1 00:00:04,811 --> 00:00:06,973 Hello, everyone. 2 00:00:06,973 --> 00:00:10,797 Welcome to the DNA repair interest group video 3 00:00:10,797 --> 00:00:14,674 conferences. Today we are pleased to feature 2 4 00:00:14,674 --> 00:00:16,950 distinguished speaker, Dr. 5 00:00:16,950 --> 00:00:21,203 Mahina Monster and Dr. Rafael Pavani. Both are the recipients 6 00:00:21,203 --> 00:00:26,083 of the NHF Fellows Award for research act of excellence, 7 00:00:26,083 --> 00:00:30,964 which recognize outstanding scientific research by NIH, 8 00:00:30,964 --> 00:00:36,561 Each speaker will present, about approximately 2025 min, followed 9 00:00:36,561 --> 00:00:38,972 by a QA session after each. 10 00:00:38,972 --> 00:00:42,375 So please submit your questions on via chat box and 11 00:00:42,375 --> 00:00:45,979 we will, address them after each of the presentation. 12 00:00:45,979 --> 00:00:51,089 So today the 1st speaker is Dr. Mahina M who is post our fellow 13 00:00:51,089 --> 00:00:55,955 at the national issues or for environmental health science. 14 00:00:55,955 --> 00:00:59,618 Or an IHS, working under the mentorship of, 15 00:00:59,618 --> 00:01:03,963 Dr. Thomas, in the DNA replication fidelity group. 16 00:01:03,963 --> 00:01:08,708 So within the laboratory of genome integrity and structural 17 00:01:08,708 --> 00:01:16,239 biology. Originally from Bangladesh, she earned her 18 00:01:16,239 --> 00:01:24,951 MBBS degree and the complete the postgraduate training and 19 00:01:24,951 --> 00:01:28,705 Dr. Mouseer Dam pursued a Ph. At Kumamoto University in 20 00:01:28,705 --> 00:01:32,231 Japan where her dissertation focused on the role of 21 00:01:32,231 --> 00:01:33,960 DNA polymerase, Epsilon. 22 00:01:33,960 --> 00:01:38,429 In the progression of prognosis, the the metro cancer. Recurrent 23 00:01:38,429 --> 00:01:44,820 or researcher at the NIHS Center, on understanding 24 00:01:44,820 --> 00:01:51,978 the inner repair pathways particularly in the interplay 25 00:01:51,978 --> 00:01:56,244 She investigated these passwords by analyzing mutational patterns 26 00:01:56,244 --> 00:02:01,059 in engineer the east providing foundational insights into 27 00:02:01,059 --> 00:02:05,958 how cells manage any damage with a broad implications for 28 00:02:05,958 --> 00:02:12,965 Without further ado, Dr. 29 00:02:12,965 --> 00:02:16,970 Thank you for your kind interaction, and I appreciate 30 00:02:16,970 --> 00:02:24,364 the opportunity to share my work with you all today and 31 00:02:24,364 --> 00:02:30,950 I am Mahina and presenting our work from the DNA 32 00:02:30,950 --> 00:02:35,940 And today I will present our research on the whole genome 33 00:02:35,940 --> 00:02:45,568 study of nucleotide extinction repair and translation synthesis 34 00:02:45,568 --> 00:02:53,973 in sacrifices, and these sisters represents our effort 35 00:02:53,973 --> 00:02:58,786 And before diving into the specific software study, let 36 00:02:58,786 --> 00:03:03,950 me provide some background to set the context of our work. 37 00:03:03,950 --> 00:03:08,546 Here we outline the determinants of DNA replication, fidelity 38 00:03:08,546 --> 00:03:13,353 and repair pathway and DNA replication and repair 39 00:03:13,353 --> 00:03:17,964 pathways are vital for preventing mutation and 40 00:03:17,964 --> 00:03:22,506 In the pre-replication states various repair mechanisms like 41 00:03:22,506 --> 00:03:26,973 manipulated exhibition repair, base accession repair help. 42 00:03:26,973 --> 00:03:33,023 Correct any existing DNA damage before replication 43 00:03:33,023 --> 00:03:38,951 begins. And the replication fidelity stays. The. 44 00:03:38,951 --> 00:03:43,074 No, fidelity is maintained through multiple mechanisms 45 00:03:43,074 --> 00:03:47,960 including polymerase selectivity and externicless proofreading. 46 00:03:47,960 --> 00:03:52,276 And the stall replication for can lead to challenges that 47 00:03:52,276 --> 00:03:56,969 require specific responses such as translation DNA synthesis. 48 00:03:56,969 --> 00:04:01,735 And facilitated it fascinated by DNA polymerase data to bypass 49 00:04:01,735 --> 00:04:09,682 this slation. And finally, in the post replication estates, 50 00:04:09,682 --> 00:04:16,956 Missbass repair mechanisms work to correct any errors 51 00:04:16,956 --> 00:04:21,972 And my focus of interest lies in the neglected exhibition 52 00:04:21,972 --> 00:04:31,552 repair and translation synthesis pathway. How primary objective 53 00:04:31,552 --> 00:04:40,980 was to investigate how mutation in any both in linear and TLS 54 00:04:40,980 --> 00:04:44,951 9 7 if mutations. 55 00:04:44,951 --> 00:04:49,239 India, is there a versatile repair mechanism that's 56 00:04:49,239 --> 00:04:57,456 remotely conserved from bacteria to humane, the pathway 57 00:04:57,456 --> 00:05:05,972 involves over 30, 30 proteins in EU carriers and repairs 58 00:05:05,972 --> 00:05:11,910 And this diagram illustrate the coordinated action of over 59 00:05:11,910 --> 00:05:17,950 30 proteins in u carriers like red 23, red 2 and right 14. 60 00:05:17,950 --> 00:05:23,456 That works in any pathway. In the inner pathway 61 00:05:23,456 --> 00:05:28,961 red 14 that that east homologue of human is X. 62 00:05:28,961 --> 00:05:33,717 Pier plays the critical role in verifying DNA damage after 63 00:05:33,717 --> 00:05:43,767 initial recombination by write for write 23 in global 64 00:05:43,767 --> 00:05:54,954 linear or transcription couple in your pathway, then helic 65 00:05:54,954 --> 00:06:00,694 And my focus of interest is red 14 XPA in human omelet 66 00:06:00,694 --> 00:06:06,966 that verifies DNA damage and stabilize the repair complex. 67 00:06:06,966 --> 00:06:12,244 Before I say it in addition to neglected extension repair, 68 00:06:12,244 --> 00:06:16,976 my research also focuses on, translation synthesis. 69 00:06:16,976 --> 00:06:21,006 Polymer is data. Is the key player in translation 70 00:06:21,006 --> 00:06:26,510 DNA synthesis pathway. It is involved in translation 71 00:06:26,510 --> 00:06:30,957 DNA synthesis when replication by another 72 00:06:30,957 --> 00:06:35,916 And it is the complex enzyme made up, several proteins 73 00:06:35,916 --> 00:06:40,967 and rip 3 is the catalytic subunit of polymerase data. 74 00:06:40,967 --> 00:06:45,601 And this figure shows that translation repair process 75 00:06:45,601 --> 00:06:53,469 involved in DNA damage repair during replication in 76 00:06:53,469 --> 00:07:01,954 replication and assistants that process start when the 77 00:07:01,954 --> 00:07:07,104 And in instruction states rep one the submarine of polymerase 78 00:07:07,104 --> 00:07:11,536 data and other translation polymerases are recruited 79 00:07:11,536 --> 00:07:15,968 to the damage site along with the immigrant marker. 80 00:07:15,968 --> 00:07:18,022 In the extension and state polymerase data helps 81 00:07:18,022 --> 00:07:19,888 extend past the damage of geneside allowing 82 00:07:19,888 --> 00:07:20,973 replication to continue. 83 00:07:20,973 --> 00:07:25,899 And this process is part of what's called DNA 84 00:07:25,899 --> 00:07:31,206 damage. Tolerance or translation synthesis which 85 00:07:31,206 --> 00:07:36,956 helps cell cope with DNA damage during replication. 86 00:07:36,956 --> 00:07:42,417 And final step shows that DNA is successfully replicated. Pass 87 00:07:42,417 --> 00:07:47,967 the damage side marked by blue triangle. And polymer is jetta. 88 00:07:47,967 --> 00:07:53,927 Is not only essential in basic cellular responses but also 89 00:07:53,927 --> 00:08:00,354 involved in several important similar processes. Like creating 90 00:08:00,354 --> 00:08:05,951 diversity in immunoglobulin during B cell maturation. 91 00:08:05,951 --> 00:08:11,614 Go, contributing the cancer cell evolution by increasing. 92 00:08:11,614 --> 00:08:16,548 Mutrition, edging process potentially influencing 93 00:08:16,548 --> 00:08:18,964 the cellular sentences. 94 00:08:18,964 --> 00:08:22,926 And this table gives an overview of the various 95 00:08:22,926 --> 00:08:26,972 roll-up polymerase data both in Vivo and Vitro. 96 00:08:26,972 --> 00:08:32,216 Each row leads different type of damage the organism it 97 00:08:32,216 --> 00:08:36,982 affects and what polymerase jet does in response. 98 00:08:36,982 --> 00:08:42,292 This wide range of function highlights polymerit jetters 99 00:08:42,292 --> 00:08:46,697 importance. Not only in helping cell cope with 100 00:08:46,697 --> 00:08:51,964 damage but also in process like evolution and disease. 101 00:08:51,964 --> 00:08:56,969 The error rate of polymerase data is intermediate 102 00:08:56,969 --> 00:09:01,974 between DNA replication and TLS DNA polymerases. 103 00:09:01,974 --> 00:09:06,417 It is 10 to 100 for lace accurate then its fellow 104 00:09:06,417 --> 00:09:10,950 B family member in the DNA replication operators. 105 00:09:10,950 --> 00:09:18,958 But 5 to 34 more accurate than Y family TLS. 106 00:09:18,958 --> 00:09:24,502 And this slide shows the active site of PB family polymerase 107 00:09:24,502 --> 00:09:30,680 is the image highlights key conserve residue near the 108 00:09:30,680 --> 00:09:36,976 active site which are procel for the engines activity 109 00:09:36,976 --> 00:09:39,715 And below we see a comparison table of these conjugacy, 110 00:09:39,715 --> 00:09:42,336 you in B family polymerases including RB 69 and this 111 00:09:42,336 --> 00:09:43,949 position is important across B. 112 00:09:43,949 --> 00:09:51,234 Family polymerases where it is typically occupied 113 00:09:51,234 --> 00:09:58,964 by hydrophobic residue like leucine and methionine. 114 00:09:58,964 --> 00:10:04,582 And my specific focus in this study is on mutation in 115 00:10:04,582 --> 00:10:07,973 polymerase data, L, 9, 7, 9, F. 116 00:10:07,973 --> 00:10:14,699 This mutation chains leucine to filler and at position 9 79. 