The Third Exon of the Budding Yeast Meiotic Recombination Gene HOP2 Is Required for Calcium-dependent and Recombinase Dmc1-specific Stimulation of Homologous Strand Assimilation

Abstract

During meiosis in Saccharomyces cerevisiae, the HOP2 and MND1 genes are essential for recombination. A previous biochemical study has shown that budding yeast Hop2-Mnd1 stimulates the activity of the meiosis-specific strand exchange protein ScDmc1 only 3-fold, whereas analogous studies using mammalian homologs show >30-fold stimulation. The HOP2 gene was recently discovered to contain a second intron that lies near the 3'-end. We show that both HOP2 introns are efficiently spliced during meiosis, forming a predominant transcript that codes for a protein with a C-terminal sequence different from that of the previously studied version of the protein. Using the newly identified HOP2 open reading frame to direct synthesis of wild type Hop2 protein, we show that the Hop2-Mnd1 heterodimer stimulated Dmc1 D-loop activity up to 30-fold, similar to the activity of mammalian Hop2-Mnd1. ScHop2-Mnd1 stimulated ScDmc1 activity in the presence of physiological (micromolar) concentrations of Ca(2+) ions, as long as Mg(2+) was also present at physiological concentrations, leading us to hypothesize that ScDmc1 protomers bind both cations in the active Dmc1 filament. Co-factor requirements and order-of-addition experiments suggested that Hop2-Mnd1-mediated stimulation of Dmc1 involves a process that follows the formation of functional Dmc1-ssDNA filaments. In dramatic contrast to mammalian orthologs, the stimulatory activity of budding yeast Hop2-Mnd1 appeared to be specific to Dmc1; we observed no Hop2-Mnd1-mediated stimulation of the other budding yeast strand exchange protein Rad51. Together, these results support previous genetic experiments indicating that Hop2-Mnd1 specifically stimulates Dmc1 during meiotic recombination in budding yeast.

DNA Strand Exchange and RecA Homologs in Meiosis

“…This idea was supported by the finding that mutation of the S. pombe homolog of Hop2 (Meu13) reduced chromosome pairing somewhat in a spo11 mutant background and that S. cerevisiae hop2 mutants build synaptonemal complexes between nonhomologous chromosomes (Nabeshima et al 2001;Tsubouchi and Roeder 2003). However, biochemical evidence for direct interaction between Hop2-Mnd1 and strand-exchange proteins (Petukhova et al 2005), as well as evidence showing that Hop2-Mnd1 can stimulate strand exchange in purified systems Petukhova et al 2005;Enomoto et al 2006;Ploquin et al 2007;Chan et al 2014), suggest Hop2-Mnd1 functions directly at sites of meiotic recombination in vivo. The simplest explanation for the absence of significant colocalization between Dmc1 and Hop2-Mnd1 is that the dsDNA-binding activity of Hop2-Mnd1 accounts for the presence of DSB-independent foci, and only a small fraction of Hop2-Mnd1 foci are associated with DNA recombination intermediates at any given moment; for instance, the preponderance of Hop2-Mnd1 complexes are not engaged with Dmc1 (and/or Rad51) and may obscure the relatively small subset of complexes that are.…”

“…In budding yeast, Hop2 and Mnd1 are not required for Rad51-mediated meiotic recombination in the dmc1 hed1 double-mutant background (Lao et al 2013). Furthermore, neither budding yeast nor fission yeast Hop2-Mnd1 stimulates Rad51's D-loop activity in vitro (Ploquin et al 2007;Chan et al 2014). Finally, no evidence of Hop2-Mnd1 stimulation of Rad51 activity has been reported for Hop2 2/2 or Mnd1 2/2 knockout mice (Petukhova et al 2003).…”

“…However, Hop2-Mnd1 binds DNA directly, preferring dsDNA to ssDNA Enomoto et al 2006). Furthermore, the finding that preincubation of duplex DNA with Hop2-Mnd1 provides optimal stimulation of yeast Dmc1 suggested that either nonspecific dsDNA capture or alteration of the structure of duplex target might be important for the mechanism of stimulation Chan et al 2014). Indeed, affinity capture and cosedimentation experiments using the mouse and human orthologs of Hop2-Mnd1 and Dmc1, respectively, have provided evidence for homology-independent capture of dsDNA by ssDNA-Dmc1 filaments (Pezza et al 2007).…”

“…Although nonphysiologically high Ca 2þ concentrations (low millimolar) have often been utilized to increase strand-exchange efficiency (see above), a recent study shows that micromolar Ca 2þ promotes robust Dmc1 activity provided Mg 2þ is also present at physiological concentrations (Chan et al 2014). A crystal structure of an archael homolog of Dmc1 includes Ca 2þ and Mg 2þ bound to different sites in each protomer, and the same study showed Ca 2þ stabilizes an active filament conformation (Qian et al 2006).…”

“…The fact that Dmc1 foci form normally in the absence of Hop2-Mnd1 suggests that Hop2-Mnd1 acts on Dmc1 after the Mei5 -Sae3 complex exerts its influence ( Initial biochemical studies of budding yeast Hop2-Mnd1 showed that the protein stimulates Dmc1 D-loop activity only threefold ). However, this study was flawed because the Hop2 protein used lacked its normal carboxyl terminus, which is coded by a third exon in the HOP2 gene (Chan et al 2014). The third exon had not been discovered at the time the expression construct used in the earlier experiments was built.…”