Análisis del papel de los componentes de la ruta RAD6/RAD18 de Saccharomyces cerevisiae en la tolerancia al daño en el DNA durante la replicación cromosómica

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-10-2014La ruta RAD6/RAD18 de tolerancia al daño en el DNA, conservada evolutivamente, tiene un papel fundamental en el mantenimiento de la estabilidad del genoma, permitiendo el baipás de lesiones no reparadas en el DNA que impiden el movimiento de las horquillas de replicación. Esta ruta puede subdividirse en dos ramas que muestran dos estrategias de tolerancia al daño en el DNA: síntesis de DNA a través de lesiones (TLS) y un mecanismo alternativo que permite evitar el daño. TLS utiliza polimerasas especializadas de baja fidelidad que pueden replicar directamente a través de las lesiones, mediante un proceso que es frecuentemente mutagénico. Por el contrario, la otra rama de la ruta lleva a cabo el baipás de las lesiones mediante un cambio de molde transitorio, en el que la cadena naciente bloqueada utiliza la cadena no dañada y recién sintetizada de la cromátida hermana como molde para la replicación de la zona de la lesión, en un proceso que resulta libre de errores. En esta tesis, hemos analizado la contribución de las dos ramas de la ruta RAD6/RAD18 a la tolerancia a las lesiones en el DNA que bloquean la replicación cromosómica, utilizando Saccharomyces cerevisiae como modelo eucariótico y el compuesto alquilante metil metanosulfonato (MMS) como agente genotóxico. Hemos encontrado que la rama libre de errores, mediada por Rad5, tiene el papel principal en esta respuesta, mientras que las polimerasas de síntesis a través de lesiones tienen solo una contribución menor. Hemos mostrado también que Rad5 es absolutamente necesaria para la progresión de las horquillas de replicación a través de un DNA dañado por MMS, lo que indica una función directa de esta proteína en las horquillas de replicación para la tolerancia al daño en el DNA. Además, hemos encontrado que las actividades E3- ubiquitina ligasa y ATPasa/helicasa de Rad5 son necesarias para la función de esta proteína durante la replicación cromosómica en presencia de daño en el DNA, y que estas actividades operan secuencialmente, no independientemente. Apoyando su papel específico durante la replicación, hemos mostrado también que la proteína Rad5 tiene un pico de expresión durante la fase S y que forma focos nucleares en respuesta al daño en el DNA durante la fase S. Si bien todos estos resultados indican una función de Rad5 acoplada a la replicación del DNA, hemos encontrado que la expresión de esta proteína después de que la mayor parte de la replicación del genoma se haya completado revierte el efecto causado en su ausencia por el MMS durante la fase S, lo que sugiere que también es posible un modo de acción post-replicativo de Rad5. En conjunto, los resultados obtenidos en esta tesis indican que la rama libre de errores de la ruta RAD6/RAD18 de tolerancia al daño en el DNA, dependiente de Rad5, constituye un sistema eficiente que asegura la compleción de la replicación cromosómica y la viabilidad celular bajo condiciones de daño en el DNA, al mismo tiempo que minimiza el riesgo de mutagénesis, contribuyendo al mantenimiento de la integridad del genomaThe evolutionarily conserved RAD6/RAD18 pathway of DNA damage tolerance plays a crucial role in genome stability maintenance, allowing the bypass of unrepaired DNA lesions that hamper replication forks. This pathway can be subdivided into two branches that show two strategies of DNA damage tolerance: translesion DNA synthesis (TLS) and the alternative damage avoidance subpathway. TLS uses specialized, lowfidelity DNA polymerases that can replicate directly across the lesions, in a process that is frequently mutagenic. In contrast, the damage avoidance branch mediates lesion-bypass by transient template switching, in which the blocked DNA nascent strand uses the newly synthesized undamaged strand of the sister chromatid as a template for replication over the DNA lesion, in a process that results error-free. In this PhD thesis, we have analysed the contribution of the two branches of the RAD6/RAD18 pathway to the tolerance of chromosome replication-blocking DNA lesions, using Saccharomyces cerevisiae as a eukaryotic model and the alkylating compound methyl methanesulfonate (MMS) as a DNA damaging-agent. We have found that the error-free sub-pathway, mediated by Rad5, has the principal role in this cellular response, whereas translesion synthesis polymerases make only a minor contribution. We have also shown that Rad5 is absolutely required for the progression of replication forks through MMS-damaged DNA, which indicates a direct function of this protein at forks for DNA damage tolerance. Moreover, we have found that the E3-ubiquitin ligase and ATPase/helicase activities of Rad5 are necessary for the function of this protein during chromosome replication in the presence of DNA damage, and that these activities operate sequentially, not independently. Supporting its specific role during replication, we have also shown that the Rad5 protein has a peak of expression during S-phase and that it forms nuclear foci in response to DNA damage during S-phase. While all these results indicate a Rad5 function coupled to DNA replication, we have found that the expression of this protein after bulk genome replication reverts the effects caused by MMS in its absence during S-phase, suggesting that a post-replicative mode of action of Rad5 is also possible. Altogether, the results obtained in this PhD thesis indicate that the error-free, Rad5-dependent, branch of the RAD6/RAD18 pathway of DNA damage tolerance constitutes a efficient system that ensures the completion of chromosome replication and cell viability under DNA-damaging conditions while minimising the risk of mutagenesis, thereby contributing to genome integrity maintenance

