Mus81 cleavage of Holliday junctions: a failsafe for processing meiotic recombination intermediates?

Abstract

The Holliday junction (HJ) is a central intermediate of homologous recombination. Its cleavage is critical for the formation of crossover recombinants during meiosis, which in turn helps to establish chiasmata and promote genetic diversity. Enzymes that cleave HJs, called HJ resolvases, have been identified in all domains of life except eukaryotic nuclei. Controversially, the Mus81-Eme1 endonuclease has been proposed to be an example of a eukaryotic nuclear resolvase. However, hitherto little or no HJ cleavage has been detected in recombinant preparations of Mus81-Eme1. Here, we report the purification of active forms of recombinant Schizosaccharomyces pombe Mus81-Eme1 and Saccharomyces cerevisiae Mus81-Mms4, which display robust HJ cleavage in vitro, which, in the case of Mus81-Eme1, is as good as the archetypal HJ resolvase RuvC in single turnover kinetic analysis. We also present genetic evidence that suggests that this activity might be utilised as a back-up to Mus81-Eme1's main activity of cleaving nicked HJs during meiosis in S. pombe.

Figure 1

Model for Mus81-Eme1 generating crossovers and non-crossovers during meiosis by cleaving a mixture of nicked and intact HJs.

Figure 2

Purification of Mus81-Eme1 and analytical ultracentrifugation of Mus81-Mms4. (A) Summary of the purification scheme. (B) X0n and X0 cleavage activity in Mus81-Eme1 gel filtration fractions. Reactions (20 μl) contained 4 μl of the indicated gel filtration fraction, 1.1 nM of labelled junction DNA and 10 mM MgCl₂, and were analysed by native PAGE. (C) An estimation of the molecular weight of Mus81-Eme1 based on its gel filtration profile relative to known standards. The standards are thyroglobulin (∼670 kDa), bovine gamma-globulin (158 kDa), chicken ovalbumin (44 kDa) and equine myoglobin (17 kDa). V_C is the column volume, V_O is the void volume and V_R is the elution volume. (D) SDS–PAGE analysis of purified Mus81-Eme1 and Mus81^DD-Eme1. The gel is stained with Coomassie blue. Note that the identity of His-Eme1 and Mus81 was confirmed by N-terminal amino-acid sequencing. (E) g(s) profile calculated from 20 sample distribution data sets using Sedfit (Schuck and Rossmanith, 2000). The data are shown as open circular symbols with a six-peak Gaussian fit plotted over as a black line. The individual peaks found by that fit are shown as broken red lines and have values 1.5±0.1S, 2.1±0.2S, 4.2±0.03S, 7.3±0.04S, 9.2±0.1S and 14.2±0.04S. (F) Plots of apparent molecular weight against the absorbance of the sample at the midpoint of the 6000 r.p.m. profile for each dilution used. Black symbols and line at 6000 r.p.m.; red at 8000 r.p.m.; green at 10 500 r.p.m.; blue at 15 000 r.p.m. and cyan at 18 000 r.p.m.

Figure 3

Characterizing the cleavage of X0 by Mus81-Eme1. (A) The effect of MgCl₂ concentration on X0 and X0n cleavage by Mus81-Eme1. Reactions (20 μl) contained 1.1 nM of labelled junction, together with the indicated amounts of Mus81-Eme1 and MgCl₂. (B) Histogram showing the mean data from three experiments represented in (A). Error bars represent the standard deviations. (C) Comparison of the cleavage of X0, X0n and X12 by Mus81-Eme1. Reactions (20 μl) contained 1.1 nM junction DNA and the specified amounts of protein. (D) Comparison of the rate of cleavage of X0 and X0n by Mus81-Eme1. Reactions (80 μl) contained 1.3 nM labelled junction and 0.8 nM of protein. A 9 μl volume of samples was withdrawn at the stated intervals for native PAGE analysis. The data are the mean of three experiments, and error bars represent the standard deviations. (E) Single turnover kinetic analysis of X0 cleavage by Mus81-Eme1. Reactions (40 μl) contained 13.4 nM Mus81-Eme1 and 0.9 nM X0. A gel mobility shift assay confirmed that all of the X0 was bound by Mus81-Eme1 under these conditions (data not shown). The data are the mean of three experiments, and error bars represent the standard deviations.

Figure 4

Comparing the binding of X0 and X0n by Mus81-Mms4. (A) Binding of X0 and X0n with increasing concentrations of Mus81-Mms4 in the presence of EDTA. (B, C) The effect of linear double-stranded competitor DNA on X0 and X0n binding by Mus81-Mms4 in the presence of EDTA (B) and 200 μM CaCl₂ (C). In both (B) and (C), the right-hand panels show mean data from three experiments represented in the left-hand panels. Error bars represent the standard deviations.

Figure 5

The effect of arm length on X junction cleavage. (A) Native gels showing the cleavage of long-armed M1 and M1n by Mus81-Eme1 and RuvC. Reactions (20 μl) contained 1 nM junction DNA and 6.7 nM Mus81-Eme1 or 10 nM RuvC as indicated. RuvC cleavage reactions contained 10 mM MgCl₂. The bottom panel is a schematic representation of the long-armed M1(n) showing its bi-mobile core and the length of its arms. The asterisk indicates the radiolabel. (B) Same as in (A) but using short-armed M1(n). Note that the M1n short-armed junction runs as a smear because it is relatively unstable and tends to fall apart during PAGE.

Figure 6

The effect of a strand nick on cleavage site selection by Mus81-Eme1 and RuvC. (A, B) Denaturing gels showing cleavages in strands 2 and 3 of X26 and X26n made by Mus81-Eme1 and RuvC. Reactions (40 μl) contained 1 nM junction DNA and either 50 nM RuvC or 6.7 nM Mus81-Eme1. RuvC cleavage reactions contained 10 mM MgCl₂. Selected cleavage sites are numbered. (C, D) Schematic of X26(n) showing the positions of the selected Mus81-Eme1 cleavage sites shown in (A) and (B). The asterisk indicates the site of the nick in X26n. (E) Histogram of the amount of cleavage by Mus81-Eme1 at each of the 18 selected sites in strand 2 of X26 and X26n shown in (B). The data are the mean of three experiments. Error bars are omitted for clarity.

Figure 7

Comparing Mus81-Eme1 and RuvC for CO formation during meiosis in S. pombe. (A) Schematic of the cross to assess meiotic recombination at the ade6 locus. The filled circles indicate the relative positions of the M26 and L469 mutations. (B) Bar diagram showing the percentage of Ade⁺ recombinants associated with crossing over in the ura4-aim2 - ade6 - his3-aim interval for the indicated crosses. Error bars represent standard deviations about the mean values.