The influence of chromatin in DNA-RNA hybrid metabolism

The influence of chromatin in DNA-RNA hybrid metabolism Juan Carlos Martínez Cañas Tesis doctoral Universidad de Sevilla 2020 2 Tesis doctoral: Juan Carlos Martínez Cañas 4 Tesis doctoral: Juan Carlos Martínez Cañas Tesis doctoral: Juan Carlos Martínez Cañas 5 Index RESUMEN ............................................................................................................................... 15 INTRODUCTION ...................................................................................................................... 21 DNA DAMAGE RESPONSE. .................................................................................................................. 23 DNA damage repair .................................................................................................................. 25 Recognition of DSBs prior to be repaired .................................................................................. 25 DNA damage checkpoint .......................................................................................................... 30 R LOOPS. .......................................................................................................................................... 30 Transcription as a source of genome instability. ...................................................................... 32 Replication as a source of genome instability. ......................................................................... 32 Detection of R loops among the genome. ................................................................................ 34 R loops and chromatin. ............................................................................................................. 35 CHROMATIN REMODELERS .................................................................................................................. 39 SWI/SNF family. ........................................................................................................................ 39 ISWI family. ............................................................................................................................... 39 CHD family. ............................................................................................................................... 40 INO80 family. ............................................................................................................................ 40 POST-TRANSLATIONAL MODIFICATIONS OF HISTONES ............................................................................... 40 OBJECTIVES ............................................................................................................................ 43 RESULTS ................................................................................................................................. 47 CHAPTER I- CYTOSINE DEAMINATION AS A TOOL TO DETERMINE DNA-RNA HYBRID LENGTH, FREQUENCY AND DISTRIBUTION. .................................................................................................................................. 49 H-28, H3K9-23A and H4K31Q histone mutations that lead to DNA-RNA hybrids do not lead to genome instability unless AID is overexpressed. .......................................................... 51 H3K9-23A mutation does not impair replication fork progression and suppresses the replication defects of hpr .................................................................................................... 54 Nucleosome positioning is altered in H-28 and H3K9-23A ................................................. 57 R loops are similar in size regardless of whether they induce genome instability or not. ........ 59 Similar R loop length in several R loop accumulating mutants. .............................................. 64 R loops are formed at a very low frequency ............................................................................. 66 6 Tesis doctoral: Juan Carlos Martínez Cañas Bisulfite-qPCR as an alternative method to DRIP ..................................................................... 68 AID* overexpression does not affect viability of cells but induces mutations. ......................... 71 AID* overexpression induces mutations genome-wide. ........................................................... 73 AID* induced mutations occur preferentially in large genes independently of their GC content and expression levels. ............................................................................................................... 75 Histone mutants enhance R loop dependent AID-induced mutations in CAN1 gene. .............. 77 CHAPTER IIEFFECT OF THE LOSS OF CHROMATIN REMODELERS AND HISTONE MODIFIERS IN DNA-RNA HYBRID METABOLISM .................................................................................................................................... 79 Chromatin remodeling mutants do not enhanceS9.6 signal. ................................................... 81 Chromatin remodeling mutants do not lead to R loop-dependent genome instability but they enhance the sensitivity to AID overexpression. ........................................................................ 82 Chromatin remodeling mutants do not have DNA-RNA hybrid-dependent genome instability .................................................................................................................................................. 86 Rtt109 loss leads to the accumulation of DNA-RNA hybrids. ................................................... 89 Rtt109 prevents DNA-RNA hybrid accumulation through its catalytic activity. ....................... 93 R loops accumulate in rtt cells in all cell ccle phases ..................................................... 95 Histone H deposition during S phase is not decreased in rtt mutant ............................. 97 The R loop accumulation phenotpe of rtt mutant is independent of the HK acetylation state. ...................................................................................................................... 98 Mutation of H3K14 and H3K23 (Rtt109 targets) to alanine increases DNA-RNA hybrid accumulation. ........................................................................................................................... 99 The genotoic sensitiit of rtt mutant is not dependent on DNA-RNA hybrids. ........... 100 The Rad foci accumulation phenotpe of rtt mutant is partiall caused b DNA-RNA hybrids. ................................................................................................................................... 100 The SCE defect of rtt is not caused b DNA-RNA hybrid accumulation. ......................... 