DSBs are highly cytotoxic lesions that occur spontaneously during normal cellular processes, by treatment of cells with DNA damaging agents or as intermediates in programmed recombination events. If DSBs are left unrepaired, or repaired inappropriately, they can trigger mutagenic events including chromosome loss, deletions, duplications, inversions and translocations, events associated with tumorigenesis. The long-term objective of our research program is to understand how cells repair DNA double strand breaks (DSBs) to maintain genome integrity. The focus of the current proposal is the mechanism and regulation of 5'-3'end resection, which has emerged as a key regulatory step to govern repair pathway choice, and is the critical first step for homology-directed repair of DSBs. While much progress has been made in the identification of the key components of the end resection machinery, the mechanism of resection initiation is still in question.
The first aim of te proposal is to define the roles of Sae2 and Xrs2 in MRX complex functions. We identified several mre11 gain-of-function alleles, mre11-sas, that can bypass the DNA damage sensitivity of the sae2? mutant by extinguishing the DNA damage checkpoint. We plan to investigate the dynamics of MRX association with DNA ends in vivo and by single-molecule imaging, and determine the dependence on other components of the DNA damage checkpoint for suppression of sae2? by the mre11-sas alleles. The role of Xrs2 in the cellular response to DNA damage will be dissected using an MRE11-NLS fusion that bypasses the requirement for Xrs2 to transport Mre11 to the nucleus. The exonuclease-defective mre11-H59S allele will be used with sgs1? and exo1? mutations to test the model that resection initiates by a nick internal to the DNA ends.
The second aim follows up on our interesting new finding that RPA prevents repair of DSBs by microhomology-mediated end joining (MMEJ). This mutagenic repair mechanism is thought to be responsible for chromosome rearrangements in cancer cells. We will investigate the roles of RPA and Sae2 in suppression of chromosomal inverted duplications mediated by annealing between short inverted repeats.
The final aim i s to understand how the DNA damage checkpoint regulates end resection. The ssDNA formed by end resection is important to activate the DNA damage checkpoint;however, excessive ssDNA is detrimental to genome integrity and several studies indicate that the DNA damage checkpoint prevents extensive resection. We will identify the resection nucleases inhibited by Rad9 during the cell cycle and dissect the functions of Rad9 responsible for inhibition of end resection. Finally, the role of the DNA damage clamp in promoting or inhibiting end resection will be tested in the presence or absence of Rad9.

Public Health Relevance

The repair of DNA double-strand breaks (DSBs) is essential to maintain genome integrity and to guard against cancer in humans. In this proposal, genetic and physical approaches will be used to determine the roles of Sae2/CtIP in regulating the nuclease and checkpoint responses of the Mre11 complex, the role of RPA in suppressing genome rearrangements, and how the DNA damage checkpoint regulates the mechanisms used to repair DSBs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM041784-26A1
Application #
8817622
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Janes, Daniel E
Project Start
1989-04-01
Project End
2018-05-31
Budget Start
2014-09-05
Budget End
2015-05-31
Support Year
26
Fiscal Year
2014
Total Cost
$392,558
Indirect Cost
$143,759
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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