THE DNA DAMAGE CHECKPOINT REGULATES MISMATCH REPAIR COMPLEXES DURING SINGLE STRAND ANNEALING PATHWAY

By threatening genome stability, endogenous and exogenous sources have the potential to alter the fundamental building blocks of genetic information. The vast majority of lesions are single-strand DNA breaks caused by physiological processes such as DNA replication or oxidative stress during cellular metabolism. Moreover, single-strand DNA breaks can be converted to DNA double-strand breaks (DSBs), which are the most dangerous form of DNA lesions. DSBs are throught to be particularly relevant because their repair is more difficult then other types of DNA damage, potentially leading to chromosome rearrangements and loss of genetic information. Cells evolved multiple pathways to repair DNA anomalies and maintain genome integrity. Moreover, eukaryotic cells respond to DSBs by activating the DNA damage checkpoint (DDC), a surveillance network essential for coordinating a multifaceted response to DNA lesions. Each DSB repair pathway must be coordinated with a series of signalling responses that either halt cell division or cause cell death or adaptation to irreparable lesions. Despite extensive research into DNA repair pathways, many aspects of DSB processing and repair remain unknown. Failures in repair DSB or unfaithful repair can occur when specific DNA repair and/or checkpoint pathways are inactivated, or when lesions exceed the capability of DNA repair mechanisms. Among DSBs repair pathways, Single Strand Annealing (SSA) proceeds via annealing of DNA repeats, resulting in an intermediate with non– homologous 3ꞌ–ended tails (3' NHTs) that are removed by the Rad1 – Rad10 (human XPF – ERCC1) endonuclease complex together with Saw1 and Slx4. Furthermore, Slx4 with Rtt107 limits the binding of the DDC mediator Rad9/53BP1 in proximity of the DSB, reducing checkpoint signalling. We observed that the deletion of RAD9 gene rescues the defects in SSA of both rad1Δ and slx4Δ mutant cells. Therefore, we hypothesized that the DDC limits the activity of a nuclease and/or pathway in the absence of the main Rad1-axis for repair. We found that the deletion of PMS1 gene, which expresses an endonuclease involved in the mismatch repair, strongly reduced SSA repair of rad1∆rad9∆ cells by Southern blotting analysis in our genetic system. Moreover, by chromatin immunoprecipitation, rad1Δrad9Δ cells exhibited high Pms1 and Msh2 enrichment at the 3' NHTs, providing direct evidence of these mismatch repair proteins involvement in the processing of this DNA intermediate. In this thesis, the alternative pathway engaged for DSB repair by SSA in rad1∆rad9∆ cells is described and characterized, proposing a model through which Rad9/53BP1 and the DDC signalling regulate the interplay of different nucleases to process non-homologous 3ꞌ-ended DNA repair intermediates, with implication for chromosome rearrangement and genome instability in human cells. Moreover, I also contributed to characterizing a new function of Rad9/53BP1 during DSB repair by homologous recombination in yeast Saccharomyces cerevisiae. In particular, we found that Rad9 promotes crossover formation by limiting Sgs1 and Mph1 helicases during DSB repair. All of the findings presented in this thesis elucidate the molecular mechanisms aimed at preserving genome integrity, focusing on the role of Rad9/53BP1 and DDC signalling, with potential implications for cancer biology.