RESUMEN
The RecQ helicases are conserved from bacteria to humans and play a critical role in genome stability. In humans, loss of RecQ gene function is associated with cancer predisposition and/or premature aging. Recent experiments have shown that the RecQ helicases function during distinct steps during DNA repair; DNA end resection, displacement-loop (D-loop) processing, branch migration, and resolution of double Holliday junctions (dHJs). RecQ function in these different processing steps has important implications for its role in repair of double-strand breaks (DSBs) that occur during DNA replication and meiosis, as well as at specific genomic loci such as telomeres.
Asunto(s)
Bacterias/enzimología , Reparación del ADN , RecQ Helicasas/metabolismo , Enfermedad/genética , Inestabilidad Genómica , Humanos , RecQ Helicasas/química , RecQ Helicasas/genéticaRESUMEN
Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.
Asunto(s)
ADN Helicasas/metabolismo , Reparación del ADN/genética , Recombinación Homóloga , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Daño del ADN/genética , Daño del ADN/efectos de la radiación , ADN Helicasas/genética , ADN Polimerasa II/genética , ADN Polimerasa II/metabolismo , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Reparación del ADN/efectos de la radiación , Replicación del ADN/genética , Replicación del ADN/efectos de la radiación , Inestabilidad Genómica , Mutación/genética , Antígeno Nuclear de Célula en Proliferación/genética , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteína SUMO-1/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sumoilación , Rayos Ultravioleta/efectos adversosRESUMEN
Life is maintained through alternating phases of cell division and quiescence. The causes and consequences of spontaneous mutations have been extensively explored in proliferating cells, and the major sources include errors of DNA replication and DNA repair. The foremost consequences are genetic variations within a cell population that can lead to heritable diseases and drive evolution. While most of our knowledge on DNA damage response and repair has been gained through cells actively dividing, it remains essential to also understand how DNA damage is metabolized in cells which are not dividing. In this review, we summarize the current knowledge concerning the type of lesions that arise in non-dividing budding and fission yeast cells, as well as the pathways used to repair them. We discuss the contribution of these models to our current understanding of age-related pathologies.
Asunto(s)
Reparación del ADN , Mutación , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Modelos BiológicosRESUMEN
Schizosaccharomyces pombe is an ascomycete fungus that divides by medial fission; it is thus commonly referred to as fission yeast, as opposed to the distantly related budding yeast Saccharomyces cerevisiae. The reproductive lifestyle of S. pombe relies on an efficient genetic sex determination system generating a 1:1 sex ratio and using alternating haploid/diploid phases in response to environmental conditions. In this review, we address how one haploid cell manages to generate two sister cells with opposite mating types, a prerequisite to conjugation and meiosis. This mating-type switching process depends on two highly efficient consecutive asymmetric cell divisions that rely on DNA replication, repair, and recombination as well as the structure and components of heterochromatin. We pay special attention to the intimate interplay between the genetic and epigenetic partners involved in this process to underscore the importance of basic research and its profound implication for a better understanding of chromatin biology.
Asunto(s)
Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Saccharomyces cerevisiae/genética , Reproducción/genética , Replicación del ADNRESUMEN
Saccharomyces cerevisiae Srs2 helicase plays at least two distinct functions. One is to prevent recombinational repair through its recruitment by sumoylated Proliferating Cell Nuclear Antigen (PCNA), evidenced in postreplication-repair deficient cells, and a second one is to eliminate potentially lethal intermediates formed by recombination proteins. Both actions are believed to involve the capacity of Srs2 to displace Rad51 upon translocation on single-stranded DNA (ssDNA), though a role of its helicase activity may be important to remove some toxic recombination structures. Here, we described two new mutants, srs2R1 and srs2R3, that have lost the ability to hinder recombinational repair in postreplication-repair mutants, but are still able to remove toxic recombination structures. Although the mutants present very similar phenotypes, the mutated proteins are differently affected in their biochemical activities. Srs2R1 has lost its capacity to interact with sumoylated PCNA while the biochemical activities of Srs2R3 are attenuated (ATPase, helicase, DNA binding and ability to displace Rad51 from ssDNA). In addition, crossover (CO) frequencies are increased in both mutants. The different roles of Srs2, in relation to its eventual recruitment by sumoylated PCNA, are discussed.
