Pch2 prevents Mec1/Tel1-mediated Hop1 phosphorylation occurring independently of Red1 in budding yeast meiosis

Abstract

A prominent feature of meiosis in most sexually reproducing organisms is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks are repaired using intact homologous non-sister chromatids rather than sister chromatids. Budding yeast DNA damage checkpoint kinases Mec1 and Tel1 act together with the axial element protein Red1 to promote interhomolog recombination by phosphorylating another axial element protein Hop1. Mec1 and Tel1 also phosphorylate γH2A and the synaptonemal complex protein Zip1 independently of Red1 to facilitate premeiotic DNA replication and to destabilize homology-independent centromere pairing, respectively. It has been unclear why Hop1 phosphorylation is Red1-dependent. Here, we report that the pachytene checkpoint protein 2 (Pch2) specifically prevents Red1-independent Hop1 phosphorylation. Our findings reveal a new function for Pch2 in linking two axial element proteins Red1 and Hop1 thus coordinating their effects in meiotic recombination and the checkpoint network.

Figure 1. PCH2 specifically prevents Mec1/Tel1-mediated Hop1 hypophosphorylation in WT and red1Δ.

(A) Western blot time-course analyses of phosphorylated Hop1-T318, Zip1-S75, H2A-S129 and Rad54-T132 in sporulating cells were performed as recently described , . Hsp104 was used as a loading control. All experiments were repeated twice, and the results of representative sporulation time courses are shown. Hyperphosphorylated Hop1 and hypophosphorylated Hop1 are indicated with black and white arrows, respectively. (B) Quantification of phosphorylated protein in (A) (see Materials & Methods). Relative levels of phosphorylated proteins were determined by setting the level of WT “5 hr” as 1.

Figure 2. Rec8 and the catalytic subunit of PP4, Pph3, do not affect Red1-independent Hop1 phosphorylation.

(A) Western blot time-course analyses were performed as described in Figure 1. To validate the specificity of antisera against phosphorylated Hop1-T318, total meitoic cell lysates from strains carrying the wild-type (WT) HOP1 allele (at 5 hr) and the hop1^T318A mutant allele (at 5 hr) were used as positive and negative controls, respectively. The hop1^T318A variant encodes a mutant protein in which the T318 residue of Hop1 has been mutated to alanine. (B) Quantification of phosphorylated protein in (A). Relative levels of phosphorylated proteins were determined by setting the level of WT “5 hr” as 1.

Figure 3. Neither dot1Δ nor xrs2ΔN can recapitulate the effects of pch2Δ on preventing Red1-independent Hop1-T318 phosphorylation.

(A) Western blot time-course analyses were performed as described in Figure 1. (B) Quantification of phosphorylated protein in (A). Relative levels of phosphorylated proteins were determined by setting the level of red1Δ pch2Δ “5 hr” as 1.