Regulation of Hed1 and Rad54 binding during maturation of the meiosis‐specific presynaptic complex

Abstract

Most eukaryotes have two Rad51/RecA family recombinases, Rad51, which promotes recombination during mitotic double-strand break (DSB) repair, and the meiosis-specific recombinase Dmc1. During meiosis, the strand exchange activity of Rad51 is downregulated through interactions with the meiosis-specific protein Hed1, which helps ensure that strand exchange is driven by Dmc1 instead of Rad51. Hed1 acts by preventing Rad51 from interacting with Rad54, a cofactor required for promoting strand exchange during homologous recombination. However, we have a poor quantitative understanding of the regulatory interplay between these proteins. Here, we use real-time single-molecule imaging to probe how the Hed1- and Rad54-mediated regulatory network contributes to the identity of mitotic and meiotic presynaptic complexes. Based on our findings, we define a model in which kinetic competition between Hed1 and Rad54 helps define the functional identity of the presynaptic complex as cells undergo the transition from mitotic to meiotic repair.

Spontaneous self-segregation of Rad51 and Dmc1 DNA recombinases within mixed recombinase filaments

“…Genetic and biochemical data have demonstrated that Hed1 interacts specifically with Rad51 (15,39), and Hed1-GFP complements a hed1D strain, indicating that the fluorescent protein retains function (14). Consistent with these findings, we can readily detect Hed1-GFP binding to Rad51, but we are unable to detect any GFP-Hed1 association with Dmc1 ( Figure 2A) (40). Therefore, we consider Hed1-GFP to be good proxy for defining the location of Rad51 within the presynaptic complex.…”

“…To test this prediction, we sought to determine whether any Rad51 remained bound to the mixed recombinase filaments after ATP depletion by using Hed1-GFP to detect Rad51. Importantly, our previous experiments have shown that Hed1-GFP does not bind to either RPA-ssDNA or Dmc1-ssDNA, and presence of bound Hed1 is strictly dependent upon the integrity of the Rad51-ssDNA (40). Therefore, Hed1-GFP would only be able to bind to the presynaptic complexes if Rad51 remained present after ATP depletion.…”

“…We wanted to test the possibility that Dmc1 might also exert some influence over Rad51 by comparing the disassembly rates of Rad51 only, Dmc1 only, and mixed filaments containing both recombinases. For this comparison, we also took advantage of the fact that when calcium is present, Rad51 dissociates from ssDNA upon ATP depletion, but Dmc1 does not ( Figure 4A) (40).…”

Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

“…The Rad51-Binding Factor Hed1 Does Not Affect Srs2. During meiosis, Rad51 strand-exchange activity is down-regulated by the meiosis-specific Rad51-binding factor Hed1, which prevents Rad54 from associating with Rad51-ssDNA filaments (51)(52)(53). Importantly, we used GFP-tagged Hed1 in DNA curtain assays to identify the locations of Rad51 within mixed recombinase filaments containing Rad51 and Dmc1 (Fig.…”

“…Importantly, we used GFP-tagged Hed1 in DNA curtain assays to identify the locations of Rad51 within mixed recombinase filaments containing Rad51 and Dmc1 (Fig. 4B) (52). Bulk biochemical assayed revealed that Hed1-GFP has no appreciable impact on Srs2 ATP hydrolysis activity (SI Appendix, Fig.…”