Papel de factores de la transcripción y la replicación del ADN en el origen de la inestabilidad genómica.

Mantener la integridad del genoma no es tarea fácil para las células. El ADN sufre constantes ataques de agentes químicos y/o físicos de origen exógeno y endógeno, pero no sólo eso, los propios procesos celulares que emplean el ADN como sustrato también pueden generar daños y roturas. Dos de los procesos más fundamentales para la supervivencia de la célula, la replicación y la transcripción, son a su vez de las fuentes más importantes de daños en el ADN, especialmente en los casos de colisiones entre las maquinarias responsables de ambos procesos. Las colisiones entre transcripción y replicación pueden darse por diversos motivos, como la acumulación de estrés torsional en el ADN, la competencia entre polimerasas por el ADN molde en regiones del genoma con alta tasa de transcripción, o la presencia de secuencias específicas de ADN con tendencia a formar estructuras diferentes a su conformación B canónica. Dentro de esta última clasificación existe un tipo de estructura nucléica que está adquiriendo una creciente relevancia, los bucles R (más conocidos como R-loops). De origen primordialmente co-transcripcional, los R-loops están constituidos por una cadena sencilla de ADN desplazada y un híbrido de ARN:ADN formado entre el ARN y su hebra de ADN molde. Los híbridos de ARN:ADN son estructuras naturales, que presentan mayor estabilidad que la propia doble hélice de ADN, y que participan en diversos procesos celulares, como la replicación del ADN mitocondrial o el cambio de isotipo de las inmunoglobulinas. Sin embargo, la presencia de una cadena sencilla de ADN desplazada, característica de los R-loops, o la capacidad de estos de interferir con la replicación, pueden suponer riesgos para el mantenimiento de la estabilidad de los genomas. La presente tesis se plantea con la meta de avanzar en el conocimiento sobre los mecanismos que dan lugar a la formación y acumulación de los R-loops, y la de descubrir nuevos factores implicados en el mantenimiento de su homeostasis para evitar la aparición de conflictos entre la transcripción y la replicación y la inestabilidad genética que va asociada a ellos. Adicionalmente, empleando el organismo modelo S. cerevisiae, investigamos el funcionamiento de la proteína humana RECQL5, que a día de hoy se considera uno de los factores con el papel más claro y directo en la coordinación de los procesos de transcripción y replicación del ADN. Nuestros resultados apoyan la creciente noción de que la presencia de R-loops en las células es más común de lo que se pensaba inicialmente, incluso en fondos genéticos silvestres. Mediante análisis de secuenciación masiva y estudios bioinformáticos hemos encontrado que los telómeros, el ADN ribosómico (rDNA), los transposones y numerosos genes transcritos por la RNAPII son regiones con enriquecimiento de híbridos de ARN:ADN en una estirpe silvestre, patrón que se mantiene con cierta constancia en mutantes hpr1Δ. Los mutantes en este componente del complejo THO de elongación de la transcripción muestran fenotipos de acumulación de R-loops, hiperrecombinación e inestabilidad genética asociada a la transcripción. Nuestro trabajo sugiere que la diferencia puede no residir en la cantidad de híbridos que se formen en el fondo mutante, si no en ciertas características que diferencien estos R-loops de los presentes en cepas silvestres. Sin embargo, mejoras en la metodología deben ser introducidas antes de poder arrojar conclusiones más definitivas. Paralelamente hemos investigado cómo se originan los R-loops. Actualmente se considera que esencialmente son estructuras formadas durante la trascripción. No obstante, no se puede descartar la posibilidad de que un transcrito pudiera hibridar con otras regiones homólogas del genoma, generando R-loops en trans. Nuestros resultados no muestran ningún indicio de que la formación de R-loops no co-transcripcionales sea independiente de la transcripción, o que su formación tenga un impacto detectable en recombinación. Otros datos que rechazan la hipótesis de que Rad51 tenga un papel activo en la formación de R-loops en el mutante hpr1Δ. Una búsqueda de nuevos factores implicados en la homeostasis de R-loops nos llevó hasta la helicasa de ADN Mph1, FANCM en humanos. Los mutantes de levadura deficientes para esta proteína o su actividad helicasa acumulaban híbridos de ARN:ADN. A pesar de esto, las células mph1Δ no mostraron fenotipos de hiperrecombinación, ni defectos replicativos, ni interacciones genéticas con hpr1 o sen1. Serán necesarios estudios futuros para dilucidar si el papel de Mph1 en la eliminación de R-loops es directo o indirecto. Finalmente, hemos demostrado que la helicasa RECQL5 humana puede expresarse en levaduras, donde interacciona con proteínas ortólogas de aquellas humanas con las que se asocia de forma natural, como RNAPII o Rad51. Sin embargo, no hemos podido relacionar la inestabilidad genómica que observamos al expresar RECQL5 en levaduras con defectos producidos en la transcripción o en la replicación. No obstante, describimos por primera vez una relación funcional entre RECQL5 y la helicasa Srs2 de la levadura, que aporta nuevas vías para comprender el funcionamiento de ambas proteínas y sus papeles en el mantenimiento de la estabilidad genómica

