Papel del Cdc7 en la regulación de la recombinación homóloga durante la tolerancia a daños replicativos

Trabajo realizado en el Departamento de Biología Molecular, CABIMER y en el Departamento de Genética, Facultad de Biología, Universidad de Sevilla, para optar al grado de Doctor en Biología por la Lda. María José Cabello Lobato.La respuesta a daños en el ADN (DDR, DNA damage response) es una compleja red de mecanismos cuyo objetivo final es evitar la acumulación de mutaciones y reordenamientos genómicos que comprometen el correcto funcionamiento celular. Entre los principales mecanismos de la DDR se encuentran los puntos de control o ͞>checkpoints>, los mecanismos de reparación y tolerancia de daños y los programas de senescencia celular y apoptosis. En conjunto, esta respuesta funciona como una barrera que previene la inestabilidad genética, y por tanto clave para prevenir el desarrollo del cáncer. La correcta duplicación del ADN es esencial para el mantenimiento de la información genética y la progresión a lo largo del ciclo celular. La horquillas replicativas son estructuras altamente dinámicas y frágiles, tanto por la presencia de fragmentos de ADN de cadena sencilla (ssDNA) como por la descromatinización asociada con la síntesis de ADN, lo que las hace susceptibles de ser sustrato de nucleasas y otras enzimas implicadas en el procesamiento del ADN. Además en cada ciclo celular, numerosos obstáculos generados tanto por agentes endógenos y exógenos que causan lesiones en el ADN (aductos, sitios abásicos o incorporación de ribonucleótidos en vez de nucleótidos) como los propios obstáculos intrínsecos del ADN (zonas altamente compactadas, proteínas unidas, híbridos ADN-ARN, etc.) impiden el avance de la horquilla de replicación y, en algunos casos, llevan al colapso y la rotura de éstas. Por ello, las células han desarrollado diferentes mecanismos implicados en la protección, reparación y reiniciación de las horquillas, y mutaciones en estos mecanismos causan inestabilidad genética y están asociados con cáncer y numerosas enfermedades genéticas. Aunque la complejidad de estos mecanismos aumenta a lo largo de la evolución, los aspectos básicos están muy conservados desde levaduras hasta humanos. En este trabajo hemos utilizado Saccharomyces cerevisiae como organismo modelo por sus ventajas para la investigación genética y bioquímica.Peer Reviewe

Rad51 replication fork recruitment is required for DNA damage tolerance

Homologous recombination (HR) is essential for genome integrity. Recombination proteins participate in tolerating DNA lesions that interfere with DNA replication, but can also generate toxic recombination intermediates and genetic instability when they are not properly regulated. Here, we have studied the role of the recombination proteins Rad51 and Rad52 at replication forks and replicative DNA lesions. We show that Rad52 loads Rad51 onto unperturbed replication forks, where they facilitate replication of alkylated DNA by non-repair functions. The recruitment of Rad52 and Rad51 to chromatin during DNA replication is a prerequisite for the repair of the non-DSB DNA lesions, presumably single-stranded DNA gaps, which are generated during the replication of alkylated DNA. We also show that the repair of these lesions requires CDK1 and is not coupled to the fork but rather restricted to G2/M by the replicative checkpoint. We propose a new scenario for HR where Rad52 and Rad51 are recruited to the fork to promote DNA damage tolerance by distinct and cell cycle-regulated replicative and repair functions. © 2013 European Molecular Biology Organization.RG‐P and MJC‐L were recipients of pre‐doctoral training grants (FPI) from the Spanish Ministry of Science. AMM‐C was a recipient of a contract of the Juan de la Cierva program from the Spanish government. Research was funded by the Spanish Ministry of Science (BFU2006‐08336 and BFU2009‐09036) and the Junta de Andalucia (P07‐CVI‐032000).Peer Reviewe

La fosforilación de la helicasa Mcm2 por Cdc7 es necesaria para la reparación recombinacional de daños replicativos