117 00:10:14,699 --> 00:10:17,950 Why we choose this mutation? 118 00:10:17,950 --> 00:10:23,259 Because losing at position 979 is highly conserved and 119 00:10:23,259 --> 00:10:28,961 critical. Seat in the active site of DNA polymerase jeta. 120 00:10:28,961 --> 00:10:33,276 By replacing it with thinner and a similar but bulkier 121 00:10:33,276 --> 00:10:40,414 hydrophobic Am I not seed and this chains intended 122 00:10:40,414 --> 00:10:47,980 to reduce the engine's fidelity allowing us to study 123 00:10:47,980 --> 00:10:53,361 We work with 4 is a strain, a wild-type, a victory, a 124 00:10:53,361 --> 00:11:00,227 lot variant affecting polymer status function, red 40, 125 00:11:00,227 --> 00:11:06,966 delete, strain that disturb any year and combination 126 00:11:06,966 --> 00:11:12,693 We compare spontaneous mutation rate in wild tab and mutant 127 00:11:12,693 --> 00:11:24,714 stain in reported gene and we found that the mutation 128 00:11:24,714 --> 00:11:36,962 rate in double mutant strain was significantly higher 129 00:11:38,964 --> 00:11:44,004 These are next slides present a visual representation of the 130 00:11:44,004 --> 00:11:48,766 mutation spectrum we observed in the era 3 reporter gene 131 00:11:48,766 --> 00:11:50,976 across our defined state. 132 00:11:50,976 --> 00:11:55,840 The wildebeest train shows a relatively clean spectrum 133 00:11:55,840 --> 00:12:00,401 with few mutations and this primarily simple based 134 00:12:00,401 --> 00:12:02,955 substitution shown in blue. 135 00:12:02,955 --> 00:12:08,627 And the rate 14 delete chain we observed a notable increase in 136 00:12:08,627 --> 00:12:14,253 mutation frequency particularly interesting is the appearance 137 00:12:14,253 --> 00:12:19,972 of more complex in including deletion and it is shown in red. 138 00:12:19,972 --> 00:12:24,109 Are in the Ref 3 LL. Stain shows a distinct pattern 139 00:12:24,109 --> 00:12:28,694 from both wild type and red 14 delayed with a particular 140 00:12:28,694 --> 00:12:32,952 increase in insertion event highlighted with green. 141 00:12:32,952 --> 00:12:38,847 And in double metering strain, we see the significant increase 142 00:12:38,847 --> 00:12:45,721 in all types of mutation, the striking patterns suggest 143 00:12:45,721 --> 00:12:51,971 linear and TLS don't just work independently they 144 00:12:51,971 --> 00:12:57,977 Previously our lab work on reported in which is provided 145 00:12:57,977 --> 00:13:03,983 as a as a snapshot view of polymerase data metogenesis. 146 00:13:03,983 --> 00:13:08,291 Think of it as just taking a picture through a small 147 00:13:08,291 --> 00:13:21,027 window. However, in our current study we have expanded this 148 00:13:21,027 --> 00:13:33,979 dramatically by employing whole genome sequencing, allowing 149 00:13:36,982 --> 00:13:41,467 And before moving into the methodology of our experiment, 150 00:13:41,467 --> 00:13:49,218 I would like to highlight some important primary steps 151 00:13:49,218 --> 00:13:56,969 we took over for our mutation accumulation experiment 152 00:13:56,969 --> 00:14:01,595 To establish a solid baseline, we begin by analyzing data 153 00:14:01,595 --> 00:14:05,978 from previous studies that You use the reporter team. 154 00:14:05,978 --> 00:14:11,798 These analysis allowed us to calculate both the passes 155 00:14:11,798 --> 00:14:20,152 number and the expected mutation rate for each strain like for 156 00:14:20,152 --> 00:14:27,966 a 500 mutation accumulation rate 3 elap needed 160 passes 157 00:14:27,966 --> 00:14:33,972 But, we went through up to 100 passes. 158 00:14:33,972 --> 00:14:35,902 And now let's wipe through our experimental approach. 159 00:14:35,902 --> 00:14:37,976 We designed a comprehensive mutation accumulation study. 160 00:14:37,976 --> 00:14:43,972 Our experimental workflow include 1st reconstructed 161 00:14:43,972 --> 00:14:50,612 and verified DC string using PCR and sequencing and then 162 00:14:50,612 --> 00:14:57,963 strings were passes for mutation accumulation for 100 passes. 163 00:14:57,963 --> 00:15:03,444 We were start passing with single colonies from 164 00:15:03,444 --> 00:15:09,975 Omojegas deployed. Taking 2 single colonies every 48 h. 165 00:15:09,975 --> 00:15:15,036 Freezing samples at key time points. We also checked from 166 00:15:15,036 --> 00:15:20,318 time to time by markers. And did whole genome sequencing at 167 00:15:20,318 --> 00:15:25,958 18 and 50 passes to check the status of mutation accumulation. 168 00:15:25,958 --> 00:15:31,993 And finally, at 100 passes, we did whole genome sequencing 169 00:15:31,993 --> 00:15:39,895 by Let me know and over sake with high coverage sequencing 170 00:15:39,895 --> 00:15:46,979 rigorous mutation calling using mover pipeline then 171 00:15:46,979 --> 00:15:50,996 And this design allowed us to track how meditation 172 00:15:50,996 --> 00:15:55,778 accumulation over time and determine whether the 173 00:15:55,778 --> 00:16:00,959 combination of this pathway defects lead to affects 174 00:16:00,959 --> 00:16:05,649 Now let's move on to the result of our study on this 175 00:16:05,649 --> 00:16:10,969 slide. We are analyzing the mutation rate of forest teams. 176 00:16:10,969 --> 00:16:15,426 The wild-type, the red 14 delete, rate 3 LF and 177 00:16:15,426 --> 00:16:19,978 double METEN, are denoting by different colors. 178 00:16:19,978 --> 00:16:25,204 The bar graph shows the mutation rates for all mutations and 179 00:16:25,204 --> 00:16:30,589 substitution across this state. And it shows both 180 00:16:30,589 --> 00:16:36,962 the red 14 and double-mutin, exhibit higher mutation rate 181 00:16:36,962 --> 00:16:41,366 And this suggests a significant increase in mutation 182 00:16:41,366 --> 00:16:45,571 rate. When both India is disrupted and polymerize 183 00:16:45,571 --> 00:16:47,973 status function is altered. 184 00:16:47,973 --> 00:16:52,667 And in the table below we can see more detail the wild type 185 00:16:52,667 --> 00:17:05,318 chain had a baseline mutation rate of point 2, a mutation 186 00:17:05,318 --> 00:17:17,970 per deca based part generation and in comparison to rate 187 00:17:22,975 --> 00:17:28,981 alemitians significantly delivers mutation rates. 188 00:17:28,981 --> 00:17:33,867 And this slide shows a detailed comparison of mutation rates 189 00:17:33,867 --> 00:17:42,418 of various types of mutation including single-based 190 00:17:42,418 --> 00:17:50,969 perilation and instruction and complex mutation on 191 00:17:50,969 --> 00:17:55,748 SPEAKER. One types, specifically dramatic increase in complex 192 00:17:55,748 --> 00:17:59,978 mutation in double metal, strain indicated by purple. 193 00:17:59,978 --> 00:18:05,031 And this is the most interesting part of our result and tell 194 00:18:05,031 --> 00:18:09,113 us how polymerase data, particularly in a repair 195 00:18:09,113 --> 00:18:13,959 deficacy background, contributes to mutation complexity. 196 00:18:13,959 --> 00:18:15,917 And these slides summarize that. D types of complex mutation 197 00:18:15,917 --> 00:18:16,962 observed acro's define a chain. 198 00:18:16,962 --> 00:18:21,897 We categorize complex mutation into tendon base pair, 199 00:18:21,897 --> 00:18:27,344 double disk per substitution, bifurcated double base pairs 200 00:18:27,344 --> 00:18:32,978 of a state issue and here been extension, local conversion. 201 00:18:32,978 --> 00:18:38,034 And reciprocal repeat expansion contraction. And that table 202 00:18:38,034 --> 00:18:49,119 highlight the counts and mutation ratio for each 203 00:18:49,119 --> 00:19:02,974 straight, notably the double maintain, strain shows a sharp 204 00:19:02,974 --> 00:19:08,552 And this significant rise suggests that both RAT 14 205 00:19:08,552 --> 00:19:14,637 and polymerase data mutation are critically Critical in 206 00:19:14,637 --> 00:19:17,956 preventing, complex mutation. 207 00:19:17,956 --> 00:19:22,612 Now let me summarize the result. Our whole genome analysis 208 00:19:22,612 --> 00:19:28,441 revealed for critical insight resource. First, st mutation 209 00:19:28,441 --> 00:19:34,973 rate, whole genome sequencing of sacrifices services strain with 210 00:19:34,973 --> 00:19:40,840 We will increase mutation rate compared to the wild type. 211 00:19:40,840 --> 00:19:46,450 Next specific mutation that deletion of red 14 laid to 212 00:19:46,450 --> 00:19:51,957 elevates best substitution mutation while rape 3 LL. 213 00:19:51,957 --> 00:19:56,432 Ferant increase complex mutation rate. Next, synergistic 214 00:19:56,432 --> 00:20:03,978 interaction, the combination of rate 14 delete and rate 215 00:20:03,978 --> 00:20:10,976 3 11 showed the highest meditation rate across all 216 00:20:10,976 --> 00:20:15,159 Lastly, complex mutation. Complex mutation particularly 217 00:20:15,159 --> 00:20:19,951 tendon base pair substitutes and bifurcate double-based pairs. 218 00:20:19,951 --> 00:20:22,220 Ser vice, Titian, were significantly 219 00:20:22,220 --> 00:20:27,416 e levated in double maintenance 220 00:20:27,416 --> 00:20:36,968 chain. And this comprehensive picture suggests a crucial 221 00:20:36,968 --> 00:20:39,191 And our this whole genome and TLS pathways in maintaining 222 00:20:39,191 --> 00:20:41,674 genome stability. And our this whole genome analysis across 100 223 00:20:41,674 --> 00:20:42,974 passes serves as a foundational. 224 00:20:42,974 --> 00:20:44,212 And our this whole genome analysis across 100 passives 225 00:20:44,212 --> 00:20:45,327 serves as a foundational study for understanding 226 00:20:45,327 --> 00:20:45,977 polymerstitis meta genesis. 227 00:20:45,977 --> 00:20:50,626 Provide a available reference point for future 228 00:20:50,626 --> 00:20:52,951 research in this feed. 229 00:20:52,951 --> 00:20:58,412 Now I am highlighting the key implications of our findings and 230 00:20:58,412 --> 00:21:03,962 where we are headed next. Our study revealed 3 major insights. 