SEQUENCES INHIBIT THE 636 00:34:46,880 --> 00:34:47,880 REPLICATIVE ENZYMES. 637 00:34:47,880 --> 00:34:52,040 THESE ARE THE DATA I'D LIKE TO 638 00:34:52,040 --> 00:34:53,160 SHARE WITH YOU TODAY. 639 00:34:53,160 --> 00:34:54,600 THESE ARE UNPUBLISHED DATA. 640 00:34:54,600 --> 00:34:57,040 PURSE I WANT TO TALK ABOUT THE 641 00:34:57,040 --> 00:34:59,400 POLYMERASE FIDELITY AND THE WORK 642 00:34:59,400 --> 00:35:04,840 OF MY CURRENT GRADUATE STUDENT 643 00:35:04,840 --> 00:35:08,840 WHO TOOK OUR EXISTING POLYMERASE 644 00:35:08,840 --> 00:35:12,200 ASSAY AND WORKED VERY HARD TO 645 00:35:12,200 --> 00:35:15,480 LEARN HOW TO PURIFY G4 646 00:35:15,480 --> 00:35:18,840 CONTAINING SUBSTRATES AND CREATE 647 00:35:18,840 --> 00:35:21,520 THE MOLECULES FOR POLYMERASE GAP 648 00:35:21,520 --> 00:35:26,680 FILLING REACTION S AND CONTAIN 649 00:35:26,680 --> 00:35:30,080 THE G4 MOTIF AND THEN TAKE THE 650 00:35:30,080 --> 00:35:37,800 PRODUCTS AND CREATE THEM INTO 651 00:35:37,800 --> 00:35:41,600 THE MUTANTS AND THEN INDEPENDENT 652 00:35:41,600 --> 00:35:42,640 MUTANTS ARE SEQUENCES TO 653 00:35:42,640 --> 00:35:45,640 DETERMINE THE SPECIFICITY. 654 00:35:45,640 --> 00:35:51,840 SO MARY ELIZABETH CHOSE TWO G4 655 00:35:51,840 --> 00:35:52,040 MOTIF. 656 00:35:52,040 --> 00:36:06,840 ONE IS WITHIN THE L4 LOCUS. 657 00:36:06,840 --> 00:36:10,640 AND THIS HAS A TM OF AROUND 76 658 00:36:10,640 --> 00:36:10,880 DEGREES. 659 00:36:10,880 --> 00:36:17,520 SHE ALSO ANALYZED THE ONE WITH 660 00:36:17,520 --> 00:36:20,440 FIVE G TRACKS. 661 00:36:20,440 --> 00:36:23,720 THIS IS A WELL CHARACTERIZED G4 662 00:36:23,720 --> 00:36:25,840 AND IT FORMS A PARALLEL 663 00:36:25,840 --> 00:36:27,840 STRUCTURE AND IS MORE STABLE. 664 00:36:27,840 --> 00:36:32,520 THEN MARY ELIZABETH CREATED A 665 00:36:32,520 --> 00:36:41,040 MUTANT FORM THAT ONLY CONTAINS 666 00:36:41,040 --> 00:36:43,080 FOUR 4 TRACTS AND LOSING THAT 667 00:36:43,080 --> 00:36:44,240 FIFTH TRACK DID NOT CHANGE ITS 668 00:36:44,240 --> 00:36:49,200 STABILITY. 669 00:36:49,200 --> 00:36:52,960 SO USING THIS ASSAY MARY 670 00:36:52,960 --> 00:36:56,040 ELIZABETH WAS ABLE TO SHOW 671 00:36:56,040 --> 00:37:02,040 FIDELITY WAS MAINTAINED DURING 672 00:37:02,040 --> 00:37:06,880 DAP -- DNA SYNTHESIS AND WE 673 00:37:06,880 --> 00:37:12,280 HAVE POL KAPPA AND POL BETA IS 674 00:37:12,280 --> 00:37:14,800 THE LEAST ACCURATE. 675 00:37:14,800 --> 00:37:18,080 I HAVE A CLOSE UP AND 676 00:37:18,080 --> 00:37:19,760 QUANTITATED WHETHER THE ERRORS 677 00:37:19,760 --> 00:37:21,800 ARE OCCURRING WITHIN THE G4 678 00:37:21,800 --> 00:37:24,840 MOTIF VERSUS THE SURROUNDING 679 00:37:24,840 --> 00:37:25,880 INTERNAL CONTROL. 680 00:37:25,880 --> 00:37:30,840 AND YOU CAN SEE THAT FOR THE 681 00:37:30,840 --> 00:37:33,280 DIFFERENT POLYMERASES AND G4 682 00:37:33,280 --> 00:37:36,440 MOTIFS THEY'RE NOT A HOT SPOT OF 683 00:37:36,440 --> 00:37:38,040 INCREASED POLYMERASE ERRORS. 684 00:37:38,040 --> 00:37:40,080 HOWEVER, THAT DOESN'T MEAN TO 685 00:37:40,080 --> 00:37:43,200 SAY G4s ARE NOT AFFECTING THE 686 00:37:43,200 --> 00:37:43,480 POLYMERASES. 687 00:37:43,480 --> 00:37:47,000 WHAT MARY ELIZABETH OBSERVED IS 688 00:37:47,000 --> 00:37:51,240 SHE HAS AN INCREASED FREQUENCY 689 00:37:51,240 --> 00:37:53,120 IN OUR GENE IN THE THREE PRIME 690 00:37:53,120 --> 00:37:55,320 END SO WHAT YOU'RE LOOKING AT 691 00:37:55,320 --> 00:37:58,960 HERE IS A CLOSE UP OF A 692 00:37:58,960 --> 00:38:01,320 MUTATIONAL SPECTRUM OF THE 693 00:38:01,320 --> 00:38:01,640 TARGET. 694 00:38:01,640 --> 00:38:04,840 SHOWN IN RED ARE THE SEQUENCES 695 00:38:04,840 --> 00:38:08,040 OF THE G4 MOTIF SO THE G4 WINDOW 696 00:38:08,040 --> 00:38:09,720 IN THE BRACKETS. 697 00:38:09,720 --> 00:38:13,240 SHE THEN CREATED WINDOWS OF THE 698 00:38:13,240 --> 00:38:14,880 EQUIVALENT SIZE TO THE G4 MOTIF. 699 00:38:14,880 --> 00:38:19,920 I THINK VISUALLY YOU CAN SEE AS 700 00:38:19,920 --> 00:38:22,360 THE G4 BECOMES MORE STABLE THIS 701 00:38:22,360 --> 00:38:26,040 IS ADA HAS AN INCREASED 702 00:38:26,040 --> 00:38:30,040 FREQUENCY OF ERRORS WITHIN THIS 703 00:38:30,040 --> 00:38:31,840 IT THREE-PRONG BLANKING REGION. 