A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability

Abstract

During meiosis, homologous recombination repairs programmed DNA double-stranded breaks. Meiotic recombination physically links the homologous chromosomes (“homologs”), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1’s activity is regulated by its accessory factors including the heterodimeric protein Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister joint molecule intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1. Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filament lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements.

Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase

“…Rad51 strand exchange activity is responsible for the hedΔR delay in prophase I exit Rad51:Dmc1 stoichiometry in the presynaptic filament is important for normal interhomolog recombination [116]. Hed1 and Rad54 can stabilize Rad51 on the filament, raising the possibility that the problems in DNA repair observed in hedΔR are due to improperly assembled presynaptic filaments resulting from the absence of HED1 and/or the presence of Rad54 [49,117,118].…”

How strand exchange protein function benefits from ATP hydrolysis

“…Thus, homology recognition and strand exchange is driven by product stability and does not require chemical energy from ATP hydrolysis [ 16 ●● , 17 ●● ]. Analysis of Rad51 and Dmc1 mutants defective for ATPase activity found that, like RecA’s, their intrinsic hydrolytic activity is dispensable for homology search and strand exchange in vitro and in vivo [ 18 – 21 ].…”

“…The budding yeast SWI5-MEI5/SFR1 homolog, Sae3-Mei5, is meiosis-specific, and is required for Dmc1 focus formation and strand exchange [ 45 ]. While little is known about the mechanism through which Sae3-Mei5 promotes Dmc1 filament formation, an ATPase mutant of Dmc1, Dmc1-E157D, bypasses the requirement for Sae3-Mei5 in vivo , suggesting that Sae3-Mei5’s function is similar to that of the fission yeast and mammalian homologs [ 21 ].…”

Rad51 and Dmc1 have similar tolerance for mismatches in yeast meiosis

“…Forming the Dmc1 nucleoprotein filament depends on a different set of auxiliary factors that are either specifically expressed in meiosis (Sei3 and Mei5, Hop2 and Mnd1) or play only minor roles in mitotic DSB repair (most notably, Rad54's homolog, Rdh54, also known as Tid1) (39)(40)(41)(42)(43)(44).…”