231 00:21:03,962 --> 00:21:08,267 First, st INNER and TLS pathways. Don't just operate 232 00:21:08,267 --> 00:21:12,374 independently, they work together as a partner in 233 00:21:12,374 --> 00:21:14,973 maintaining genomic stability. 234 00:21:14,973 --> 00:21:20,031 Second, when, at 14 is lost, polymerase data becomes more 235 00:21:20,031 --> 00:21:24,794 error prone, suggesting that 14 helps keep polymerase 236 00:21:24,794 --> 00:21:26,952 data activity in check. 237 00:21:26,952 --> 00:21:33,065 Part the synthetic effect we observe between this pathway 238 00:21:33,065 --> 00:21:39,361 have. Important implication for understanding metrogenesis 239 00:21:39,361 --> 00:21:41,967 and cancer development. 240 00:21:41,967 --> 00:21:46,263 Looking ahead, we plan to challenge this pathways 241 00:21:46,263 --> 00:21:51,584 with exogenous DNA damage to further under their cooperative 242 00:21:51,584 --> 00:21:53,979 role in genome production. 243 00:21:53,979 --> 00:21:59,394 And as we look to the future, my resource aspiration extend 244 00:21:59,394 --> 00:22:10,684 beyond art. Polymerance data though classified as a low 245 00:22:10,684 --> 00:22:21,973 fidelity laser studied member compared to other fellow 246 00:22:21,973 --> 00:22:25,426 And with humans inevitable journey into the space 247 00:22:25,426 --> 00:22:29,469 exploration, understanding how these essential 248 00:22:29,469 --> 00:22:33,952 polymers function in space the environment becomes 249 00:22:33,952 --> 00:22:38,088 Just as Mark Whitney's the Martian had added to 250 00:22:38,088 --> 00:22:43,973 Survivor on Mars. Our DNA European mechanism including 251 00:22:43,973 --> 00:22:49,968 polymerase data will also need to function effectively 252 00:22:49,968 --> 00:22:54,548 And before I conclude, I would like to acknowledge 253 00:22:54,548 --> 00:22:58,710 past my deepest thanks to our DNA replication 254 00:22:58,710 --> 00:23:03,982 viability group at Anais, particularly my supervisor Dr. 255 00:23:03,982 --> 00:23:08,376 Tom Kankill our former lab member our number cities for 256 00:23:08,376 --> 00:23:13,048 Harm mentorship special thanks to Scott Lohan for sequence 257 00:23:13,048 --> 00:23:17,963 statical analysis and supporting me throughout the research. 258 00:23:17,963 --> 00:23:22,782 Thank you to all. My lab mates, especially Sarah 259 00:23:22,782 --> 00:23:30,614 Mars, I'm also grateful to our collaborators at AIDS 260 00:23:30,614 --> 00:23:39,951 especially Adam Barkwter, the UNC Chapel Hill high throughout 261 00:23:39,951 --> 00:23:45,624 Former members whose foundational work helped shape 262 00:23:45,624 --> 00:23:51,814 this project and thank you all for your attention. I am 263 00:23:51,814 --> 00:23:54,966 happy to take now question. 264 00:23:54,966 --> 00:23:59,574 Thank you very much for, for the wonderful presentation, 265 00:23:59,574 --> 00:24:04,482 for the audience, if you have any questions, please type in 266 00:24:04,482 --> 00:24:06,978 the, in the, in the chat box. 267 00:24:06,978 --> 00:24:10,264 Also the panelists, if you have any questions, so 268 00:24:10,264 --> 00:24:13,952 please. Come off mute and and ask a question directly. 269 00:24:13,952 --> 00:24:19,149 Oh, we already have one question from, Bruce Temple. What, do 270 00:24:19,149 --> 00:24:21,960 you think that ARE is acting on? 271 00:24:21,960 --> 00:24:28,019 And here is the base devastation is, in, based 272 00:24:28,019 --> 00:24:31,970 substitution is increased in. 273 00:24:31,970 --> 00:24:39,978 Yeah. 274 00:24:39,978 --> 00:24:48,281 And you collaborate a little on how the. In the manian cells, 275 00:24:48,281 --> 00:24:51,956 how TLS cooperates with N. 276 00:24:51,956 --> 00:24:55,960 Sorry, can you? He's taking the question. 277 00:24:55,960 --> 00:25:01,459 If you if you could elaborate on how TLS and N. 278 00:25:01,459 --> 00:25:04,969 Cooperate in mammalian cells. 279 00:25:04,969 --> 00:25:05,970 We checking is. 280 00:25:05,970 --> 00:25:09,974 Human cells. 281 00:25:09,974 --> 00:25:17,982 We checked in in ESL, but we did not take in in, mammalian cell. 282 00:25:17,982 --> 00:25:21,953 We will take it here. 283 00:25:21,953 --> 00:25:23,896 There's 1 more question in the chat. From Wolf Dietrich 284 00:25:23,896 --> 00:25:24,956 here. Very nice presentation. 285 00:25:24,956 --> 00:25:32,964 Did you have a chance to analyze structural variations? 286 00:25:32,964 --> 00:25:37,969 No, I yet not. 287 00:25:37,969 --> 00:25:43,675 I would think to, Analyzed that's that 288 00:25:43,675 --> 00:25:46,978 to have a. Thank you. 289 00:25:46,978 --> 00:25:51,330 Were spontaneous mutations, I gather. I guess it 290 00:25:51,330 --> 00:25:55,954 would. Bruce, was getting at is there any pattern? 291 00:25:55,954 --> 00:26:04,796 In the mutations that you found. That might implicate 292 00:26:04,796 --> 00:26:12,971 some sort of DNA damage, spontaneous DNA damage. 293 00:26:12,971 --> 00:26:13,972 And you're not. 294 00:26:13,972 --> 00:26:18,977 Oh, sorry. 295 00:26:18,977 --> 00:26:19,978 And, and, repeat the equipment. 296 00:26:19,978 --> 00:26:23,951 Okay. And I think was elaborating on what Bruce 297 00:26:23,951 --> 00:26:28,153 might have meant about what lesions, specifically 298 00:26:28,153 --> 00:26:29,954 asking which damage. 299 00:26:29,954 --> 00:26:30,955 We are a second. 300 00:26:30,955 --> 00:26:38,469 In, future, we are taking, the cis partin and UV radiation 301 00:26:38,469 --> 00:26:41,966 we are are trying to test. 302 00:26:41,966 --> 00:26:47,972 Okay. 303 00:26:47,972 --> 00:26:48,973 Okay. 304 00:26:48,973 --> 00:26:50,152 Well, what you have shown is spontaneous, spontaneous 305 00:26:50,152 --> 00:26:51,323 mutation. Do you have any idea what might be causing 306 00:26:51,323 --> 00:26:51,976 these spontaneous mutations? 307 00:26:51,976 --> 00:26:52,977 Oh. 308 00:26:52,977 --> 00:27:04,956 So such as base. Lots of basis or whatever. 309 00:27:04,956 --> 00:27:10,962 I will think about it. 310 00:27:10,962 --> 00:27:17,969 As continuous. 311 00:27:17,969 --> 00:27:19,987 Okay, I think perhaps in interest of time 312 00:27:19,987 --> 00:27:20,972 we need to move on. 313 00:27:20,972 --> 00:27:22,974 There's 1 more question there. 314 00:27:22,974 --> 00:27:27,979 Oh, okay, sorry. 315 00:27:27,979 --> 00:27:28,980 Yeah, too. 316 00:27:28,980 --> 00:27:34,132 Actually, 2 now. Do you think? Do you think that the 317 00:27:34,132 --> 00:27:39,451 it's riff 3, the, the, the, will affect upcoming DNTP 318 00:27:39,451 --> 00:27:41,960 or DNA binding affinity. 319 00:27:41,960 --> 00:27:47,966 I think. 320 00:27:47,966 --> 00:27:52,425 I have to know these. At this moment I did 321 00:27:52,425 --> 00:27:54,973 not know, I will think. 322 00:27:54,973 --> 00:27:59,397 Okay, and then, Nila Mahashi wants to know why the L. 9 323 00:27:59,397 --> 00:28:03,982 7 9 if shows lower mutation rates compared to wild type. 324 00:28:03,982 --> 00:28:06,951 But this has increased in double mutants. 325 00:28:06,951 --> 00:28:11,956 Yeah, this is the. 326 00:28:11,956 --> 00:28:19,375 Ill-, web 3 is the, actually low militant. And what in the when 327 00:28:19,375 --> 00:28:28,456 the red 14 is a Is, is it, sleep as some, best serviceitation 328 00:28:28,456 --> 00:28:37,982 and make a gap and this gap is a are not, in normal replication 329 00:28:37,982 --> 00:28:45,065 And what this the wild step petition is. 330 00:28:45,065 --> 00:28:48,960 More than the others. 331 00:28:48,960 --> 00:28:52,326 Alright, so for the sake of time, we have to move 332 00:28:52,326 --> 00:28:55,967 on and to, to the next, to the second speaker today. 333 00:28:55,967 --> 00:29:00,609 Doctor, you can stop sharing and, Dr. Pavani, you can 334 00:29:00,609 --> 00:29:02,974 start sharing your slides. 335 00:29:02,974 --> 00:29:05,541 So I will introduce Dr. Pavani, who is a molecular 336 00:29:05,541 --> 00:29:08,287 biologist and dedicate to elucidating complexities of 337 00:29:08,287 --> 00:29:10,982 a genome integrity with a focus on DNA replication. 338 00:29:10,982 --> 00:29:14,887 Repair and the molecular mechanism underlying various 339 00:29:14,887 --> 00:29:24,429 diseases. During his doctoral study, Dr. Pavani completed 340 00:29:24,429 --> 00:29:33,972 the internet internship at the University of attendee at 341 00:29:33,972 --> 00:29:35,461 His PhD work results in several publications and drug discovery. 342 00:29:35,461 --> 00:29:36,975 His PhD work results in several publications and drug discovery. 343 00:29:36,975 --> 00:29:41,665 His PhD work results in several publications and already 344 00:29:41,665 --> 00:29:46,580 earned him the Outstanding Physics Award in the University 345 00:29:46,580 --> 00:29:48,953 of South, It was on 19, Dr. 346 00:29:48,953 --> 00:29:52,957 As a postdoctoral fellow under the mentorship of 347 00:29:52,957 --> 00:29:54,959 Dr. Andrea. On his way. 348 00:29:54,959 --> 00:29:58,864 His research has a significant advance the understanding of 349 00:29:58,864 --> 00:30:04,769 genome instability in human cells and notably he discovered 350 00:30:04,769 --> 00:30:10,975 that the loss of the XL one is synthetically lethal with BRCA 351 00:30:10,975 --> 00:30:14,010 Additionally, Dr. Pavani conducted a highest resolution 352 00:30:14,010 --> 00:30:16,751 analysis of the formation and the fate of collab, 353 00:30:16,751 --> 00:30:17,982 the replication fork. 354 00:30:17,982 --> 00:30:22,047 Revealing that such events can lead to both single, single, 355 00:30:22,047 --> 00:30:28,159 and, and, and, the, the, the, the, You demonstrated that the 356 00:30:28,159 --> 00:30:33,965 magazine involved in repairing this solution differ from 357 00:30:33,965 --> 00:30:40,972 Double stream breaks. So without further ado, Dr. 358 00:30:40,972 --> 00:30:41,973 Yes, we can. 359 00:30:41,973 --> 00:30:43,989 Hi, can you hear me? Is everything? Yeah, nice. 360 00:30:43,989 --> 00:30:44,976 So, I'm Rafael Pavani. 