105 rtt leads to the spontaneous accumulation of DSBs that are not induced b the presence of DNA-RNA hybrids. ............................................................................................................... 111 Deletion of HPR gene in an rtt background leads to a snergistic effect on genotoic sensitivity. ............................................................................................................................... 112 DISCUSSION ......................................................................................................................... 115 Tesis doctoral: Juan Carlos Martínez Cañas 7 I- NEW INSIGHTS FROM HISTONE MUTANTS AND THE DISTINGUISHABLE DNA-RNA HYBRIDS ACCUMULATED IN THEM. ........................................................................................................................................... 117 IINEW CHROMATIN CHANGING FACTORS INVOLVED IN DNA-RNA HOMEOSTASIS..................................... 121 CONCLUSSIONS .................................................................................................................... 127 MATERIALS AND METHODS .................................................................................................. 131 YEAST STRAINS, PLASMID AND PRIMERS ............................................................................................... 133 EXPERIMENTS ................................................................................................................................. 141 REFERENCES ......................................................................................................................... 147 8 Tesis doctoral: Juan Carlos Martínez Cañas Tesis doctoral: Juan Carlos Martínez Cañas 9 Index of Figures Introduction Figure I 1. Factors leading to DNA damage .......................................................... 24 Figure I 2. Model of DSB repair by NHEJ and HR pathways ................................. 26 Figure I 3. Homologous recombination by SDSA and DSBR ................................ 29 Figure I 4. Model of an R loop ............................................................................... 31 Results Figure R 1. Histone mutants, which accumulate DNA-RNA hybrids, do not present genome instability unless AID is overexpressed ................................................... 52 Figure R 2. Study of the genotoxic sensitivity of the indicated strains after their growth in medium containing different concentrations of hydroxyurea .................. 54 Figure R 3. H3K9-23A h a   a   1 mutant ................................................................................................................... 56 Figure R 4. Possible conformations of DNA affect its sensitivity to sodium bisulfite generating different products that can be detected by PCR .................................. 60 Figure R 5. Detection of R loops by PCR in different regions of the GCN4 gene .. 61 Figure R 6. R loop length is similar between WT and R loop accumulating mutants .............................................................................................................................. 63 Figure R 7. R loop length is similar in R loop accumulating mutants ..................... 65 Figure R 8. R loops are formed at a very low frequency ....................................... 67 Figure R 9. Scheme of absolute R loop quantification method .............................. 68 Figure R 10. Comparison of bisulfite-qPCR assay with DRIP ............................... 70 Figure R 11. AID* overexpression does not affect viability but induces mutations in CAN1 gene ............................................................................................................ 72 Figure R 12. AID* overexpression induces mutations genome-wide ..................... 74 Figure R 13. Meta-analysis of mutations detects AID*-induced enrichment in larger genes .................................................................................................................... 76 16 Tesis doctoral: Juan Carlos Martínez Cañas Tesis doctoral: Juan Carlos Martínez Cañas 17 RESUMEN El genoma, el conjunto de genes de un organismo o especie, debe copiarse de forma fidedigna de célula a célula. Sin embargo, existen situaciones que comprometen la estabilidad del genoma dando lugar a la aparición de mutaciones, roturas en el ADN, reordenamientos cromosómicos, etc...que en último lugar pueden desencadenar la muerte celular o incluso la aparición de tumores. La relevancia del mantenimiento de la estabilidad del genoma se hace patente en el hecho de que la evolución ha desarrollado complejos mecanismos de reparación y respuesta al daño en el ADN para prevenir la inestabilidad genética. Sin embargo, el ADN no está exento al ataque de numerosos factores que pueden condicionar su estabilidad, desde agentes exógenos hasta el propio metabolismo celular. En concreto, se conoce que los procesos de transcripción y replicación del ADN son una fuente importante de inestabilidad genética. Aunque existen diversos motivos por los que estos dos mecanismos pueden generar daño en el ADN, uno de los más estudiados es la formación de R loops. Estos son estructuras formadas por un híbrido de ADN-ARN que desplaza la otra hebra del ADN dejándola en forma de cadena sencilla, más susceptible al ataque de determinados agentes como la enzima AID. Aunque los híbridos de ADN-ARN desempeñan papeles fisiológicos en las células, su acumulación puede potenciar la inestabilidad genómica. Un claro ejemplo de esto lo constituyen los mutantes del complejo THO, un complejo proteico que interviene en la formación de la ribonucleopartícula uniéndose al ARNm en formación y facilitando su exportación fuera del núcleo. En ausencia de dichos factores, la desprotección del ARNm permite su unión al ADN molde, dando lugar a una acumulación co-transcripcional de híbridos de ADN-ARN que generan altos niveles de inestabilidad genética, principalmente detectada como recombinación entre secuencias repetidas. Asimismo, existen proteínas 18 Tesis doctoral: Juan Carlos Martínez Cañas implicadas en la eliminación de los híbridos una vez formados, como las RNasas H y ciertas helicasas. Es importante destacar que en el genoma de los eucariotas, el ADN no se encuentra desnudo en el núcleo de la célula sino que se asocia con determinadas proteínas dando lugar a la estructura de la cromatina cuyas unidades básicas son los nucleosomas. Cada uno de ellos está formado por un octámero proteico que contiene dos copias de cada una de las cuatro histonas H3, H4, H2A y H2B. La colas amino-terminales de las histonas sufren modificaciones post-traduccionales como metilaciones, acetilaciones, fosforilaciones o ADP-ribosilaciones que pueden tener consecuencias tanto para la estructura de la