Repair of DNA double-strand breaks (DSBs) protects cells and organisms, as well as their genome integrity. Since DSB repair occurs in the context of chromatin, chromatin must be modified to prevent it from inhibiting DSB repair. Evidence supports the role of histone modifications and ATP-dependent chromatin remodeling in repair and signaling of chromosome DSBs. The key questions are, then, what the nature of chromatin altered by DSBs is and how remodeling of chromatin facilitates DSB repair. Here we report a chromatin alteration caused by a single HO endonuclease-generated DSB at the Saccharomyces cerevisiae MAT locus. The break induces rapid nucleosome migration to form histone-free DNA of a few hundred base pairs immediately adjacent to the break. The DSB-induced nucleosome repositioning appears independent of end processing, since it still occurs when the 5-to-3 degradation of the DNA end is markedly reduced. The tetracyclinecontrolled depletion of Sth1, the ATPase of RSC, or deletion of RSC2 severely reduces chromatin remodeling and loading of Mre11 and Yku proteins at the DSB. Depletion of Sth1 also reduces phosphorylation of H2A, processing, and joining of DSBs. We propose that RSC-mediated chromatin remodeling at the DSB prepares chromatin to allow repair machinery to access the break and is vital for efficient DSB repair.
Elimination of a double-strand break (DSB) flanked by direct repeat sequences is mediated by single-strand annealing (SSA), which relies on a distinct set of gene products involving recombination, mismatch repair, and nucleotide excision repair. Here, we screened for yeast mutants defective in SSA with a plasmid-based SSA assay coupled to a barcode microarray readout. The screen identified Yal027Wp/Saw1 (single-strand annealing weakened 1) and Slx4 besides other known SSA proteins. Saw1 interacts physically with Rad1/Rad10, Msh2/Msh3, and Rad52 proteins, and cells lacking SLX4 or SAW1 accumulate recombination intermediates blocked at the Rad1/Rad10-dependent 3' flap cleavage step. Slx4 and Saw1 also contribute to the integrity of ribosomal DNA arrays. Saw1 mutants that fail to interact with Rad1, but retain interaction with Rad52 and Msh2, are defective in 3' flap removal and SSA repair. Deletion of SAW1 abolished association of Rad1 at SSA intermediates in vivo. We propose that Saw1 targets Rad1/Rad10 to Rad52-coated recombination intermediates.
Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.
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