704 00:38:31,840 --> 00:38:34,160 THE IT DATA IS QUANTITATED FOR 705 00:38:34,160 --> 00:38:37,560 YOU HERE AND ANALYZED 706 00:38:37,560 --> 00:38:39,440 STATISTICALLY AND FOR POL ADA 707 00:38:39,440 --> 00:38:42,920 AND KAPPA WE SEE THE CHANGE IN 708 00:38:42,920 --> 00:38:44,040 DISTRIBUTION. 709 00:38:44,040 --> 00:38:45,920 AGAIN AN EXCESS OF ERRORS IN THE 710 00:38:45,920 --> 00:38:50,840 FLANKING REGION FOR THE TWO G4 711 00:38:50,840 --> 00:38:54,920 MOTIFS THAT FORM STABLE PARALLEL 712 00:38:54,920 --> 00:38:57,320 STRUCTURES. 713 00:38:57,320 --> 00:39:00,640 WE ALSO SAW A GREAT BREADTH OF 714 00:39:00,640 --> 00:39:02,920 POLYMERASE ERRORS FOR ANYONE WHO 715 00:39:02,920 --> 00:39:06,240 STUDIES THIS IS AN ENZYME THAT 716 00:39:06,240 --> 00:39:08,040 MOSTLY MAKES SUBSTITUTIONS. 717 00:39:08,040 --> 00:39:10,680 IT'S KIND OF BORING BUT WE 718 00:39:10,680 --> 00:39:12,280 STARTED TO SEE QUITE A RANGE 719 00:39:12,280 --> 00:39:17,680 THEN OF DIFFERENT TYPES OF 720 00:39:17,680 --> 00:39:20,040 MUTATIONS WITHIN THE G4 MOTIF 721 00:39:20,040 --> 00:39:22,840 AND MANY OF THESE INCLUDED BOTH 722 00:39:22,840 --> 00:39:26,760 THE WHAT WE WOULD EXPECT OF THE 723 00:39:26,760 --> 00:39:32,200 G TRACTS AND ALSO THE LOOPS. 724 00:39:32,200 --> 00:39:40,640 HAS TO ARE LARGER INSERTIONS AND 725 00:39:40,640 --> 00:39:41,960 WE'RE SEEING GREAT INCREASE IN 726 00:39:41,960 --> 00:39:44,040 THE FREQUENCY OF LARGER 727 00:39:44,040 --> 00:39:44,800 DELETIONS. 728 00:39:44,800 --> 00:39:46,880 BUT POL DELTA ALSO MAKES AN 729 00:39:46,880 --> 00:39:49,040 INTERESTING NEW CLASS OF 730 00:39:49,040 --> 00:39:51,960 MUTATIONS WHICH I HAVE NOT 731 00:39:51,960 --> 00:39:54,840 OBSERVED PREVIOUSLY IN MY 732 00:39:54,840 --> 00:39:56,720 STUDIES IN WHICH THERE'S 733 00:39:56,720 --> 00:39:59,560 INSERTIONS HERE IN THE UPPER 734 00:39:59,560 --> 00:40:01,560 CASE IN THE MIDDLE OF THE G4 735 00:40:01,560 --> 00:40:04,680 TRACK SO THESE INSERTION 736 00:40:04,680 --> 00:40:05,880 MUTATIONS ACTUALLY SERVE TO 737 00:40:05,880 --> 00:40:13,800 EXPAND THE G4 MOTIF. 738 00:40:13,800 --> 00:40:15,960 THIS IS IN THE PARALLEL 739 00:40:15,960 --> 00:40:17,920 STRUCTURE G4s. 740 00:40:17,920 --> 00:40:19,760 WE MENTIONED DELETION MUTATION. 741 00:40:19,760 --> 00:40:22,760 THIS IS NOT AN ERROR WE NORMALLY 742 00:40:22,760 --> 00:40:25,720 SEE WITH POL ADA BUT WHEN 743 00:40:25,720 --> 00:40:28,680 SYNTHESIZING THROUGH THE STABLE 744 00:40:28,680 --> 00:40:32,000 G4s WE SEE LARGE DELETION 745 00:40:32,000 --> 00:40:35,840 BASICALLY REMOVE THE BLANKING 746 00:40:35,840 --> 00:40:37,960 SEQUENCE OF THE TK GENE. 747 00:40:37,960 --> 00:40:41,640 WHAT DOES THIS MEAN 748 00:40:41,640 --> 00:40:42,000 BIOLOGICALLY? 749 00:40:42,000 --> 00:40:45,360 TO GET TO THE QUESTIONS OF THE 750 00:40:45,360 --> 00:40:46,680 BIOLOGICAL CONSEQUENCES OF 751 00:40:46,680 --> 00:40:50,520 POLYMERASE ERRORS WE AGAIN 752 00:40:50,520 --> 00:40:56,040 WORKED WITH OUR COLLABORATORS 753 00:40:56,040 --> 00:41:04,720 AND HE TOOK ALL OF THE MUE TAPTS 754 00:41:04,720 --> 00:41:06,160 AND ANALYZED THEM FOR THE 755 00:41:06,160 --> 00:41:13,120 QUADRANT SCORE SO AGAIN QUADRANT 756 00:41:13,120 --> 00:41:16,400 IS A MACHINE ALGORITHM BASED ON 757 00:41:16,400 --> 00:41:18,840 DATA FROM G4 SEQ AND 758 00:41:18,840 --> 00:41:30,320 INCORPORATES THE FEATURES AND WE 759 00:41:30,320 --> 00:41:30,720 MRO 760 00:41:30,720 --> 00:41:49,840 PLOT THE CHANGE YOU SEE THE 761 00:41:49,840 --> 00:41:50,840 ALTERED DISTRIBUTION AND SOME OF 762 00:41:50,840 --> 00:41:53,080 THE MUTANTS HAVE ELIMINATED THE 763 00:41:53,080 --> 00:42:02,840 ABILITY TO FORM A G4. 764 00:42:02,840 --> 00:42:08,040 THESE MUTANTS THAT ELIMINATE THE 765 00:42:08,040 --> 00:42:08,880 G4 POTENTIAL INCLUDE 766 00:42:08,880 --> 00:42:10,720 SUBSTITUTION OR FRIENDSHIPS THAT 767 00:42:10,720 --> 00:42:14,000 CHANGE THE G TRACK AND THE LOOPS 768 00:42:14,000 --> 00:42:15,560 OR THE LARGE DELETIONS THAT 769 00:42:15,560 --> 00:42:18,400 REMOVE THE G4. 770 00:42:18,400 --> 00:42:21,920 SO WHAT ABOUT THE OTHER HALF? 771 00:42:21,920 --> 00:42:26,840 TWO G4s INHIBIT HUMAN POLYMERASE 772 00:42:26,840 --> 00:42:28,160 DELTA. 