cromatina como para todos los procesos básicos del ADN, incluida la formación de híbridos de ADN-ARN. Estudios previos en el laboratorio identificaron ciertas mutaciones en las colas amino terminales de las histonas H3 y H4 que Estos híbridos a diferencia de los que se acumulan otros mutantes como los del complejo THO, no dan lugar a un incremento de la inestabilidad genómica, ni por un aumento de la recombinación entre secuencias repetidas ni tampoco mediante una acumulación de focos de Rad52. En esta tesis profundizamos en el estudio de dichos mutantes de histonas, analizando los niveles de pérdida de heterozigosidad o pérdida de cromosomas y hemos conseguido medir la longitud y la frecuencia de los R loops gracias a la puesta a punto de una técnica basada en el uso de bisulfito sódico in vitro. Además, hemos utilizado una versión hiper-mutagénica de la AID con el fin de poder mapear los híbridos en todo el genoma in vivo. Hemos observado que los R loops que se acumulan en los mutantes de histonas tienen la misma longitud que aquellos que están presentes en cepas silvestres u otros mutantes que dan lugar a la acumulación de híbridos como los del complejo THO. Los resultados de esta tesis, por tanto, nos han llevado a concluir que la longitud de los R loops no es un factor determinante en la generación de inestabilidad genética mediada a través de estas estructuras. Tesis doctoral: Juan Carlos Martínez Cañas 19 Por último, para identificar nuevos factores de la cromatina que tengan un papel en el metabolismo de los R loops, hemos realizado una búsqueda para identificar mutantes en remodeladores de cromatina o modificadores de histonas. Hemos encontrado que la deleción de la acetil-transferasa Rtt109 provoca un incremento de R loops dependiente de su actividad catalítica, y gracias al análisis de distintas mutaciones en las histonas H3 y H4 hemos asociado dicho incremento con dianas concretas de entre todas las descritas para Rtt109 in vivo o in vitro. Por otra parte, dado que Rtt109 tiene un papel en la reparación de roturas de doble cadena del ADN, previamente descrito en nuestro laboratorio, hemos estudiado la relación entre los fenotipos de defecto en la reparación y la acumulación de híbridos de ADN-ARN, tanto en ausencia de Rtt109 como en los mutantes individuales en sus dianas. Los resultados de esta tesis nos indican que la acumulación de híbridos de ADN-ARN no es un impedimento para la correcta reparación de roturas de doble cadena, ni tampoco son la causa de todo el daño presente en ausencia de Rtt109. No obstante, concluimos que la persistente acumulación de roturas de doble cadena podría favorecer la formación de híbridos de ADN-ARN. De esta forma, el trabajo manifiesta la relevancia del contexto cromatínico en el que se encuentra el ADN en la formación de R loops. 20 Tesis doctoral: Juan Carlos Martínez Cañas Tesis doctoral: Juan Carlos Martínez Cañas 21 INTRODUCTION 22 Tesis doctoral: Juan Carlos Martínez Cañas Tesis doctoral: Juan Carlos Martínez Cañas 23 Genetic instability The genome contains all the information needed to form from a simple bacteria to a complex human being and it is essential to preserve it from generation to generation in order to keep individuals and species alive through time. Genome can vary between distinct generations as a natural consequence of the metabolism of DNA. This process of changes in the DNA is referred as genome instability and although is needed for evolution, many factors, including exogenous and endogenous agents, can enhance it, resulting in genetic alterations which can be classified according to the type of event stimulated and the cell cycle stage: chromosomal instability, micro- and minisatellite instability, mutations, etc. This events can provoke the loss of genes, chromosome rearrangements, cancer and cell death. However, cells posses several mechanisms to preserve the integrity of the genome, starting for the machinery itself in charge of copying it, avoiding the appearing of mutations, and finishing with proteins involved in the repair of the DNA damage once is formed, such as histones and their modifications, and also non chromatin-bound factors. DNA damage response. DNA damage can be produced by factors outside of the cell and among the exogenous factors which can lead to genome instability we can remark the exposure to UV and ionizing radiations, environmental carcinogens and chemicals. Moreover, cellular processes also constitute endogenous sources of genome instability such as, reactive oxygen species generated by metabolism, transcription and also DNA replication (Figure I1). All of them differ in the type of DNA damage generated and the subsequent cellular response activated. To counteract all these lesions which could lead to genetic instability, cells have evolved specialized systems that detect the damage, signal its presence and mediate the repair in 24 Tesis doctoral: Juan Carlos Martínez Cañas order to safeguard the stability of the genome. This is orchestrated by a highly conserved network of proteins generically termed as DNA damage response (DDR), which organize multiple cellular events that although independent, cells have to coordinate in space and time to preserve DNA integrity. Figure I 1. Factors leading to DNA damage DDR is composed of sensor, transducers and effectors. Upon the generation of a DNA lesion, the DDR coordinates the response by activating two parallel routes: 1) DNA repair pathways that deal with the restoration of the DNA lesion, and 2) DNA damage checkpoint, a cell cycle blockage that restrains mitosis until DNA damage has been fixed. Figure I1. Factors leading to DNA damage. Positive and negative supercoiling generated during replication impedes the replication fork progression and makes DNA damage agents easy to access DNA, respectively. Hybridization of mRNA (red line) to its DNA template (R loop) exposes ssDNA, which is more sensible to the attack of DNA damage agent. Replication (green arrows and circles) and transcription (red arrow and circle) conflicts are also a source of genetic instability. Ionizing radiation (IR) and UV can provoke interstrand-crosslink (purple line). All of these DNA damage agent can lead to the formation of SSB. and DSBs. Tesis doctoral: Juan Carlos Martínez Cañas 25 DNA damage repair The most important repair pathways are base excision repair (BER), for oxidative damage to DNA bases and single-strand breaks (SSBs), nucleotide excision repair (NER) to remove bulky adducts, mismatch repair (MMR) for errors occurring during DNA replication, and homologous recombination (HR) or non-homologous end joining (NHEJ) to repair double-strand breaks (DSBs), which are the most deleterious lesions in the DNA, since a single DSB can lead to cell death (Rich et al., 