| DNA double strand break repair is essential for cell survival and genome stability. In eukaryotes, cells possess two conserved mechanisms to repair double strand breaks (DSBs): homologous recombination (HR) and non-homologous end joining (NHEJ). Each pathway requires a distinct set of repair enzymes and accessibility to substrate DNA is important for successful repair events. In eukaryotes, genomic DNA consists of a basic repeating unit called nucleosome, and forms highly ordered chromatin structure. Cells have evolved essential mechanisms to overcome this natural barrier for DNA transactions: post-translational modification of histone tails or chromatin remodeling by ATP-dependent chromatin remodelers. RSC, a member of SWI/SNF like chromatin remodeling complex, is involved in many nuclear processes such as gene expression, sister chromatid cohesion and faithful chromosome segregations. Furthermore, subunits of RSC complex rsc30 and rsc8 carboxyl terminal deletion mutants have been identified as novel end-joining factors. The goal of this dissertation is to investigate the role of RSC complex in NHEJ and HR.;To reveal the precise functions of RSC in NHEJ, I have examined the association of a core ATPase subunit of RSC, Sth1, at the HO break site by chromatin immunoprecipitation. I discovered that Sth1 begins to associate rapidly at DSB sites and its binding increased to four fold at the break site in donorless yeast strain. Notably, Sth1 association was reduced in the absence of Mre11, Ku, and rsc30 suggesting these repair factors stimulate recruitment of RSC to the break site. Collectively, these results support the model that RSC directly participates in NHEJ repair.;Although RSC is clearly involved in NHEJ, evidences suggeste that RSC may also function in HR. Severe gamma-ray sensitivity of mutants lacking nonessential RSC genes such as RSC2, RSC7, and HTL1 cannot be explained by NHEJ defect, due to the fact that the deletion of Ku70 does not enhance sensitivity the cells to irradiation. Additionally, RSC physically interacts with Mre11, which functions in both NHEJ and HR. To investigate the role of RSC in HR repair, I first determined sensitivity profiles of all available RSC mutants to a variety of genotoxic stress including high temperature and multiple DSB causing agents. I found that several rsc mutants fail to grow at high temperature and exhibit signs of chromosome instability. I also discovered that many rsc mutants are hypersensitive to DSB causative agents. Furthermore, RSC protects cells from genotoxic damage in Rad59-dependent manner. The results are further confirmed by the physical interactions between Rsc1 and Rsc2 with Rad59 in yeast two hybrid and biochemical assays.;Defects in DNA damage checkpoint activation can lead to sensitivity to DSB. Therefore, I tested if rsc mutants were deficient in the establishment of damage-induced checkpoint arrest by detecting the Rad53 activation and Ddc2-GFP focus formation in the absence of rsc using immunoblot assays and live cell imaging assay, respectively. I found that DNA damage checkpoint signaling is intact in rsc mutants.;I systematically investigated whether RSC is required for specific types of HR including mating type switching gene conversion, single strand annealing, break induced recombination repair and ectopic gene conversion. The results suggested that RSC is not defective in any of these HR pathways. Next, I tested whether RSC functions in recombination between sisters using pulse field gel electrophoresis and unequal sister chromatid exchange using the specific genetic assay. I found that recombination between sister chromatids is severely defective in rsc7 mutant. Furthermore, end resection was significantly delayed in rsc mutants, and the association of Mre11 at the break site was dramatically reduced in ChIP assays and live cell imaging assays. I also found that cohesin subunits, Smc-1 and Mcd-1, are not efficiently associated at DNA break in the absence of Rsc2 or Rsc7. Collectively, I propose that RSC promotes recombination between sister chromatids by facilitating the damage-induced loading of cohesions at DNA breaks. |