773 00:42:28,160 --> 00:42:34,840 THIS IS A PROJECT OF SUSAN HILE 774 00:42:34,840 --> 00:42:37,520 AND STUDIED THIS FOR L1 SEQUENCE 775 00:42:37,520 --> 00:42:39,040 AS WELL AS ANOTHER MOTIF BASED 776 00:42:39,040 --> 00:42:40,720 ON THE QUADRANT SCORE THESE ARE 777 00:42:40,720 --> 00:42:42,840 NOT EXPECTED TO BE VERY STABLE 778 00:42:42,840 --> 00:42:47,280 STRUCTURES AND THIS IS CONFIRMED 779 00:42:47,280 --> 00:42:54,040 ANTI-PARALLEL STRUCTURES AND 780 00:42:54,040 --> 00:42:58,640 KNOW G4 MOTIF WITHIN THE L1 781 00:42:58,640 --> 00:43:01,840 SEQUENCE THIS HAS A FAIRLY HIGH 782 00:43:01,840 --> 00:43:03,160 QUADRANT SCORE EXPECTED TO BE 783 00:43:03,160 --> 00:43:06,760 STABLE AND INDEED LOOKING AT THE 784 00:43:06,760 --> 00:43:12,160 THERMO STABILITY IN OUR REACTION 785 00:43:12,160 --> 00:43:15,560 BUFFERS AND CIRCULAR 786 00:43:15,560 --> 00:43:17,160 CONFIGURATION IS EXPECTED TO BE 787 00:43:17,160 --> 00:43:21,480 A STABLE STRUCTURE AND FORM 788 00:43:21,480 --> 00:43:22,080 INSTRA STRAND STRUCTURES. 789 00:43:22,080 --> 00:43:24,240 THE QUESTION IS HOW DO THEY 790 00:43:24,240 --> 00:43:25,640 IMPACT THE DNA POLYMERASES? 791 00:43:25,640 --> 00:43:28,640 THE SYSTEM IS THIS SAME. 792 00:43:28,640 --> 00:43:31,040 AN EX TENSE SYSTEM WHERE WE LOOK 793 00:43:31,040 --> 00:43:41,040 AT THE G4s WITHIN THE SINGLE 794 00:43:41,040 --> 00:43:48,760 STRANDED VECTORS AND LOOKING AT 795 00:43:48,760 --> 00:43:50,720 THE UNSTABLE AND STABLE 796 00:43:50,720 --> 00:43:53,000 STRUCTURES AND IF YOU LOOK AT 797 00:43:53,000 --> 00:43:56,160 THE L1 SEQUENCE SUSAN ALSO 798 00:43:56,160 --> 00:43:57,440 ANALYZED POLYMERASE PROGRESSION 799 00:43:57,440 --> 00:44:00,520 THROUGH THIS SEQUENCE OF THE G 800 00:44:00,520 --> 00:44:02,040 RICH AND C RICH STRANDS. 801 00:44:02,040 --> 00:44:04,480 WHAT I HOPE YOU CAN SEE IS THIS 802 00:44:04,480 --> 00:44:06,520 IT VERY PRONOUNCED PAUSING OF 803 00:44:06,520 --> 00:44:09,800 DELTA AT THE BASE OF THE G4 AND 804 00:44:09,800 --> 00:44:12,160 THE DATA ARE QUANTITATED HERE 805 00:44:12,160 --> 00:44:16,840 WHERE ALL THE G4s DO HAVE 806 00:44:16,840 --> 00:44:18,440 SOMEWHAT INCREASED TERMINATION 807 00:44:18,440 --> 00:44:20,680 BUT THE STABLE G4 IS THE HIGHEST 808 00:44:20,680 --> 00:44:22,480 AMOUNT AND THAT IS NOT SEEN ON 809 00:44:22,480 --> 00:44:32,040 THE COMPLIMENTARY STRAND. 810 00:44:32,040 --> 00:44:38,440 WE'RE ASKING DO THE TWO 811 00:44:38,440 --> 00:44:39,200 POLYMERASES AT THE REPLICATION 812 00:44:39,200 --> 00:44:43,760 FOR DELTA AND EPSILON REMAIN 813 00:44:43,760 --> 00:44:49,920 THROUGH A G4 CONSEQUENCE. 814 00:44:49,920 --> 00:44:57,000 THIS IS A CLOSE UP OF PAPER FROM 815 00:44:57,000 --> 00:45:02,440 THE COLLABORATORS AND THE G4 816 00:45:02,440 --> 00:45:06,520 MOTIF HAVE A LARGE DISTRIBUTION 817 00:45:06,520 --> 00:45:09,240 OF INCREASED INTERPULSE DURATION 818 00:45:09,240 --> 00:45:10,640 STRANDS AND THE IT OTHER DOESN'T 819 00:45:10,640 --> 00:45:13,640 SHOW THAT TYPE OF DISTRIBUTION. 820 00:45:13,640 --> 00:45:16,760 WE SAW WHERE THE G STRAND HAS 821 00:45:16,760 --> 00:45:18,160 MORE INHIBITION THAN THE C 822 00:45:18,160 --> 00:45:19,440 STRAND. 823 00:45:19,440 --> 00:45:23,800 TO LOOK AT THIS QUESTION SUSAN 824 00:45:23,800 --> 00:45:26,640 HAS DEVELOPED A SYSTEM LOOKING 825 00:45:26,640 --> 00:45:28,960 AT A VARIETY OF G4 MOTIF. 826 00:45:28,960 --> 00:45:32,160 SO IN ADDITION TO THE THREE WE 827 00:45:32,160 --> 00:45:34,840 LOCKED AT WE ALSO ASKED OUR 828 00:45:34,840 --> 00:45:36,880 GENOMICS COLLABORATORS WHAT ARE 829 00:45:36,880 --> 00:45:40,320 THE MOST ABUN DAND STABLE MOTIFS 830 00:45:40,320 --> 00:45:41,640 PREDICTED STABLE MOTIFS IN THE 831 00:45:41,640 --> 00:45:42,840 HUMAN GENOME. 832 00:45:42,840 --> 00:45:44,040 THE IT DATA ARE SHOWN ON THE 833 00:45:44,040 --> 00:45:48,440 RIGHT. 834 00:45:48,440 --> 00:45:51,520 THESE ARE THE NUMBER OF G4 835 00:45:51,520 --> 00:45:54,680 MOTIFS AN YOU SEE GREAT 836 00:45:54,680 --> 00:45:57,280 ENRICHMENT WITHIN THE TWO MOBILE 837 00:45:57,280 --> 00:46:00,880 ELEMENTS AND THE L1PA ELEMENTS. 