2000). The choice of the DSB repair pathway depends on the 5' end resection needed for HR which rely on the cell cycle stage: HR is active during S/G2 phases while NHEJ is more likely to occur in G1 and also in G2 phases. However, both routes share a previous common step of recognition of the DSB by the DNA- damage checkpoint.. Recognition of DSBs prior to be repaired When a DSB occurs, the DNA ends interact directly with the MR(X)N complex (Grenon et al., 2001), composed of Mre11, Rad50, and Xrs2 in yeast. This complex recruits Tel1 transducer kinase triggering checkpoint activation (Lee and Paull, 2005, Mantiero et al., 2007, Paull and Lee, 2005). Although the activation of Tel1 seems to be sufficient to induce a checkpoint response, the repair of persistent DSBs, through the formation of RPA-coated single-stranded DNA (ssDNA) (Zou and Elledge, 2003), require the activation of Mec1, arresting cells in G2 phase. Tel1 stimulates processing of DSB, which consists on the 5' end resection nearby a DSB. Since this resection process lead to the repair of DSBs by HR (Frank-Vaillant and Marcand, 2002), we will see it more in detail when we describe this repair pathway. After the activation of the checkpoint, it is produced an amplification of the DNA damage signal, being phosphorylation of serine 128 of the histone H2A the most important and is carried out by the checkpoint kinases Tel1 and Mec1 (Downs et al., 2000). Once the DNA damage is signaled, this can be repaired by the two main pathways: NHEJ and HR (Figure I2). 32 Tesis doctoral: Juan Carlos Martínez Cañas DNA-RNA helicase Sen1, that separate RNAs from their DNA templates. Moreover, negative supercoiling during replication and transcription also favor R loop formation. In this cases, topoisomerases are very important to remove the torsional stress and thus, reducing the possibility of R loop formation. Transcription as a source of genome instability. Although transcription machinery or any of the related structures formed during this process can represent a blockage to the replication progression, the transcription per se can constitute another source of DNA damage. Sequences highly expressed are more prone to be damaged by mutations and recombination events, phenomena known as transcription-associated mutation (TAM) and transcriptionassociated recombination (TAR), respectively. It is thought that these genome instability phenotypes are due to a more accessibility of the non-transcribed strand (NTS) to DNA damage agents than the transcribed strand (TS) although it seems most likely that the strand asymmetric sensitivity observed is due to the NTS, but not the TS is single stranded. In this sense, R loops could explain the differences between the damage sensitivity of both strands. Replication as a source of genome instability. During the copy of the genome, the replication machinery can encounter with some obstacles which must overcome to ensure the correct transmission of the genomic information to daughter cells. Among all this obstacles we can remark the presence of topological stress, the structure of chromatin, different proteins bound to the DNA, the presence of DNA damage and non-B DNA structures and the transcription machinery itself. As replication progresses, the replication forks themselves induce conformational changes in the topology of DNA, opening it behind the replication forks (negative supercoiling) and closing it ahead of the replication forks (positive supercoiling). In one hand, the negative supercoiling, makes DNA more accessible to the attack by different agents, while the positive supercoiling could impede the progression of Tesis doctoral: Juan Carlos Martínez Cañas 33 the replication machinery and favor its stalling. However, cells count with topoisomerases (I and II) which relieve the topological stress creating a SSB or a DSB, respectively by passing of one DNA molecule through the other. The presence of DNA damage and also non-histone proteins bound to the DNA could lead to the replication fork stalling by impeding its moving ahead these obstacles. Nevertheless, there is a battery of proteins including helicases and DNA repair pathways to eliminate them from the DNA. In addition, DNA is bound to histone proteins, forming the chromatin, which can be postranslationally modified by acetylation, methylation, phosphorylation, etc, resulting in chromatin structures that can favor or hinder its replication. Conflicts between replication and transcription machineries deserve a mention apart. When replication and transcription occur at the same time, these two machineries can collide since the replication fork cannot progress past an elongating RNA polymerase. Depending on the direction of these processes, collisions can be classified in head-on collisions, when replication forks and RNA polymerase move in a convergent opposite direction, and co-directional collisions, when both machineries move in the same direction. Some studies in yeast revealed that head-on collision are more deleterious for the genome integrity than co-directional collisions, since only the first ones could be able to pause the progression of replication forks inducing recombination (Prado and Aguilera, 2005). Part of the different genome instability phenotypes observed in the two kind of collisions may be attributed to R loops (Garcia-Rubio et al., 2018, Hamperl et al., 2017, Lang et al., 2017). In artificial collision systems, R loops are only detected in the head-on collision (Hamperl et al., 2017, Lang et al., 2017). However, R loops can be stabilized by Yra1 overexpression and can be detected even in co-directional collisions (Garcia-Rubio et al., 2018). It is not clear how R loops can cause the blockage of replication forks and several mechanisms have been developed to explain it. One possibility is that the retention 34 Tesis doctoral: Juan Carlos Martínez Cañas of RNA polymerase or the recruitment of DNA-RNA binding proteins could create an obstacle to the replication fork progression. Another possibilities include the negative supercoiling generated by R loops between replication and transcription machineries or a compaction of the chromatin associated to R loops. In order to counteract these collisions, cells posses different mechanisms to avoid or reduce their formation. In bacteria, for example, genes are co-directly oriented with the replication origins. In yeast, there are replication block carriers at rDNA locus and in mammals replication and transcription take place at different time points, since loci highly transcribed are replicated late in S phase and vice versa. Detection of R loops among the genome. Since the clear relation between R loop and genome instability, several strategies has evolved from the past few years