361 00:30:44,976 --> 00:30:49,733 I'm a postdoc at Indonesian's Vi's lab and I want 1st 362 00:30:49,733 --> 00:30:53,951 to thank Karen and Shozang for the invitation. 363 00:30:53,951 --> 00:30:57,891 That's so I can present my work for you guys today. I'm going 364 00:30:57,891 --> 00:31:01,807 to talk about a specific type of DNA devil strand break that 365 00:31:01,807 --> 00:31:03,961 is coupled with replication for. 366 00:31:03,961 --> 00:31:08,825 So, different types of Devil Strand break can happen inside 367 00:31:08,825 --> 00:31:13,151 a cell. And, the, most well -studied one, we'll call 368 00:31:13,151 --> 00:31:17,975 here canonical grades outside the context of replication. 369 00:31:17,975 --> 00:31:21,722 And these breaks are usually produced by external factors 370 00:31:21,722 --> 00:31:25,305 such as ionizing radiation or restriction enzymes. And 371 00:31:25,305 --> 00:31:28,953 they give rise to double -winded double strand breaks. 372 00:31:28,953 --> 00:31:32,349 You can see here left hand and the right hand of a double 373 00:31:32,349 --> 00:31:38,004 strand break. And as I said, this breaks were very well 374 00:31:38,004 --> 00:31:43,968 studied and basically almost everything we know about DNA 375 00:31:43,968 --> 00:31:48,330 However, another type of break can happen and this And it can 376 00:31:48,330 --> 00:31:52,666 happen as replication forks. For example, when a replication 377 00:31:52,666 --> 00:31:54,979 fork fits a lesion called Nick. 378 00:31:54,979 --> 00:31:59,127 Is a single strand break, DNA interruption and one 379 00:31:59,127 --> 00:32:00,952 of the single trends. 380 00:32:00,952 --> 00:32:04,595 And when the fork hits this lesion, which will collapse 381 00:32:04,595 --> 00:32:08,418 the replication fork, you can lose replay some components 382 00:32:08,418 --> 00:32:09,961 such as the CMG helix. 383 00:32:09,961 --> 00:32:13,234 And according to the current models, this will give rise 384 00:32:13,234 --> 00:32:16,430 to a single when the double strength break. So one end 385 00:32:16,430 --> 00:32:17,969 only different from the . 386 00:32:17,969 --> 00:32:21,548 One This is what the current model said, okay? And this 387 00:32:21,548 --> 00:32:31,635 breaks are very important because they are considered the 388 00:32:31,635 --> 00:32:42,960 primary source of the endogenous that was trend breaks actually 389 00:32:42,960 --> 00:32:45,598 they enhance this amount of necks. That will be 390 00:32:45,598 --> 00:32:48,966 unrepaired. So you increase the chances of collapsed forks. 391 00:32:48,966 --> 00:32:51,479 And although this lesions are very important, they are still 392 00:32:51,479 --> 00:32:55,133 not well understood. So, based on the knowledge we know in 393 00:32:55,133 --> 00:32:58,976 the canonical breaks, when you have a double strength break, 394 00:32:58,976 --> 00:33:06,481 Connomial of the Zen joining that is basically like gating 395 00:33:06,481 --> 00:33:13,031 the 2 ends of the double strand break and this can 396 00:33:13,031 --> 00:33:16,961 give rise to small deletions. 397 00:33:16,961 --> 00:33:19,534 But when sales interest phase and move on, they 398 00:33:19,534 --> 00:33:22,416 prefer to use another type of pathway that is called 399 00:33:22,416 --> 00:33:23,968 a model does recombination. 400 00:33:23,968 --> 00:33:27,654 The main difference here is one of the 1st steps. So the 401 00:33:27,654 --> 00:33:34,303 breaks are processed in a process called end reception 402 00:33:34,303 --> 00:33:40,952 where one of the trends is chopped off forming this 3 403 00:33:40,952 --> 00:33:44,058 This process is very accurate and I will talk 404 00:33:44,058 --> 00:33:47,475 in the next lighting more detail about it. But if 405 00:33:47,475 --> 00:33:50,962 this breaks are respected, but HR is not working. 406 00:33:50,962 --> 00:33:53,635 So we have to another, 2 other pathways called 407 00:33:53,635 --> 00:33:56,686 single strand annealing and micromology mediated and 408 00:33:56,686 --> 00:33:59,971 joining they are considered to be alternative pathways. 409 00:33:59,971 --> 00:34:03,506 And, but still they need the 2 wins of a double strength 410 00:34:03,506 --> 00:34:09,904 break. So, I'm all of those recombination pathway 411 00:34:09,904 --> 00:34:16,954 starts when the breaks are respected, as I mentioned, 412 00:34:16,954 --> 00:34:21,678 Once the brakes are respected, a protein co RPA binds to the 413 00:34:21,678 --> 00:34:23,961 single strand DNA overhangs. 414 00:34:23,961 --> 00:34:25,920 And the complex of Braca one, P 2 and BRCA 2, we recruit rad 415 00:34:25,920 --> 00:34:26,964 51 to this single stranded DNA. 416 00:34:26,964 --> 00:34:34,441 Once RAD. 51 is loaded, it will search for the homology 417 00:34:34,441 --> 00:34:37,975 of the sister chromatids. 418 00:34:37,975 --> 00:34:41,173 And, will invade forming this structure. We call D 419 00:34:41,173 --> 00:34:44,788 -loop. And DNA synthesis will start until it reaches the 420 00:34:44,788 --> 00:34:47,952 knowledge with the second end of the break here. 421 00:34:47,952 --> 00:34:51,856 And then the D loop is displaced. The, the, the, and 422 00:34:51,856 --> 00:34:53,958 the repair is now complete. 423 00:34:53,958 --> 00:34:56,877 However, is a little bit different when we think 424 00:34:56,877 --> 00:35:01,827 about breaks associated with replication as the the 425 00:35:01,827 --> 00:35:06,971 literature suggests these are one end that breaks so 426 00:35:06,971 --> 00:35:10,699 And actually the repair of one-ended brakes is not very 427 00:35:10,699 --> 00:35:16,058 well understood. There is a pathway in East called 428 00:35:16,058 --> 00:35:21,952 breaking this replication where the one ended break is 429 00:35:21,952 --> 00:35:25,067 And this, deal loop can synthesize more than 430 00:35:25,067 --> 00:35:28,959 hundreds of KBs and go into the end of the chromosome. 431 00:35:28,959 --> 00:35:32,261 But however this pathway is not a very good idea 432 00:35:32,261 --> 00:35:36,019 to be a primary pathway to repair lesions because it's 433 00:35:36,019 --> 00:35:37,968 a highly mutagenic process. 434 00:35:37,968 --> 00:35:42,028 We've conserved DNA sentences. And, is it still unclear if 435 00:35:42,028 --> 00:35:45,712 this pathway is the primary path we use it to repair 436 00:35:45,712 --> 00:35:47,978 collapsed forks during S phase? 437 00:35:47,978 --> 00:35:51,346 So we thought that there was a need to study this lesion, 438 00:35:51,346 --> 00:35:54,457 especially in mammalian cells, whether we don't have 439 00:35:54,457 --> 00:35:55,953 much knowledge about it. 440 00:35:55,953 --> 00:35:59,828 So we in our lab, we developed a couple of years 441 00:35:59,828 --> 00:36:04,163 ago one technique, that we can, a map double strength 442 00:36:04,163 --> 00:36:05,963 breaks in the genome. 443 00:36:05,963 --> 00:36:09,527 I'm not entering the details because of time, but here's an 444 00:36:09,527 --> 00:36:12,970 example of a double strength break, canonical 1, 2 ends. 445 00:36:12,970 --> 00:36:15,809 And we basically bind adapters to the free ends and after 446 00:36:15,809 --> 00:36:19,332 the processing. We capture this, this, regions where the 447 00:36:19,332 --> 00:36:22,980 break was and then we can have like negative strand reads 448 00:36:22,980 --> 00:36:29,097 So if just for to give you an example here, here 449 00:36:29,097 --> 00:36:34,959 is a let's say this is a double-strand break. 450 00:36:34,959 --> 00:36:38,462 So we'll bind adapter to the left side and to the right side 451 00:36:38,462 --> 00:36:41,966 and the reads will always come from the 5 prime to 3 prime. 452 00:36:41,966 --> 00:36:44,437 So in a canonical double strength break, here is a 453 00:36:44,437 --> 00:36:45,970 case where it's not respected. 454 00:36:45,970 --> 00:36:49,899 You see a sharp peak on the right side with positive 455 00:36:49,899 --> 00:36:54,694 strand reads this. Strange is being sequenced and on 456 00:36:54,694 --> 00:36:59,950 the left side you have the 5 prime to 3 prime by showing 457 00:36:59,950 --> 00:37:03,303 And if the process of reception happens. 3 prime 458 00:37:03,303 --> 00:37:07,121 overhangs are there but during the end sick processing 459 00:37:07,121 --> 00:37:08,959 enzymes will cleave here. 460 00:37:08,959 --> 00:37:13,391 Because we need blunt ends to bind the adapters and the peaks 461 00:37:13,391 --> 00:37:17,968 instead of being sharp they will spread throughout the region. 462 00:37:17,968 --> 00:37:21,649 So this is the pattern of a receptive brick and this 463 00:37:21,649 --> 00:37:24,975 is a pattern of a blunt double strength break. 464 00:37:24,975 --> 00:37:28,542 Then we ask it, okay, can we use this technique to 465 00:37:28,542 --> 00:37:33,885 identify collapsed forks? So, in theory, yes, because here, 466 00:37:33,885 --> 00:37:38,956 for example, is a leading a strand neck, so when a fork 467 00:37:38,956 --> 00:37:42,875 And then, the adapter will bind here to this region and the 468 00:37:42,875 --> 00:37:47,964 reads will come from 5 prime to 3 prime generating a negative 469 00:37:47,964 --> 00:37:52,970 strength reads and if it's respected as I mentioned we will 470 00:37:52,970 --> 00:37:55,288 What about a lagging collapse in a right moving fork? So 471 00:37:55,288 --> 00:37:59,529 here is the same thing. The the fork will hit the lesion, it 472 00:37:59,529 --> 00:38:03,981 will break and then an adapter will bind adapters here to this 473 00:38:03,981 --> 00:38:09,540 So independent of we are if the lesion is on the leading 474 00:38:09,540 --> 00:38:14,451 or the lagging strand we predict that If, fork is 475 00:38:14,451 --> 00:38:19,964 moving to the right, you have negative strength reads. 