Homologous recombination (HR) is a double-strand break DNA repair pathway that preserves chromosome structure. To repair damaged DNA, HR uses an intact donor DNA sequence located elsewhere in the genome. After the double-strand break is repaired, DNA sequence information can be transferred between donor and recipient DNA molecules through different mechanisms, including DNA crossovers that form between homologous chromosomes. Regulation of DNA sequence transfer is an important step in effectively completing HR and maintaining genome integrity. For example, mitotic exchange of information between homologous chromosomes can result in loss-of-heterozygosity (LOH), and in higher eukaryotes, the development of cancer. The DNA motor protein Rdh54 is a highly conserved DNA translocase that functions during HR. Several existing phenotypes in rdh54Δ strains suggest that Rdh54 may regulate effective exchange of DNA during HR. In our current study, we used a combination of biochemical and genetic techniques to dissect the role of Rdh54 on the exchange of genetic information during DNA repair. Our data indicate that RDH54 regulates DNA strand exchange by stabilizing Rad51 at an early HR intermediate called the displacement loop (D-loop). Rdh54 acts in opposition to Rad51 removal by the DNA motor protein Rad54. Furthermore, we find that expression of a catalytically inactivate allele of Rdh54, rdh54K318R, favors non-crossover outcomes. From these results, we propose a model for how Rdh54 may kinetically regulate strand exchange during homologous recombination.

Homologous recombination (HR) is a double-strand break DNA repair pathway that preserves chromosome structure. To repair the damaged recipient DNA, HR requires an intact donor DNA sequence located elsewhere in the genome. After the double-strand break is repaired, DNA sequence information can be transferred between donor and recipient DNA molecules through different mechanisms, including DNA crossovers that form between homologous chromosomes. Regulating this transfer of information is an important step in effectively completing HR and maintaining genome integrity. For example, mitotic exchange of information between homologous chromosomes can result in loss-of-heterozygosity (LOH) in diploid organisms, and in higher eukaryotes, the development of cancer. The DNA motor protein Rdh54 is a highly conserved DNA translocase that functions during HR but has a limited role in repairing DNA. Instead, several existing phenotypes in rdh54∆ strains suggest that Rdh54 may regulate the flow of information between donor and recipient DNA molecules post DNA repair. In our current study, we used a combination of biochemical and genetic techniques to dissect the role of Rdh54 on the exchange of genetic information after DNA repair. Our data indicates that RDH54 regulates DNA sequence exchange between chromosomes by limiting the disruption of Rad51 at an early HR intermediate called the displacement loop (D-loop). Rdh54 also protects Rad51 filaments, acting in opposition to Rad51 removal by the DNA motor protein Rad54. Furthermore, we find that expression of a catalytically inactivate allele of Rdh54, rdh54K318R, displays a different distribution of information exchange outcomes than rdh54∆ cells. From these results, we propose a model for how Rdh54 may effectively regulate information transfer during homologous recombination.

Rdh54 is a conserved DNA translocase that participates in homologous recombination (HR), DNA checkpoint adaptation, and chromosome segregation. Saccharomyces cerevisiae Rdh54 is a known target of the Mec1/Rad53 signaling axis, which globally protects genome integrity during DNA metabolism. While phosphorylation of DNA repair proteins by Mec1/Rad53 is critical for HR progression little is known about how specific post translational modifications alter HR reactions. Phosphorylation of Rdh54 is linked to protection of genomic integrity but the consequences of modification remain poorly understood. Here, we demonstrate that phosphorylation of the Rdh54 C-terminus by the effector kinase Rad53 down regulates Rdh54 activity resulting in a 50% decrease in translocation velocity on dsDNA as revealed by single molecule imaging. Genetic assays reveal that loss of phosphorylation reduces interhomolog gene conversion, increases break induced replication, and decreases crossover outcomes. Our data highlight Rad53 as a key regulator of HR intermediates through activation and attenuation of Rdh54 motor function.