Temporal regulation of the Mus81-Mms4 endonuclease ensures cell survival under conditions of DNA damage

The structure-specific Mus81-Eme1/Mms4 endonuclease contributes importantly to DNA repair and genome integrity maintenance. Here, using budding yeast, we have studied its function and regulation during the cellular response to DNA damage and show that this endonuclease is necessary for successful chromosome replication and cell survival in the presence of DNA lesions that interfere with replication fork progression. On the contrary, Mus81-Mms4 is not required for coping with replicative stress originated by acute treatment with hydroxyurea (HU), which causes fork stalling. Despite its requirement for dealing with DNA lesions that hinder DNA replication, Mus81-Mms4 activation is not induced by DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. This post-replicative mode of action of Mus81-Mms4 limits its nucleolytic activity during S-phase, thus avoiding the potential cleavage of DNA substrates that could cause genomic instability during DNA replication. At the same time, it constitutes an efficient fail-safe mechanism for processing DNA intermediates that cannot be resolved by other proteins and persist after bulk DNA synthesis, which guarantees the completion of DNA repair and faithful chromosome replication when the DNA is damagedSpanish Ministry of Economy and Competitiveness [BFU2010-16989 and Consolider Ingenio CSD2007-00015 to J.A.T.]; Fundación Ramón Areces (Institutional Grant to the Centro de Biologıa Molecular Severo Ochoa); Spanish Ministry of Economy and Competitiveness (predoctoral fellowships to M.V.V and M.A.O-B.); Universidad Autónoma de Madrid (predoctoral fellowship to M.G-F.); Consejo Superior de Investigaciones Cientıficas (JAE-Doc contract to M.S.). Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2010-16989 and Consolider Ingenio CSD2007-00015]Peer Reviewe

Pif1-Family helicases support fork convergence during DNA replication termination in eukaryotes

The convergence of two DNA replication forks creates unique problems during DNA replication termination. In E. coli and SV40, the release of torsional strain by type II topoisomerases is critical for converging replisomes to complete DNA synthesis, but the pathways that mediate fork convergence in eukaryotes are unknown. We studied the convergence of reconstituted yeast replication forks that include all core replisome components and both type I and type II topoisomerases. We found that most converging forks stall at a very late stage, indicating a role for additional factors. We showed that the Pif1 and Rrm3 DNA helicases promote efficient fork convergence and completion of DNA synthesis, even in the absence of type II topoisomerase. Furthermore, Rrm3 and Pif1 are also important for termination of plasmid DNA replication in vivo. These findings identify a eukaryotic pathway for DNA replication termination that is distinct from previously characterized prokaryotic mechanisms

Rad5 plays a major role in the cellular response to DNA damage during chromosome replication

© 2014 The Authors. The RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired lesions that block replication forks. It is subdivided into two branches: translesion DNA synthesis, which is frequently error prone, and the error-free DNA-damage-avoidance subpathway. Here, we show that Rad5HLTF/SHPRH, which mediates the error-free branch, has a major role in the response to DNA damage caused by methyl methanesulfonate (MMS) during chromosome replication, whereas translesion synthesis polymerases make only a minor contribution. Both the ubiquitin-ligase and the ATPase/helicase activities of Rad5 are necessary for this cellular response. We show that Rad5 is required for the progression of replication forks through MMS-damaged DNA. Moreover, supporting its role during replication, this protein reaches maximum levels during S phase and forms subnuclear foci when replication occurs in the presence of DNA damage. Thus, Rad5 ensures the completion of chromosome replication under DNA-damaging conditions while minimizing the risk of mutagenesis, thereby contributing significantly to genome integrity maintenance.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; grants BFU2010-16989, BFU2013-43766Peer Reviewe