The long-term objective of this research program is to understand the molecular mechanisms of homologous recombination in eukaryotes. Homologous recombination plays two essential roles during the life cycle of most organisms. It is required to repair lethal lesions in DNA, such as double-strand breaks, and it is essential for the pairing and segregation of homologous chromosomes during meiosis. The importance of these functions is evidenced by increased mutagenesis, and mitotic and meiotic aneuploidy in the absence of recombination. Since many human cancer-prone syndromes are associated with increased genome instability, an understanding of the mechanisms of recombination is likely to be important in understanding these diseases. Our goals are to identify the genes and proteins required for homologous recombination in the yeast, Saccharomyces cerevisiae. The resection of DNA ends to generate single-stranded DNA is an essential step in recombination. Mre11p is a nuclease and is required to process meiosis-specific double-strand breaks (DSBs), but the Mre11 nuclease activity is not required for processing HO-induced DSBs in mitotic cells. Nucleases that are redundant with Mre11 will be identified using genetic screens or by screening a library of yeast GSTfusion proteins. The Mre11 complex will be purified from mitotic and meiotic cells to identify novel associated factors with Mre11 and for biochemical characterization. We developed a colony color-sectoring assay to identify genes involved in mitotic recombination. Using this assay we characterized a RAD51-independent recombination pathway that requires the Rad52 homologue, Rad59. RAD51-independent recombination occurs by two major pathways, break-induced replication (BIR) and single-strand annealing (SSA). The role of RAD59 in BIR and oligonucleotide-directed gene targeting will be determined. Residues of Rad59 important for DNA repair and RAD51-independent recombination will be identified. Studies with the Rad52/Rad59 complex will address the role of the complex in strand annealing and strand invasion to prime DNA synthesis. Other genes that function in the RAD51 -independent recombination pathway will be identified using the colony-color sectoring assay and by interaction of their products with Rad59 in a two hybrid screen

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041784-15
Application #
6625817
Study Section
Genetics Study Section (GEN)
Program Officer
Anderson, Richard A
Project Start
1989-04-01
Project End
2006-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
15
Fiscal Year
2003
Total Cost
$406,593
Indirect Cost
Name
Columbia University (N.Y.)
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Gnügge, Robert; Oh, Julyun; Symington, Lorraine S (2018) Processing of DNA Double-Strand Breaks in Yeast. Methods Enzymol 600:1-24
Gnügge, Robert; Symington, Lorraine S (2017) Keeping it real: MRX-Sae2 clipping of natural substrates. Genes Dev 31:2311-2312
Ruff, Patrick; Donnianni, Roberto A; Glancy, Eleanor et al. (2016) RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication. Cell Rep 17:3359-3368
Wei, Jia; Zhang, Yixiao; Yu, Tai-Yuan et al. (2016) A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation. Cell Discov 2:16044
Symington, Lorraine S (2016) Mechanism and regulation of DNA end resection in eukaryotes. Crit Rev Biochem Mol Biol 51:195-212
Ciccia, Alberto; Symington, Lorraine S (2016) Stressing Out About RAD52. Mol Cell 64:1017-1019
Oh, Julyun; Al-Zain, Amr; Cannavo, Elda et al. (2016) Xrs2 Dependent and Independent Functions of the Mre11-Rad50 Complex. Mol Cell 64:405-415
Chen, Huan; Donnianni, Roberto A; Handa, Naofumi et al. (2015) Sae2 promotes DNA damage resistance by removing the Mre11-Rad50-Xrs2 complex from DNA and attenuating Rad53 signaling. Proc Natl Acad Sci U S A 112:E1880-7
Deng, Sarah K; Chen, Huan; Symington, Lorraine S (2015) Replication protein A prevents promiscuous annealing between short sequence homologies: Implications for genome integrity. Bioessays 37:305-13
Deng, Sarah K; Yin, Yi; Petes, Thomas D et al. (2015) Mre11-Sae2 and RPA Collaborate to Prevent Palindromic Gene Amplification. Mol Cell 60:500-8

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