838 00:46:00,880 --> 00:46:04,560 SO WE CREATED ANOTHER MOTIF THAT 839 00:46:04,560 --> 00:46:14,200 IS ONE THAT CORRESPONDS TO THE 840 00:46:14,200 --> 00:46:17,680 ELEMENT THEY'RE PROMINENT IN 841 00:46:17,680 --> 00:46:21,760 THE HUMAN GENOME AND WANTED TO 842 00:46:21,760 --> 00:46:24,360 LOOK AT G4s AND WE CHOSE ONE OF 843 00:46:24,360 --> 00:46:28,840 THE OVER SEQUENCES THAT WERE 844 00:46:28,840 --> 00:46:30,840 DESCRIBED SEVERAL YEARS AGO. 845 00:46:30,840 --> 00:46:33,840 THIS WAS ONE OF THOSE SHOWN TO 846 00:46:33,840 --> 00:46:39,760 BE FUNCTIONALLY WORK AS AN 847 00:46:39,760 --> 00:46:47,400 ORIGIN IN THE SEQUENCE. 848 00:46:47,400 --> 00:46:50,240 AND WE COLLABORATED WITH A 849 00:46:50,240 --> 00:46:54,640 FORMER POST-DOC AND WE WANT IT 850 00:46:54,640 --> 00:47:01,120 LOCK AT THE REPLICATIVE ENZYMES 851 00:47:01,120 --> 00:47:05,200 AND ASK THE QUESTION WHAT IS TO 852 00:47:05,200 --> 00:47:08,200 THE SYNTHESIS THROUGH THE G4 853 00:47:08,200 --> 00:47:09,960 MOTIF AND WE'LL QUANTIFY 854 00:47:09,960 --> 00:47:11,960 PRODUCTS BECAUSE THESE ARE GOING 855 00:47:11,960 --> 00:47:12,600 TO COMPLIP ALL THE WAY AROUND 856 00:47:12,600 --> 00:47:13,960 OUR CIRCLE. 857 00:47:13,960 --> 00:47:15,920 THESE ARE SMALL CIRCLE. 858 00:47:15,920 --> 00:47:20,240 WE'RE GOING TO ASK HOW MANY 859 00:47:20,240 --> 00:47:24,040 PRODUCTS ARE INHIBITED AND HOW 860 00:47:24,040 --> 00:47:33,440 CAN SYNTHESIZE PAST THE G4 AND 861 00:47:33,440 --> 00:47:35,440 WE HAVE TWO REACTIONS ONE FOR 862 00:47:35,440 --> 00:47:37,400 THE G RICH AND ONE FOR THE C 863 00:47:37,400 --> 00:47:39,240 RICH STRAND AND WHAT WE'RE DOING 864 00:47:39,240 --> 00:47:45,640 IS WE'RE SETTING UP A SYSTEM 865 00:47:45,640 --> 00:47:48,520 MIMIC WHAT IS GOING ON WHERE THE 866 00:47:48,520 --> 00:47:51,840 POL DELTA WOULD BE ON ONE STRAND 867 00:47:51,840 --> 00:47:53,920 THE G RICH STRAND AND POL 868 00:47:53,920 --> 00:47:56,040 EPSILON IS FOUND ON THE C RICH 869 00:47:56,040 --> 00:47:56,280 STRAND. 870 00:47:56,280 --> 00:48:01,320 YOU CAN SEE IN EITHER 871 00:48:01,320 --> 00:48:04,840 ORIENTATION, BOTH THE ENZYMES 872 00:48:04,840 --> 00:48:07,240 ARE GREATLY INHIBITED BY THIS 873 00:48:07,240 --> 00:48:08,240 PARTICULAR SEQUENCE I'M SHOWING 874 00:48:08,240 --> 00:48:16,080 HERE. 875 00:48:16,080 --> 00:48:20,200 THIS EX TENTS PRECEDING THE 876 00:48:20,200 --> 00:48:20,480 CONSEQUENCE. 877 00:48:20,480 --> 00:48:21,800 WE BELIEVE WE'RE GETTING 878 00:48:21,800 --> 00:48:23,320 STRUCTURE FORM IN THE 879 00:48:23,320 --> 00:48:23,920 DOUBLE-STRANDED TEMPLATE AND 880 00:48:23,920 --> 00:48:32,080 BECAUSE WE'RE LOOKING AT THE 881 00:48:32,080 --> 00:48:33,880 MULTI SUB UNIT ENZYMES WE'RE 882 00:48:33,880 --> 00:48:38,840 SEEING INHIBITION OF THE 883 00:48:38,840 --> 00:48:41,960 POLYMERASES AS THEY APPROACH THE 884 00:48:41,960 --> 00:48:42,400 G4. 885 00:48:42,400 --> 00:48:44,240 THESE IS PRELIMINARY DATA SO FAR 886 00:48:44,240 --> 00:48:50,240 JUST TO GIVE AN EXAMPLE OF WHAT 887 00:48:50,240 --> 00:48:57,080 WE'RE DEVELOPING. 888 00:48:57,080 --> 00:48:59,480 AND DELTA GETS THE C RICH STRAND 889 00:48:59,480 --> 00:49:02,360 AND YOU CAN SEE THE 890 00:49:02,360 --> 00:49:04,760 DETERMINATION THE PARTITIONING 891 00:49:04,760 --> 00:49:08,040 VALUE IS QUITE BIASSED TOWARDS 892 00:49:08,040 --> 00:49:09,720 TERMINATION ON THE G RICH 893 00:49:09,720 --> 00:49:10,000 STRAND. 894 00:49:10,000 --> 00:49:11,280 WE SEE THE SAME SITUATION IN THE 895 00:49:11,280 --> 00:49:15,400 OPPOSITE SO IF THE FORK IS GOING 896 00:49:15,400 --> 00:49:17,840 IN THE OTHER DIRECTION NOW DELTA 897 00:49:17,840 --> 00:49:24,520 GETS THE G RICH STRAND AND ALSO 898 00:49:24,520 --> 00:49:26,240 HAS A HIGHER PARTITIONING VALUE 899 00:49:26,240 --> 00:49:33,040 COMPARED TO THE RANDOM CONTROL. 900 00:49:33,040 --> 00:49:37,440 WE WANT TO TAKE THE DATA ONE 901 00:49:37,440 --> 00:49:39,320 STEP FURTHER TO DEVELOP A 902 00:49:39,320 --> 00:49:41,960 REPLICATION UNCOUPLING INDEX TO 903 00:49:41,960 --> 00:49:44,400 GET AT THE QUESTION OF THIS 904 00:49:44,400 --> 00:49:47,360 DISCONNECT OF POLYMERASES ON THE 905 00:49:47,360 --> 00:49:49,640 TWO STRANDS AS THEY'RE 906 00:49:49,640 --> 00:49:51,200 SYNTHESIZING THROUGH A G4. 907 00:49:51,200 --> 00:49:54,320 WE TAKE THE PARTITIONING VALUE 908 00:49:54,320 --> 00:49:56,480 FOR EPSILON COMPARED TO DELTA ON 909 00:49:56,480 --> 00:49:58,880 THE C RICH STRAND AND THAT WOULD 910 00:49:58,880 --> 00:50:02,160 BE OUR LEADING STRAND UNCOUPLING 911 00:50:02,160 --> 00:50:04,440 INDEX. 