Pch2 is a hexameric ring ATPase that remodels the chromosome axis protein Hop1

Abstract

In budding yeast the pachytene checkpoint 2 (Pch2) protein regulates meiotic chromosome axis structure by maintaining the domain-like organization of the synaptonemal complex proteins homolog pairing 1 (Hop1) and molecular zipper 1 (Zip1). Pch2 has also been shown to modulate meiotic double-strand break repair outcomes to favor recombination between homologs, play an important role in the progression of meiotic recombination, and maintain ribosomal DNA stability. Pch2 homologs are present in fruit flies, worms, and mammals, however the molecular mechanism of Pch2 function is unknown. In this study we provide a unique and detailed biochemical analysis of Pch2. We find that purified Pch2 is an AAA+ (ATPases associated with diverse cellular activities) protein that oligomerizes into single hexameric rings in the presence of nucleotides. In addition, we show Pch2 binds to Hop1, a critical axial component of the synaptonemal complex that establishes interhomolog repair bias, in a nucleotide-dependent fashion. Importantly, we demonstrate that Pch2 displaces Hop1 from large DNA substrates and that both ATP binding and hydrolysis by Pch2 are required for Pch2-Hop1 transactions. Based on these and previous cell biological observations, we suggest that Pch2 impacts meiotic chromosome function by directly regulating Hop1 localization.

Keywords: AAA proteins; hexameric ATPase; meiosis.

Fig. 1.

Purification of Pch2 and mutants. (A) Alignment of Pch2 amino acid sequences from S. cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens. Walker A and Walker B motifs are highlighted (shading). The alignment was generated using T-Coffee (67, 68). (B) Purification of wild-type and mutant Pch2, His₆–Hop1, and His₆–Dmc1. Dmc1, His₆–Dmc1; EQ, GST–Pch2–E399Q; GA, GST–Pch2–G319A; Hop1, His₆–Hop1; Pch2, untagged Pch2 after removal of GST tag; WT, wild-type GST–Pch2. See Materials and Methods and SI Materials and Methods for details.

Fig. 2.

Pch2 and GST–Pch2 forms hexameric rings in the presence of nucleotides. (A) Elution profiles of the Pch2 protein from a Superdex 200 size-exclusion column in the absence and presence of 2 mM ATPγS. The expected elution volumes (Ve) for different oligomeric forms of Pch2 are indicated by dashed lines. (B, Left) Representative negative staining electron micrograph of Pch2 in the presence of ATPγS. Black arrows indicate ring-shaped particles representing top views of Pch2 hexameric rings. (B, Upper and Lower Right) The projection structure of Pch2 hexameric rings in the presence of ATPγS. Upper Right shows the 2D averages and Lower Right the same average is displayed after sixfold rotational symmetry has been imposed. (C, Left) Representative negative-staining electron micrograph of GST–Pch2 in the presence of ATPγS. Black arrows indicate ring-shaped particles representing top views of GST–Pch2 hexameric rings. (C, Upper and Lower Right) The projection structure of GST–Pch2 hexameric rings in the presence of ATPγS. Upper Right shows the 2D averages and Lower Right the same average is displayed after sixfold rotational symmetry has been imposed. Densities at the vertices of the triangle represent GST dimers.

Fig. 3.

Spore viability of pch2 mutants in the csm4Δ/csm4Δ background. Dashed lines indicate the spore viabilities of csm4Δ/csm4Δ PCH2/PCH2 (64%) and csm4Δ/csm4Δ pch2Δ/pch2Δ (31%) (42, 43). *P < 0.05 compared with csm4Δ/csm4Δ PCH2/PCH2; ^†P < 0.05 compared with csm4Δ/csm4Δ pch2Δ/pch2Δ. See Table 2 for details.

Fig. 4.

ATPase and ATPγS binding activity of Pch2 and mutants. (A) ATPase activity of GST–Pch2 and Pch2. Proteins were present at 6 nM and error bars represent SDs from four experiments. The K_m and K_cat values are listed in Table 3. (B) ATPase activity of GST–Pch2, GST–Pch2–G319A, and GST–Pch2–E399Q. GST–Pch2 was present at 6 nM, and GST–Pch2–G319A and GST–Pch2–E399Q were present at 40 nM. Error bars represent SDs obtained from two to four experiments. (C) ATPγS binding activity of Pch2 and mutants. E399Q, GST–Pch2–E399Q; G319A, GST–Pch2–G319A; WT, GST–Pch2. All reactions contained 100 nM wild-type or mutant Pch2 and 12 µM ³⁵S-labeled ATPγS. Error bars represent SDs from two experiments. See SI Materials and Methods for details. (D) ATPase activity of Pch2 (12 nM) in the presence of 0.5, 2, or 8 µM (concentration in nucleotides) of the indicated DNA substrates. Error bars represent SDs from two experiments.

Fig. 5.

Pch2 interacts with Hop1. (A and B) In vitro-binding assays performed with purified Hop1 and GST–Pch2 or GST–Pch2–E399Q in the absence (A) or presence (B) of the indicated nucleotide (200 µM). (C–E). Hop1 (41.7 nM) binding to a 69-bp ³²P-dsDNA substrate (1.15 µM, concentration in nucleotides) was determined in filter-binding assays. In C–E, error bars represent SDs from at least three repetitions. (C) Hop1 DNA-binding activity in the presence of different amounts of GST–Pch2 (8.3, 16.7, and 41.7 nM) and 200 µM ATP. (D) Hop1 DNA-binding activity in the presence of GST–Pch2 (16.7 nM) and the indicated nucleotides. (E) Hop1 DNA-binding activity in the presence of indicated GST–Pch2 mutant proteins (16.7 nM) and 200 µM ATP. (F) Acrylamide gel EMSA. Hop1 (110 nM) was incubated with 69-bp ³²P-dsDNA (1.38 µM, concentration in nucleotides) in the presence of ATP and in the presence or absence of 25 nM GST–Pch2, and the amount of DNA bound was analyzed by EMSA.

Fig. 6.

Pch2-mediated dissociation of Hop1 from DNA. (A) Hop1 titration. Reactions (25 μL) in Buffer A [20 mM Tris (pH 7.5), 0.01 mM EDTA, 2 mM MgCl₂, 40 µg/mL BSA, 0.1 mM DTT, 75 mM NaCl, 9% glycerol], 60 ng BamHI-digested pUC19 (2.7 kb), and 0, 80, 120, 160, 200, and 240 nM Hop1 were incubated at 30 °C for 20 min, after which they were loaded onto an agarose gel (0.7%) and analyzed as described in SI Materials and Methods. (B) Reactions (25 μL) in Buffer A with 60 ng BamHI-digested pUC19 (2.7 kb) and 200 nM Hop1 were incubated at 30 °C for 10 min, after which 200 nM GST–Pch2 and 300 µM ATP or 50 µM ATPγS or 300 µM ADP were added as indicated. Reactions were then continued for 5 min at 30 °C, and loaded onto a 0.7%-agarose gel. (C) Reactions (25 μL) in Buffer A with 60 ng BamHI-digested pUC19 (2.7 kb) and 200 nM Hop1 were incubated at 30 °C for 10 min, after which 200 nM GST–Pch2 and 300 µM ATP were added as indicated in the presence of 40 ng 1.3-kb trap DNA. Reactions were then continued for 10 min at 30 °C, after which they were loaded onto a 0.7%-agarose gel and analyzed as before.