476 00:38:19,964 --> 00:38:23,945 And if moved to the left, positive trend reads since these 477 00:38:23,945 --> 00:38:27,753 breaks are one-ended breaks. But to distinguish what is 478 00:38:27,753 --> 00:38:31,976 leading and lagging, we need to target uni directional fork. 479 00:38:31,976 --> 00:38:36,036 So we develop a system to study this type of lesions. So 1st 480 00:38:36,036 --> 00:38:39,859 we needed to map the origin of replications in mammalian 481 00:38:39,859 --> 00:38:43,954 cells and we did that by using a technique called EDU SICK. 482 00:38:43,954 --> 00:38:47,958 We are Sells in g 1 and we synchronously release then 483 00:38:47,958 --> 00:38:51,962 in S phase in the presence of EDU and video calling. 484 00:38:51,962 --> 00:38:52,497 Did you is a timid in analog that will be incorporated 485 00:38:52,497 --> 00:38:52,963 into the DNA and a video calling is a polymer. 486 00:38:52,963 --> 00:38:58,167 DNA polymerase inhibitor. So the fido calling treatment 487 00:38:58,167 --> 00:39:05,317 will allow the origins to fire but they don't wellongate so 488 00:39:05,317 --> 00:39:11,982 we can distinguish here this nice peaks and every peak 489 00:39:11,982 --> 00:39:16,033 We select one origin zone. And, here is okay see because another 490 00:39:16,033 --> 00:39:19,957 technique so that the direction of replication is very clear. 491 00:39:19,957 --> 00:39:23,980 So if we put Nick's here in these squares, you will know 492 00:39:23,980 --> 00:39:29,522 exactly that if you put a Nick here on this square, you will 493 00:39:29,522 --> 00:39:34,972 target mainly right moving forks and if you next year will 494 00:39:34,972 --> 00:39:38,309 So if you unique, different strands, you'll be targeting 495 00:39:38,309 --> 00:39:39,977 leading or lagging strands. 496 00:39:39,977 --> 00:39:43,101 And we developed an inducible, system. Just 497 00:39:43,101 --> 00:39:46,951 about DETAIL NICE has 9 NICE to produce this Knicks. 498 00:39:46,951 --> 00:39:50,487 So our experimental setting is basically putting this 499 00:39:50,487 --> 00:39:54,855 Knicks near to the origins that we fire and the forks 500 00:39:54,855 --> 00:39:59,964 will synchronously hit these lesions and then we will analyze 501 00:39:59,964 --> 00:40:04,468 So we get a synchronous sales. We arrest any g 1, add the 502 00:40:04,468 --> 00:40:08,973 docks to induce the niche and then release into S phase. 503 00:40:08,973 --> 00:40:13,518 And when we did that, at the leading breaks, so with the 504 00:40:13,518 --> 00:40:17,982 leading mix will collapse for leading strength breaks. 505 00:40:17,982 --> 00:40:21,180 We see exactly what we predict negative trend reads 506 00:40:21,180 --> 00:40:25,365 in the right moving forks. And positive trend reads 507 00:40:25,365 --> 00:40:29,960 in the left moving forks, which is expected and is very 508 00:40:29,960 --> 00:40:32,857 Showing that's a collapse at 4. We generate one-ended brakes. 509 00:40:32,857 --> 00:40:38,039 However, to our surprise when we did that targeting the lagging 510 00:40:38,039 --> 00:40:42,973 strength, instead of this clear pick one end peak, we see 2 511 00:40:42,973 --> 00:40:48,644 So it seems the lagging brakes generate 2 ended breaks and 512 00:40:48,644 --> 00:40:54,184 this is very surprising because it's different from what 513 00:40:54,184 --> 00:40:56,954 the current model suggests. 514 00:40:56,954 --> 00:41:00,216 And when we zooming at this breaks, as I mentioned, we can 515 00:41:00,216 --> 00:41:01,959 detect with seconds reception. 516 00:41:01,959 --> 00:41:05,896 We can measure the reception length. So reception at 517 00:41:05,896 --> 00:41:09,508 collapsed forks is around 5 KB for both leading 518 00:41:09,508 --> 00:41:10,968 or lagging breaks. 519 00:41:10,968 --> 00:41:14,337 And of course, the 1st question that arrive at is like, where 520 00:41:14,337 --> 00:41:17,523 does the second end of the double strength break at these 521 00:41:17,523 --> 00:41:18,976 lagging breaks come from? 522 00:41:18,976 --> 00:41:23,069 And then we thought about 2 possibilities. One possibility 523 00:41:23,069 --> 00:41:26,950 is you'll have a lagging neck, the fork will collapse. 524 00:41:26,950 --> 00:41:30,184 CMG with the helices will be unloaded and when a 525 00:41:30,184 --> 00:41:33,957 converging fork hits this lesion it will also collapse. 526 00:41:33,957 --> 00:41:37,016 So it's a double collapse and forming this double-ended 527 00:41:37,016 --> 00:41:39,963 double-strand break. I don't have time to show here. 528 00:41:39,963 --> 00:41:43,367 We tested this model. We blocked the conversion for 529 00:41:43,367 --> 00:41:46,879 and we basically see no difference. So we think that 530 00:41:46,879 --> 00:41:48,972 this is not the correct model. 531 00:41:48,972 --> 00:41:53,780 So we are like, okay, there is a second, type of model that 532 00:41:53,780 --> 00:41:55,979 can explain these results. 533 00:41:55,979 --> 00:42:00,422 But this requires that the CMG helix kind of ignores the 534 00:42:00,422 --> 00:42:05,473 lesion on the lagging strand is important to point that the CMG 535 00:42:05,473 --> 00:42:07,958 travels on the leading strand. 536 00:42:07,958 --> 00:42:13,458 So this way, CMG can simply by pass and since the lagging 537 00:42:13,458 --> 00:42:15,966 strands is discontinuous. 538 00:42:15,966 --> 00:42:19,725 It can just continue since the sizing DNA after the break. 539 00:42:19,725 --> 00:42:23,203 And then the replication itself will form this to end 540 00:42:23,203 --> 00:42:24,975 the double strength break. 541 00:42:24,975 --> 00:42:28,445 And can we test that? It's very hard to test this in 542 00:42:28,445 --> 00:42:31,982 Vivo because the lack of reagents for, for the CMGs. 543 00:42:31,982 --> 00:42:35,124 However, our collaborator at Harvard Medical School, 544 00:42:35,124 --> 00:42:38,305 Johannes Walter, He has a very interesting system to 545 00:42:38,305 --> 00:42:39,957 study this type of things. 546 00:42:39,957 --> 00:42:43,082 Because they use channel pose extracts. They are 547 00:42:43,082 --> 00:42:46,117 very active and functional. And they can label 548 00:42:46,117 --> 00:42:47,965 proteins within this tract. 549 00:42:47,965 --> 00:42:51,342 So they hear, for example, label the genes that is part 550 00:42:51,342 --> 00:42:54,628 of the CMG helix and they also label the fan one that 551 00:42:54,628 --> 00:42:57,975 is a protein that travels with the replication forks. 552 00:42:57,975 --> 00:43:02,164 So this way they can measure the CMG progression and also 553 00:43:02,164 --> 00:43:06,170 DNA synthesis. And it's very nice to see that they can 554 00:43:06,170 --> 00:43:09,953 basically see a replication origin in a tetra DNA. 555 00:43:09,953 --> 00:43:10,616 And then we ask then, okay, so can we put Nick's in 556 00:43:10,616 --> 00:43:10,954 the middle of the system? 557 00:43:10,954 --> 00:43:17,487 They say yes, so we asked them to use the NICE CAS 9 DTNA that 558 00:43:17,487 --> 00:43:23,774 we use in our system and targets the leading or the lagging 559 00:43:23,774 --> 00:43:26,970 strand of a replication fork. 560 00:43:26,970 --> 00:43:31,475 So this is the 1st result. When we have a Nick, on the 561 00:43:31,475 --> 00:43:33,977 leading strand here, the CAS. 562 00:43:33,977 --> 00:43:37,756 90 cases in magenta. You can see here the origin just fired 563 00:43:37,756 --> 00:43:44,933 h ere. Can see that CMG progresses 564 00:43:44,933 --> 00:43:56,967 until we reach as the NIC and the chasm and it's stalled 565 00:43:56,967 --> 00:44:00,283 strand it falls off together with the cas line forming 566 00:44:00,283 --> 00:44:03,444 a 1-ended break. However, when they did that in the 567 00:44:03,444 --> 00:44:06,977 lagging strength, they observe a very different outcome. 568 00:44:06,977 --> 00:44:11,353 Instead of the CMG, stalling at the Kas. 9 next site and at 569 00:44:11,353 --> 00:44:15,929 the neck, CMG just bypassed the lesion, ignoring the presence 570 00:44:15,929 --> 00:44:17,955 of the chasm and the neck. 571 00:44:17,955 --> 00:44:22,406 And, this is, will generate this 2 and a double strand 572 00:44:22,406 --> 00:44:24,962 break via replication by pass. 573 00:44:24,962 --> 00:44:29,466 So, and this make a lot of sense with our model. And 574 00:44:29,466 --> 00:44:33,971 not only that, you can see that after CMG bypassed. 575 00:44:33,971 --> 00:44:35,934 There is a gap forming here where, where the, where 576 00:44:35,934 --> 00:44:38,040 the blue collar is. So we interpret this as the breaks 577 00:44:38,040 --> 00:44:38,976 that started to happen. 578 00:44:38,976 --> 00:44:44,669 Are being now respected, consistent with the 2 end break. 579 00:44:44,669 --> 00:44:50,625 We found it in our So basically in this structural part The 580 00:44:50,625 --> 00:44:53,957 leading strand brakes using CAS. 581 00:44:53,957 --> 00:44:56,900 19 A they behave at the current models. Showing a 582 00:44:56,900 --> 00:44:59,963 formation of single when the double strand breaks. 583 00:44:59,963 --> 00:45:03,545 However, if you target the lagging strand and this can 584 00:45:03,545 --> 00:45:07,293 happen maybe in different contexts. Replication can just 585 00:45:07,293 --> 00:45:10,974 bypass this lesion forming a two- double strand break. 586 00:45:10,974 --> 00:45:14,478 So the second part of the talk, we investigated also for 587 00:45:14,478 --> 00:45:17,981 collapse repair. And basically we ask it if we collect. 588 00:45:17,981 --> 00:45:24,259 Samples at later time points. Do we see the brakes being 589 00:45:24,259 --> 00:45:27,958 resolved? And the answer is yes. 590 00:45:27,958 --> 00:45:28,893 Here is wild type cells. Here's a release 6 h. You 591 00:45:28,893 --> 00:45:29,960 see a very nice signal in the legging or leading breaks. 