912 00:50:04,440 --> 00:50:08,200 AND YOU CAN SEE THAT THE G4 913 00:50:08,200 --> 00:50:17,800 MOTIF FROM IS QUITE BIAS. 914 00:50:17,800 --> 00:50:20,760 POL EPSILON HAS QUITE A BIT AND 915 00:50:20,760 --> 00:50:22,840 WE CAN COMPARE THE POLYMERASE 916 00:50:22,840 --> 00:50:25,640 REPLICATION THROUGH WHAT SHOULD 917 00:50:25,640 --> 00:50:27,480 BE CONSIDERED A NORMAL TEM 918 00:50:27,480 --> 00:50:30,680 POLITICAL REGION AND YOU CAN SEE 919 00:50:30,680 --> 00:50:33,720 THAT BOTH OF THE IT NOBLE 920 00:50:33,720 --> 00:50:38,520 ELEMENTS AND VA AND L1, G4s HAVE 921 00:50:38,520 --> 00:50:39,800 INCREASED UNCOUPLING INDEX. 922 00:50:39,800 --> 00:50:43,480 SO WHAT I'VE TOLD YOU FOR OUR 923 00:50:43,480 --> 00:50:46,840 RECENT RESEARCH LOOKING AT G4s 924 00:50:46,840 --> 00:50:50,840 IN THE GENOME HOW THE 925 00:50:50,840 --> 00:50:52,240 POLYMERASES NAVIGATE THROUGH, 926 00:50:52,240 --> 00:50:53,840 WE'VE LOOKED AT MUTATIONS AND 927 00:50:53,840 --> 00:50:57,240 FOUND SO FAR THE HUMAN 928 00:50:57,240 --> 00:50:57,880 POLYMERASE ERROR FREQUENCY 929 00:50:57,880 --> 00:51:02,160 WITHIN THE G4 WILL NOT GREATLY 930 00:51:02,160 --> 00:51:02,840 ELEVATED. 931 00:51:02,840 --> 00:51:06,560 THE G4s THEMSELVES DON'T HAVE A 932 00:51:06,560 --> 00:51:07,600 HAD THE SPOT TO THE POLYMERASE 933 00:51:07,600 --> 00:51:12,520 BUT THE PRESENCE OF THE G4 934 00:51:12,520 --> 00:51:14,400 INCREASES ERRORS IN THE BLANKING 935 00:51:14,400 --> 00:51:14,840 REGION. 936 00:51:14,840 --> 00:51:17,240 THE ERRORS WITHIN THE G4 ARE 937 00:51:17,240 --> 00:51:18,640 EXPECTED TO INCREASE AND 938 00:51:18,640 --> 00:51:20,200 DECREASE STABILITY OVER 939 00:51:20,200 --> 00:51:21,360 EVOLUTIONARY TIME THERE SHOULD 940 00:51:21,360 --> 00:51:22,960 BE A MAINTENANCE OF HE IT G4s 941 00:51:22,960 --> 00:51:24,920 MONTREAL 942 00:51:28,720 --> 00:51:29,920 MAINTENANCE AND IN THE GENOME 943 00:51:29,920 --> 00:51:34,120 AND WHAT IS OBSERVED IN THE GERM 944 00:51:34,120 --> 00:51:37,560 LINE ARE PROBABLY DUE TO 945 00:51:37,560 --> 00:51:39,960 INHIBITING THE PAIR OR 946 00:51:39,960 --> 00:51:43,240 INCREASING THE IT DNA DAMAGE. 947 00:51:43,240 --> 00:51:45,920 FOR REPLICATION WE OBSERVED SOME 948 00:51:45,920 --> 00:51:51,840 BUT NOT ALL G4s DO REPLICATIVE 949 00:51:51,840 --> 00:51:54,640 DNA SYNTHESIS AND HAVE PAUSING 950 00:51:54,640 --> 00:51:57,920 BY THE DELTA AND THE ENZYMES ON 951 00:51:57,920 --> 00:51:59,760 THE G RICH STRAND AND THAT 952 00:51:59,760 --> 00:52:01,360 UNCOUPLING INDEX IS GREATEST 953 00:52:01,360 --> 00:52:04,080 WHEN THE G4s ARE IN THE LEADING 954 00:52:04,080 --> 00:52:06,840 STRAND CONFIRMATION MEANING 955 00:52:06,840 --> 00:52:08,000 EPSILON GETS THE G RICH STRAND. 956 00:52:08,000 --> 00:52:10,720 WHAT WE WANT TO DO NOW OF COURSE 957 00:52:10,720 --> 00:52:14,520 IS LOOK AT HOW OTHER REPLICATION 958 00:52:14,520 --> 00:52:16,720 PROTEINS CAN ASSIST IN 959 00:52:16,720 --> 00:52:18,440 SYNTHESIZING THROUGH THESE G4s 960 00:52:18,440 --> 00:52:19,240 THAT ARE EXPECTED TO BE 961 00:52:19,240 --> 00:52:23,480 INHIBITORY. 962 00:52:23,480 --> 00:52:24,520 SO IN THE LAST TWO MINUTES, WHAT 963 00:52:24,520 --> 00:52:27,920 I WANTED TO TELL YOU WAS JUST TO 964 00:52:27,920 --> 00:52:30,840 GIVE YOU AN IDEA OF THE OTHER 965 00:52:30,840 --> 00:52:33,320 PROJECTS IN MY LABORATORY AND 966 00:52:33,320 --> 00:52:34,920 THAT THEY ARE SUBCULTURE BASED 967 00:52:34,920 --> 00:52:39,440 AND THEY'RE TRYING TO ASK THE 968 00:52:39,440 --> 00:52:41,400 QUESTION WHEN DO THESE DITORS 969 00:52:41,400 --> 00:52:43,960 BECOME THREATS TO HUMAN GENOME 970 00:52:43,960 --> 00:52:44,240 STABILITY. 971 00:52:44,240 --> 00:52:46,280 THE REPLICATION STRESS WE'RE 972 00:52:46,280 --> 00:52:49,840 FOCUSSING ON ARE THINGS THAT WE 973 00:52:49,840 --> 00:52:53,000 KNOW WILL IMPACT DNA POLYMERASES 974 00:52:53,000 --> 00:52:58,520 SUCH AS THE DNTP LEVELS THAT MAY 975 00:52:58,520 --> 00:53:02,240 BE LOWERED BY REDUCTASE. 976 00:53:02,240 --> 00:53:03,880 I MENTIONED EXHAUSTION IS 977 00:53:03,880 --> 00:53:05,200 SOMETHING THEY'RE IT INTERESTED 978 00:53:05,200 --> 00:53:08,240 IN LOCKING AT THE EFFECT ON 979 00:53:08,240 --> 00:53:08,440 DITORS. 