Asunto(s)
ADN Helicasas/genética , ADN Helicasas/metabolismo , Reparación del ADN , Antígeno Nuclear de Célula en Proliferación/metabolismo , Recombinación Genética , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN/ultraestructura , ADN Helicasas/química , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN/genética , Eliminación de Gen , Mutación , Recombinasa Rad51/ultraestructura , RecQ Helicasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Supresión Genética , Rayos UltravioletaRESUMEN
The genetics of quiescence is an emerging field compared to that of growth, yet both states generate spontaneous mutations and genetic diversity fueling evolution. Reconciling mutation rates in dividing conditions and mutation accumulation as a function of time in non-dividing situations remains a challenge. Nitrogen-starved fission yeast cells reversibly arrest proliferation, are metabolically active and highly resistant to a variety of stresses. Here, we show that mutations in stress- and mitogen-activated protein kinase (S/MAPK) signaling pathways are enriched in aging cultures. Targeted resequencing and competition experiments indicate that these mutants arise in the first month of quiescence and expand clonally during the second month at the expense of the parental population. Reconstitution experiments show that S/MAPK modules mediate the sacrifice of many cells for the benefit of some mutants. These findings suggest that non-dividing conditions promote genetic diversity to generate a social cellular environment prone to kin selection.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Mitosis , Mutación , Nitrógeno/fisiología , Schizosaccharomyces/genética , Schizosaccharomyces/fisiología , Técnicas de Cocultivo , ADN/metabolismo , Citometría de Flujo , Variación Genética , Genotipo , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Schizosaccharomyces pombe/genética , Análisis de Secuencia de ADN , Transducción de Señal , Procesos EstocásticosRESUMEN
Genetic and molecular studies have indicated that an epigenetic imprint at mat1, the sexual locus of fission yeast, initiates mating type switching. The polar DNA replication of mat1 generates an imprint on the Watson strand. The process by which the imprint is formed and maintained through the cell cycle remains unclear. To understand better the mechanism of imprint formation and stability, we characterized the recruitment of early players of mating type switching at the mat1 region. We found that the switch activating protein 1 (Sap1) is preferentially recruited inside the mat1M allele on a sequence (SS13) that enhances the imprint. The lysine specific demethylases, Lsd1/2, that control the replication fork pause at MPS1 and the formation of the imprint are specifically drafted inside of mat1, regardless of the allele. The CENP-B homolog, Abp1, is highly enriched next to mat1 but it is not required in the process. Additionally, we established the computational signature of the imprint. Using this signature, we show that both sides of the imprinted molecule are bound by Lsd1/2 and Sap1, suggesting a nucleoprotein protective structure defined as imprintosome.
RESUMEN
To maintain life across a fluctuating environment, cells alternate between phases of cell division and quiescence. During cell division, the spontaneous mutation rate is expressed as the probability of mutations per generation (Luria and Delbrück, 1943; Lea and Coulson, 1949), whereas during quiescence it will be expressed per unit of time. In this study, we report that during quiescence, the unicellular haploid fission yeast accumulates mutations as a linear function of time. The novel mutational landscape of quiescence is characterized by insertion/deletion (indels) accumulating as fast as single nucleotide variants (SNVs), and elevated amounts of deletions. When we extended the study to 3 months of quiescence, we confirmed the replication-independent mutational spectrum at the whole-genome level of a clonally aged population and uncovered phenotypic variations that subject the cells to natural selection. Thus, our results support the idea that genomes continuously evolve under two alternating phases that will impact on their size and composition.
