In humans, homologous recombination (HR) performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. HR is responsible for the repair of DNA double-strand breaks induced by ionizing radiation (IR) and cross-linking agents (CLA), which are commonly used in tumor therapy. Malfunction of HR causes genome instability leading to cancer and various chromosomal abnormalities such as Down's and other syndromes. Our long-term goal is to understand the molecular mechanisms of HR in human cells. We will use biochemical and in vitro reconstitution approaches for analysis of the enzymatic machinery of HR. Initiated at DNA breaks, HR promotes a search for homologous sequences and subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for the repair. The invasion produces a cross-stranded structure, known as Holliday junction (HJ). HJ possesses a remarkable ability to branch migrate (BM) along the DNA axis. BM i) affects the amount of genetic material contributed by each parent, ii) causes dissociation of HR intermediates, and iii) rescues stalled replication forks through their regression. We discovered recently that hRad54, an important HR protein, catalyzes an ATP dependent BM of HJ. hRad54 is a motor protein that promotes HJ recognition, couples energy consumption with mechanical motion, and promotes migration of HJs. Previously, it was shown that hRad54 stimulates DNA strand exchange activity of hRad51, a key protein of HR. Here we want to understand how these two essential activities of hRad54, BM and stimulation of hRad51, are coordinated (Aim 1). Bloom's syndrome helicase (BLM) is another eukaryotic protein that promotes BM of HJ. Paradoxically, hRad54 and BLM mutants show dissimilar phenotypes in human cells. We will investigate the molecular basis for this difference (Aim 2). We will identify the protein domains which support critical hRad54 functions: BM and hRad51 stimulation (Aim 3). Resolution of HJ remains the most mysterious stage of HR. hRad54 protein physically interacts with Mus81/Eme1, a structure- specific endonuclease, which cleaves HJs. Here we will determine the effect of this interaction on the resolution of HJ by Mus81/Eme1 (Aim 4).

Public Health Relevance

OF THE PROJECT In humans, the system of homologous recombination performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. Homologous recombination is responsible for the repair of DNA double-strand breaks induced by ionizing radiation and cross-linking agents, which are commonly used in tumor therapy. Knowledge of the mechanisms of homologous recombination leads to more efficient approaches for cancer treatment.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA100839-09
Application #
8258794
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Pelroy, Richard
Project Start
2003-04-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
9
Fiscal Year
2012
Total Cost
$259,019
Indirect Cost
$90,395
Name
Drexel University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
002604817
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Bugreev, Dmitry V; Pezza, Roberto J; Mazina, Olga M et al. (2011) The resistance of DMC1 D-loops to dissociation may account for the DMC1 requirement in meiosis. Nat Struct Mol Biol 18:56-60
Bugreev, Dmitry V; Yu, Xiong; Egelman, Edward H et al. (2007) Novel pro- and anti-recombination activities of the Bloom's syndrome helicase. Genes Dev 21:3085-94
Mazina, Olga M; Rossi, Matthew J; Thomaa, Nicolas H et al. (2007) Interactions of human rad54 protein with branched DNA molecules. J Biol Chem 282:21068-80
Bugreev, Dmitry V; Hanaoka, Fumio; Mazin, Alexander V (2007) Rad54 dissociates homologous recombination intermediates by branch migration. Nat Struct Mol Biol 14:746-53
Bugreev, Dmitry V; Golub, Efim I; Stasiak, Alicja Z et al. (2005) Activation of human meiosis-specific recombinase Dmc1 by Ca2+. J Biol Chem 280:26886-95
Mazina, Olga M; Mazin, Alexander V (2004) Human Rad54 protein stimulates DNA strand exchange activity of hRad51 protein in the presence of Ca2+. J Biol Chem 279:52042-51
Bugreev, Dmitry V; Mazin, Alexander V (2004) Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc Natl Acad Sci U S A 101:9988-93