980 00:53:08,440 --> 00:53:15,000 MY LAB STUDIED DEFICIENCIES OF 981 00:53:15,000 --> 00:53:17,680 POLYMERASES AND I WANTED TO SHOW 982 00:53:17,680 --> 00:53:28,920 YOU ONE LAST BIT OF DATA 983 00:53:28,920 --> 00:53:30,680 ONCAGENE DATA AND I WANTED TO 984 00:53:30,680 --> 00:53:33,040 SHARE IT WITH YOU AND THEY'VE 985 00:53:33,040 --> 00:53:34,760 BEEN RECENTLY PUBLISHED AND 986 00:53:34,760 --> 00:53:37,480 THESE ARE THE DATA OF GRADUATE 987 00:53:37,480 --> 00:53:48,320 STUDENT WHO JUST DEFENDED LAST 988 00:53:48,320 --> 00:53:48,640 YE 989 00:53:48,640 --> 00:53:53,480 YEAR AND THIS WAS A MUTANT FORM 990 00:53:53,480 --> 00:53:58,920 OF H RAS AND DID IT IN 991 00:53:58,920 --> 00:53:59,680 FIBROBLASTS. 992 00:53:59,680 --> 00:54:06,400 SO OUR EXPECTATION IS ONCA GENE 993 00:54:06,400 --> 00:54:07,920 EXPRESSION WAS UP REGULATE DNA 994 00:54:07,920 --> 00:54:09,200 POLYMERASES BECAUSE PREVIOUSLY 995 00:54:09,200 --> 00:54:11,520 MY LAB HAD SHOWN IN A 996 00:54:11,520 --> 00:54:14,040 PUBLICATION THAT IF YOU TREAT 997 00:54:14,040 --> 00:54:16,880 CELLS WITH AN HU YOU GET UP 998 00:54:16,880 --> 00:54:20,720 REGULATION AT THE PROTEIN LEVEL 999 00:54:20,720 --> 00:54:23,440 OF DNA POLYMERASE BETA THIS IS 1000 00:54:23,440 --> 00:54:29,920 AN INDEPENDENT MECHANISM AND 1001 00:54:29,920 --> 00:54:32,640 IT'S FOR CHROMATIN FRACTION AND 1002 00:54:32,640 --> 00:54:34,840 WHAT HE SAW WAS THE OPPOSITE. 1003 00:54:34,840 --> 00:54:37,760 I REPEATED SOME OF THESE 1004 00:54:37,760 --> 00:54:39,240 EXPERIMENTS AND IT'S QUITE 1005 00:54:39,240 --> 00:54:41,200 SURPRISING TO ALL OF US IN THAT 1006 00:54:41,200 --> 00:54:46,880 WHAT WE OBSERVED WAS THE DNA 1007 00:54:46,880 --> 00:54:51,200 POLYMERASES ARE RAPIDLY DEPLETED 1008 00:54:51,200 --> 00:54:53,440 UPON ONCA GENE REPLICATION AND 1009 00:54:53,440 --> 00:54:55,480 WE RECENTLY PUBLISHED THIS AND 1010 00:54:55,480 --> 00:54:59,040 YOU CAN SEE THIS RAPID DOWN 1011 00:54:59,040 --> 00:55:06,000 REGULATION OF A DA, CKAPPA AND 1012 00:55:06,000 --> 00:55:10,840 THE POL D3 FOR THOSE WHO STUDY 1013 00:55:10,840 --> 00:55:14,320 BREAK INDUCED REPAIR AND 1014 00:55:14,320 --> 00:55:17,200 POLYMERASE SELFA. 1015 00:55:17,200 --> 00:55:19,520 I HAVE TAKE THEN THE CULTURES 1016 00:55:19,520 --> 00:55:22,040 OUT AND IS THE CELLS ARE RESTED 1017 00:55:22,040 --> 00:55:29,680 AND THEY DID NOT TOTAL LLY SENESCE 1018 00:55:29,680 --> 00:55:32,520 YOU CAN CONTINUE THESE FOR WEEKS 1019 00:55:32,520 --> 00:55:34,800 AND TO ME IT'S QUITE PROVOCATIVE 1020 00:55:34,800 --> 00:55:38,640 THERE COULD BE A WINDOW OF 1021 00:55:38,640 --> 00:55:40,040 VULNERABILITY AS FAR AS GENOME 1022 00:55:40,040 --> 00:55:42,760 INSTABILITY DUE TO THE ALTERED 1023 00:55:42,760 --> 00:55:44,640 DNA REPLICATION PROGRAM IN 1024 00:55:44,640 --> 00:55:46,880 RESPONSE TO ONCA GENE 1025 00:55:46,880 --> 00:55:47,920 ACTIVATION. 1026 00:55:47,920 --> 00:55:49,200 THANK YOU ALL VERY MUCH FOR YOUR 1027 00:55:49,200 --> 00:55:49,440 ATTENTION. 1028 00:55:49,440 --> 00:55:52,640 I THINK I'VE MENTIONED EVERYONE 1029 00:55:52,640 --> 00:55:54,880 ON THE PENN STATE TEAM WHO 1030 00:55:54,880 --> 00:55:58,720 HELPED WITH THE STUDY AND I HAVE 1031 00:55:58,720 --> 00:55:59,440 MY FUNDING SOURCES. 1032 00:55:59,440 --> 00:56:00,760 THANK YOU AGAIN AND I'LL BE 1033 00:56:00,760 --> 00:56:06,800 HAPPY TO TAKE ANY QUESTIONS. 1034 00:56:06,800 --> 00:56:12,280 >> THANK YOU, KRISTIN. 1035 00:56:12,280 --> 00:56:13,520 VERY WONDERFUL TALK. 1036 00:56:13,520 --> 00:56:16,760 I APPRECIATE YOUR GOING TO THE 1037 00:56:16,760 --> 00:56:17,200 BACKGROUND. 1038 00:56:17,200 --> 00:56:22,840 THERE'S A FEW QUESTIONS HERE. 1039 00:56:22,840 --> 00:56:38,120 ONE FROM THE UP BOX DOES DNA 1040 00:56:38,120 --> 00:56:41,040 REPAIRS REPEAT AND IS IT STALLED 1041 00:56:41,040 --> 00:56:46,880 AND ARE TRIPPLEXES MUTOGENIC. 1042 00:56:46,880 --> 00:56:50,360 >> THAT'S A GREAT QUESTION. 1043 00:56:50,360 --> 00:56:50,960 THANK YOU. 1044 00:56:50,960 --> 00:56:57,360 WE HAVE LOOKED AT TRIPLEX DNAs. 1045 00:56:57,360 --> 00:57:02,240 THIS WOULD BE IN POLY PURIN 1046 00:57:02,240 --> 00:57:05,880 TRACKS WHEN THE MICROSATELLITE 1047 00:57:05,880 --> 00:57:10,600 TRACKS BECOME SUCH THE SINGLE 1048 00:57:10,600 --> 00:57:13,280 STRAND SNAPS BACK AND THE SINGLE 1049 00:57:13,280 --> 00:57:16,440 STRAND SNAPS BACK TO PERFORM THE 1050 00:57:16,440 --> 00:57:16,680 TRIPLEX. 