Genome stability relies on a network of interacting systems that detect and repair disturbances in the integrity of DNA. A central pathway that processes single-stranded DNA gaps and potentially lethal double-strand breaks in DNA that can occur during DNA replication employs a mechanism that repairs these lesions by homologous recombination. Primary components of this pathway in eukaryotes include Rad51 whose function is to search for DNA sequence homology and promote strand exchange, plus factors that enable, enhance, and control Rad51's activity. Among this constellation of Rad51-interacting proteins the master regulator is BRCA2, the product of a breast cancer susceptibility gene. BRCA2 in turn, is completely dependent upon its key interacting partner, Dss1, for activity. Through the concerted workings of this hierarchical complex comprised of Dss1, BRCA2, and Rad51, the homologous recombination system is harnessed to maintain stability and preserve the integrity of the genome. We seek to understand how BRCA2 functions in governing recombinational repair of DNA. We use the microbe Ustilago maydis as a model system for experimentation because it has a well-conserved BRCA2-homolog, Brh2, and is amenable to biochemical analysis and molecular genetic manipulations. The powerful attributes of this system open the way for gaining insight into BRCA2's molecular mechanism through avenues not immediately approachable in the vertebrate systems. Our primary objectives are to investigate the role of the two different DNA-binding regions of Brh2, to examine how Brh2 controls the activity of Rad51, to investigate how Brh2 serves in repair of DNA by homologous recombination by means beyond its established role in mediating delivery of Rad51 to single-stranded DNA, and to map a second DNA-binding region in human BRCA2. Our long-term goal is to provide knowledge of the function of BRCA2 and gain insight into how its dysfunction can lead to initiation of tumorigenic transformation.

Public Health Relevance

Initiating progression to tumorigenesis can be provoked when the cellular systems responsible for maintaining genome stability are compromised. Repairing damaged DNA by homologous recombination comprises one of these system dedicated to preserving genomic integrity. An important gene that regulates homologous recombination encodes the BRCA2 protein, mutations in which cause early-onset breast cancer. This is a proposal to learn about the fundamental action of how BRCA2 works in regulating homologous recombination by studying the orthologous or equivalent protein in the setting of a simple, genetically amenable microbe..

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079859-07
Application #
8461952
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Janes, Daniel E
Project Start
2007-07-12
Project End
2015-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
7
Fiscal Year
2013
Total Cost
$325,355
Indirect Cost
$132,837
Name
Weill Medical College of Cornell University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Zhou, Qingwen; Holloman, William K (2014) Dual DNA-binding domains shape the interaction of Brh2 with DNA. DNA Repair (Amst) 22:104-11
Kojic, Milorad; Holloman, William K (2012) Brh2 domain function distinguished by differential cellular responses to DNA damage and replication stress. Mol Microbiol 83:351-61
Holloman, William K (2011) Unraveling the mechanism of BRCA2 in homologous recombination. Nat Struct Mol Biol 18:748-54
Kojic, Milorad; Zhou, Qingwen; Fan, Jie et al. (2011) Mutational analysis of Brh2 reveals requirements for compensating mediator functions. Mol Microbiol 79:180-91
Zhou, Qingwen; Kojic, Milorad; Holloman, William K (2009) DNA-binding Domain within the Brh2 N Terminus Is the Primary Interaction Site for Association with DNA. J Biol Chem 284:8265-73
Mazloum, Nayef; Holloman, William K (2009) Second-end capture in DNA double-strand break repair promoted by Brh2 protein of Ustilago maydis. Mol Cell 33:160-70
Mazloum, Nayef; Holloman, William K (2009) Brh2 promotes a template-switching reaction enabling recombinational bypass of lesions during DNA synthesis. Mol Cell 36:620-30
Zhou, Qingwen; Mazloum, Nayef; Mao, Ninghui et al. (2009) Dss1 regulates interaction of Brh2 with DNA. Biochemistry 48:11929-38
Holloman, William K; Schirawski, Jan; Holliday, Robin (2008) The homologous recombination system of Ustilago maydis. Fungal Genet Biol 45 Suppl 1:S31-9
Mazloum, Nayef; Zhou, Qingwen; Holloman, William K (2008) D-loop formation by Brh2 protein of Ustilago maydis. Proc Natl Acad Sci U S A 105:524-9

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