La AAA+ ATPasa Mgs1/WRNIP1 y su relación con la tolerancia al daño en el DNA durante la replicación cromosómica

Abstract

DNA damage tolerance mechanisms are essential for coping with lesions of the genetic material that remain unrepaired during S phase and interfere with replication forks. These mechanisms contribute to the completion of chromosome replication in every cell cycle, so they have a very important role in maintaining genomic integrity. In eukaryotic cells, the tolerance to DNA damage is mainly carried out by the RAD6/RAD18 pathway, which is divided into two branches that define two different modes of action: translesion DNA synthesis, performed by specialized polymerases and frequently mutagenic, and template switching, normally error-free, mediated by the E3 ubiquitin-ligase Rad5 in Saccharomyces cerevisiae (HLTF and SHPRH in human cells) and the E2 ubiquitinconjugating complex Ubc13-Mms2. In this doctoral thesis, as part of the analysis of factors that could modulate the tolerance to DNA damage, we found that the removal of the AAA+ ATPasa Mgs1 of S. cerevisiae (WRNIP1 in human cells, RarA/MgsA in bacteria), or its catalytic activity, significantly suppress the sensitivity of cells lacking Rad5 to treatment with DNAdamaging agents, such as methyl methanesulfonate (MMS), or replicative stress, such as hydroxyurea (HU). The elimination of Mgs1 in rad5D cells, or the inactivation of its ATPase activity, facilitate an alternative pathway to template switching that allows chromosomal replication under genotoxic stress conditions that would block replication forks due to the absence of Rad5. This replication depends on the polymerase d and the modification of PCNA in the Lys164 residue, and is mediated by a Rad52- and Rad59- dependent mode of homologous recombination. This alternative pathway requires the participation of Esc2 and Elg1, which would make possible the unloading of the anti-recombinase Srs2 and SUMO-PCNA from replication forks in mgs1Drad5D cells, allowing recombination locally. The presence of Mgs1 in cells lacking Rad5 prevents this recombination, so that replication forks are blocked due to the absence of both template switching and another pathway that allows tolerating DNA lesions to complete chromosome replication. This situation correlates with an accumulation of Elg1 in chromatin in rad5D cells, which could be a cause or a consequence of the stabilization of PCNA and Srs2 at forks. In RAD5+ cells, and under conditions of DNA damage or replicative stress, Mgs1 could contribute to inhibit homologous recombination at damaged or stalled replication forks, thus facilitating template switching as the principal mechanism of DNA damage tolerance during S phase