Trabajo presentado en el XXXVI Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Madrid del 4 al 6 de septiembre de 2013La correcta duplicación del ADN es esencial para el mantenimiento de la información genética y la progresión a lo largo del ciclo celular, por lo que lesiones en el ADN que interfieren con el avance de las horquillas de replicación suponen una amenaza importante para la viabilidad celular. Las células disponen de mecanismos de tolerancia a estos daños que priorizan el avance de las horquillas sobre la reparación de los daños. Estos mecanismos pueden introducir o no mutaciones, dependiendo de que utilicen la cadena dañada (TLS; translesion synthesis) o la cromátida hermana (TS; template switching) como molde. En ambos casos el proceso está asociado a la acumulación de ADN de cadena sencilla (ssDNA) que debe ser reparado. Las proteínas de recombinación homóloga Rad52 y Rad51 participan en la ruta de TS mediante funciones replicativas y reparacionales reguladas a lo largo del ciclo. Rad52 y Rad51 son reclutados a las horquillas en ausencia de daño y facilitan el avance de éstas a través del ADN dañado, quedando unidas a los fragmentos de ssDNA cuya reparación promueven una vez terminada la replicación. En este trabajo hemos estudiado el papel de la quinasa de fase S Cdc7 en la tolerancia a daños replicativos generados por el agente alquilante MMS. La fosforilación de la helicasa replicativa MCM2-7 por Cdc7 es esencial para la iniciación de la replicación. Adicionalmente, Cdc7 participa en los procesos de tolerancia a daño asociados a la ruta de TLS, aunque los mecanismos moleculares mediante los que actúa se desconocen. Presentamos evidencias de que Cdc7 participa también en la ruta recombinacional de tolerancia a daños replicativos mediante un mecanismo que requiere la fosforilación de MCM2 y que regula la unión de Rad52 al DNA dañado. Estos datos abren la posibilidad de que Cdc7 actúe como un interruptor molecular que controle las rutas de TLS y de TS, y por tanto la acumulación de mutaciones durante la respuesta a daños en el ADN, de especial importancia en los procesos tumorales.Peer reviewe

Cdc7, Rad53 and Asf1 stabilize Rad52 binding to replicative DNA damage

Póster presentado en AbCam Meeting >Mechanism of Recombination: 50th Anniversary Meeting of the Holliday Model>, celebrado en Alicante del 19 al 23 de mayo de 2014The response to DNA lesions that impair the advance of replication forks relies on DNA damage tolerance (DDT) mechanisms, which facilitate fork bypass across the lesions and repair of the ssDNA fragments generated during this process. One of these mechanisms is Homologous Recombination (HR), which uses the information of an intact template to repair DNA breaks. We have recently shown that the recombination proteins Rad52 and Rad51 contribute to tolerate replicative DNA damage through replicative and repair activities, which are regulated during the cell cycle. In S phase, Rad52 and Rad51 travel with the fork and facilitate replication bypass across the lesion by unknown mechanisms. Repriming of DNA synthesis downstream of the lesion leaves Rad52 and Rad51 loaded at the ssDNA lesions left behind the fork, which are repaired at HR centers formed in G2/M (González-Prieto et al. 2013, EMBO J 32:1307). Here, we report a role for the kinase Cdc7 in promoting the loading/stabilization of Rad52 to the ssDNA lesions generated upon treatment with the alkylating agent methyl-methane sulfonate (MMS). This effect is partially mediated by phosphorylation of Mcm2. In addition, we show that Rad53, which requires the kinase activity of Cdc7 for full activation, is also required for Rad52 binding to DNA damage. Notably, Mec1 does not share this function. In addition, we have observed that the histone chaperone Asf1, which forms a complex with Rad53, is also required for Rad52 binding to replicative DNA damage. This role is independent of histone H3K56 acetylation and chromatin assembly, but also of the interactions between Asf1 with Rad53 and histones. We are currently exploring the mechanisms by which Cdc7, Rad53 and Asf1 stabilize Rad52 at replicative lesions during S phase.Peer Reviewe

In Vivo Binding of Recombination Proteins to Non-DSB DNA Lesions and to Replication Forks

Homologous recombination (HR) has been extensively studied in response to DNA double-strand breaks (DSBs). In contrast, much less is known about how HR deals with DNA lesions other than DSBs (e.g., at single-stranded DNA) and replication forks, despite the fact that these DNA structures are associated with most spontaneous recombination events. A major handicap for studying the role of HR at non-DSB DNA lesions and replication forks is the difficulty of discriminating whether a recombination protein is associated with the non-DSB lesion per se or rather with a DSB generated during their processing. Here, we describe a method to follow the in vivo binding of recombination proteins to non-DSB DNA lesions and replication forks. This approach is based on the cleavage and subsequent electrophoretic analysis of the target DNA by the recombination protein fused to the micrococcal nuclease.This work was supported by grants BFU2012-38171 and BFU2015-63698-P from the Spanish government.Peer reviewe

