Chromosomal breaks are repaired by high fidelity homologous recombination (HR) or by low fidelity nonhomologous end joining (NHEJ). Both pathways are essential in maintaining genome stability as even a minor deficiency in DSB repair pathways can result in cancer or other severe diseases. The initial processing of DNA double strand breaks (DSBs) to single strands, a process termed DNA end resection, is the critical first step of HR needed for loading of damage response and repair proteins. Resection is tightly controlled in the cell cycle and determines the usage of high fidelity HR and lower fidelity NHEJ for the repair of DSBs. As resection controls the fidelity of DSB repair it is imperative to define the mechanisms that execute or regulate resection. In yeast and human cells, the MRN (Mre11-Rad50-Nbs1) complex initiates resection, whereas Exo1 or Sgs1-Dna2 mediates extensive resection. The objective of this proposal is to define how the initial and two long-range resection pathways are controlled. We will establish new assays to follow DSB end resection in fission yeast. The fission yeast system provides a number of features well conserved with human. In the first aim, we will use newly designed assays in fission yeast to define a new function of Rad52 protein in controlling extensive resection, to elucidate the role of noncoding RNA in resection and recombination, and to compare resection within dense heterochromatin and loose euchromatin. In the second aim we will investigate how different types of stress (proteotoxic, osmotic, mitochondrial) affect DSB end resection and the fidelity of DSB repair. Crosstalk between stress response pathways and DSB repair enzymes will be investigated. A combination of genetic, molecular biology and biochemical approaches will be used to define the precise mechanism of resection and resection regulation. Our findings are relevant to human disease, cancer, drug resistance and genome evolution, and will provide a framework for future investigations of the initial steps of homologous recombination and the relationship between stress and the fidelity of DNA repair in other organisms including human.
The major goal of this project is to understand how cells regulate initial step of chromosomal breaks repair, a critical process for proper maintenance of the genome, for signaling and repair of DNA damage, and for the choice between high and low fidelity repair. All enzymes investigated here are well conserved in evolution, so the proposed research in the model organism is highly relevant to our understanding of the DNA repair processes in humans.
|Yu, Yang; Pham, Nhung; Xia, Bo et al. (2018) Dna2 nuclease deficiency results in large and complex DNA insertions at chromosomal breaks. Nature 564:287-290|