Asunto(s)
Mutación , Schizosaccharomyces/genética , Variación Biológica Poblacional , Schizosaccharomyces/fisiología , Selección Genética , Factores de TiempoRESUMEN
In budding yeast, loss of topoisomerase III, encoded by the TOP3 gene, leads to a genomic instability phenotype that includes slow growth, hyper-sensitivity to genotoxic agents, mitotic hyper-recombination, increased chromosome missegregation, and meiotic failure. Slow growth and other defects of top3 mutants are suppressed by mutation of SGS1, which encodes the only RecQ helicase in S. cerevisiae. sgs1 is epistatic to top3, suggesting that the two proteins act in the same pathway. To identify other factors that function in the Sgs1-Top3 pathway, we undertook a genetic screen for non-sgs1 suppressors of top3 defects. We found that slow growth and DNA damage sensitivity of top3 mutants are suppressed by mutations in RAD51, RAD54, RAD55, and RAD57. In contrast, top3 mutants show extreme synergistic growth defects with mutations in RAD50, MRE11, XRS2, RDH54, and RAD1. We also analyzed recombination at the SUP4-o region, showing that in a rad51, rad54, rad55, or rad57 background top3Delta does not increase recombination to the same degree as in a wild-type strain. These results suggest that the presence of the Rad51 homologous recombination complex in a top3 background facilitates creation of detrimental intermediates by Sgs1. We present a model wherein Rad51 helps recruit Sgs1-Top3 to sites of replicative damage.
Asunto(s)
ADN-Topoisomerasas de Tipo I/genética , Recombinación Genética/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas , Daño del ADN , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN , ADN-Topoisomerasas , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mutación , Recombinasa Rad51 , Proteína Recombinante y Reparadora de ADN Rad52 , RecQ Helicasas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Eukaryotic DNA polymerase δ (Pol δ) activity is crucial for chromosome replication and DNA repair and thus, plays an essential role in genome stability. In Saccharomyces cerevisiae, Pol δ is a heterotrimeric complex composed of the catalytic subunit Pol3, the structural B subunit Pol31, and Pol32, an additional auxiliary subunit. Pol3 interacts with Pol31 thanks to its C-terminal domain (CTD) and this interaction is of functional importance both in DNA replication and DNA repair. Interestingly, deletion of the last four C-terminal Pol3 residues, LSKW, in the pol3-ct mutant does not affect DNA replication but leads to defects in homologous recombination and in break-induced replication (BIR) repair pathways. The defect associated with pol3-ct could result from a defective interaction between Pol δ and a protein involved in recombination. However, we show that the LSKW motif is required for the interaction between Pol3 C-terminal end and Pol31. This loss of interaction is relevant in vivo since we found that pol3-ct confers HU sensitivity on its own and synthetic lethality with a POL32 deletion. Moreover, pol3-ct shows genetic interactions, both suppression and synthetic lethality, with POL31 mutant alleles. Structural analyses indicate that the B subunit of Pol δ displays a major conserved region at its surface and that pol31 alleles interacting with pol3-ct, correspond to substitutions of Pol31 amino acids that are situated in this particular region. Superimposition of our Pol31 model on the 3D architecture of the phylogenetically related DNA polymerase α (Pol α) suggests that Pol3 CTD interacts with the conserved region of Pol31, thus providing a molecular basis to understand the defects associated with pol3-ct. Taken together, our data highlight a stringent dependence on Pol δ complex stability in DNA repair.
Asunto(s)
Dominio Catalítico , ADN Polimerasa III/metabolismo , Reparación del ADN , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Alelos , Secuencia de Aminoácidos , ADN Polimerasa III/genética , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Genes Letales , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Recombinación Genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
DNA polymerases play a central role during homologous recombination (HR), but the identity of the enzyme(s) implicated remains elusive. The pol3-ct allele of the gene encoding the catalytic subunit of DNA polymerase delta (Poldelta) has highlighted a role for this polymerase in meiotic HR. We now address the ubiquitous role of Poldelta during HR in somatic cells. We find that pol3-ct affects gene conversion tract length during mitotic recombination whether the event is initiated by single-strand gaps following UV irradiation or by site-specific double-strand breaks. We show that the pol3-ct effects on gene conversion are completely independent of mismatch repair, indicating that shorter gene conversion tracts in pol3-ct correspond to shorter extensions of primed DNA synthesis. Interestingly, we find that shorter repair tracts do not favor synthesis-dependent strand annealing at the expense of double-strand-break repair. Finally, we show that the DNA polymerases that have been previously suspected to mediate HR repair synthesis (Polepsilon and Poleta) do not affect gene conversion during induced HR, including in the pol3-ct background. Our results argue strongly for the preferential recruitment of Poldelta during HR.
