Fission yeast switches mating type by a replication-recombination coupled process

Abstract

Fission yeast exhibits a homothallic life cycle, in which the mating type of the cell mitotically alternates in a highly regulated fashion. Pedigree analysis of dividing cells has shown that only one of the two sister cells switches mating type. It was shown recently that a site- and strand-specific DNA modification at the mat1 locus precedes mating-type switching. By tracking the fate of mat1 DNA throughout the cell cycle with a PCR assay, we identified a novel DNA intermediate of mating-type switching in S-phase. The time and rate of appearance and disappearance of this DNA intermediate are consistent with a model in which mating-type switching occurs through a replication-recombination coupled pathway. Such a process provides experimental evidence in support of a copy choice recombination model in Schizosaccharomyces pombe mating-type switching and is reminiscent of the sister chromatid recombination used to complete replication in the presence of certain types of DNA damage.

Fig. 1. Schematic representation of the mating-type loci on chromosome II. (A) The mat1 locus contains either the P (white box) or M (gray box) mating-type alleles, mat2 contains the P and mat3 the M loci located distal to the centromere (CEN II). The P and M DNA regions have no homology and are 1104 and 1128 bp, respectively. The BamHI and EcoRI restriction sites in the three cassettes are shown. The H1 (59 bp) and H2 (135 bp) homology boxes are common to all cassettes, whereas the H3 (57 bp) box is common to only the silent mat2P and mat3M cassettes (Kelly et al., 1988). The asterisk indicates the position of the DNA modification at mat1. The horizontal arrows, labeled P1–4 and P′1, indicate the position and the orientation of the PCR primers. The positions of the deletions in mat1M, smt0 and mat1M, Δmat2-3 mutant strains are indicated. (B) Hypothetical intrachromosomal folding allowing mat1P switching to mat1M. The PCR primers are indicated and the gene conversion is represented by a cross connecting P1 with P3 or P4 primers. (C) Same as in (B) except that the hypothetical intrachromosomal folding allows mat1M switching to mat1P.

Fig. 2. Identification of a DNA species connecting mat1 with mat2P or mat3M loci. (A) PCR amplification products using a wild-type homothallic genomic DNA as template, followed by endonuclease digestion and resolution by agarose gel electrophoresis. PCR primers and molecular weight markers are indicated. Twenty-five cycles of amplification were used with the P1–P2 primer pair and 30 cycles with the P1–P3 and P1–P4 pairs. (B) Expected size of the PCR products with and without enzymatic restriction.

Fig. 3. Identification of a gene conversion intermediate. (A) Agarose gel electrophoresis analysis of PCR amplification products. The genomic DNAs were isolated from wild-type, mat1M, smt0 and mat1M, Δmat2-3 strains, as indicated. The P1 primer was replaced by the P′1 primer (see Figure 1), and M indicates the molecular weight markers. Twenty-five cycles of amplification were used with the P′1–P2 primer pair and 30 cycles with the P′1–P3 and P′1–P4 pairs. (B) Native acrylamide gel electrophoresis analysis. The genomic DNA used is the same as above, and the P′1 primer was ³²P labeled to reduce the number of amplification cycles. Only 20 cycles were used with the P′1–P2 primer pair and 25 cycles with the P′1–P3 and P′1–P4 pairs.

Fig. 4. The gene conversion DNA intermediate appears at the end of S–phase and is resolved in early G₂-phase. (A) Autoradiogram of the PCR products resolved in a polyacrylamide gel under native conditions. The three primer pairs were described in Figure 1 and the genomic DNA templates were obtained from synchronized homothallic cdc25 strain. The low level of DNA intermediate products (P1–P3 and P1–P4) found at all times during the cell cycle indicates the degree of synchrony of the cdc25 cell population. Twenty PCR cycles were used with the P1–P2 primers, whereas 21 cycles were used with P1–P3 and 22 cycles with P1–P4. (B) The relative DNA concentration of the PCR product shown in (A) was plotted as a function of time. As expected, the P1–P2 PCR product roughly doubles during S-phase. The P1–P3 and P1–P4 PCR products appear at 120 min in the middle of S-phase and disappear in early G₂-phase. The P1–P2 PCR products were reduced 2–fold to facilitate the comparison. (C) DNA content of the synchronized cdc25 population measured by flow cytometry. The cell population arrested in G₂–M is released at time 0 and enters anaphase at ∼30 min and S–phase between 60 and 80 min (see the text for details). Septum formation is concomitant with S-phase and is followed by cytokinesis at the 140 min time point. The second mitosis begins after the 220 min time point (data not shown). The drifting of the 2C peak at 0 and 180 min is the result of the division of elongated cells.

Fig. 5. Analysis of the gene conversion intermediates in swi mutant strains. The panels show autoradiograms of the PCR products, resolved through a native polyacrylamide gel, of genomic DNA isolated from wild-type and the three classes of swi mutant strains, as indicated. Twenty cycles were used with the P1–P2 primer pair, which serves as internal control, and 25 cycles were used with the P1–P3 and P1–P4 pairs.

Fig. 6. Replication–recombination coupled model. Only the mating-type switches from P to M using the leading strand synthesis are represented for clarity. The H1, H2 and H3 boxes are indicated and the DNA modification on the mat1 upper strand is symbolized by an asterisk. The modified mat1P^* locus in G₁-phase (a). The interrupted replication fork arriving from the right provided the 3′-OH invading strand (b). A double-stranded DNA end is formed transitorily (c). Strand invasion in the H1 homology box of the opposite silent cassette, mat3M in this example (d). DNA synthesis is initiated and the newly synthesized strand is displaced from its template, forming a migrating D-loop (Ferguson and Holloman, 1996) (e). When copy synthesis has passed the H2–H3 boxes (DNA intermediates used as template in the PCR assay), an intramolecular secondary structure can be formed (f), recognition by the swi4/8 gene products may stop DNA synthesis, followed by resolution mediated by the swi9/10 gene products (see the text for details). Upon resolution, DNA synthesis can proceed, giving one unswitched chromatid (mat1P^*) capable of switching again during the next replication and one switched-intact chromatid (mat1M), which will be modified asymmetrically (^*) during the next replication (z).