A Mutation in Rad54 that Affects PCNA Interaction Results in Decreased Homologous Recombination Events Following Double‐Strand Breaks

Abstract

The Rad54 protein has multiple roles in the double‐strand break repair pathway of homologous recombination (HR). When DNA is damaged and the repair mechanisms (HR) are hindered, the DNA can remain nonfunctional, or worse, lead to maladies such as cancer. It is essential to understand the genes regulating HR in order to treat these disorders. We looked into one such critical mechanism in this project. Rad54 interacts with PCNA, the eukaryotic DNA replication clamp, but the function of this interaction is unknown. We hypothesize that this interaction is important in the steps of HR that require DNA synthesis. To determine the function of this interaction in Saccharomyces cerevisiae, a series of point mutants of rad54 were made and one, K102A/R103A (rad54‐KR/AA), was found to be defective for this interaction. The PCNA interaction is about 5‐fold reduced in the rad54‐KR/AA mutant protein, as measured by microscale thermophoresis. In addition, this mutant protein was tested in vitro for ATPase activity and the ability to bind other recombination proteins, notably the Rad51 recombinase. The ATPase activity and Rad51 interaction are intact, suggesting it is a true separation‐of‐function mutation. This phenotypic impact of this PCNA interaction mutation is being examined in vivo using assays that measure HR at the mating type locus. Strand invasion and repair synthesis are being tested using a PCR‐based assay, still in progress. The ability to complete recombination and switch mating types is known to require Rad54, so we are testing the rad54‐KR/AA mutant by inducing breaks at the MAT locus, plating cells and determining the number of cells that have switched mating type. So far we have found that the percentage of rad54‐KR/AA cells that can switch mating type is considerably lower than that of the wild type. This indicates that the Rad54‐PCNA interaction is required to efficiently repair double strand breaks. The fact that the Rad54‐KR/AA mutant protein is still capable of performing the switch albeit at a less frequent rate indicates that while that protein region is important for interaction with PCNA and for repair, it is not essential‐ it does not cripple the entire mechanism. In the PCR‐based assay, we expect to see less HR synthesis product because switching is reduced. This would indicate that rad54 KR/AA mutant protein region plays an important part in PCNA interactions‐ which are vital for HR; these interactions being a critical step in the final steps of synthesis and ultimately repair. When we know more about how these three components (Rad54, Rad51, and PCNA) all work together to repair damaged DNA, it will be a critical development in the field of genetic repair. Once we know the physiology of this damage, we stand a better chance of finding improved treatments to homologous recombination disorders, and possibly even cancer as a whole.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.