Asunto(s)
ADN Polimerasa III/metabolismo , ADN de Hongos/metabolismo , Ácidos Nucleicos Heterodúplex/metabolismo , Recombinación Genética , Saccharomyces cerevisiae/enzimología , Disparidad de Par Base/efectos de la radiación , Intercambio Genético/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de la radiación , ADN Polimerasa II/metabolismo , Reparación del ADN/efectos de la radiación , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Homocigoto , Pérdida de Heterocigocidad/efectos de la radiación , Viabilidad Microbiana/efectos de la radiación , Mitosis/efectos de la radiación , Modelos Genéticos , Proteína 2 Homóloga a MutS/metabolismo , Polimorfismo de Longitud del Fragmento de Restricción , Radiación Ionizante , Recombinación Genética/efectos de la radiación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Saccharomyces cerevisiae Srs2 helicase was shown to displace Rad51 in vitro upon translocation on single-stranded DNA. This activity is sufficient to account for its antirecombination effect and for the elimination of otherwise dead-end recombination intermediates. Roles for the helicase activity are yet unknown. Because cells lacking Srs2 show increased incidence of mitotic crossovers, it was postulated that Srs2 promotes synthesis-dependent strand annealing (SDSA) by unwinding the elongating invading strand from the donor strand. We report here that synthetic DNA structures that mimic D loops are good substrates for the Srs2 helicase activity, that Srs2 translocates on RPA-coated ssDNA, and, furthermore, that the helicase activity is largely stimulated by the presence of Rad51 nucleoprotein filaments on double-stranded DNA. These properties strongly support the idea that Srs2 actively prevents crossovers by promoting SDSA.
Asunto(s)
Intercambio Genético/fisiología , ADN Helicasas/metabolismo , ADN de Hongos/metabolismo , ADN de Cadena Simple/metabolismo , Recombinasa Rad51/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ADN Helicasas/genética , ADN de Hongos/genética , ADN de Cadena Simple/genética , Mitosis/fisiología , Recombinasa Rad51/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In vegetative cells, most recombination intermediates are metabolized without an association with a crossover (CO). The avoidance of COs allows for repair and prevents genomic rearrangements, potentially deleterious if the sequences involved are at ectopic locations. We have designed a system that permits to screen spontaneous intragenic recombination events in Saccharomyces cerevisiae and to investigate the CO outcome in different genetic contexts. We have analyzed the CO outcome in the absence of the Srs2 and Sgs1 helicases, DNA damage checkpoint proteins as well as in a mutant proliferating cell nuclear antigen (PCNA) and found that they all contribute to genome stability. Remarkably high effects on COs are mediated by srs2Delta, mrc1Delta and a pol30-RR mutation in PCNA. Our results support the view that Mrc1 plays a specific role in DNA replication, promoting the Srs2 recruitment to PCNA independently of checkpoint signaling. Srs2 would prevent formation of double Holliday junctions (dHJs) and thus CO formation. Sgs1 also negatively regulates CO formation but through a different process that resolves dHJs to yield non-CO products.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Intercambio Genético , ADN Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromosomas Fúngicos/genética , Daño del ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/genética , Endonucleasas/metabolismo , Genes Fúngicos/genética , Mitosis , Modelos Genéticos , Mutación/genética , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , RecQ Helicasas , Saccharomyces cerevisiae/citologíaRESUMEN
Toxic recombination events are detected in vegetative Saccharomyces cerevisiae cells through negative growth interactions between certain combinations of mutations. For example, mutations affecting both the Srs2 and Sgs1 helicases result in extremely poor growth, a phenotype suppressed by mutations in genes that govern early stages of recombination. Here, we identify a similar interaction involving double mutations affecting Sgs1 or Top3 and Mus81 or Mms4. We also find that the primary DNA structures that initiate these toxic recombination events cannot be double-strand breaks and thus are likely to be single-stranded DNA. We interpret our results in the context of the idea that replication stalling leaves single-stranded DNA, which can then be processed by two competing mechanisms: recombination and nonrecombination gap-filling. Functions involved in preventing toxic recombination would either avoid replicative defects or act on recombination intermediates. Our results suggest that Srs2 channels recombination intermediates back into the gap-filling route, whereas Sgs1Top3 and Mus81Mms4 are involved in recombination andor in replication to allow replication restart.