An appropriate cellular response to DNA damage, which includes damage recognition, cell cycle checkpoint activation, and repair, is crucial for the maintenance of genome integrity. Since perturbation of this response can jeopardize cell function and survival, it is important to understand the underlying molecular mechanism. Cohesin is an essential multiprotein complex that associates with DNA during interphase and is involved in the establishment and maintenance of sister chromatid cohesion. Genetic evidence suggests that cohesin also plays a role in DNA double strand break (DSB) response/repair. However, it was unclear whether this was due to general perturbation of cohesion, or whether cohesin has a specialized role at the damage site. Recently, my laboratory has developed a method to introduce damage at a discrete site in the cell nucleus using laser microirradiation, allowing subsequent immunofluorescent detection of factors recruited to the damage site. This system provides a unique tool to study the kinetics of, and the requirement(s) for, factor recruitment/modification at the sites of DNA damage in vivo. Using this system, we demonstrated that human cohesin is recruited to the damage in both an S/G2-specific and the DSB repair factor Mrel l/Rad50- dependent manner. Based on these results, we hypothesize that cohesin has a specialized role in cell cycle- specific DSB response/repair, most likely in homologous recombination repair (HR), by establishing the local sister chromatid cohesion at the damage sites. The objective of this project is Loexamine the mechanism of cohesin recruitment to DSB damage to address its role at the damage sites.
Specific aims are 1) analysis of cohesin recruitment to DNA damage using the laser system complemented by chromatin crosslinking and immunoprecipitation (CHIP) of restriction enzyme-induced DNA breaks, 2) biochemical dissection of the domain(s) within the cohesin complex that is required for DNA damage recognition/targeting, and 3) identification and characterization of proteins that dictate DNA damage recognition by cohesin, including the factors important for the establishment of sister chromatid cohesion for mitosis, using RNAi and/or mutant cells and protein interaction assays. Understanding the role of cohesin in DNA repair will provide an important insight into the mechanism of the DNA damage response in mammalian cells and may provide a novel marker or a potential therapeutic target for cancer diagnosis and treatment.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA100710-04
Application #
7228422
Study Section
Special Emphasis Panel (ZRG1-CDF-2 (90))
Program Officer
Pelroy, Richard
Project Start
2004-07-01
Project End
2009-04-30
Budget Start
2007-05-21
Budget End
2008-04-30
Support Year
4
Fiscal Year
2007
Total Cost
$231,763
Indirect Cost
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Tuzon, Creighton T; Spektor, Tanya; Kong, Xiaodong et al. (2014) Concerted activities of distinct H4K20 methyltransferases at DNA double-strand breaks regulate 53BP1 nucleation and NHEJ-directed repair. Cell Rep 8:430-8
Kong, Xiangduo; Ball Jr, Alexander R; Pham, Hoang Xuan et al. (2014) Distinct functions of human cohesin-SA1 and cohesin-SA2 in double-strand break repair. Mol Cell Biol 34:685-98
Wu, Nan; Kong, Xiangduo; Ji, Zhejian et al. (2012) Scc1 sumoylation by Mms21 promotes sister chromatid recombination through counteracting Wapl. Genes Dev 26:1473-85
Kong, Xiangduo; Stephens, Jared; Ball Jr, Alexander R et al. (2011) Condensin I recruitment to base damage-enriched DNA lesions is modulated by PARP1. PLoS One 6:e23548
Ball Jr, Alexander R; Yokomori, Kyoko (2011) Damage site chromatin: open or closed? Curr Opin Cell Biol 23:277-83
Zeng, Weihua; Ball Jr, Alexander R; Yokomori, Kyoko (2010) HP1: heterochromatin binding proteins working the genome. Epigenetics 5:287-92
Gomez-Godinez, Veronica; Wu, Tao; Sherman, Adria J et al. (2010) Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation. Nucleic Acids Res 38:e202
Stephens, Jared; Mohanty, Samarendra K; Genc, Suzanne et al. (2009) Spatially sculpted laser scissors for study of DNA damage and repair. J Biomed Opt 14:054004
Yusufzai, Timur; Kong, Xiangduo; Yokomori, Kyoko et al. (2009) The annealing helicase HARP is recruited to DNA repair sites via an interaction with RPA. Genes Dev 23:2400-4
Kong, Xiangduo; Mohanty, Samarendra K; Stephens, Jared et al. (2009) Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells. Nucleic Acids Res 37:e68

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