Yra1-bound RNA–DNA hybrids cause orientation-independent transcription– replication collisions and telomere instability

R loops are an important source of genome instability, largely due to their negative impact on replication progression. Yra1/ALY is an abundant RNA-binding factor conserved from yeast to humans and required for mRNA export, but its excess causes lethality and genome instability. Here, we show that, in addition to ssDNA and ssRNA, Yra1 binds RNA–DNA hybrids in vitro and, when artificially overexpressed, can be recruited to chromatin in an RNA– DNA hybrid-dependent manner, stabilizing R loops and converting them into replication obstacles in vivo. Importantly, an excess of Yra1 increases R-loop-mediated genome instability caused by transcription–replication collisions regardless of whether they are codirectional or head-on. It also induces telomere shortening in telomerase-negative cells and accelerates senescence, consistent with a defect in telomere replication. Our results indicate that RNA–DNA hybrids form transiently in cells regardless of replication and, after stabilization by excess Yra1, compromise genome integrity, in agreement with a two-step model of R-loop-mediated genome instability. This work opens new perspectives to understand transcription-associated genome instability in repair-deficient cells, including tumoral cells.European Research Council ERC2014 AdG669898 TARLOOPMinisterio de Economía y Competitividad BFU2016-75058-PJunta de Andalucía PA12- BIO123

The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage

RNA-DNA hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1’s helicase activity plays a crucial role in responding to persistent RNA-DNA hybrids

RNA polymerase II contributes to preventing transcription-mediated replication fork stalls

Transcription is a major contributor to genome instability. A main cause of transcription‐associated instability relies on the capacity of transcription to stall replication. However, we know little of the possible role, if any, of the RNA polymerase (RNAP) in this process. Here, we analyzed 4 specific yeast RNAPII mutants that show different phenotypes of genetic instability including hyper‐recombination, DNA damage sensitivity and/or a strong dependency on double‐strand break repair functions for viability. Three specific alleles of the RNAPII core, rpb1‐1, rpb1‐S751F and rpb9∆, cause a defect in replication fork progression, compensated for by additional origin firing, as the main action responsible for instability. The transcription elongation defects of rpb1‐S751F and rpb9∆ plus our observation that rpb1‐1 causes RNAPII retention on chromatin suggest that RNAPII could participate in facilitating fork progression upon a transcription‐replication encounter. Our results imply that the RNAPII or ancillary factors actively help prevent transcription‐associated genome instability.. Research was funded by grants from the Spanish Ministry of Economy and Competitiveness (Consolider 2010 CSD2007-0015 and BFU2010-16372), the Junta de Andalucía (CVI4567) and the European Union (FEDER). IF-A and JL-B were recipient of predoctoral training grants from the Spanish Ministry of Economy and Competitiveness and the Instituto Carlos III, respectively.Peer Reviewe

The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage

<div><p>RNA-DNA hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1’s helicase activity plays a crucial role in responding to persistent RNA-DNA hybrids.</p></div

Mph1 forms foci at telomeres during senescence, when RNA-DNA hybrids accumulate.

