The general goal of the proposed research is to identify and define pathways that regulate homologous recombination as a DNA repair and DNA damage tolerance pathway in somatic cells. The fundamental approach is to use the budding yeast Saccharomyces cerevisiae as a lead organism and to establish novel mechanisms, which will be transferred by proof-of-principle experiments to humans to validate the general significance of the original paradigms.
The Specific Aims are: 1. Determine the mechanism of anti-recombination and crossover regulation by Srs2. Srs2 is an anti- recombinase that acts by dissociating Rad51 from ssDNA. In addition, Srs2 has been demonstrated genetically to limit crossover formation. Sub-aim A: We will determine the mechanism of crossover regulation by Srs2. We will determine the roles of Rad55-Rad57 and PCNA and their post-translational modifications on the Srs2 anti-recombination and anti-crossover activity. Sub-aim B: We will extend these findings to humans and determine which proteins proposed to exert Srs2 function are involved in crossover control. 2. Determine the mechanism of activation of Mus81-Mms4. Mus81-Mms4 is a structure-selective endonuclease that processes HR-dependent joint molecules. We have reconstituted in vitro the Cdc5 (Polo kinase)-mediated activation of Mus81-Mms4 and developed an experimental plan to differentiate between different mechanistic models of direct activation of the Mus81-Mms4 catalytic activity. Sub-aim A: We will establish the mechanism of activation using genetic and biochemical approaches in yeast. Sub-aim B: We will conduct proof-of-principle experiments to show that also human MUS81-EME1 is directly activated by Polo- kinase-mediated phosphorylation.
Homologous recombination is a central DNA pathway that addresses complex DNA damage such as DNA double-stranded breaks or interstrand crosslinks, as well as stalled or broken replication forks. Such lesions are induced by ionizing radiation and other common modalities of DNA damaged-based anti-cancer therapy. The work in this proposal will establish novel paradigms for the regulation of homologous recombination that will lead to an improved mechanistic understanding of this critical DNA repair pathway. Such knowledge is fundamental is developing biology-based approaches to improve efficacy and reduce side effects of DNA damage-based anti-tumor therapy.
Showing the most recent 10 out of 34 publications