592 00:45:29,960 --> 00:45:37,692 And after, when we collect 12 h time points, we see that 593 00:45:37,692 --> 00:45:41,972 these breaks are largely gone. 594 00:45:41,972 --> 00:45:44,914 And then I said, okay, we can start testing some 595 00:45:44,914 --> 00:45:47,978 factors involved in a double strain brick repair. 596 00:45:47,978 --> 00:45:51,486 And of course, we started with ride 51. So we depleted 597 00:45:51,486 --> 00:45:54,958 route 51 VS IRNA and we perform at our experiment and 598 00:45:54,958 --> 00:45:58,956 very different from what we observed in the wild type cells. 599 00:45:58,956 --> 00:46:03,061 Under route 51 the pollution the brakes are persistent 600 00:46:03,061 --> 00:46:07,129 And not only that, both lagging or leading strengths, 601 00:46:07,129 --> 00:46:10,968 but not only that, the reception increased a lot. 602 00:46:10,968 --> 00:46:14,483 So from 5 KB that I mentioned to you before, now the brakes 603 00:46:14,483 --> 00:46:15,973 show 40 KB of reception. 604 00:46:15,973 --> 00:46:20,588 So The lack of red 51 generates hyper recepted and unrepaired 605 00:46:20,588 --> 00:46:33,057 breaks. And what is very curious is when you look at the leading 606 00:46:33,057 --> 00:46:43,967 breaks that in wild type are mainly one-ended breaks at 607 00:46:43,967 --> 00:46:47,760 I will get back to this in a minute. And of course, we 608 00:46:47,760 --> 00:46:51,975 needed to test if BIR is really a thing at Collapsed Forks. 609 00:46:51,975 --> 00:46:56,396 And when we did, when we depleted factors involved in 610 00:46:56,396 --> 00:46:58,982 BIR, such as POLY TREE or RAD. 611 00:46:58,982 --> 00:47:02,617 52, we don't see any effect in the resolution of the 612 00:47:02,617 --> 00:47:09,291 DNA breaks. So we concluded that It's seeing the BIR is 613 00:47:09,291 --> 00:47:15,966 not the primary pathway to repair collapsed forks, but 614 00:47:15,966 --> 00:47:19,515 So let's go back to this very interesting thing that 615 00:47:19,515 --> 00:47:23,247 we found at the leading one -ended breaks that are now 616 00:47:23,247 --> 00:47:24,975 converted to 200 breaks. 617 00:47:24,975 --> 00:47:28,237 How this happened? So to understand better this we did 618 00:47:28,237 --> 00:47:31,982 another experiment that we call a strength specific RPA chip. 619 00:47:31,982 --> 00:47:35,940 So if you remember, while RPA is the protein that binds 620 00:47:35,940 --> 00:47:37,954 to the single trend of DNA. 621 00:47:37,954 --> 00:47:41,243 And we observe it, mainly our pay bound to one 622 00:47:41,243 --> 00:47:44,961 strength, which is consistent with a 1-ended break. 623 00:47:44,961 --> 00:47:48,757 And what is interesting is we here is the next site. 624 00:47:48,757 --> 00:47:53,821 So before the the next site. We can see RPA here that is 625 00:47:53,821 --> 00:47:58,975 consistent with our pay bound to the single stranded DNA 626 00:47:58,975 --> 00:48:03,205 But we also CRP signal after the break. And around 3 KB and 627 00:48:03,205 --> 00:48:08,351 this is although it is small it's very reproducible when 628 00:48:08,351 --> 00:48:13,957 we interpret this as a strand invasion followed by sharp DNA 629 00:48:13,957 --> 00:48:18,008 If this is really straining, and when we deplete route 51, 630 00:48:18,008 --> 00:48:19,963 this signal should be gone. 631 00:48:19,963 --> 00:48:23,430 And that's exactly what happened. You don't see 632 00:48:23,430 --> 00:48:26,970 now like positive strand reads after the break. 633 00:48:26,970 --> 00:48:30,917 However, you see a new signal emerging on the other side of 634 00:48:30,917 --> 00:48:36,101 the brake. And this is totally consistent with what we observe 635 00:48:36,101 --> 00:48:40,951 at the NSIC, that is a two -ended break being formed with 636 00:48:40,951 --> 00:48:44,076 Offer to end the double strength break. And this is 637 00:48:44,076 --> 00:48:48,130 not only RAD 51 deficient cells, but when we do this 638 00:48:48,130 --> 00:48:52,963 experiment in BRCA one deficient cells or in BRCA 2 deficient 639 00:48:52,963 --> 00:48:57,312 So it's HR deficiencies characteristic. One end breaks 640 00:48:57,312 --> 00:48:59,970 are converted to 2 ended breaks. 641 00:48:59,970 --> 00:49:04,081 And of course, we ask it if the conversion forks are responsible 642 00:49:04,081 --> 00:49:08,031 for this conversion, since the lagging as I mentioned to you, 643 00:49:08,031 --> 00:49:11,982 the conversion forks are not responsible for the 200 breaks. 644 00:49:11,982 --> 00:49:15,796 So the way we did this, we, created additional guides 645 00:49:15,796 --> 00:49:17,954 that we call blocking guides. 646 00:49:17,954 --> 00:49:21,868 Oh, not enter in detail here, but here you can see the, we 647 00:49:21,868 --> 00:49:23,960 put this blocking guides here. 648 00:49:23,960 --> 00:49:27,084 And the idea is that they will collapse the other 649 00:49:27,084 --> 00:49:30,624 replication for the converging fork. So in this way you 650 00:49:30,624 --> 00:49:33,970 don't allow the conversion for to heat this lesion. 651 00:49:33,970 --> 00:49:38,602 So basically we have here if you just use the guide lead to 652 00:49:38,602 --> 00:49:41,062 right that is a leading guide y 653 00:49:41,062 --> 00:49:45,982 ou will see this nice 200 breaks in RPA chip as I showed you. 654 00:49:45,982 --> 00:49:48,160 But what happens if we block th 655 00:49:48,160 --> 00:49:54,318 e converging for? The prediction is if the conversion for is 656 00:49:54,318 --> 00:49:59,963 really involved in creating the double when the double 657 00:49:59,963 --> 00:50:03,499 However, if conversion fork is not responsible for the 658 00:50:03,499 --> 00:50:06,970 2 ended breaks, then the signal should be unchanged. 659 00:50:06,970 --> 00:50:10,828 And, we see a huge decrease in the signal and this is 660 00:50:10,828 --> 00:50:14,501 very reproducible. So it seems that the converting 661 00:50:14,501 --> 00:50:18,982 forks are responsible for the creation of a 2 winded breaks. 662 00:50:18,982 --> 00:50:23,007 Leading sites in HR deficient cells. And of course, when, 663 00:50:23,007 --> 00:50:27,894 so when we, route to one recruitment is efficient, the 664 00:50:27,894 --> 00:50:32,963 single and the double stream breaks are converted to to 665 00:50:32,963 --> 00:50:36,021 And another interesting observation, of course, we 666 00:50:36,021 --> 00:50:40,153 did as a control but show at interesting results when 667 00:50:40,153 --> 00:50:43,974 we blocked the converging forks in at one end it 668 00:50:43,974 --> 00:50:48,097 Here if you zoom in you can see the pattern I showed 669 00:50:48,097 --> 00:50:51,982 before. Now this pattern of RPA increases a lot. 670 00:50:51,982 --> 00:50:54,816 And we interpret this as there was a transition for short track 671 00:50:54,816 --> 00:50:57,591 gene conversion, a little bit of synthesis after the break to 672 00:50:57,591 --> 00:50:58,955 a long track gene conversion. 673 00:50:58,955 --> 00:51:05,184 So that is a A pathway that resembles BIR found in East. 674 00:51:05,184 --> 00:51:10,803 So we think that maybe if you block the conversion 675 00:51:10,803 --> 00:51:15,972 fork, now a process such as BIR is activated. 676 00:51:15,972 --> 00:51:19,718 So the summary of this part is if you have a 1 ended break. 677 00:51:19,718 --> 00:51:25,045 If you have a converging fork available, the you have a 678 00:51:25,045 --> 00:51:30,954 strand invasion, a little bit of DNA synthesis, but it seems 679 00:51:30,954 --> 00:51:32,649 To rescue here, it to emerge and a holiday junction 680 00:51:32,649 --> 00:51:35,863 will be formed and resolved later. However, if you don't 681 00:51:35,863 --> 00:51:38,962 have the conversion fork, now it seems that a pathway 682 00:51:38,962 --> 00:51:46,186 We didn't test that yet. But is very different from 683 00:51:46,186 --> 00:51:53,977 what we observed in the presence of, conversion forks. 684 00:51:53,977 --> 00:51:58,151 And if you have a 1 ended break, but in the case you don't have 685 00:51:58,151 --> 00:52:03,136 a strain invasion because HR is deficient then the conversion 686 00:52:03,136 --> 00:52:07,957 fork will hit this lesion and also break creating a double 687 00:52:07,957 --> 00:52:11,930 And what is nice about this is, in the HR deficient tumors, 688 00:52:11,930 --> 00:52:17,353 we, one of the prominent characteristic of these tumors 689 00:52:17,353 --> 00:52:22,972 is micromology mediated and joining scars that basically 690 00:52:22,972 --> 00:52:27,532 So We think that this happened in HR deficient tumors. And also 691 00:52:27,532 --> 00:52:33,103 we, Broca one as I mentioned is one important protein involved 692 00:52:33,103 --> 00:52:37,954 in a model of recombination and play major roles in 2 693 00:52:37,954 --> 00:52:43,165 One is control of end reception and the other is a route 51 694 00:52:43,165 --> 00:52:47,800 recruitment. So we are interested in knowing if, one 695 00:52:47,800 --> 00:52:52,969 contributes to these 2 steps in HR at Collapse and Forks. 696 00:52:52,969 --> 00:52:55,937 One we did and sick, it was very surprising. We don't 697 00:52:55,937 --> 00:52:59,278 see any difference between a break in wild type cells or in 698 00:52:59,278 --> 00:53:00,977 bracket on the fishing cells. 699 00:53:00,977 --> 00:53:04,173 So since the Braca one in this case of collapsed fork 700 00:53:04,173 --> 00:53:05,982 is dispensable for reception. 701 00:53:05,982 --> 00:53:09,694 And then we said, okay, let's get the same guide 702 00:53:09,694 --> 00:53:13,531 RNA and put in our wild type CAS 9, to generate a 703 00:53:13,531 --> 00:53:15,959 canonical double strand break. 