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 #
5R01CA100839-10
Application #
8453415
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
2013-05-01
Budget End
2014-04-30
Support Year
10
Fiscal Year
2013
Total Cost
$243,478
Indirect Cost
$84,971
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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Shahar, Or David; Kalousi, Alkmini; Eini, Lital et al. (2014) A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair. Nucleic Acids Res 42:5689-701
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Mazina, Olga M; Rossi, Matthew J; Deakyne, Julianna S et al. (2012) Polarity and bypass of DNA heterology during branch migration of Holliday junctions by human RAD54, BLM, and RECQ1 proteins. J Biol Chem 287:11820-32
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Bugreev, Dmitry V; Rossi, Matthew J; Mazin, Alexander V (2011) Cooperation of RAD51 and RAD54 in regression of a model replication fork. Nucleic Acids Res 39:2153-64

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