Biochemical Characterization of MET1-RAD51 Interaction in Homologous Recombination
Date
2021-08
Authors
Durham, Jesse
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Abstract
Fanconi anemia (FA) is a human genetic disorder that causes organ defects, physical abnormalities, bone marrow failure, and increased risk of malignancy. These phenotypes result from an impaired response to DNA damage due to the dysfunction of a cluster of FA proteins. Among these, human FANCM (budding yeast ortholog, Mph1) functions in DNA replication fork (RF) repair and DNA double-strand break (DSB) repair by homologous recombination (HR). In a recent study, the homolog of human ZGRF1 in Saccharomyces cerevisiae, Mte1, was found to protect the Rad51-made D-loop, essential for HR, from disassociation by Mph1. Our preliminary data showed that Mte1 interacts with Rad51 in vitro. However, the biological role of this interaction remains unknown. The goal of this project was to examine the effects of this interaction on homologous recombination. Two different Rad51 binding site mutants of Mte1 were created and the Rad51 interaction of each mutant was evaluated via an in vitro pull down assay. A Mte1 mutant containing the phenylalanine to alanine mutation at position 169 showed a reduction in Rad51 binding at 150 mM and 200 mM salt concentrations. Electrophoretic mobility shift assays (EMSAs) confirmed that our mutants had the same binding affinity to DNA as the wild type, indicating that our mutation had no effect on DNA binding. Importantly, we found that wild type Mte1 stimulates Rad51-mediated D-loop formation in vitro, suggesting Mte1 could promote homologous recombination. The stimulation of D-loop formation was dependent on Mte1-Rad51 interaction, as evidenced by Mte1-F169A’s impairment of stimulation on Rad51-mediated D-loop formation. These findings suggest that the interaction between Mte1:Rad51 does play a role in the formation of D-loop structures and impairment of this interaction causes a reduction in D-loop formation.
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Keywords
homologous recombination, D-loop formation
Citation
Durham, J. (2021). Biochemical characterization of MET1-RAD51 interaction in homologous recombination (Unpublished thesis). Texas State University, San Marcos, Texas.