Nucleosome dynamics regulates DNA processing

DSBs, if not repaired properly, pose a serious threat to genome integrity. Improperly repaired DSBs can lead to loss of genetic material, to chromosomal duplications or translocations and to carcinogenesis1. The yeast Mre11–Rad50–Xrs2 (MRX) complex facilitates the recognition of DNA ends and commitment to repair by homologous recombination. Subsequently, the nucleolytic processing of the ends results in a 3′ single-stranded DNA (ssDNA) intermediate that is bound by replication protein A (RPA) to provide the signal for DNA damage–checkpoint activation2. The Rad52 protein helps displace RPA from ssDNA to promote assembly of a polymer of the Rad51 recombinase protein. The Rad51–ssDNA nucleoprotein filament then performs a search for a homologous DNA sequence to initiate errorfree repair3. Recent genetic studies have identified two redundant pathways for DNA end resection during homologous recombination, carried out by the yeast Sgs1–Dna2 and Exo1 enzymes4–6. In addition to DSB processing, Dna2 has an essential role during DNA replication, and Exo1 is involved in DNA mismatch repair (MMR), meiotic crossovers and the processing of stalled replication forks and improperly capped telomeres7–12. Recently, in vitro studies have demonstrated that efficient resection of DNA by the yeast Sgs1–Dna2 pathway requires a large contingent of proteins, including the MRX complex, RPA and the Top3–Rmi1 complex13. In contrast, Exo1 is sufficient to resect double-stranded DNA (dsDNA) ends in vitro14,15. The components of both S. cerevisiae resection pathways are conserved among eukaryotes, and defects in the human homologs of Sgs1 (BLM, WRN and RECQ4) have been linked with disease pathologies resulting in cancer predisposition and premature aging16. ATP-dependent chromatin-remodeling enzymes use the energy from ATP hydrolysis to disrupt histone-DNA contacts, which results in nucleosome sliding, eviction and/or histone exchange. In S. cerevisiae, a large number of remodeling enzymes, including RSC, SWI/SNF, INO80, SWR-C and Fun30, are recruited to chromatin regions adjacent to an HO endonuclease–induced DSB17–23. RSC appears to catalyze the eviction or mobilization of nucleosomes directly adjacent to the DSB, promoting the recruitment of the MRX complex and subsequent DNA processing18. The Ino80 complex is also required for efficient DNA resection, though the Fun30 enzyme plays a more dominant part in these events22,23. The Swr1 and Ino80 complexes regulate the dynamic incorporation of the histone variant H2A.Z within DSB chromatin, and H2A.Z has been reported to also affect DNA processing efficiency24,25. Although these ATP-dependent chromatinremodeling enzymes have been linked to DSB processing, it is not yet clear how they might facilitate this process. Here, to determine how chromatin structure affects DNA processing pathways, we use a combination of assays on in vitro–reconstituted chromatin and studies of yeast gene-deletion mutants. We find that the helicase activity of yeast Sgs1 and its human homolog, BLM, is reduced on nucleosomal substrates and that efficient resection by the Sgs1–Dna2–dependent machinery requires a nucleosome-free gap adjacent to the DSB. We also report that resection by Exo1 is blocked by nucleosomes and that processing activity can be partially restored by removal of the H2A–H2B dimers or incorporation of the histone variant H2A.Z. The SWR1-dependent incorporation of H2A.Z is found to also have a role in Exo1-dependent resection in vivo. Our study suggests that these two DNA processing pathways require distinct chromatin remodeling events to navigate chromatin structure, indicating complex interactions between chromatin dynamics and DNA repair.