<p><b>A.</b> Both wild type and <i>tlc1</i> cells were derived from the <i>TLC1/tlc1</i> heterozygous diploid yAL95 and grown for approximately 60 population doublings (PD) before RNA was extracted from exponentially growing cells. Following reverse transcription with a telomeric sequence, TERRA levels were analysed at the indicated telomeres via qPCR with subtelomeric specific primer pairs (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007136#sec009" target="_blank">methods</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007136#pgen.1007136.s006" target="_blank">S1 Table</a> for details). Three biological replicates were used for each genotype. Error bars indicate 95% confidence intervals. * represents significance relative to wild type determined by Student’s t-test (P<0.05). <b>B.</b> Both wild type and <i>tlc1</i> cells were grown for approximately 60 population doublings. ChIP with the S9.6 antibody that specifically recognizes RNA-DNA hybrids followed by qPCR. Error bars represent 95% confidence intervals, *, significance relative to wild type as determined by a Student’s t-test (P<0.05). <b>C.</b> Representative images for senescing cells showing co-localization between Mph1-YFP, Rad52-yEmRFP and Cdc13-CFP. Scale bar, 3 μm. Quantification of Mph1 (<b>D</b>.) and Cdc13-foci (<b>E</b>.). RNase H1 was expressed from a plasmid (pBB39).</p

The SMC5/6 complex regulates Mph1 at RNA-DNA hybrids.

<p><b>A.</b> Yeast haploid cells with the indicated genotypes were generated following tetrad dissection of ySLG419. Cells were grown overnight in liquid YPD at 23°C and spotted in 10-fold serial dilutions at the indicated temperatures on YPD agar. Digital images were acquired following 2 days of incubation. <b>B.</b> Haploids were derived from dissection of ySLG419 and ten-fold serial dilutions were spotted. Images were taken after 2 days. <b>C.</b> <i>MPH1</i>-dependent accumulation of Rad52 foci in <i>rnh201 smc6-9</i> mutants. Spontaneous Rad52-mCherry foci were quantified in wild type (yBL1052), <i>mph1</i> (YBL1051), <i>smc6-9</i> (yBL1047), <i>rnh201</i> (yBL1053), <i>mph1 smc6-9</i> (yBL1050), <i>rnh201 smc6-9</i> (yBL1048), and <i>mph1 smc6-9 rnh201</i> (yBL1049) after shifting an exponentially growing culture (OD₆₀₀ = 0.3) from 25°C to 30°C for 3 hours in SC medium supplemented with 100 μg/ml adenine. Upper panel: Representative images of Rad52 foci. Arrowheads indicate foci. Scale bar, 3 μm. Lower panel: Quantification of Rad52 foci. Two replicates of 200–600 cells were examined. Error bars indicate 95% confidence intervals. *, significance relative to the wild type determined by Fisher’s exact test (P < 0.05). <b>D.</b> The heterozygous diploid strain yBL1022 was micro-dissected, and the haploid offsprings with the indicated genotype were spotted in 10-fold serial dilutions onto YPD-agar and incubated for 2 days at 30°C. <b>E.</b> <i>smc6-9 rnh201 mph1</i> was transformed with either empty vector or <i>MPH1</i> expression plasmids (<i>WT</i> allele or helicase-dead mutants). The transformants were spotted on selective media and grown for three days at 25°C or for two days at 30°C. <b>F.</b> The indicated genotypes were derived via tetrad dissection of ySLG115. Cells were diluted daily (every 24 hours) to OD₆₀₀ 0.01 before the density was re-determined and cells were re-diluted. 6 biological replicates were used for every genotype indicated. Error bars represent SEM.</p

Mph1’s fork reversal activity might allow dissolution of RNA-DNA hybrids.

<p>When replication forks are paused at an RNA-DNA hybrid (either an R-loop as depicted, or consecutively incorporated ribonucleotides), Mph1 gets recruited to the stalled fork, potentially through its C-terminal interaction domain with RPA. Mph1 may directly remove the RNA-DNA hybrid (in the case of an R-loop) through its helicase activity, or promote the resolution by other factors (e.g. the RNase H enzymes as depicted). The Smc5/6 complex negatively regulates Mph1’s pro-recombinogenic activity at RNA-DNA hybrids to prevent toxic recombination intermediates from accumulating (see text for detailed explanation). Persistent R-loops at shortened telomeres may represent a natural scenario where such a regulation occurs.</p