DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can trigger mutagenic events such as chromosome loss, deletions, duplications or translocations, events associated with tumorigenesis. Two mechanistically distinct pathways have evolved to repair DSBs: homologous recombination (HR) and non- homologous end joining (NHEJ). An alternative mechanism of NHEJ has recently been described that is independent ofthe canonical end joining factors, utilizes microhomologies (MH) to align the ends and is associated with deletions and chromosome translocations. HR initiates by the nucleolytic degradation ofthe 5'strands of DSBs to yield 3'single-stranded DNA (ssDNA) tails, a process referred to as end resection. Replication protein A (RPA) is initially bound to the ssDNA, and then displaced by Rad51 to form a nucleoprotein filament that catalyzes homologous pairing and strand invasion. Studies in budding yeast have shown that the conserved Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex, together with Sae2/CtlP, initiates end resection while more extensive processing of the 5'strands requires the 5'-3'exonuclease, Exol, or the combined activities ofthe Sgsl/BLM helicase and Dna2 endonuclease. We hypothesize that the initiation of end processing by the Mre11-Rad50-Xrs2/NBS1 complex and Sae2/CtlP prevents repair by NHEJ and promotes HR;however, end resection has the potential to reveal MH internal to the break site that can be used to align ends for mutagenic MH mediated end joining (MMEJ). While HR is likely to be favored if cells form long tracts of ssDNA to assemble Rad51 nucleoprotein filaments, short ssDNA tracts might be less efficiently used for HR and directed to the MMEJ pathway. The first two aims of this project will address the role of resection initiation, in particular the role of Sae2/CtlP, extensive resection, RPA binding and assembly of the Rad51 recombinase in repair pathway choice in budding yeast. To carry out these studies we have developed novel genetic assays that will allow us to distinguish between different modes of repair. For the third aim we plan to use physical assays to monitor DNA end resection of a site-specific DSB in mouse cells to determine the domains of CtlP required for end resection, and interplay with NHEJ and 53BP1.

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 how the processing of DSBs by Sae2/CtlP, and cell cycle regulation of this process, influence the pathways used to repair DSBs.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
1P01CA174653-01A1
Application #
8608845
Study Section
Special Emphasis Panel ()
Project Start
Project End
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
1
Fiscal Year
2014
Total Cost
$304,630
Indirect Cost
$114,236
Name
Columbia University
Department
Type
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Symington, Lorraine S (2016) Mechanism and regulation of DNA end resection in eukaryotes. Crit Rev Biochem Mol Biol 51:195-212
Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa et al. (2016) Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors. Elife 5:
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
Aparicio, Tomas; Baer, Richard; Gottesman, Max et al. (2016) MRN, CtIP, and BRCA1 mediate repair of topoisomerase II-DNA adducts. J Cell Biol 212:399-408
Reczek, Colleen R; Shakya, Reena; Miteva, Yana et al. (2016) The DNA resection protein CtIP promotes mammary tumorigenesis. Oncotarget 7:32172-83
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
Yamamoto, K; Lee, B J; Li, C et al. (2015) Early B-cell-specific inactivation of ATM synergizes with ectopic CyclinD1 expression to promote pre-germinal center B-cell lymphomas in mice. Leukemia 29:1414-24
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; Yin, Yi; Petes, Thomas D et al. (2015) Mre11-Sae2 and RPA Collaborate to Prevent Palindromic Gene Amplification. Mol Cell 60:500-8
Sato, Mai; Rodriguez-Barrueco, Ruth; Yu, Jiyang et al. (2015) MYC is a critical target of FBXW7. Oncotarget 6:3292-305

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