to detect them. The most widely used is the S9.6 antibody, which can bind specifically to DNA-RNA hybrids. It was originally isolated by Carrico and colleagues in 1985. Mouse (BALB/c) B-cell was fused with Mouse (BALB/c) Sp2/0Ag14 myeloma to produce monoclonal antibodies against RNA-DNA hybrids. Animals were immunized with RNA-DNA hybrids formed by using single stranded PhiX174 DNA as template for E. coli DNA dependent RNA polymerase (Boguslawski et al., 1986). Many labs have taken advantage of this specificity and have used it to map DNA-RNA hybrids among the genome in yeast (Chan et al., 2014, El Hage et al., 2014, Wahba et al., 2016) and mammals (Ginno et al., 2013, Ginno et al., 2012, Nadel et al., 2015, Sanz et al., 2016, Stork et al., 2016). Most of methods using S9.6 antibody capture DNA-RNA hybrids from digested DNA followed by sequencing of captured DNA (DRIP-seq) or RNA (DRIP- c-Seq (Chedin, 2016). A few authors have revealed that S9.6 antibody also presents high specificity for dsRNAs (Phillips et al., 2013, Hartono et al., 2018), which could reduce the DNA-RNA immunoprecipitation efficiency and have modified the original protocol adding an RNase III in vitro treatment prior to immunoprecipitation with S9.6 (Hartono et al., 2018, Svikovic et al., 2019), since this enzyme degrades dsRNAs. Alternative techniques to S9.6 antibody has been Tesis doctoral: Juan Carlos Martínez Cañas 35 developed such as the used of inactive RNase H (Chen et al., 2017), which is able to bind DNA-RNA hybrids without resolving them or the use of bisulfite, which converts specifically cytosines into uraciles of the ssDNA (Ginno et al., 2012, Yu et al., 2003, Huang et al., 2006, Garcia-Pichardo et al., 2017). Apart from S9.6 antibody and inactive RNase H enzyme, there is and additional tool to indirectly detect R loops, the Active-Induced Deaminase (AID) which is able to deaminate cytosines of ssDNA generating uracils. This enzyme is present in mammals and it is thought to trigger somatic hypermutation and class switch recombination in an R loop dependent manner (Muramatsu et al., 2000, Revy et al., 2000). Several studies have overexpressed AID in vivo in order to enhance a R loop-dependent genome instability phenotype (Garcia-Pichardo et al., 2017, Gomez-Gonzalez and Aguilera, 2009, Mischo et al., 2011, Garcia-Benitez et al., 2017). There is an interesting variant of this enzyme called superAID or AID* which is more mutagenic (Wang et al., 2009) and have been used in some studies to reproduce mutations found in cancers and discover regions of the genome more prone to be mutated (Taylor et al., 2013, Taylor et al., 2014), although it has never been used to map R loops among the genome. Independently of the method used to detect R loops, RNase H is a key element in all these techniques which helps to decipher if the signal measured comes or not from DNA-RNA hybrids. RNase H can also be per se a good tool to detect R loops since it is able to suppress genome instability phenotypes such as growth defects (Gomez-Gonzalez et al., 2009, Feldman and Peterson, 2019). R loops and chromatin. Although it is not clear the mechanisms surrounding the R loop-induced genome instability, many studies have related the R loop formation with changes in chromatin state. There are many observations suggesting that R loops locally open the chromatin structure, possibly by regulating nucleosome occupancy, positioning 36 Tesis doctoral: Juan Carlos Martínez Cañas and/or turnover. DNA-RNA hybrids alter the B-structure of DNA and prevent nucleosome wrapping in vitro (Dunn and Griffith, 1980), and in vivo, loss of FACT complex, which swaps nucleosomes around the RNA polymerase during transcription elongation, causes R loop accumulation suggesting that nucleosome redeposition during transcription prevents R loop formation. It is though that R loops can also trigger the recruitment of chromatin modifying enzymes to alter the chromatin state. After the mapping of R loops throughout the genome, a set of histone modification have been associated to R loops, finding out that their accumulation correlates with transcription active marks such as H3K3me1, H3K4me3 and H3 acetylation (H3K27ac), supporting the idea that R loops are formed in a open chromatin. However, another studies correlate R loop formation with chromatin compaction. THO mutants in yeast are characterized by a high genome instability associated by R loop formation. This increase in R loop accumulation is accompanied with higher levels of H3S10P (a mark typically associated with condensed chromosomes during mitosis) outside of mitosis, and the overexpression of RNase H suppresses H3S10P signal (Castellano-Pozo et al., 2013). In this context of R loop causing chromatin compaction, other study has shown that R loops can promote chromatin condensation at the 3' end of genes causing transcription termination (Skourti-Stathaki et al., 2014) Tesis doctoral: Juan Carlos Martínez Cañas 37 Chromatin Genomic DNA is not naked inside cells, but it is wrapped 146 bp around nucleosomes which are formed by two copies of histones H2A, H2B, H3 and H4, constituting the chromatin. The consecutive nucleosomes are bound by naked DNA non-histone provided forming the chromatin fiber. A second level of organization would imply the histone H1 which binds nucleosomes one above another to form the 30 nm fiber. Finally, DNA can pack even more, generating the chromosomes observed during metaphase. According to its activity, there are two types of chromatin: heterocromatin, which is highly condensed and inactive, and the euchromatin, slightly compacted, rich in transcribed genes and for this reason active. However, what defines the state of chromatin are the basic units of nucleosomes: histones and their post-translational modifications (PTMs). In addition, chromatin can also be affected by altering whole nucleosomes through chromatin remodelers. In both processes, histone chaperons can play a role helping to chromatin modifiers and remodelers to carry out their functions. 