Regulation of Initial Steps of Chromosomal Breaks Repair

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Recombination is essential in maintaining genome stability, and even minor deficiency in recombinational double-strand break (DSB) repair pathways results in cancer or other severe diseases. The initial processing of DNA DSBs to single strands, a process termed DNA end resection, is the critical first step of homologous recombination needed for the loading of damage response and repair proteins. Resection is tightly controlled in the cell cycle and determines the usage of homologous recombination versus nonhomologous end joining for repair. In yeast and human cells, the Mre11-Rad50-Nbs1/Xrs2 complex initiates resection, whereas Exo1 or Sgs1- Dna2 mediates extensive resection. Systematic studies of resection have thus far only been done in euchromatin, whereas we propose to study it in three different types of silenced heterochromatin. We designed many new assays to examine resection within transcriptionally silent chromatin in fission yeast (Aim #1). The fission yeast system provides an excellent model organism for this study as chromatin features are well conserved with those in human cells. In Aim #2 we focus on control of one of the most mutagenic pathways of DSB repair called Break Induced Replication (BIR). BIR is normally used for the repair of a single-end DSB. Here the goal is to understand how this pathway is suppressed during the repair of two-ended DSBs. We focus on the role of ssDNA annealing by Rad52 and synchronous resection of two ends of a DSB by the Mre11-Rad50-Xrs2 complex. In Aim #3 we focus on the resection-independent function of Dna2 nuclease/helicase during homologous recombination.