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 #
5P01CA098993-10
Application #
8725060
Study Section
Special Emphasis Panel (ZCA1)
Project Start
Project End
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
10
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Type
DUNS #
City
Burlington
State
VT
Country
United States
Zip Code
05405
Galick, Heather A; Marsden, Carolyn G; Kathe, Scott et al. (2017) The NEIL1 G83D germline DNA glycosylase variant induces genomic instability and cellular transformation. Oncotarget 8:85883-85895
Silva, Michelle C; Bryan, Katie E; Morrical, Milagros D et al. (2017) Defects in recombination activity caused by somatic and germline mutations in the multimerization/BRCA2 binding region of human RAD51 protein. DNA Repair (Amst) 60:64-76
Robey-Bond, Susan M; Benson, Meredith A; Barrantes-Reynolds, Ramiro et al. (2017) Probing the activity of NTHL1 orthologs by targeting conserved amino acid residues. DNA Repair (Amst) 53:43-51
Maher, R L; Marsden, C G; Averill, A M et al. (2017) Human cells contain a factor that facilitates the DNA glycosylase-mediated excision of oxidized bases from occluded sites in nucleosomes. DNA Repair (Amst) 57:91-97
Cannan, Wendy J; Rashid, Ishtiaque; Tomkinson, Alan E et al. (2017) The Human Ligase III?-XRCC1 Protein Complex Performs DNA Nick Repair after Transient Unwrapping of Nucleosomal DNA. J Biol Chem 292:5227-5238
Zhou, Jia; Chan, Jany; Lambelé, Marie et al. (2017) NEIL3 Repairs Telomere Damage during S Phase to Secure Chromosome Segregation at Mitosis. Cell Rep 20:2044-2056
Marsden, Carolyn G; Jensen, Ryan B; Zagelbaum, Jennifer et al. (2016) The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype. PLoS Genet 12:e1006208
Lee, Andrea J; Wallace, Susan S (2016) Visualizing the Search for Radiation-damaged DNA Bases in Real Time. Radiat Phys Chem Oxf Engl 1993 128:126-133
Cannan, Wendy J; Pederson, David S (2016) Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin. J Cell Physiol 231:3-14
Silva, Michelle C; Morrical, Milagros D; Bryan, Katie E et al. (2016) RAD51 variant proteins from human lung and kidney tumors exhibit DNA strand exchange defects. DNA Repair (Amst) 42:44-55

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