Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1

Abstract

During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.

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

“…This hypothesis is supported by the observation that Dmc1 foci formation is compromised in vivo when Rad51 is absent (7,18), the finding that Rad51 is downregulated by Hed1 and Mek1 during meiosis (14,16,17), and the finding that Rad51 strand invasion activity is not required during meiosis (13). 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.…”

“…Despite these many commonalities, Dmc1 is the recombinase responsible for performing strand invasion during meiosis (13). In contrast, Rad51 is actively downregulated by Hed1-and Mek1-mediated inhibition of its interactions with Rad54, which is a cofactor that is required for Rad51 strand invasion activity (11,(14)(15)(16)(17). However, Rad51 is required for normal progression through meiosis and promotes Dmc1 foci formation, consistent with a role for Rad51 as a Dmc1 accessory factor (6,7,13,18).…”

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

“…Genetic and biochemical studies have shown that Hed1 interacts with Rad51 and inhibits strand exchange activity by preventing Rad54 from associating with the presynaptic complex (Tsubouchi & Roeder, 2006;Busygina et al, 2008Busygina et al, , 2012Callender et al, 2016).…”

“…In support of this model, we find that Rad54 and Hed1 binding interactions are mutually exclusive, and once established, these binding interactions are effectively irreversible over the time scales that might be relevant to meiotic progression ( Fig 7B). Reactivation of Rad51 coincides with inactivation of Mek1, and as a result, Rad54 phosphorylation and Hed1 phosphorylation are lost, and Hed1 is degraded (Callender et al, 2016;Argunhan et al, 2017;Prugar et al, 2017). Interestingly, Rad51 plays a late role in meiotic prophase by repairing residual DSBs after disassembly of the synaptonemal complex (Argunhan et al, 2017;Prugar et al, 2017).…”

“…Rad54 is a required cofactor for Rad51 strand invasion activity in vivo; therefore, Hed1 binding downregulates the activity of Rad51 in meiosis. Hed1 is also phosphorylated by Mek1, thereby promoting Hed1 stability (Callender et al, 2016). Hed1 is also phosphorylated by Mek1, thereby promoting Hed1 stability (Callender et al, 2016).…”