Rdh54 stabilizes Rad51 at displacement loop intermediates to regulate genetic exchange between chromosomes

Abstract

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.

Disparate roles forC. elegansDNA translocase paralogs RAD-54.L and RAD-54.B in meiotic prophase germ cells

“…Based on prior studies identifying several disparate biochemical activities for RAD54 family translocases, we speculate that RAD-54.B might potentially function in multiple capacities at DSBR sites. For example, it might affect the structure of DSBR intermediates by modulating D-loop size or D-loop maturation, as proposed for yeast Rdh54/Tid1 (Keymakh et al, 2022;Shah et al, 2020), and/or it might modulate the activity of RAD-54.L in promoting RAD-51 removal. RAD-54.B functioning in such capacities at DSBR sites may underlie the modest difference in CO distribution observed in the rad-54.B mutant and the contribution of RAD-54.B to meiotic success in a rad-54.L partial loss-of-function background.…”

“…Moreover, additional functions for S. cerevisiae Rdh54 and mammalian RAD54B have also been demonstrated in mitotically dividing cells where DMC1 is absent and RAD51 is the only recombinase. For example, yeast Rdh54 has been implicated in limiting the size of Rad51-mediated D-loop intermediates in vivo (Keymakh et al, 2022;Shah et al, 2020), and biochemical evidence indicates that the two RAD54 paralogs may occupy different sites on assembled presynaptic filaments (Crickard et al, 2020a), suggesting that both RAD54 paralogs may be deployed in different ways at the same DSBR site. Further, loss of either RAD54 paralog causes increased sensitivity to DNA damaging agents both in mouse embryonic stem cells and mice, with loss of both paralogs causing stronger sensitivity (Wesoly et al, 2006).…”

The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

“…This assay also contains markers to report on chromosome loss. We have previously used this assay and found that rad54 Δ /rad54 Δstrains are severely defective in this assay and result in limited recombination (27).…”

“…The WT strain used in this assay and the procedure for diploid formation are described here (27). The genotypes for modifying these strains can be found in Supplemental Table 2 .…”

“…The end point of the homology search is the extension of D-loops, which allows the transition to the DNA repair and resolution phase of HR (23). In Saccharomyces cerevisiae, the steps in the homology search are regulated by several DNA translocases and helicases, including Rad54 (24), Rdh54 (14,(25)(26)(27), and Srs2 (13,(28)(29)(30)(31)(32). These ATP-dependent motors control intermediates, including Rad51 filaments (28,29,32,33) and D-loop structures (13,33,34).…”