Using Schizosaccharomyces pombe meiosis to analyze DNA recombination intermediates

Abstract

The fission yeast Schizosaccharomyces pombe has many biological characteristics that make it an ideal model organism for the study of meiosis. A nearly synchronous meiosis is one of the most important. Under certain environmental and genetic conditions, large cultures of S. pombe can be induced to undergo meiosis in a timely and predictable manner that allows for changes in the DNA to be observed and analyzed by gel electrophoresis. Initiation of meiotic recombination via programmed DNA double-strand breaks, the formation of joint molecule recombination intermediates, and the resolution of these intermediates into crossover DNA products can all be seen with consistent timing during the progression of a synchronous meiotic induction. The timing of recombination events, the genetic requirements for the formation and disappearance of recombination intermediates, and the analysis of the DNA structures of those intermediates allow a comparison of meiotic recombination in fission yeast with that in the only other species similarly studied, the budding yeast Saccharomyces cerevisiae.

Fig. 1

Pulsed-field gel electrophoresis (PFGE) of S. pombe meiotic DNA to assay DSBs. A pat1-114 rad50S haploid strain was induced for meiosis (Section 3.1.1 and 3.2). (A) PFGE of whole chromosomes (labeled 1, 2, and 3) (Section 3.3.1.1), stained with EtBr (0.5 μg/mL). The bracket on the right highlights the broken chromosomal DNA. (B) PFGE of DNA from the same induction digested with NotI, stained with EtBr (left panel) (Section 3.3.1.2). The size markers are Lambda Ladder PFG markers (NEB). A Southern blot of the gel (right panel) was hybridized with a 1 kb probe specific to the left end of the 501 kb NotI fragment J (arrowhead). DNA fragments broken at the meiotic DSB hotspot mbs1 are indicated with an asterisk. (C) PFGE of DNA from the same induction digested with EagI, stained with EtBr (left panel) (Section 3.3.1.2). The size markers are Mid Range 1 (left) and Low Range PFG markers (right) (NEB). A Southern blot of the gel (right panel) was hybridized with a 1 kb probe specific to the left end of the 60.3 kb EagI fragment (arrowhead) of the NotI fragment J. DNA fragments broken at the meiotic DSB hotspot mbs1 are indicated with an asterisk. In each panel W indicates the wells into which the plugs with DNA were placed.

Fig. 2

Standard gel electrophoresis of S. pombe meiotic DNA to assay crossover DNA. (A) The DNA construct to assay JMs and crossover DNA at mbs1. The PmlI and XbaI sites are heterozygous in the diploid strain and flank the DSB hotspot mbs1. Digestion at the flanking PvuII sites in combination with PmlI and XbaI digestion produces parental DNA 9.2 kb long (parent 1; P1) and 6.8 kb long (parent 2; P2). Recombination (meiotic crossing-over or conversion of one marker) produces DNA 11.2 kb long (recombinant 1; R1) and 4.8 kb long (recombinant 2; R2). (B) Southern blot of the gel described in Section 3.3.2, probed as depicted in Fig. 2A. Note that incomplete digestion produces a fragment identical to R1. Therefore, quantitation of recombination is 2(R2)/[P1+P2+2(R2)]. Leftmost lane contains a 1 kb ladder; the bottom marker is 4 kb. (Modified from Ref. (11).)

Fig 3

2D gel electrophoresis of DNA recombination intermediates at mbs1. (A) The left cartoon is a representation of the migration of different DNA forms during 2D electrophoresis, digested with a single restriction enzyme (Section 3.3.3.1). The left image is a Southern blot of DNA from a pat1-114 mus81Δ diploid strain, which was meiotically induced for 2.5 h. The branched intermediates are replication structures. The right image is from the same induction at 5 h. The X-shaped spikes are HJs arising from meiotic recombination (indicated by arrow). Both time points were digested with PvuII and probed as in Fig. 2A. (B) The left cartoon depicts the migration of different DNA forms from a diploid strain with heterozygous flanking restriction markers (Fig. 2A) triply digested and run in a 2D gel. The right image is of DNA prepared at 4.5 h after induction of the strain in (A), digested with PvuII, PmlI, and XbaI, and assayed by 2D gel electrophoresis (Section 3.3.3.2). The IH HJs run as two species (due to their asymmetric structure) at a position between the two IS HJ spikes (highlighted by bracket). See Ref. (11) for further explanation. The black arrowhead points to a partial digestion fragment. (Modified from Ref. (11)).