Pch2 acts through Xrs2 and Tel1/ATM to modulate interhomolog bias and checkpoint function during meiosis

Abstract

Proper segregation of chromosomes during meiosis requires the formation and repair of double-strand breaks (DSBs) to form crossovers. Repair is biased toward using the homolog as a substrate rather than the sister chromatid. Pch2 is a conserved member of the AAA(+)-ATPase family of proteins and is implicated in a wide range of meiosis-specific processes including the recombination checkpoint, maturation of the chromosome axis, crossover control, and synapsis. We demonstrate a role for Pch2 in promoting and regulating interhomolog bias and the meiotic recombination checkpoint in response to unprocessed DSBs through the activation of axial proteins Hop1 and Mek1 in budding yeast. We show that Pch2 physically interacts with the putative BRCT repeats in the N-terminal region of Xrs2, a member of the MRX complex that acts at sites of unprocessed DSBs. Pch2, Xrs2, and the ATM ortholog Tel1 function in the same pathway leading to the phosphorylation of Hop1, independent of Rad17 and the ATR ortholog Mec1, which respond to the presence of single-stranded DNA. An N-terminal deletion of Xrs2 recapitulates the pch2Δ phenotypes for signaling unresected breaks. We propose that interaction with Xrs2 may enable Pch2 to remodel chromosome structure adjacent to the site of a DSB and thereby promote accessibility of Hop1 to the Tel1 kinase. In addition, Xrs2, like Pch2, is required for checkpoint-mediated delay conferred by the failure to synapse chromosomes.

Figure 1. Pch2 and Rad17 prevent intersister repair.

(A) Southern blot of 2D gel analysis of joint molecules in indicated strains 9 hr after transfer to SPM. (B) Quantitation of interhomolog double Holliday junctions (IH-dHJs) and intersister double Holliday junctions (IS-dHJs) as a percent of total DNA isolated from synchronized meiotic cultures at the indicated times after transfer to SPM (A). (C) Southern blot of 1D gel analysis of DSB turnover in indicated strains. The slow-migrating DSB species at late time points in dmc1Δ rad17Δ and dmc1Δ pch2Δ rad17Δ are likely DNA hairpin structures . Rad17 may be involved in limiting formation of these structures. (D) Quantitation of DSBs (% total DNA) and percentage of cells that have completed at least the first meiotic division (post-MI) from the time course shown in (C).

Figure 2. Pch2 and Rad17 promote Hop1 and Mek1 activation.

(A) Western blot analysis of WT, rad17Δ, pch2Δ and pch2Δ rad17Δ at indicated time points after transfer to SPM using α-Hop1 antibody. Pgk1 Western blot was used as the loading control. The phosphorylated isoforms of Hop1 are detectable as slow-moving species. (B) Mek1–3HA immunoprecipitates from WT, rad17Δ, pch2Δ and pch2Δ rad17Δ at indicated time points were analyzed by Western blot using α-phospho-Akt substrate (recognizing pT327 of Mek1) and α-HA antibodies. *IgG heavy chain. Cell lysates were analyzed by Western blot using α-HA and α-Pgk1 antibodies.

Figure 3. Meiotic progression analysis in single and double mutant strains.

(A–F) Percentage of cells that have completed at least the first meiotic division (post-MI) in indicated strains. All data were from the same time course experiment.

Figure 4. Hop1 phosphorylation in various mutants.

(A–D) Hop1 phosphorylation was analyzed in indicated strains using α-Hop1 antibody similar to Figure 2A.

Figure 5. Pch2 interacts with putative BRCT repeats of Xrs2.

(A–C) Two-hybrid spot assay of Pch2 and MRX complex components. Transformants carrying both LexA DNA binding domain (LexA)- and Gal4 activation domain (Gal4AD)-fusions were spotted on SC-Leu-Trp (left) and SC-Leu-Trp-His plus 1mM 3-AT (right) plates. (D–F) Mapping of the Pch2-interacting region of Xrs2. Xrs2(1–313)-fha: Two amino acids (S47 and T50) in the FHA domain were altered to alanine, shown as “AA” in (D). (G–H) Percentage of cells that have completed at least the first meiotic division (post-MI) in indicated strains. xrs2ΔN-13myc encodes 13myc-tagged Xrs2(314–854). Data were from the same time course experiment.

Figure 6. xrs2ΔN-13myc phenocopies pch2Δ.

(A–C) Hop1 phosphorylation was analyzed in indicated strains using α-Hop1 antibody similar to Figure 2A. (D) Southern blot of 1D gel analysis of DSBs in sae2Δ and sae2Δ xrs2ΔN-13myc. (E) Percentage of cells that have completed at least the first meiotic division (post-MI) in indicated strains. (F) Model; see text for details.