38 Tesis doctoral: Juan Carlos Martínez Cañas Table I 1. List of chromatin remodelers and homologues Table I1. List of chromatin remodelers and their homologues in yeast, fly and humans. From (Clapier and Cairns, 2009) Tesis doctoral: Juan Carlos Martínez Cañas 39 Chromatin remodelers Chromatin remodelers are tools present in cells from yeast to humans (Table I1) which couple the energy released from the ATP hydrolysis with changes in the nucleosomes, sliding octamers of histones across the DNA, changing the conformation of nucleosomal DNA or modifying the compositions of octamers. They can be classified in four different families: SWI/SNF, ISWI, CHD and INO80. All of these families posses and ATPase domain split in 2 parts (DExx and HELIC) where there is an insertion whose size depends on the specific family of chromatin remodeler. Moreover, they also differentiate in the domains adjacent to the ATPase domain, which confers the specificity in their functions. SWI/SNF family. It was the first family described in two independent genetic screenings for altered gene expression involved in regulating mating type switching (SWI) and sucrose fermentation in yeast (Sucrose Non-Fermenting). T There are two SWI/SNF ATPases in yeast: Swi2/Snf2, which is part of SWI/SNF complex, and Sth1, from the RSC complex. They all carry out their function through the bromodomain targeted to acetylation marks present in the tail of histones. Among the functions of this chromatin remodeler family, most of studies relate it with the activation of gene transcription (Armstrong and Emerson, 1998), although others works suggest they could also contribute to gene repression (Trouche et al., 1997). They can also participate in replication initiation and nucleotide excision repair (Flanagan and Peterson, 1999, Hara and Sancar, 2003, Gaillard et al., 2003) ISWI family. This family was name as Imitating SWItch due to was homologue to SWI/SNF family only in it ATPase domain. Members of this family are involve in nucleosome assembly and spacing and they exert their function through HANDSANT-SLIDE domains which allows to bind DNA. They are involved in transcription suppression (Vary et al., 2003, Mellor and Morillon, 2004) and also facilitates the de novo nucleosome assembly. In yeast there are two ISWI ATPases: Isw1 and Isw2 40 Tesis doctoral: Juan Carlos Martínez Cañas forming part of four different complexes whose additional members provide them specificity to their targets CHD family. This family contains tandem chromodomains at the N-terminal region, a DNA- binding domain at the C-terminal region that binds to A-T rich regions preferentially and helicase/ATPase like-SNF2 domains (Chromodomain - Helicase - DNA binding). This family participates in nucleosome spacing and deposition. In yeast there is only one CHD member, Chd1 which is able to bind to methylated histone H3 with unknown functions (Pray-Grant et al., 2005). It also regulates transcription elongation (Simic et al., 2003) and prevents histone exchange maintaining chromatin structure with Isw1 (Smolle et al., 2012) INO80 family. There are two different member in this family: Swr1 and Ino80, which gives name to the family, since it is responsible for regulation of inositol-responsive gene expression. They present a Helicase-SANT (HSA) domain essential for actin and ARP (actin related proteins) binding. The members of this family are involved in histone variant exchange, such as variant H2A.Z deposition. They can bind to the replication forks and Holliday junctions, interfere in nucleosome eviction (Krogan et al., 2003, Mizuguchi et al., 2004, Tsukuda et al., 2005, van Attikum et al., 2007) and also is believed to participate in DNA repair (Morrison et al., 2004) Post-translational modifications of histones Histones regulate the state of chromatin through several modifications, which in addition, can alter the function of many other factors altering the recruitment of proteins and complexes and, for so, this PTMs can have a role in multiple cellular processes such as transcription and replication of DNA as well as its repair when damaged. There are many PTMS such as, ubiquitylation, sumoylation, deimination, etc the most important for the development of this thesis are acetylation ( and deacetylation), methylation (and demethylation) and phosphorylation. Tesis doctoral: Juan Carlos Martínez Cañas 41 Acetylation of histones occurs at their lysine residues and is catalyzed by histone acetyltransferases (HATs). These enzymes transfer an acetyl group from acetyl- CoA cofactor to  -amino group of the lysine side chain. This reaction has as a consequence the neutralization of charges between DNA and histones weakening their interactions. For this reason, it is though that acetylation would have a role in transcription activation, since it relaxes chromatin, making it more accessible to transcription factors (euchromatin). Moreover, this acetylation mark provides a specific bind site for bromodomain containing proteins, such as SWI/SNF chromatin remodelers. Apart from their function in transcription activation, there are another acetylation marks involved in DNA repair, such as H3K14 acetylation (Wang et al., 2012) or in replication-coupled nucleosome assembly, such as H3K56 acetylation (Masumoto et al., 2005). Acetylation of lysines is reversible, in a reaction catalyzed by histone deacetylases (HDACs). As it returns the positive charge to histones, it is thought that this reaction reinforces the interaction between DNA and histones, which leads to chromatin compaction (heterochromatin) and transcription repression since it difficulties the access to DNA by transcription factors. Another histone PTM is methylation of lysines and arginines which can be mono-, di-, or trimethylated and mono- or dimethylated, respectively. This reaction is catalyzed by histone methyltransferases (HMTs) and did not cause the loss of histone charge. Its function is carried out by recruiting chromodomain-containing proteins which alters the chromatin structure. Depending on the interacting partners and the methylated site, can either activate or repress transcription. For example, H3K4me3 leads to transcription activation (Howe et al., 2017), whereas H3K23me3 leads to transcription repression (Boros et al., 2014). Serines, threonines and also tyrosines are susceptible to be phosphorylated or dephosphorylated by kinases or phosphatases, respectively. It seems that histone phosphorylation may be a common feature of signal transduction pathways, in response to cell cycling or cellular stress. One of the most phosphorylated marks 128 Tesis doctoral: Juan Carlos Martínez Cañas Tesis doctoral: Juan Carlos Martínez Cañas 129 Conclusions 1. The DNA-RNA hybrid accumulation inH31-28, H3K9-23A and H4K31Q histone mutations does not lead to LOH or plasmid loss, in contrast to previously reported DNA-RNA hybrid accumulating mutants, such as 1. 2. The H3K9-23A mutation suppresses the replication fork progression impairment of 1. This supports previous results that indicate that histone H3 lysines 9, 14, 18 or 23 residues are required for the genetic instability associated with DNA-RNA hybrids. In this thesis, we establish that H3K9A, and not H3K14A or H3K23A, is the mutation responsible of this suppression. 3. The H31-28 and H3K9-23A mutations alter the pattern of nucleosome positioning along the genome. This is specific for these particular substitutions, not being observed in H3K9-23R. Notably, we have observed no changes in nucleosome positioning for H4K31Q, indicating that changes in nucleosome positioning are not the cause of the DNA-RNA hybrid accumulation observed. 