Rad51 protein is the eukaryotic representative of the RecA/Rad51 fannlly of DNA strand transferase enzymes. Homologous DNA strand exchanges catalyzed by RadSI are critical for Homology-Dlrected DNA Repair (HDR) and therefore for genome stability. To promote HDR, RadSI must first assemble onto single stranded DNA In the form of a presynaptic filament. Filament assembly allosterically activates RadSI to catalyze ATP hydrolysis, to search for homology in a sister chromosome, and to perform DNA strand exchange reactions. There Is compelling evidence that defects in the assembly and activity of RadSI presynaptic filaments are linked to human cancer. The overall goal of Project 3 Is to understand how specific changes in the structure, function, and molecular interactions of RadSI can lead to genomic instability and cancer.
The SPECIFIC AIMS of Project 3 are: (1) To test the hypothesis that key amino acid residues at the filament interface and in the ATPase active site of RadSI control the allosteric transitions that couple the ATPase catalytic cycle to DNA strand exchange. Using yeast RadSI as a model, the catalytic and allosteric mechanisms of RadSI will be probed using a combination of site-directed mutagenesis, biochemical and biophysical analyses, and structural biology methods. (2) To test the hypothesis that tumor-derived variants of human hRADSI protein have altered biochemical and/or regulatory properties. hRADSI variants identified in Project 1 will be characterized biochemically alongside wild-type hRADSI to identify any changes in DNA binding or catalytic properties, or in key protein-protein interactions. (3) To test the hypothesis that interactions between hRADSI and DNA polymerase beta (Pol-beta) help recruit hRADSI onto ssDNA generated as a result of abortive base excision repair (BER). hRADSI:Pol-beta interactions will be characterized biochemically and disrupted by mutagenesis to assess their importance for DNA repair functions. Interesting mutants from Aims 2-3 will be exported to Projects 1 and 4 for in vivo and chromatin studies. This project will provide rigorous models for the structure, function, and assembly of RadSI presynaptic filaments, and for potential cross-talk between HDR and BER pathways, in normal vs, tumor cells, which will be useful for predicting cancer susceptibility and for developing new cancer treatments.

Public Health Relevance

The human RAD51 protein plays critical roles in DNA repair pathways that protect cells from mutations and from cancer. In this project we will isolate variant forms of RADSI that are found in human cancer cells, and we will study how these RAD51 variants differ from normal RAD51, Information from this project will help us to design better diagnosis and treatment strategies for cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA098993-09
Application #
8543550
Study Section
Special Emphasis Panel (ZCA1-RPRB-0)
Project Start
Project End
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
9
Fiscal Year
2013
Total Cost
$325,592
Indirect Cost
$89,516
Name
University of Vermont & St Agric College
Department
Type
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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Wallace, Susan S (2014) Base excision repair: a critical player in many games. DNA Repair (Amst) 19:14-26
Nelson, Shane R; Dunn, Andrew R; Kathe, Scott D et al. (2014) Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases. Proc Natl Acad Sci U S A 111:E2091-9
Lubula, Mulu Y; Poplawaski, Amanda; Glass, Karen C (2014) Crystallization and preliminary X-ray diffraction analysis of the BRPF1 bromodomain in complex with its H2AK5ac and H4K12ac histone-peptide ligands. Acta Crystallogr F Struct Biol Commun 70:1389-93
Prakash, Aishwarya; Carroll, Brittany L; Sweasy, Joann B et al. (2014) Genome and cancer single nucleotide polymorphisms of the human NEIL1 DNA glycosylase: activity, structure, and the effect of editing. DNA Repair (Amst) 14:17-26
Sjolund, Ashley; Nemec, Antonia A; Paquet, Nicolas et al. (2014) A germline polymorphism of thymine DNA glycosylase induces genomic instability and cellular transformation. PLoS Genet 10:e1004753
Lee, Andrea J; Warshaw, David M; Wallace, Susan S (2014) Insights into the glycosylase search for damage from single-molecule fluorescence microscopy. DNA Repair (Amst) 20:23-31
Prakash, Aishwarya; Eckenroth, Brian E; Averill, April M et al. (2013) Structural investigation of a viral ortholog of human NEIL2/3 DNA glycosylases. DNA Repair (Amst) 12:1062-71
Liu, Minmin; Doublie, Sylvie; Wallace, Susan S (2013) Neil3, the final frontier for the DNA glycosylases that recognize oxidative damage. Mutat Res 743-744:4-11
Odell, Ian D; Wallace, Susan S; Pederson, David S (2013) Rules of engagement for base excision repair in chromatin. J Cell Physiol 228:258-66

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