1051 00:57:16,680 --> 00:57:20,200 THEY DO BECOME DETOURS WHEN 1052 00:57:20,200 --> 00:57:21,120 THEY'RE LONG ENOUGH. 1053 00:57:21,120 --> 00:57:22,800 SOMETHING WE'RE VERY INTERESTED 1054 00:57:22,800 --> 00:57:25,760 IN BECAUSE WE ARE NOW STUDYING 1055 00:57:25,760 --> 00:57:29,640 THE GGGA SEQUENCES THAT ARE TOO 1056 00:57:29,640 --> 00:57:37,720 SHORT TO FORM A TRIPLEX SO WE 1057 00:57:37,720 --> 00:57:43,240 MONITORED THEM BY CHANGING PH 1058 00:57:43,240 --> 00:57:46,640 AND USING THIS AND THE QUESTION 1059 00:57:46,640 --> 00:57:49,840 WHETHER THEY'RE HOT SPOTS FOR 1060 00:57:49,840 --> 00:57:54,640 MUTOGENESIS I WOULD DIRECT YOU 1061 00:57:54,640 --> 00:58:09,800 TO -- WE HAVE NOT STUDIED THE 1062 00:58:09,800 --> 00:58:17,440 MUTOGENESIS AS WELL AS SHE HAS. 1063 00:58:17,440 --> 00:58:23,280 >> THANKS, KRISTIN, VERY NICE 1064 00:58:23,280 --> 00:58:25,240 TALK. 1065 00:58:25,240 --> 00:58:41,760 ANY ON THE INHIBITED POL DELTA? 1066 00:58:41,760 --> 00:58:50,840 OUR HYPOTHESIS WOULD BE WE HAVE 1067 00:58:50,840 --> 00:58:54,960 NOT BEEN ABLE TO SET UP OUR DUEL 1068 00:58:54,960 --> 00:58:59,960 POLYMERASE REACTION WITH PCNA 1069 00:58:59,960 --> 00:59:04,960 WITH POL IOATA IT'S A 1070 00:59:04,960 --> 00:59:06,240 BIOCHEMIST'S CHALLENGE TO GET 1071 00:59:06,240 --> 00:59:09,360 THE BUFFER SYSTEM TO GET THE 1072 00:59:09,360 --> 00:59:10,360 ACTIVITIES HAPPENING TOGETHER TO 1073 00:59:10,360 --> 00:59:15,160 CO -- DO THE STUDIES. 1074 00:59:15,160 --> 00:59:19,280 WE'RE INTERESTED IN THOSE TYPES 1075 00:59:19,280 --> 00:59:24,360 OF ANALYSES. 1076 00:59:24,360 --> 00:59:30,440 >> IN THE HSV2K ASSAY HAVE YOU 1077 00:59:30,440 --> 00:59:32,840 LOOKED AT THE IN VITRO REACTION 1078 00:59:32,840 --> 00:59:35,520 AFFECTS FREQUENCIES AND 1079 00:59:35,520 --> 00:59:35,760 SPECTRUM? 1080 00:59:35,760 --> 00:59:37,320 >> WE HAVE NOT DONE THAT TYPE OF 1081 00:59:37,320 --> 00:59:58,680 ANALYSIS YET. 1082 00:59:58,680 --> 00:59:59,360 >> THOSE INVOLVED IN THE 1083 00:59:59,360 --> 01:00:01,200 POLYMERASE DELTA 1084 01:00:03,520 --> 01:00:06,960 BETA, KPV AND KPD ARE YOU 1085 01:00:06,960 --> 01:00:07,800 CONSIDERED THEM AND HAVE YOU 1086 01:00:07,800 --> 01:00:09,480 LOOKED AT THEM? 1087 01:00:09,480 --> 01:00:13,240 >> I WANT TO LOOK AT MORE HE LA 1088 01:00:13,240 --> 01:00:15,720 CASES. 1089 01:00:15,720 --> 01:00:19,400 TO DATE WE'VE ONLY LOOKED AT ONE 1090 01:00:19,400 --> 01:00:26,800 IN COLLABORATION WITH A LAB. 1091 01:00:26,800 --> 01:00:30,200 I'M INTERESTED IN MORE OF THE 1092 01:00:30,200 --> 01:00:33,480 RG4 PROJECT, FOR EXAMPLE. 1093 01:00:33,480 --> 01:00:38,440 I ACTUALLY HADN'T CONSIDERED 1094 01:00:38,440 --> 01:00:44,560 WHAT YOU SUGGEST ED IT HAS AN 1095 01:00:44,560 --> 01:00:45,760 ACTIVE ROLE AND I NEVER 1096 01:00:45,760 --> 01:00:48,320 CONSIDERED THAT SO THAT WOULD BE 1097 01:00:48,320 --> 01:00:53,720 SOMETHING I'D BE INTERESTED IN 1098 01:00:53,720 --> 01:00:55,240 DOING, YES. 1099 01:00:55,240 --> 01:00:57,560 >> WE FOUND IT INVOLVED IN 1100 01:00:57,560 --> 01:00:59,120 TRANSCRIPTION AS WELL REPAIR AND 1101 01:00:59,120 --> 01:01:00,160 PATIENTS HAVE MANY DIFFERENT 1102 01:01:00,160 --> 01:01:01,120 FEATURES AND IT'S HARD TO 1103 01:01:01,120 --> 01:01:05,920 EXPLAIN THEM ALL. 1104 01:01:05,920 --> 01:01:09,040 THE NEUROLOGIC SYSTEM AND 1105 01:01:09,040 --> 01:01:09,760 DYSREGULATION. 1106 01:01:09,760 --> 01:01:13,560 AND YOU'VE BEEN TALKING ABOUT 1107 01:01:13,560 --> 01:01:18,720 POL ADA. 1108 01:01:18,720 --> 01:01:28,800 SO ANDREAS, WOW, FEW MORE 1109 01:01:28,800 --> 01:01:29,080 QUESTIONS. 1110 01:01:29,080 --> 01:01:30,880 FOR REPLICATION WAS THERE ANY 1111 01:01:30,880 --> 01:01:37,040 OTHER DIFFERENCE BETWEEN THE POL 1112 01:01:37,040 --> 01:01:41,200 DELTA AND EPSILON CASES AND 1113 01:01:41,200 --> 01:01:45,760 LOOKING AT MAGNESIUM 1114 01:01:45,760 --> 01:01:46,760 CONCENTRATION. 1115 01:01:46,760 --> 01:01:49,160 >> EXCELLENT QUESTION. 