Abstract
Displacement loops (D-loops) are critical intermediates formed during homologous recombination. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54/Tid1 is also present in somatic cells where its function is less understood. While Rdh54/Tid1 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54. Here, we show that Rdh54/Tid1 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54/Tid1 uniquely restricts the length of Rad51-Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast. We propose that Rdh54/Tid1 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54/Tid1 acts as a physical roadblock to Rad54 translocation, limiting D-loop formation and D-loop length.
Highlights
Homologous recombination (HR) is a universal DNA repair pathway that uses an intact homologous donor for the repair of double-stranded DNA breaks (DSBs), stalled or collapsed forks and interstrand crosslinks (Kowalczykowski, 2015; Wright et al, 2018)
Using a novel Displacement loops (D-loops) Mapping Assay, we further demonstrate that Rdh54/Tid1 uniquely restricts the length of Rad51-Rad54-mediated D-loops
The dynamic nature of D-loops endowed by these enzymes prevents concomitant invasions, either of both broken ends in the same donor molecule leading to double Holliday Junctions (Wright et al, 2018; Piazza and Heyer, 2019), or of a single end into two different donors leading to multi-invasions (MI) (Piazza and Heyer, 2018; Piazza et al, 2017), inhibiting downstream covalent alterations of the donors mediated by structure-selective endonucleases (SSEs)
Summary
Homologous recombination (HR) is a universal DNA repair pathway that uses an intact homologous donor for the repair of double-stranded DNA breaks (DSBs), stalled or collapsed forks and interstrand crosslinks (Kowalczykowski, 2015; Wright et al, 2018). The dynamic nature of D-loops endowed by these enzymes prevents concomitant invasions, either of both broken ends in the same donor molecule leading to double Holliday Junctions (dHJ) (Wright et al, 2018; Piazza and Heyer, 2019), or of a single end into two different donors leading to multi-invasions (MI) (Piazza and Heyer, 2018; Piazza et al, 2017), inhibiting downstream covalent alterations of the donors mediated by structure-selective endonucleases (SSEs) Both crossovers and MI-induced rearrangements increase in mph, sgs1-top3-rmi, and srs mutants (Ira et al, 2003; Piazza et al, 2019; Piazza et al, 2017; Prakash et al, 2009; Prakash et al, 2009). Together these findings highlight an antagonistic relationship between the two Swi2/Snf ATPases and their function in HR
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