Defective histone supply causes condensin-dependent chromatin alterations, SAC activation and chromosome decatenation impairment

The structural organization of chromosomes is essential for their correct function and dynamics during the cell cycle. The assembly of DNA into chromatin provides the substrate for topoisomerases and condensins, which introduce the different levels of superhelical torsion required for DNA metabolism. In particular, Top2 and condensin are directly involved in both the resolution of precatenanes that form during replication and the formation of the intramolecular loop that detects tension at the centromeric chromatin during chromosome biorientation. Here we show that histone depletion activates the spindle assembly checkpoint (SAC) and impairs sister chromatid decatenation, leading to chromosome mis-segregation and lethality in the absence of the SAC. We demonstrate that histone depletion impairs chromosome biorientation and activates the Aurora-dependent pathway, which detects tension problems at the kinetochore. Interestingly, SAC activation is suppressed by the absence of Top2 and Smc2, an essential component of condensin. Indeed, smc2-8 suppresses catenanes accumulation, mitotic arrest and growth defects induced by histone depletion at semi-permissive temperature. Remarkably, SAC activation by histone depletion is associated with condensin-mediated alterations of the centromeric chromatin. Therefore, our results reveal the importance of a precise interplay between histone supply and condensin/Top2 for pericentric chromatin structure, precatenanes resolution and centromere biorientationSpanish Ministry of Science [BFU2009-09036, BFU2012- 38171]; Spanish Government [to M.M.-P., M.J.C.-L.]. Funding for open access charge: Spanish Ministry of Science [BFU2012-38171].Peer Reviewe

Cdc7-dependent interactions of Mcm2-7 with G1-loaded Rad51 and Rad52 regulate the recombinational response to replicative damage

Resumen del póster presentado a la Conferencia Mechanisms of Recombination, celebrada en Alicante (España) del 16 al 20 de mayo de 2016.The response to DNA lesions that impair the advance of replication forks relies on DNA damage tolerance (DDT) mechanisms, which facilitate fork bypass across the lesions and repair of the ssDNA fragments generated during this process. One of these mechanisms is Homologous Recombination (HR), which uses the information of an intact template to repair the ssDNA fragments. An important mechanistic difference with the replication-independent process of double-strand break (DSB) repair by HR is that Rad52 and Rad51 travel with the forks and their loading into the ssDNA lesions is coupled to DNA replication. We show in Saccharomyces cerevisiae that Rad51 interacts physically with the replicative helicase MCM2-7 regardless of the presence of DNA damage, and that this interaction requires the continuous activity during S phase of the cell-cycle master kinase Cdc7. Remarkably, even though Rad51 and MCM2-7 are recruited independently to chromatin, they display similar kinetics of DNA binding during the cell cycle. In the absence of DNA damage they accumulate in G1 and are released as replication is completed, whereas in the presence of the alkylating agent methyl-methane sulfonate (MMS) they remain bound to chromatin, suggesting that the helicase MCM2-7 plays a role in DDT. The loading/stabilization of Rad51 in response to MMS requires the kinase activity of Cdc7. Likewise, the binding of Rad52 to ssDNA lesions generated upon treatment with MMS is impaired in a thermosensitive cdc7-4 allele at semi-permissive temperature, being this effect independent of the roles of Cdc7 on replication initiation, checkpoint activation or translesion synthesis. Instead, it is partially mediated by phosphorylation of Mcm2 at serines 164 and 170. However, neither cdc7-4 nor a phosphomimetic mcm2AA affect the association between Rad51 and MCM2-7, suggesting that Cdc7 maintains Rad52/Rad51/MCM2-7 at chromatin in response to DNA damage through a different mechanism. Consistent with these results a reduction in the kinase activity of Cdc7 affects the kinetics of sister-chromatid junctions and Rad52 repair in response to MMS. Overall, these results uncover a novel role for Cdc7 in the recombinational branch of DDT by regulating the binding of Rad51 to chromatin in coordination with the helicase MCM2-7.Peer Reviewe

A mechanism for the recruitment of recombination proteins during DNA damage tolerance

