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The tumour suppressor SLX4 plays multiple roles in the maintenance of genome stability, acting as a scaffold for structure-specific endonucleases and other DNA repair proteins. It directly interacts with the mismatch repair (MMR) protein MSH2 but the significance of this interaction remained unknown until recent findings showing that MutSβ (MSH2-MSH3) stimulates in vitro the SLX4-dependent Holliday junction resolvase activity. Here, we characterize the mode of interaction between SLX4 and MSH2, which relies on an MSH2-interacting peptide (SHIP box) that drives interaction of SLX4 with both MutSβ and MutSα (MSH2-MSH6). While we show that this MSH2 binding domain is dispensable for the well-established role of SLX4 in interstrand crosslink repair, we find that it mediates inhibition of MutSα-dependent MMR by SLX4, unravelling an unanticipated function of SLX4., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The DNA damage response (DDR) preserves the genetic integrity of the cell by sensing and repairing damages after a genotoxic stress. Translesion Synthesis (TLS), an error-prone DNA damage tolerance pathway, is controlled by PCNA ubiquitination. In this work, we raise the question whether TLS is controlled locally or globally. Using a recently developed method that allows to follow the bypass of a single lesion inserted into the yeast genome, we show that (i) TLS is controlled locally at each individual lesion by PCNA ubiquitination, (ii) a single lesion is enough to induce PCNA ubiquitination and (iii) PCNA ubiquitination is imperative for TLS to occur. More importantly, we show that the activation of the DDR that follows a genotoxic stress does not increase TLS at individual lesions. We conclude that unlike the SOS response in bacteria, the eukaryotic DDR does not promote TLS and mutagenesis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In order to explore the mechanisms employed by living cells to deal with DNA alterations, we have developed a method by which we insert a modified DNA into a specific site of the yeast genome. This is achieved by the site-specific integration of a modified plasmid at a chosen locus of the genome of Saccharomyces cerevisiae, through the use of the Cre/lox recombination system. In the present work, we have used our method to insert a single UV lesion into the yeast genome, and studied how the balance between error-free and error-prone lesion bypass is regulated. We show that the inhibition of homologous recombination, either directly (by the inactivation of Rad51 recombinase) or through its control by preventing the polyubiquitination of PCNA (ubc13 mutant), leads to a strong increase in the use of Trans Lesion Synthesis (TLS). Such regulatory aspects of DNA damage tolerance could not have been observed with previous strategies using plasmid or randomly distributed DNA lesions, which shows the advantage of our new method. The very robust and precise integration of any modified DNA at any chosen locus of the yeast genome that we describe here is a powerful tool that will enable the exploration of many biological processes related to replication and repair of modified DNA., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The genome of all organisms is constantly threatened by numerous agents that cause DNA damage. When the replication fork encounters an unrepaired DNA lesion, two DNA damage tolerance pathways are possible: error-prone translesion synthesis (TLS) that requires specialized DNA polymerases, and error-free damage avoidance that relies on homologous recombination (HR). The balance between these two mechanisms is essential since it defines the level of mutagenesis during lesion bypass, allowing genetic variability and adaptation to the environment, but also introduces the risk of generating genome instability. Here we report that the mere proximity of replication-blocking lesions that arise in Escherichia coli's genome during a genotoxic stress leads to a strong increase in the use of the error-prone TLS. We show that this increase is caused by the local inhibition of HR due to the overlapping of single-stranded DNA regions generated downstream of the lesions. This increase in TLS is independent of SOS activation, but its mutagenic effect is additive with the one of SOS. Hence, the combination of SOS induction and lesions proximity leads to a strong increase in TLS that becomes the main lesion tolerance pathway used by the cell during a genotoxic stress.

The RecBCD complex is a key factor in DNA metabolism. This protein complex harbors a processive nuclease and two helicases activities that give it the ability to process duplex DNA ends. These enzymatic activities make RecBCD a major player in double strand break repair, conjugational recombination and degradation of linear DNA. In this work, we unravel a new role of the RecBCD complex in the processing of DNA single-strand gaps that are generated at DNA replication-blocking lesions. We show that independently of its nuclease or helicase activities, the entire RecBCD complex is required for recombinational repair of the gap and efficient translesion synthesis. Since none of the catalytic functions of RecBCD are required for those processes, we surmise that the complex acts as a structural element that stabilizes the blocked replication fork, allowing efficient DNA damage tolerance., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)