Análisis de factores implicados en la respuesta celular al daño en el adn durante la replicación comosómica en saccharomyces cerevisiae

Resumen

Eukaryotic genomes are especially vulnerable during the S phase of the cell cycle, when chromosomes must be replicated. Chromosome replication is a complex process and, in addition, numerous exogenous and endogenous agents damage the DNA and interfere inevitably with the progression and stability of replication forks, challenging genome integrity. Failures to protect DNA replication forks or the inability to process stalled forks to finish chromosome duplication lead to genomic instability, a hallmark of cancer and other diseases. To cope with these problems, eukaryotic cells activate a DNA damage response that promotes the stabilization of replication forks, the repair or tolerance of DNA lesions, and the resumption of DNA synthesis after fork blocks. In this PhD Thesis, we have analysed the contribution of several pathways and proteins to the maintenance of genome stability during chromosome replication in the presence of DNA damage, using the budding yeast Saccharomyces cerevisiae as a model. We have shown that when the DNA template is damaged by the alkylating agent methyl methanesulfonate, base excision repair, homologous recombination and DNA damage tolerance pathways, together with a functional S-phase checkpoint, are essential for efficient progression of DNA replication forks and cell survival. In the absence of base excision repair, replication forks stall reversibly in cells exposed to MMS. This repair reaction is necessary to eliminate the DNA lesions that impede fork progression, and has to be coordinated with recombination and damage tolerance activities to avoid fork collapse and allow forks to resume and complete chromosome replication. We have also studied the role of the conserved heterodimeric nuclease Mus81- Mms4 from S. cerevisiae in the response to DNA damage during chromosomal replication. We have shown that this complex is required to maintain cell viability during S phase in the presence of DNA lesions caused by MMS. Cells lacking this nuclease fail to complete chromosome replication when they are treated with this compound, which would explain the loss of cell viability under these conditions. On the contrary, Mus81-Mms4 is not required to resume DNA synthesis when forks stall due to dNTP depletion by the action of hydroxyurea. Problems originated by exposure to MMS during S phase in the absence of Mus81-Mms4 are mostly reversible after inducing the expression of this complex. Moreover, Mus81-Mms4 can work after bulk DNA synthesis and therefore its function can be uncoupled from chromosomal replication. In addition, the regulatory subunit Mms4 binds to chromatin only when the DNA is damaged, which could help to understand how this nuclease is regulated. This work also shows that the Mus81-Mms4 nuclease cooperates with the Yen1 resolvase inthe response to DNA damage during S phase.