1116 01:01:49,160 --> 01:01:51,160 SUSAN HILE WORKED HARD ON MAKING 1117 01:01:51,160 --> 01:01:51,920 THE REACTION CONDITIONS 1118 01:01:51,920 --> 01:01:59,560 IDENTICAL FOR THE DELTA AND EP 1119 01:01:59,560 --> 01:02:01,720 -- EPSILON AND ALL REACTIONS 1120 01:02:01,720 --> 01:02:05,440 CONTAINED AND PCNA AND RSC WE 1121 01:02:05,440 --> 01:02:08,640 KNOW THE RFC WAS ACTIVE IN 1122 01:02:08,640 --> 01:02:12,080 LOADING THE PCNA. 1123 01:02:12,080 --> 01:02:14,160 WE TOOK CONDITIONS ON THE G RICH 1124 01:02:14,160 --> 01:02:16,160 AND C RICH STRAND AND USED 1125 01:02:16,160 --> 01:02:18,200 CONDITIONS WHERE THE ENZYMES ALL 1126 01:02:18,200 --> 01:02:20,040 GOT UP TO THE STARTING LINE SO 1127 01:02:20,040 --> 01:02:21,760 THEY'RE RUNNING START WITH WERE 1128 01:02:21,760 --> 01:02:24,720 THE SAME, REV YOUR ENGINES, ALL 1129 01:02:24,720 --> 01:02:26,720 THE MOLECULES ARE AT THE 1130 01:02:26,720 --> 01:02:28,360 STARTING LINE AND GO THROUGH THE 1131 01:02:28,360 --> 01:02:30,960 G4 AND THAT'S HOW WE CAME UP 1132 01:02:30,960 --> 01:02:33,120 WITH OUR BIOCHEMICAL APPROACH TO 1133 01:02:33,120 --> 01:02:37,280 ASK THIS STRAND DIFFERENTIAL. 1134 01:02:37,280 --> 01:02:40,360 WE HAVE NOT YET ATTEMPTED TO ADD 1135 01:02:40,360 --> 01:02:41,080 ANY MORE PROTEINS TO THE 1136 01:02:41,080 --> 01:02:43,080 REACTION BUT IT'S SOMETHING WE 1137 01:02:43,080 --> 01:02:43,720 DEFINITELY HAVE ON THE BOOKS TO 1138 01:02:43,720 --> 01:02:50,560 DO. 1139 01:02:50,560 --> 01:02:54,040 >> THANK YOU. 1140 01:02:54,040 --> 01:02:56,760 OKAY, TELOMERES ARE SEQUENCES 1141 01:02:56,760 --> 01:02:58,160 WOULD EXPECT HIGH MUTATION 1142 01:02:58,160 --> 01:03:02,360 FREQUENCIES AND IS THERE DATA ON 1143 01:03:02,360 --> 01:03:02,560 THIS? 1144 01:03:02,560 --> 01:03:07,360 >> I WOULD DIRECT YOU TO PATTY 1145 01:03:07,360 --> 01:03:10,360 OPRE SCA'S LAB AND SOMETHING 1146 01:03:10,360 --> 01:03:13,360 THEY'RE LOOKING AT AND I'M NOT 1147 01:03:13,360 --> 01:03:15,760 IN THE TELOMERE BIOLOGY FIELD. 1148 01:03:15,760 --> 01:03:19,160 WE CHOSE OUR G4s TO BE THOSE 1149 01:03:19,160 --> 01:03:24,160 THAT ARE MOST ABUNDANT IN THE 1150 01:03:24,160 --> 01:03:27,320 GENOME THE NOBLE ELEMENTS IN THE 1151 01:03:27,320 --> 01:03:31,840 OTHER G4s AND HAVE NOT LOOKED AT 1152 01:03:31,840 --> 01:03:37,360 TELOMERIC REPEATS IN MY LAB. 1153 01:03:37,360 --> 01:03:41,640 >> AND HAVE YOU TESTED 1154 01:03:41,640 --> 01:03:43,320 POLYMERASE LEVELS IN AN ONCA 1155 01:03:43,320 --> 01:03:44,480 GENE EXPERIMENTAL SYSTEM? 1156 01:03:44,480 --> 01:03:45,680 >> WE HAVE. 1157 01:03:45,680 --> 01:03:48,640 THAT WAS PART OF MY STUDENT'S 1158 01:03:48,640 --> 01:03:48,960 THESIS. 1159 01:03:48,960 --> 01:03:51,520 THIS IS SPECIFIC -- WELL, WE 1160 01:03:51,520 --> 01:03:57,480 LOOKED AT TWO. 1161 01:03:57,480 --> 01:04:01,760 ATRAS AND CMEK AND IT DOES NOT 1162 01:04:01,760 --> 01:04:12,960 INDUCE THE SAME POLYMERASE 1163 01:04:12,960 --> 01:04:14,400 DEPLETION AND WE CAN USE A 1164 01:04:14,400 --> 01:04:17,680 PATHWAY BUT I TOLD MY STUDENTS 1165 01:04:17,680 --> 01:04:19,160 THIS WILL IT BE ANOTHER 10 YEARS 1166 01:04:19,160 --> 01:04:20,360 FIGURE OUT THE MECHANISM AND HE 1167 01:04:20,360 --> 01:04:22,800 NEEDED TO GRADUATE SO WE HAVE 1168 01:04:22,800 --> 01:04:25,040 THE OTHER ONCA GENES IN THE 1169 01:04:25,040 --> 01:04:26,760 FREEZER BUT WE DIDN'T DO THEM 1170 01:04:26,760 --> 01:04:26,920 YET. 1171 01:04:26,920 --> 01:04:28,160 GREAT QUESTION AND SOMETHING WE 1172 01:04:28,160 --> 01:04:30,280 ABSOLUTELY WANT TO TRY TO FIGURE 1173 01:04:30,280 --> 01:04:31,360 OUT WHAT'S GOING ON WITH THE 1174 01:04:31,360 --> 01:04:36,400 SYSTEM. 1175 01:04:36,400 --> 01:04:38,640 >> WELL, THANK YOU SO MUCH. 1176 01:04:38,640 --> 01:04:40,640 AS IT BEEN A WONDERFUL SEMINAR. 1177 01:04:40,640 --> 01:04:45,880 AND HAVE A HAPPY HOLIDAY AND 1178 01:04:45,880 --> 01:04:52,960 NICE BEGINNING OF WINTER NOW. 1179 01:04:52,960 --> 01:04:54,680 THIS THE WINTER SOLSTICE AND WE 1180 01:04:54,680 --> 01:04:56,200 HAVE PEOPLE CLAPPING HERE. 1181 01:04:56,200 --> 01:04:58,400 >> THANK YOU FOR THE INVITATION. 1182 01:04:58,400 --> 01:05:00,880 IT WAS MY PLEASURE AND THANK YOU 1183 01:05:00,880 --> 01:05:02,760 ALL FOR LISTENING AND HAVE A 1184 01:05:02,760 --> 00:00:00,000 HAPPY AND HEALTHY NEW YEAR.