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.The recombination proteins Rad52 and Rad51 help the fork to pass through blocking lesions and to fill in the gaps of ssDNA generated during the process of lesion bypass. Their recruitment to the ssDNA lesions has obligatorily to occur during S phase. Here we show that Rad52 and Rad51 display the same kinetics of chromatin binding as the helicase Mcm2-7: they accumulate in G1, are released during replication, and remain bound to chromatin in the presence of replicative blocking lesions. This coordinated response requires Cdc7-dependent physical interactions between Rad51/Rad52 and the Mcm2-7 helicases that are not at the fork. Accordingly, reducing Cdc7 activity impairs sister-chromatid junction formation and ssDNA filling by recombination. Therefore, the loading of Rad51 and Rad52 in G1, together with a Mcm2-7-mediated mechanism that couples the fork advance with Rad52/Rad51 binding to replicative damage, provides a strategy to ensure the filling of the ssDNA lesions through an error-free repair mechanism.Peer reviewe

Rad51 and Rad52 supply to replicative DNA lesions relies on cell cycle regulated interactions with the Mcm2-7 helicase

Resumen del póster presentado a la EMBO Conference: The DNA damage response in cell physiology and disease, celebrada en Atenas (Grecia) del 2 al 6 de octubre de 2017.The recombination proteins Rad51 and Rad52 help the fork to bypass blocking lesions and fill in the gaps of ssDNA generated during this process of DNA damage tolerance (DDT). Their recruitment to the ssDNA lesions must occur during S phase. Here we show that Rad51, Rad52 and the Mcm2-7 helicase display similar patterns of chromatin binding: they accumulate in G1, are released during replication and remain bound to chromatin in the presence of replicative blocking lesions. Moreover, their binding to chromatin during S phase requires the kinase activity of Cdc7. This kinetics of chromatin binding is coordinated through physical interactions between Rad51 and Rad52 with Mcm2-7. These interactions are prevented at the pre-replication complex and at the replication fork, suggesting that they occur with the excess of Mcm2-7 helicases that are loaded in G1. Importantly, Cdc7-mediated Mcm2-7/Rad51/Rad52 accumulation at chromatin is required for ssDNA filling by recombination, supporting the relevance of these interactions and providing new functions for Cdc7 in DDT. We propose that chromatin recruitment of Rad51/Rad52 in G1, together with a mechanism mediated by Mcm2-7 to supply Rad51/Rad52 to replicative ssDNA lesions, provide a strategy for ensuring that ssDNA lesions are filled via an error-free repair mechanism.Peer Reviewe

Physical interactions of Rad51 and Rad52 with Mcm2-7 coordinate their binding to chromatin during the cell cycle and In response to DNA damage

Resumen del póster presentado a la 28th International Conference on Yeast Genetics and Molecular Biology (ICYGMB), celebrada en Praga (Czech Republic) del 27 de agosto al 1 de septiembre de 2017.The recombination proteins Rad51 and Rad52 help the fork to bypass blocking lesions and fill in the gaps of single-stranded DNA (ssDNA) generated during this process of DNA damage tolerance (DDT). In contrast to DNA double-strand breaks, Rad51 and Rad52 recruitment to the ssDNA lesions must occur during S phase. Here we show that Rad51 and Rad52 physically interact with the replicative helicase Mcm2-7 in G1. These interactions are lost during replication unless cells divide in the presence of replicative blocking lesions. They occur mostly in chromatin but are prevented at the pre-RC and at the replication forks, suggesting that Rad51 and Rad52 interact with the excess of Mcm2-7 helicases loaded in G1 and spread to the vicinity of the replication origins. Indeed, Mcm2-7 and Rad51 accumulate at a nuclease-insoluble chromatin fraction enriched in replication factors. Notably, these interactions coordinate the kinetics of chromatin binding of Mcm2-7, Rad51 and Rad52, which accumulate in G1, are released during S/G2 and are maintained in the presence of replicative DNA damage. This chromatin binding behavior is remarkable because homologous recombination is inactive in G1 and active during S/G2. Interestingly, the kinase activity of Cdc7 is required to preserve both the integrity of the Mcm2-7/Rad51/Rad52 complexes and the presence of these factors at chromatin during S/G2. Our results suggest novel roles for Cdc7 and Mcm2-7 in the regulation of the location of recombination proteins during DDT.Peer Reviewe