The influence of chromatin in DNA-RNA hybrid metabolism Juan Carlos Martínez Cañas Tesis doctoral Universidad de Sevilla 2020 2 3 4 INDEX Introduction............................................................................................................ 21 1. Sources of DNA damage. ............................................................................... 23 1.1. Replication as a source of genome instability. ........................................ 25 1.2. Transcription as a source of genome instability. ..................................... 29 1.3. Transcription-replication conflicts as a source of genome instability. ...... 31 2. R loops ........................................................................................................... 34 2.1. Factors involved in R loop formation. ...................................................... 36 2.2. The state of the RNA. .............................................................................. 37 2.3. The state of the DNA. .............................................................................. 37 2.4. R loops and genome instability. .............................................................. 39 2.5. Detection of R loops throughout the genome. ......................................... 42 3. Chromatin. ...................................................................................................... 44 3.1. Histone post-translational modifications. ................................................. 45 3.2. Chromatin remodelers. ............................................................................ 48 3.3. Chromatin and DSB repair. ..................................................................... 49 3.4. Chromatin and R loops. .......................................................................... 50 Objectives.............................................................................................................. 55 Results…………………………………………………………………………………….57 Chapter I-Cytosine deamination as a tool to determine DNA-RNA hybrid length, frequency and distribution. .................................................................................... 59 1. H3∆1-28, H3K9-23A and H4K31Q histone mutations that lead to DNA-RNA hybrids do not lead to genome instability unless AID is overexpressed. ............ 61 2. H3K9-23A mutation does not impair replication fork progression and suppresses the replication defects of hpr1∆. ..................................................... 64 3. R loop formation in histone mutants is not related to an altered pattern of nucleosome positioning. .................................................................................... 67 5 4. R loops are similar in size regardless of whether they induce genome instability or not. ................................................................................................. 70 5. Similar R loop length in several R loop-accumulating mutants. .................. 75 6. R loops are formed at a very low frequency. .............................................. 77 7. Bisulfite-qPCR as an alternative method to DRIP ....................................... 79 8. Exploring the use of AID* deaminase in vivo to detect and map R loops. .. 82 9. AID* overexpression induces mutations genome-wide preferentially in large genes and independently of their GC content and expression levels. ............... 86 Chapter II- Effect of the loss of chromatin remodelers and histone modifiers in DNA-RNA hybrid metabolism. ............................................................................... 93 1. Chromatin remodeling mutants do not enhance S9.6 signal. ..................... 95 2. Chromatin remodeling mutants do neither lead to genome instability nor to enhanced sensitivity to AID overexpression ...................................................... 96 3. Rtt109 loss leads to the accumulation of DNA-RNA hybrids. ................... 100 4. Effect of AID* overexpression in rtt109∆ mutant. ...................................... 104 5. Rtt109 prevents DNA-RNA hybrid accumulation through its catalytic activity. ……………………………………………………………………………………106 6. DNA-RNA hybrids accumulate in rtt109∆ cells in all cell cycle phases. .... 108 7. Histone H3 deposition during S phase is not decreased in rtt109∆ mutant ……………………………………………………………………………………109 8. DNA-RNA accumulation in rtt109∆ is independent of the H3K56 acetylation state. ................................................................................................................ 111 9. Mutation of H3K14 and H3K23 (Rtt109 targets) to alanine increases DNA- RNA hybrid accumulation. ............................................................................... 113 10. The genotoxic sensitivity of rtt109∆ mutant is not dependent on DNA-RNA hybrids. ............................................................................................................ 114 6 11. The Rad52 foci accumulation phenotype of rtt109∆ mutant is partially caused by DNA-RNA hybrids. .......................................................................... 115 12. H3K9A mutation is able to suppress the genetic instability of hpr1∆. .... 118 13. The SCR defect of rtt109∆ is not caused by DNA-RNA hybrid accumulation. ................................................................................................... 118 14. rtt109∆ leads to the spontaneous accumulation of DSBs that are not induced by the presence of DNA-RNA hybrids. ............................................... 126 15. Deletion of HPR1 gene in an rtt109∆ background leads to a synergistic effect on genotoxic sensitivity. ......................................................................... 127 Discussion……………………………………………………………………………….129 1. New insights from histone mutants leading to DNA-RNA hybrid accumulation without genetic instability. .................................................................................... 131 2. Setting up the bisulfite deamination assay in yeast reveals that DNA-RNA hybrid length is not a relevant factor for the generation of genetic instability. ..... 132 3. Detection and mapping DNA-RNA hybrids by newly developed S9.6- independent methodologies based on cytosine deamination in vitro and in vivo. 134 4. A model to explain the different phenotypes of R loop-accumulating histone and hpr1∆ mutants. ............................................................................................. 138 5. R loop or related phenotypes not detected in yeast chromatin remodeler mutants. .............................................................................................................. 140 6. A screening among histone modifiers identifies Rtt109 as a new factor leading to DNA-RNA hybrid accumulation. ...................................................................... 141 7. The lack of acetylation of H3K14 and H3K23 contribute to the DNA-RNA hybrid accumulation in rtt109∆. ........................................................................... 142 8. Defective SCR contributes to the DNA-RNA hybrid accumulation of rtt109∆. ………………………………………………………………………………………143 9. A model to explain the DNA-RNA hybrid accumulation of rtt109∆. .............. 146 Conclusions ......................................................................................................... 151 7 Materials and Methods…………………………………………………………………153 Yeast strains, plasmids, primers and antibodies. ............................................. 155 Media used in this study. ................................................................................. 164 Techniques ...................................................................................................... 164 Bacterial transformation. ....................................................................................................... 164 Yeast transformation and gene disruption. ............................................................................ 164 Drop assay. ............................................................................................................................. 164 Plasmid loss assay. ................................................................................................................. 165 Recombination assay. ............................................................................................................. 165 Loss of heterozygocity (LOH) at MAT locus. ........................................................................... 165 Cell viability assay. .................................................................................................................. 166 AID*-induced mutation assay. ................................................................................................ 166 Bioinformatic analyses of AID*-induced mutations. .............................................................. 166 Rad52 foci detection. .............................................................................................................. 167 Cell culture synchronization and FACS analysis. ..................................................................... 167 Protein extraction and western blot. ...................................................................................... 168 Chromosome spreads. ............................................................................................................ 169 CTAB DNA extraction. ............................................................................................................. 169 2D-gel