Determining how a chromatin remodeling protein modulates RAD52 function
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Date
2023-05
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The Ohio State University
Abstract
Cancer is a debilitating genetic disease that affects countless individuals and families every day. In addition to mutation and changes in gene expression, mistakes can occur during S-phase that can cause DNA double-strand breaks (DSBs). These breaks can cause gross chromosomal re-arrangements including intra-chromosomal deletions (ICDs) Homologous recombination (HR) is a repair mechanism for DSBs that involves copying and using the intact DNA sequence from the undamaged homologous chromosome to repair the broken chromosome, generally resulting in little to no change in genetic information. Certain HR pathways like Single-Strand Annealing (SSA) are mutagenic and result ICDs. Therefore, it is possible for SSA to delete cell-cycle regulators that are a vital part of controlling cell division. Several proteins are involved in the complex process of DSB repair. The RAD52 gene sits at the crossroads of all HR repair pathways and is conserved from yeast to humans. Another critical part of DSB repair is chromatin remodeling, the removal of histones to allow DSB repair proteins to access the break site. KAT5, a lysine acetyltransferase, is a histone remodeler that helps to remove nucleosomes prior to DSB repair. Our groups have identified physical and genetic interactions between KAT5 and RAD52 suggesting a direct role for KAT5 in HR. However, the nature of the physical interaction between these two proteins has not been explored. This project studied this interaction both biochemically and computationally. To begin the biochemical experiments, RAD52 and Mst1 (yeast homolog of KAT5) needed to be sufficiently overexpressed and purified, and a protocol involving column chromatography was developed and optimized. After enough purified protein was produced, the two were incubated together and run via size-exclusion chromatography to test for complex formation. The result of these runs suggests a monomer-monomer interaction between RAD52 and KAT5. However, lack of yield prevented further analysis of the complex using gel electrophoresis. The computational portion of this project involved homology modeling and protein-protein docking. Several combinations of RAD52 and KAT5 were submitted to three different programs to observe potential interfaces, and further analysis of specific residues was also performed. Two of the programs used could not model specific combinations submitted, while the third docked everything. Both the biochemical and computational results provide groundwork for further research to take place. Continued study of the interaction between RAD52 and Mst1 biochemically will allow the specifics of the complex to be confirmed. The future goal of this project is to determine how these proteins interact with each other and the effect of interaction loss on HR repair. Both KAT5 and RAD52 mutations including complete deletions have been identified in cancer cells making this project immediately tractable.
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Keywords
RAD52, cancer, protein-protein docking, mutation, KAT5 (Mst1), DNA double-strand break repair