The long-term objective of this research program is to understand the molecular mechanisms of homologous recombination (HR) in a model eukaryote, the budding yeast Saccharomyces cerevisiae. HR plays an essential role in the maintenance of genome integrity by repairing cytotoxic lesions, such as double-strand breaks (DSBs), and it is essential for the pairing and segregation of homologous chromosomes during meiosis. The importance of these functions is evidenced by increased mutagenesis, chromosome rearrangements, and mitotic and meiotic aneuploidy in the absence of HR. Since many human cancer prone syndromes are associated with increased genome instability, an understanding of the mechanisms of HR is likely to be important in understanding these diseases. HR initiates at single-stranded DNA (ssDNA) formed by 5'-3' resection of DSBs, or at stalled replication forks. The single-stranded DNA formed functions as a sensor for the DNA damage response and is the substrate for Rad51 catalyzed strand exchange between the ssDNA bound by Rad51 and homologous duplex DNA. Until recently, the identity of the resection nuclease(s) was unknown. Our studies suggest a two-step mechanism for the 5'-3' resection of DNA ends: first the Mre11 complex and Sae2 remove short oligonucleotides from the 5'end, second, Exo1 or Sgs1 and Dna2 carry out extensive resection of the tailed intermediate. Having identified the critical factors involved in DNA end processing the aims of the new proposal are: (1) To determine the order of assembly of the resection proteins at DSBs, how end processing is regulated during the cell cycle, the contribution of each protein in processing different types of ends and how the DNA damage response regulates end resection. (2) The rates of spontaneous and DSB-induced recombination and chromosome loss will be measured in nuclease-defective mutants to determine the role of resection in maintenance of genome integrity. (3) The role of each of the identified resection nucleases in telomere length homeostasis, G-tail formation, degradation of uncapped telomeres and survival in the absence of telomerase will be determined.

Public Health Relevance

The repair of DNA double-strand breaks by homologous recombination is essential to maintain genome integrity and to guard against cancer in humans. In this proposal, genetic, cell biology and physical approaches will be used to determine the molecular mechanisms of DSB processing and cell cycle regulation of this process, and the role of end resection in telomere maintenance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041784-24
Application #
8247754
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Janes, Daniel E
Project Start
1989-04-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
24
Fiscal Year
2012
Total Cost
$500,326
Indirect Cost
$189,565
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
Symington, Lorraine S (2014) End resection at double-strand breaks: mechanism and regulation. Cold Spring Harb Perspect Biol 6:
Eissler, Christie L; Mazón, Gerard; Powers, Brendan L et al. (2014) The Cdk/cDc14 module controls activation of the Yen1 holliday junction resolvase to promote genome stability. Mol Cell 54:80-93
Deng, Sarah K; Gibb, Bryan; de Almeida, Mariana Justino et al. (2014) RPA antagonizes microhomology-mediated repair of DNA double-strand breaks. Nat Struct Mol Biol 21:405-12
Symington, Lorraine S; Rothstein, Rodney; Lisby, Michael (2014) Mechanisms and regulation of mitotic recombination in Saccharomyces cerevisiae. Genetics 198:795-835
Lee, Andrew H; Symington, Lorraine S; Fidock, David A (2014) DNA repair mechanisms and their biological roles in the malaria parasite Plasmodium falciparum. Microbiol Mol Biol Rev 78:469-86
Mazon, Gerard; Symington, Lorraine S (2013) Mph1 and Mus81-Mms4 prevent aberrant processing of mitotic recombination intermediates. Mol Cell 52:63-74
Chen, Huan; Lisby, Michael; Symington, Lorraine S (2013) RPA coordinates DNA end resection and prevents formation of DNA hairpins. Mol Cell 50:589-600
Klein, Hannah L; Symington, Lorraine S (2012) Sgs1--the maestro of recombination. Cell 149:257-9
Mott, Christina; Symington, Lorraine S (2011) RAD51-independent inverted-repeat recombination by a strand-annealing mechanism. DNA Repair (Amst) 10:408-15
Symington, Lorraine S; Gautier, Jean (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247-71

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