4. The length of R loops, as determined by the bisulfite-modification assay at the GCN4 gene, ranges from 38 bp to 513 bp with an average of 142 bp. Strikingly, R loop length is similar in wild-type and several R-loop accumulating strains, regardless of whether they produce genome instability or not. This indicates that DNA-RNA hybrid length is not relevant for the generation of genetic instability. Bisulfite-modification followed by qPCR can be used as a new tool, as an alternative to S9.6-based methodologies such as DRIP, to detect relative variations in DNA-RNA hybrids at specific genomics locations. Using this method we estimate that the frequency of 130 Tesis doctoral: Juan Carlos Martínez Cañas DNA-RNA hybrids is low, since we found no positive hits at the GCN4 gene among 500 yeast colonies analyzed. 5. Overexpression of a hyper-mutator AID induces mutations genome-wide, preferentially in cytosines of large genes and in an R loop-dependent manner. However, the low number of induced mutations makes this tool insufficient to map R loops genome-wide. 6. The loss of the Snf2, Isw1 or Isw2 catalytic subunits of the SWI/SNF and ISWI family of chromatin remodelers does not enhance R loop accumulation or R loop-dependent genome instability. 7. The loss of the Rtt109 acetyl-transferase leads to an accumulation of DNARNA hybrids in all cell cycle phases.Rtt109 prevents DNA-RNA hybrid accumulation through its catalytic activity but not through its reported role of H3K56 acetylation. 8. Among all the reported substrates of Rtt109, H3K14 and H3K23 are relevant for its role preventing the accumulation of DNA-RNA hybrids, since H3K14A and H3K23A mutations increase DNA-RNA hybrids and H3K14Q and H3K23Q acetyl-mimetic mutants prevent hybrid accumulation in the absence of Rtt109. 9. We favor a model in which the persistence of unrepaired DSBs leads to the observed accumulation of DNA-RNA hybrids in 109, since the previously reported repair phenotypes of defective repair in v in 109 are not dependent on hybrids. Tesis doctoral: Juan Carlos Martínez Cañas 131 MATERIALS AND METHODS 132 Tesis doctoral: Juan Carlos Martínez Cañas Tesis doctoral: Juan Carlos Martínez Cañas 133 Yeast strains, plasmid and primers Yeast strain Genotype Source H3WT MAT 3200 20 20 163 30 150 can1::MFA1pr-HIS3 hht1-hhf1::NatMX4 hht2-hhf2::[H3]- URA3 (Dai et al., 2008) H31-28 MAT 3200 20 20 163 30 150 can1::MFA1pr-HIS3 hht1-hhf1::NatMX4 hht2-2::[1- 28]-URA3 H3K9-23A MAT 3200 20 20 163 30 150 can1::MFA1pr-HIS3 hht1-hhf1::NatMX4 hht2-hhf2::[K9- 23A]-URA3 H4WT MAT 3200 20 20 163 30 150 can1::MFA1pr-HIS3 hht1-hhf1::NatMX4 hht2-hhf2::[H4]- URA3 H4K31Q MAT 3200 20 20 163 30 150 can1::MFA1pr-HIS3 hht1-hhf1::NatMX4 hht2- hhf2::[K31Q]-URA3 H31 MAT 3200 20 20 163 30 150 can1::MFA1pr-HIS3 hht1-hhf1::NatMX4 hht2-hhf2::[H3]- URA3 1 This study BY4743 MAT/MAT 3/ 3 20/ 20 15/MET15 20/LYS2 30/ 30 This study YDIH-4B MAT/M 3200/3 20/ 20 20/ 20 163/ 163 30/30 150/ 150 1::MFA1-HIS3/CAN1 hht1-hhf1::NatMX4/ hht1-hhf1::NatMX4 hht2hhf2::[H3]-URA3/ HHT2-HHF2 D. García- Pichardo YDI28-11C MATa/M 3200/3 20/ 20 20/ 20 163/ TRP1 30/30 150/ 150 1::MFA1-HIS3/ CAN1 hht1-hhf1::NatMX4/ hht1-1::NMX4 22::[1-28]-URA3/hht2- 2::[1-28]-URA3 D. García- Pichardo 134 Tesis doctoral: Juan Carlos Martínez Cañas YDIK9-2D MAT/M 3200/3 20/ 20 20/ 20 163/ TRP1 30/30 150/ 150 1::MFA1-HIS3/ can1::MFA1pr-HIS3 hht1- hhf1::NatMX4/ hht1hhf1::NatMX4 hht2-hhf2::[K9-23A]- URA3/ hht2-hhf2::[K9-23A]-URA3 D. García- Pichardo YDIK31-9A MAT/M 3200/3 20/ 20 20/ 20 163/ TRP1 30/30 150/150 1::MFA1-HIS3/ can1::MFA1pr- HIS3 hht1-hhf1::NatMX4/ hht1hhf1::NatMX4 hht2- hhf2::[K31Q]-URA3/hht2-hhf2::[K31Q]-URA3 D. García- Pichardo YDIH-18B MAT/M 3/3 20/ 20 20/ 20 3/3 150/ MET15 CAN1/ CAN1 1- hhf1::NatMX4/ hht1-hhf1::NatMX4 1::TRP1/1::TRP1 D. García- Pichardo W303-1A MAT D ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 R. Rothstein U678-4C MAT D ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1hpr1::HIS3 Piruat and Aguilera, 1998 DD379 W303-1A 24::TRP1 Kanellis et al., 2003 WHR24-3B W303-1A hpr1::HIS3 rad24::TRP1 (Gomez- Gonzalez et al., 2009) K923H MAT 3200 20 20 163 30 150 can1::MFA1pr-HIS3 hht1-hhf1::NatMX4 hht2-hhf2::[K9- 23A]-URA3 hpr1::Hyg This study K923HR MAT 3200 20 20 163 30 150 can1::MFA1pr-HIS3 hht1-hhf1::NatMX4 hht2-hhf2::[K9- 23A]-URA3 hpr1::Hyg rad24::Kan This study BY4741 MATa his31 leu20 met150 ura30 EUROSCARF Ybp249 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 bar1 RAD5 B. Pardo Tesis doctoral: Juan Carlos Martínez Cañas 135 Yeast strain Genotype Source WH101R5 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 bar1 RAD5 hpr1-101 (hpr1-L586P) This study YMFT1 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 bar1 RAD5 mft1::Kan This study thp1 WEIII-36 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 bar1 pob3-7 (Herrera- Moyano et al., 2014) XEI-13 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 lys2 met15 trp1-1 ura3-1 spt16-11 (Herrera- Moyano et al., 2014) YTOP1 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 bar1 RAD5 mft1::Hyg This study SEN-R MATa ade2-1 can1-100 his3-11,15 leu2-3,112 lys2 met15 trp1-1 ura3-1 sen1-1 M. San Martín RNH-R MATa ade2-1 can1-100 his3-11,15 leu2-3,112 lys2 met15 trp1-1 ura3-1 rnh1::Kan rnh201::Kan M. San Martín YDIH-CAN MAT/M 3200/3 20/ 20 20/ 20 163/ 163 30/30 150/ 150 CAN1/CAN1 1-hhf1::NatMX4/ hht1- hhf1::NatMX4 hht2hhf2::[H3]-URA3/ HHT2-HHF2 This study YDI28-CAN MAT/M 3200/3 20/ 20 20/ 20 163/ TRP1 30/30 150/ 150 CAN1/ CAN1 1-hhf1::NatMX4/ hht1- 1::NMX4 22::[1-28]-URA3/hht2-2::[1- 28]-URA3 This study YDIK9-CAN MAT/M 3200/3 20/ 20 20/ 20 163/ TRP1 30/30 150/ 150 CAN1/CAN1 1-hhf1::NatMX4/ hht1hhf1::NatMX4 hht2-hhf2::[K9-23A]-URA3/ hht2- hhf2::[K9-23A]-URA3 This study 136 Tesis doctoral: Juan Carlos Martínez Cañas Yeast strain Genotype Source YDIK31-CAN MAT/M 3200/3 20/ 20 20/ 20 163/ TRP1 30/30 150/150 CAN1/CAN1 1-hhf1::NatMX4/ hht1hhf1::NatMX4 hht2-hhf2::[K31Q]-URA3/hht2- hhf2::[K31Q]-URA3 This study YSNF2 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 bar1 RAD5 snf2::Kan This study YISW1 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 bar1 RAD5 isw1::Kan This study YISW2 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 bar1 RAD5 isw2::Kan This study YPL001W MATa his31 leu20 met150 ura30 1::K EUROSCARF YEL056W MATa his31 leu20 met150 ura30 2::K EUROSCARF YDR448W MATa his31 leu20 met150 ura30 2::K EUROSCARF YGR252W MATa his31 leu20 met150 ura30 5::K EUROSCARF YBR081C MATa his31 leu20 met150 ura30 7::K EUROSCARF YLR055C MATa his31 leu20 met150 ura30 8::K EUROSCARF YLL002W MATa his31 leu20 met150 ura30 109::K EUROSCARF YDR359C MATa his31 leu20 