704 00:53:15,959 --> 00:53:20,021 And when this happens, the effect of reception is very 705 00:53:20,021 --> 00:53:24,572 clear. So we conclude that one is important for reception at 706 00:53:24,572 --> 00:53:28,972 canonical doubles and breaks, but not at collapsed forks. 707 00:53:28,972 --> 00:53:32,512 However, if we get the later time points, the the BRCA one 708 00:53:32,512 --> 00:53:35,778 the efficient cells indeed show at the fact he repair 709 00:53:35,778 --> 00:53:38,982 the brakes are persistent different from wild type. 710 00:53:38,982 --> 00:53:42,846 And if Broca one doesn't work in the reception control, 711 00:53:42,846 --> 00:53:44,954 why does cells have a defect? 712 00:53:44,954 --> 00:53:48,494 Maybe Broca one is important for RAD. 51 loading. 713 00:53:48,494 --> 00:53:51,961 And indeed, when we did chipsik, we see a huge. 714 00:53:51,961 --> 00:53:52,621 The fact in Rad. 51 loading in Braca one deficient cells. So 715 00:53:52,621 --> 00:53:52,962 BRCA one is important for RAD. 716 00:53:52,962 --> 00:53:58,990 51 loading at collapsed forks and not reception. A protein 717 00:53:58,990 --> 00:54:05,276 that is very important in this Brca one context is 53 BP one 718 00:54:05,276 --> 00:54:11,981 because it's the pollution risk is embryonic letality in mouse. 719 00:54:11,981 --> 00:54:14,852 Rescue Park inhibitor sensitivity and this is 720 00:54:14,852 --> 00:54:18,018 thought to be achieved because of the restoration 721 00:54:18,018 --> 00:54:19,956 of this end reception defect. 722 00:54:19,956 --> 00:54:23,620 But since that collapsed forks, Braca one doesn't play any 723 00:54:23,620 --> 00:54:28,179 role apparently at reception of it's 50 TB one like what is 724 00:54:28,179 --> 00:54:32,969 the road that's 50 trivial one affects the repair of collapse 725 00:54:32,969 --> 00:54:38,240 51, sorry, 50 TB one, in BRCA on the efficient cells 726 00:54:38,240 --> 00:54:40,977 and we did the same essay. 727 00:54:40,977 --> 00:54:45,471 And now the brakes are largely repaired again. So, to 3 VP 728 00:54:45,471 --> 00:54:47,951 one affects the repair somehow. 729 00:54:47,951 --> 00:54:52,342 But how, right? And then when we did ride 51 chips sick 730 00:54:52,342 --> 00:54:56,532 at this BRCA 150 triple on the fission cells, we see 731 00:54:56,532 --> 00:55:00,964 the restoration of route 51 filaments and recruitment. 732 00:55:00,964 --> 00:55:04,605 So it seems that, 53 BP one acts by counteracting RAD. 733 00:55:04,605 --> 00:55:09,208 51 filament formation at collapsed forks and had nothing 734 00:55:09,208 --> 00:55:13,977 to do with the reception as it was described in canonical 735 00:55:13,977 --> 00:55:16,439 So the take home message of my talk is collapsed 736 00:55:16,439 --> 00:55:19,663 forks can generate one or 200 double friend breaks 737 00:55:19,663 --> 00:55:22,952 depending on whether the Knicks on the leading are 738 00:55:22,952 --> 00:55:25,993 All of this recombination is fundamental for repairing 739 00:55:25,993 --> 00:55:29,119 collapsed forks and it seems that the BIR or long track 740 00:55:29,119 --> 00:55:31,961 gene conversion that is the name they use in the. 741 00:55:31,961 --> 00:55:36,218 Sells seems to be an alternative pathway when converging forks 742 00:55:36,218 --> 00:55:43,538 are blocked. And in the absence of HR, even an icon deleting 743 00:55:43,538 --> 00:55:50,980 a strength that in wild type generates one end at breaks can 744 00:55:50,980 --> 00:55:54,318 Another very interesting thing is reception at Collapsed 745 00:55:54,318 --> 00:55:57,954 Forces, Braca One, Independence, Distinct for Braco One Row. 746 00:55:57,954 --> 00:56:01,851 That canonical breaks. And 53 BP and efficiency risk is route 747 00:56:01,851 --> 00:56:05,281 51 recruitment at Collapsed Forks and this is totally 748 00:56:05,281 --> 00:56:06,963 independent of reception. 749 00:56:06,963 --> 00:56:09,694 We we have we dig more into the mechanism but I don't 750 00:56:09,694 --> 00:56:12,458 have time to show here. So here is our paper that was 751 00:56:12,458 --> 00:56:13,970 published couple months ago. 752 00:56:13,970 --> 00:56:18,488 So I'll invite you to read if you're interested in this 753 00:56:18,488 --> 00:56:22,719 topic. And also there are 2 other groups that reach 754 00:56:22,719 --> 00:56:26,950 somehow similar conclusions but independent of us. 755 00:56:26,950 --> 00:56:29,894 The one is in East and another from Lorraine Simon 756 00:56:29,894 --> 00:56:33,963 to Lab and another one. In Harvard, for Ralph Scholuleb, 757 00:56:33,963 --> 00:56:37,961 here in the median cells, so I invite you to also take 758 00:56:37,961 --> 00:56:41,266 And with that I finished my talk so I really thank 759 00:56:41,266 --> 00:56:44,968 Andre Nissan's bike for all the support and mentorship. 760 00:56:44,968 --> 00:56:48,686 And all the members of the lab, especially Vino Tree 761 00:56:48,686 --> 00:56:51,975 party that it was doing this project with me. 762 00:56:51,975 --> 00:56:54,694 Almost since the beginning and very, very helpful. And 763 00:56:54,694 --> 00:56:57,288 these people highlighted here are the people in the 764 00:56:57,288 --> 00:56:59,983 lab that work it in some experiments in the project. 765 00:56:59,983 --> 00:57:04,662 And thanks our collaborators, Johannes Walter, Raj, 766 00:57:04,662 --> 00:57:06,956 Better Chaka and Wei Wu. 767 00:57:06,956 --> 00:57:10,960 And thanks very much for your attention. 768 00:57:10,960 --> 00:57:11,931 Thank you. I thank you, Rafael. What a wonderful 769 00:57:11,931 --> 00:57:12,962 presentation. And I let's move to the, QA session. 770 00:57:12,962 --> 00:57:21,983 We already have a bunch of questions, flowing in. So the 771 00:57:21,983 --> 00:57:26,976 1st question is, is a from Dr. 772 00:57:26,976 --> 00:57:31,600 Huang to single end in the DSP and double-stranded as we have 773 00:57:31,600 --> 00:57:33,950 different repair efficiencies. 774 00:57:33,950 --> 00:57:38,283 Actually no, because the single one that that was turn breaks 775 00:57:38,283 --> 00:57:40,724 a nd double and the devil's friend 776 00:57:40,724 --> 00:57:44,961 breaks they We think they are repairing the same kinetics. 777 00:57:44,961 --> 00:57:47,251 Our results suggest that so somehow after we see the maximum 778 00:57:47,251 --> 00:57:50,698 intensity 6 h after the release, but they largely disappear 779 00:57:50,698 --> 00:57:53,970 in both leading or lagging after 12 h, but they largely 780 00:57:53,970 --> 00:58:03,980 So we think they are basically repaired with similar kinetics. 781 00:58:03,980 --> 00:58:07,296 Okay, okay, this question from Michael Lifton. In the fork 782 00:58:07,296 --> 00:58:11,050 Brock experiment the fork blocks themselves seem to induce breaks 783 00:58:11,050 --> 00:58:12,955 But they appeared to be 2 ended. 784 00:58:12,955 --> 00:58:19,272 Yep. Right, I think he's talking about this particular slide, 785 00:58:19,272 --> 00:58:24,967 right? And, yes, we target the leading strands, right? 786 00:58:24,967 --> 00:58:26,339 And, yes, we targets the leading strand of the replication 787 00:58:26,339 --> 00:58:30,058 fork 2 with this blocking next strand of the replication 788 00:58:30,058 --> 00:58:33,976 for 2 if this blocking next because we wanted to make sure 789 00:58:33,976 --> 00:58:38,181 And here I need to remind you that this is a HR deficient, 790 00:58:38,181 --> 00:58:41,951 so we expect this 2 ended breaks, right, even here. 791 00:58:41,951 --> 00:58:45,661 But maybe also he's talking here and the explanation for 792 00:58:45,661 --> 00:58:49,149 that, although they target the leading strand of the 793 00:58:49,149 --> 00:58:50,960 replication for Thank you. 794 00:58:50,960 --> 00:58:55,709 We know that the Knicks are not made in all the cells. So for 795 00:58:55,709 --> 00:58:57,967 example, if you have a Nick. 796 00:58:57,967 --> 00:59:02,036 Here, right? If you don't have this initial nick in a cell, but 797 00:59:02,036 --> 00:59:03,973 you have the blocking guides. 798 00:59:03,973 --> 00:59:07,487 Then the fork that will break here will be the 1st fork 799 00:59:07,487 --> 00:59:11,172 another second so this will be a lagging strand break and 800 00:59:11,172 --> 00:59:12,982 not a leading strand break. 801 00:59:12,982 --> 00:59:17,222 So does that make sense? Because not all the Knicks 802 00:59:17,222 --> 00:59:21,018 are efficient. So, 50, we estimate the 50% of 803 00:59:21,018 --> 00:59:22,959 the cells have Knicks. 804 00:59:22,959 --> 00:59:24,391 So some cells we only have the blocking next 805 00:59:24,391 --> 00:59:26,106 but not the initial Nick. And then this fork will be 806 00:59:26,106 --> 00:59:26,963 the one hitting the neck. 807 00:59:26,963 --> 00:59:31,252 But the blocking was very efficient because we see 808 00:59:31,252 --> 00:59:40,769 differences in the 1st break. So there are a lot of cells 809 00:59:40,769 --> 00:59:49,952 that have all of them, but those results when you look 810 00:59:49,952 --> 00:59:53,682 Okay, Michael has a follow-up question. Well, the different 811 00:59:53,682 --> 00:59:57,902 question is, have you looked at the effect of BRCA one 812 00:59:57,902 --> 01:00:01,964 in 53 BP one mutations on resection at DSPs induced 813 01:00:01,964 --> 01:00:05,549 No, we haven't looked at that. It's a very good 814 01:00:05,549 --> 01:00:09,972 idea actually. Just to, we, didn't compare the mutations. 815 01:00:09,972 --> 01:00:12,753 Like canonical that was and breaks with collapsing 816 01:00:12,753 --> 01:00:15,978 forks. But, I think there are other people that did that. 817 01:00:15,978 --> 01:00:19,530 So there is a paper in nature communications. I believe 818 01:00:19,530 --> 01:00:23,048 they did this type of things and they show differences 819 01:00:23,048 --> 01:00:26,956 in the mutation of collapsed forks versus canonical breaks. 