Mechanism and regulation of DNA double-strand break repair

染色体双链断裂（DSBs）是在正常细胞中自发发生的细胞毒性病变

Chromosomal double strand breaks (DSBs) are cytotoxic lesions that occur spontaneously during normal cell metabolism or following treatment of cells with DNA-damaging agents. If unrepaired or repaired inappropriately, DSBs can lead to profoundly detrimental events, such as chromosome loss, deletions, or translocations. Defects in the repair of DSBs cause genomic instability, manifested as immunological, development or neurological defects, and predisposition to cancer. The toxicity of DSBs is exploited for radiation and chemotherapy, as well as targeted therapies directed against specific DNA repair proteins. Thus, understanding the mechanisms of DSB repair is of fundamental importance and has practical application for development of new therapeutics and uncovering pathways to resistance. Homologous recombination (HR), one of the two main mechanisms to repair DSBs, employs extensive homology and templated DNA synthesis to restore the broken chromosome. In addition to its role in repairing DSBs, HR has emerged as a prominent mechanism to restart stalled or collapsed replication forks. The overall goal of our research program is to decipher the mechanisms of homology dependent DSB repair, using the yeast Saccharomyces cerevisiae as a model system. The first part of our program builds on our previous studies showing that the conserved Mre11-Rad50-Xrs2 (MRX) complex initiates end resection, the first step of HR, while more extensive processing of the 5 terminated strands is catalyzed by Exo1 or Dna2- Sgs1. Specifically, we will use S1-seq and MNase-seq methodologies to determine whether chromatin remodeling precedes or is coupled to end resection, and will determine which resection mechanisms are impacted by loss of RSC, SNF, INO80 and Fun30 chromatin remodelers. The second part of our program focuses on the repair single-end DSBs produced by collapse of replication forks. We recently developed an efficient system to create a site-specific replication fork collapse using Cas9 nickase and have shown that cell survival is completely dependent on the MRX complex and Rad51. This system will allow us to define the elusive functions of MRX in sister-chromatid recombination and to identify novel factors that participate in collapsed fork repair. In the long term we believe that mechanisms under investigation in this proposal will provide new insight into genomic instability caused by replication stress and how chromatin structure modulates DSB repair.