met150 ura30 1::K EUROSCARF YOR023C MATa his31 leu20 met150 ura30 1::K EUROSCARF YBL052C MATa his31 leu20 met150 ura30 3::K EUROSCARF YNL330C MATa his31 leu20 met150 ura30 3::K EUROSCARF YOL004W MATa his31 leu20 met150 ura30 3::K EUROSCARF YMR263W MATa his31 leu20 met150 ura30 30::K EUROSCARF YMR075W MATa his31 leu20 met150 ura30 1::K EUROSCARF YNL021W MATa his31 leu20 met150 ura30 1::K EUROSCARF YPR068C MATa his31 leu20 met150 ura30 1::K EUROSCARF YKR029C MATa his31 leu20 met150 ura30 set3::Kan EUROSCARF YBR175W MATa his31 leu20 met150 ura30 3::K EUROSCARF YJL168C MATa his31 leu20 met150 ura30 set2::Kan EUROSCARF Tesis doctoral: Juan Carlos Martínez Cañas 137 Yeast strain Genotype Source YDR440W MATa his31 leu20 met150 ura30 1::K EUROSCARF YDR096W MATa his31 leu20 met150 ura30 1::K EUROSCARF YER169W MATa his31 leu20 met150 ura30 1::K EUROSCARF YER051W MATa his31 leu20 met150 ura30 1::K EUROSCARF YJR119C MATa his31 leu20 met150 ura30 2::K EUROSCARF Plasmid Description Source pWJ1344 YCp containing Rad52::YFP fusion under its own promoter (LEU2 marker) (Feng et al., 2007) pWJ1314 YCp containing Rad52::YFP fusion under its own promoter (TRP1 marker) (Alvaro et al., 2007) pRS413GAL Ycp pRS413 with the GAL1 promoter and the CYC1-terminator (Gavalda et al., 2016) pRS313-GAL::RNH1 YCp pRS313 containing the GALp::RNH1 fusion (Gavalda et al., 2016) p414GAL Ycp pRS414 with the GAL1 promoter and the CYC1-terminator (Garcia-Rubio et al., 2003) p414GALAID YCp pRS313 containing the GALp::AID fusion (Gomez-Gonzalez and Aguilera, 2007) pLlacZ-GALAID YCp pSCH204 containing GALp::AID fusion (Garcia-Pichardo et al., 2017) pRS217GAL Ycp pRS317 with the GAL1 promoter and the CYC1-terminator (Garcia-Pichardo et al., 2017) pRS317-GAL::RNH1 YCp pRS317 containing the GALp::RNH1 fusion (Garcia-Pichardo et al., 2017) p317RTT109wt Ycp pRS317 with wild-type RTT109 gene (Han et al., 2007) p317D89A Ycp pRS317 with RTT109 gene mutated at aspartate 89 to alanine (Han et al., 2007) p317D89N Ycp pRS317 with RTT109 gene mutated at aspartate 89 to asparagine (Han et al., 2007) 144 Tesis doctoral: Juan Carlos Martínez Cañas DNA isolated by CTAB method was digested with NdeI, NotI and XhoI. The bisulfite modification assay was performed essentially as described(Yu et al., 2003). Genomic DNA was diluted in 42 µl of distilled water with 17.5 µl of 20 mM hydroquinone and 460.5 µl of 2.5 M sodium bisulfite (pH 5.2). The mixture was sealed with mineral oil in a 500 µl microcentrifuge tube and incubated for 16 hr at 37 ºC in the dark. Bisulfite-treated DNA was purified with the Wizard SV Gel and PCR Clean-Up System (Promega) according to  aa . P b-treated DNA was desulfonated with 0.3 M NaOH at 37 ºC for 15 min. Desulfonated DNA was recovered by ethanol precipitation and resuspended in TE (pH 8.0). Bisulfite-modified DNA was used as a template for PCR with  a a  a ,  a a  a  a  ,  sequence of which matched the conversions anticipated owing to deamination of C to U in either the transcribed (TS) or nontranscribed (NTS) strand. PCR bands were purified from agarose gels with the Wizard SV Gel and PCR Clean-Up System (Promega) and cloned in pGEMT-easy. Independent clones were sequenced. Only molecules with more than four consecutive expected C to T changes were considered to determine the R-loop length. DRIP assay. Cells coming from exponential groth in YPD or SC depending of the experiment were pelleted by centrifugation and resuspended in 2.4 ml of spheroplasting buffer (1 M sorbitol, 2 mM TrisHCl pH 8.0,100 mM EDTA pH 8.0, 0.1% v/v beta-mercapto-ethanol, 2mg/ml zymoliase). Samples were incubated at 30 ºC during 30 minutes. After centrifugation, pellet was resuspended in 1.125 ml of solution I (0.8 mM GuHCl, 30 mM TrisHCl pH 8.0, 30 mMEDTA pH 8.0, 5% Tween 20, 0.5% Triton X-100) together with 40 µl of 10 mg/ml RNase A and incubated at 37 ºC during 30 minutes. Then, 75 µl of 20 mg/ml proteinase K were added and samples stood at 50 ºC for 1 hour. DNA was purified by chloroformisoamyl alcohol (24:1) and precipitated with 1 volume of isopropanol. With the help of a glass pasteur pipette, dNA was transferred to a new eppendorf where resuspended in 150 µl of 1X TE (1 mM TrisHCl pH 7.5, 0.5 mM EDTA pH 8.0) and digested overnight with 50 U of HindIII, EcoRI, BsrGI, XbaI and SspI. Half of theDNA was treated with 3 µl of RNase H (New England BioLabs) overnight 37 ºC as RNaseH control. Both samples were incubated with S9.6 antibody- Dynabeads Protein A (Invitrogen) complexes (previously Tesis doctoral: Juan Carlos Martínez Cañas 145 incubated overnight at 4 ºC) during 2.5 hours at 4º C. Samples were then washed 3 times with 1x binding buffer (10 mM NaPO4pH 7.0, 0.14 M NaCl, 0.05% Triton X-100). DNAwas eluted in 100 mL elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5 % SDS) treated 45 min with 7 µl of 20 mg/ml proteinase K at 55 ºC and purified with Quiagen DNA purification kit. Real-time quantitative PCR was performed using iTaq universal SYBR Green (Biorad) with a 7500 Real-Time PCR machine (Applied Biosystems). Chromatin immunoprecipitation (ChIP). ChIP was performed as described (Hecht et al., 1999) with some modifications. For cell extract preparation, pellets were resuspended in 500 µl of lysis buffer (50 mM HEPESKOH pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (1x Complete Protease Inhibitor Cocktail (Roche) and 1 mM PMSF).The chromatin was sonicated alternating 1 min high intensity and 1 min rest pulses for 15 min in Bioruptor sonication equipment. Samples were centrifuged for 15 min at 13000 rpm to eliminate cell debris. 20 µl of supernatant were processed as Input and 280 µl were immunoprecipitated. The immunoprecipitation was performed overnight at 4 ºC using Dynabeads Protein A (Invitrogen) previously incubated with the antibody for 4 hr rotating at 4 ºC. Beads were washed and chromatin was eluted in 250 µl elution buffer (50 mM Tris-HCl pH 7.4, 10 mM EDTA, 1% SDS) at 65 ºC for 10 min., treated with 6 µl of 50 mg/ml pronase for 1 hr at 42 ºC and decrosslinked for 6 hr at 65 ºC. Quiagen DNA purification kit was used to clean DNA. Real-time quantitative PCR was performed using iTaq universal SYBR Green (Biorad) with a 7500 Real-Time PCR machine (Applied Biosystems). SCE intermediates analysis. Analysis of the SCE intermediates was performed as described in Ortega et al. (2019). Cells transformed with pTINV-HO were grown in SRaf lacking tryptophan for mantaining plasmid until exponential growth. Galactose at 2 % was added to induced HO endonuclease overexpression and samples were collected after indicated time points. 146 Tesis doctoral: Juan Carlos Martínez Cañas DNA was extracted by phenol-chloroform-isoamyl alcohol (25:24:1), and precipitated in isopropanol. 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