820 01:00:26,956 --> 01:00:32,417 I don't remember right now if they specifically showed between 821 01:00:32,417 --> 01:00:37,967 53 VPP and efficiency, BROCOM, and the efficient cells or not. 822 01:00:37,967 --> 01:00:41,342 I need to remind, but some people look at mutations 823 01:00:41,342 --> 01:00:44,565 that collapsed forks or. Canonical double string 824 01:00:44,565 --> 01:00:45,975 breaks but we didn't 825 01:00:45,975 --> 01:00:50,300 Okay, there is another question from Nicolas Hawk. 826 01:00:50,300 --> 01:00:52,982 So, mentioned, very nice talk. 827 01:00:52,982 --> 01:00:58,057 Can you comments on the weather? Depletion for port d 828 01:00:58,057 --> 01:01:03,962 3 or other expected bir lt ggc factor effects the BR like RPA 829 01:01:03,962 --> 01:01:06,963 chip signal beyond brake size. 830 01:01:06,963 --> 01:01:11,529 Yeah, that's a very, very good question. And yes, the answer 831 01:01:11,529 --> 01:01:15,599 is yes. In our supplementary figure, I don't remember 832 01:01:15,599 --> 01:01:19,976 which number, we showed that the if we depleted party 3. 833 01:01:19,976 --> 01:01:24,592 We decrease this, RPA signal after the break. So it seems 834 01:01:24,592 --> 01:01:29,734 the party tree really helps the extension of RPA and the D-loop 835 01:01:29,734 --> 01:01:34,957 extension right and consequently the RPA loading at the brakes. 836 01:01:34,957 --> 01:01:38,847 So yes, this long track gene conversion seems to be 837 01:01:38,847 --> 01:01:42,965 dependent on party tree. I think that's the question. 838 01:01:42,965 --> 01:01:47,371 Okay. Diego Louis Rubiro says very nice presentation. In your 839 01:01:47,371 --> 01:01:54,272 opinion, could tilless enzymes like Real Riv One and Paul Zeta 840 01:01:54,272 --> 01:02:00,950 play a role in converting one -ended DSPs into 2 ended DSPs 841 01:02:00,950 --> 01:02:01,951 Convergence. 842 01:02:01,951 --> 01:02:06,956 I think it might be possible, though we, I don't have a 843 01:02:06,956 --> 01:02:11,961 clear answer for you because The N 6 only captured DA. 844 01:02:11,961 --> 01:02:13,768 Double friend break so My some other things might 845 01:02:13,768 --> 01:02:16,391 happen there but we then we cannot see it So I don't have 846 01:02:16,391 --> 01:02:18,968 an answer for that, but I believe this is a replication 847 01:02:18,968 --> 01:02:25,310 So I think there is a pathway competition at this replication 848 01:02:25,310 --> 01:02:35,135 lesions. So I believe it is very possible that this 849 01:02:35,135 --> 01:02:44,961 protest can play a role, although our results only 850 01:02:44,961 --> 01:02:46,928 If, we didn't play with Rev one. Or was that to make sure 851 01:02:46,928 --> 01:02:47,964 that they contribute somehow. 852 01:02:47,964 --> 01:02:54,971 So it's a thing that we need to test. 853 01:02:54,971 --> 01:02:59,664 Okay, there is another question from Alexandra Nail. Are there 854 01:02:59,664 --> 01:03:07,380 differences in BRCA one post -translational modification 855 01:03:07,380 --> 01:03:14,957 that contribute to this role in reception in canonical 856 01:03:14,957 --> 01:03:18,995 That's also a very good question and I don't know we didn't 857 01:03:18,995 --> 01:03:22,965 explore any post translation or modifications in Brakawa. 858 01:03:22,965 --> 01:03:27,539 We have an hypothesis that if you look at papers that 859 01:03:27,539 --> 01:03:32,975 characterized the proteins that travel with replication forks. 860 01:03:32,975 --> 01:03:37,109 A lot of, proteins such as x, 1, MRE, 11, they travel 861 01:03:37,109 --> 01:03:38,981 with replication forks. 862 01:03:38,981 --> 01:03:42,813 So we think this reception control is independent of Braca 863 01:03:42,813 --> 01:03:46,821 one because the proteins are already there so they are right 864 01:03:46,821 --> 01:03:48,958 away available to do reception. 865 01:03:48,958 --> 01:03:52,994 So you I think Broca the role of Braca one at Canonical Double 866 01:03:52,994 --> 01:03:56,966 Strand breaks is actually the proteins are not there, right? 867 01:03:56,966 --> 01:04:00,370 The brake just happened. So there is a competition to 868 01:04:00,370 --> 01:04:05,036 which pathway to go but when it's coupled with replication 869 01:04:05,036 --> 01:04:08,978 forks a lot of HR proteins are already traveling 870 01:04:08,978 --> 01:04:12,930 So we feel that this is a prior section environment because 871 01:04:12,930 --> 01:04:16,460 it's a pro HR environment since all the proteins are 872 01:04:16,460 --> 01:04:17,953 already concentrated. 873 01:04:17,953 --> 01:04:20,956 In that. 874 01:04:20,956 --> 01:04:24,612 It makes sense. We have one more question in the 875 01:04:24,612 --> 01:04:27,963 chat from Marcela, Hi Rafael, amazing work. 876 01:04:27,963 --> 01:04:31,967 Are you planning to test this technique in TLS knockout cells? 877 01:04:31,967 --> 01:04:36,883 I think it's very similar to Diego, right? Yeah, we 878 01:04:36,883 --> 01:04:42,346 haven't planned to do that, but I think it's a very good 879 01:04:42,346 --> 01:04:44,980 idea since 2 of you asked. 880 01:04:44,980 --> 01:04:48,752 But we are not sure if, as I mentioned, right, using the 881 01:04:48,752 --> 01:04:52,491 techniques we have in the lab and seek, we are not sure 882 01:04:52,491 --> 01:04:55,958 if the depletion of these lesions will be visible. 883 01:04:55,958 --> 01:05:00,414 Right? If something, if there is a consequence. We don't 884 01:05:00,414 --> 01:05:07,836 know if it will be visible in our end sick, but maybe 885 01:05:07,836 --> 01:05:14,977 if you do like amplicon seek or mutation analysis, 886 01:05:14,977 --> 01:05:18,981 Alright, do we have questions from any of the panelists? 887 01:05:18,981 --> 01:05:22,425 I want to comment on these 2 excellent talks and 888 01:05:22,425 --> 01:05:26,047 clearly there people are thinking of combining the 889 01:05:26,047 --> 01:05:29,959 translation synthesis with the, the double scan rate. 890 01:05:29,959 --> 01:05:32,962 So thank you so much. Yes. 891 01:05:32,962 --> 01:05:36,829 Okay, we do have one more question. I think we have 892 01:05:36,829 --> 01:05:40,975 some time for it. Yeah, from Nathan McGill, very sorry 893 01:05:40,975 --> 01:05:42,972 for the mispronunciation. 894 01:05:42,972 --> 01:05:45,648 Could any of the NC Grees be a reverse forked 895 01:05:45,648 --> 01:05:48,978 structure localizing to the reverse branch of the fork? 896 01:05:48,978 --> 01:05:53,774 If a fork re model is the depleted. Smock, smok Al 897 01:05:53,774 --> 01:05:58,954 one, well HTLF, give a different insect read profile. 898 01:05:58,954 --> 01:06:00,956 Perhaps you can read, in the checks. The That's it. 899 01:06:00,956 --> 01:06:05,172 Yeah. These were amazing questions here. Seriously, 900 01:06:05,172 --> 01:06:09,965 amazing questions. And this question totally makes sense. 901 01:06:09,965 --> 01:06:16,678 I am almost asking if Nathan was the reviewer tree, okay, because 902 01:06:16,678 --> 01:06:22,978 they ask that in our manuscripts and We did, HLTF depletion. 903 01:06:22,978 --> 01:06:26,331 So we, we don't think we are identify reverse forks and we 904 01:06:26,331 --> 01:06:27,950 have some reasons for that. 905 01:06:27,950 --> 01:06:32,982 How you are right, the polarity of the signal will be similar. 906 01:06:32,982 --> 01:06:36,959 So we cannot distinguish between a reverse fork. 907 01:06:36,959 --> 01:06:40,388 Or a canon or a doubles can break. However, we 908 01:06:40,388 --> 01:06:43,966 know that route. 51, for example, is important. 909 01:06:43,966 --> 01:06:47,087 For reverse forks to happen is fundamental protein to 910 01:06:47,087 --> 01:06:50,413 reverse replication forks. And instead of decreasing the 911 01:06:50,413 --> 01:06:53,976 signal, actually increase the signal when we deplete route. 912 01:06:53,976 --> 01:06:58,776 51. And the same is applied to HLTF when we do HLTF 913 01:06:58,776 --> 01:07:03,953 efficiency, we don't see any difference in the signal. 914 01:07:03,953 --> 01:07:06,925 So we really don't think and also doesn't explain 915 01:07:06,925 --> 01:07:10,364 why the leading and lagging would be different since the 916 01:07:10,364 --> 01:07:11,961 reverse forks would show. 917 01:07:11,961 --> 01:07:15,045 I think if these were reverse forks, they would show only 918 01:07:15,045 --> 01:07:17,967 one end it breaks and not to this 2 end break button. 919 01:07:17,967 --> 01:07:23,023 So we really think we are looking only at the NA. 920 01:07:23,023 --> 01:07:27,977 Double train breaks here and not reverse forts. 921 01:07:27,977 --> 01:07:31,155 Okay, we have a question from David Orin. Have you tested 922 01:07:31,155 --> 01:07:34,315 the effects of BLM and Vernus, which have been suggested 923 01:07:34,315 --> 01:07:35,951 to have a role in reception? 924 01:07:35,951 --> 01:07:39,761 We haven't tested it was too many things to do it wasn't at 925 01:07:39,761 --> 01:07:43,959 least to you know understand the nucleases But this is for sure. 926 01:07:43,959 --> 01:07:50,633 A future goal to understand the new places that play 927 01:07:50,633 --> 01:07:53,969 a role there at collapse. 928 01:07:53,969 --> 01:07:57,329 Hey, I think, there's no more question. Karen, do 929 01:07:57,329 --> 01:07:58,974 you want to wrap up or? 930 01:07:58,974 --> 01:07:59,975 No, you go ahead. 931 01:07:59,975 --> 01:08:06,299 Okay, so, again, I would like to, thank both of the, presenter 932 01:08:06,299 --> 01:08:08,951 for their wonderful talk. 933 01:08:08,951 --> 01:08:12,883 It's truly appreciated in the from the point of view of 934 01:08:12,883 --> 01:08:14,957 DNA repaired interest group. 935 01:08:14,957 --> 01:08:17,669 And again, we are looking forward for more 936 01:08:17,669 --> 01:08:20,963 interactions and more of your wonderful art, work. 937 01:08:20,963 --> 01:08:26,969 Thank you very much.