Molecular and Cellular Dissection of Mating-Type Switching Steps in Schizosaccharomyces pombe

Abstract

A strand-specific imprint (break) controls mating-type switching in fission yeast. By introducing a thiamine repressible promoter upstream of the mat1 locus, we can force transcription through the imprinted region, erasing the imprint and inhibiting further mating-type switching, in a reversible manner. Starting from a synchronized, virgin M-cell population, we show that the site-and strand-specific break is formed when DNA replication intermediates appear at mat1 during the first S phase. The formation of the break is concomitant with a replication fork pause and binding of the Swi1 protein at mat1 until early G 2 and then rapidly disappears. Upon its formation, the break remains stable throughout the cell cycle and triggers mating-type switching during the second S phase. Finally, we have recreated the mating-type switching pedigree at the molecular and single-cell levels, allowing for the first time separation between the establishment of imprinting and its developmental fate.The fission yeast Schizosaccharomyces pombe possesses a remarkable system for changing its mating type (MT) (reviewed in references 4 and 13). Homothallic MT switching in fission yeast, similar to that in budding yeast, is a genetically programmed event in haploid cells initiated by a site-specific lesion at the mat1 locus. This lesion is then repaired by homologous donor sequences, mat2-P and mat3-M, located on the same chromosome. These loci are maintained in a silent chromatin state, preventing their transcription or recombination, but they can serve as donors of genetic information at mat1 by recombination (7,15,25). The MT switching pattern, as determined several years ago by pedigree analyses at the single-cell level, follows two strict rules, as depicted in Fig. 1A (17, 28, 34). The first, termed the one-in-four rule, indicates that two consecutive divisions are necessary to produce one switched cell among four granddaughter cells. The first division produces two cells with identical MTs, but with different switching potentials, termed unswitchable (u) and switchable (s), such that only the s cell produces one switched cell after the second division. The second, termed the consecutive rule, indicates that the sister of this switched cell is able to switch during subsequent cell divisions. The newly switched cell is unswitchable, and will follow the same fate as the unswitchable (Mu or Pu) cells (where M stands for minus and P stands for plus). Therefore, an exponential culture-or well-grown colony contains not two but four cell types, with P or M MT information, and with two switching potentials, u and s, found in equal proportions (1 Pu:1 Mu:1 Ps:1 Ms). Cells from opposite MTs are able to conjugate in G 1 and sporulate when grown under sporulating conditions. The integrity of this pedigree and switching is conveniently verified by staining cells within a colony with iodine vapors, a marker of spore-containing colonies, such that wild-type homothallic cells stain darkly, whereas mutant cells exhibit a streaky or white phenotype (3...

A Unique DNA Recombination Mechanism of the Mating/Cell-type Switching of Fission Yeasts: a Review

“…As the DSB is an artifact of the DNA extraction procedure because of the presence of a fragile site at the imprint, the level of the single-stranded nick is a better quantitation of the imprint. This method indicates that nearly 50% of the chromosomes are nicked, in agreement with the strand-segregation model (32,34).…”

“…One suggestion is that the broken end invades mat2 or mat3, and extends the 3′-OH end by the copy-choice mechanism (29,30). Another possibility is that repair is initiated by template switching through the replicationrecombination coupled process (32,34,47,48), similar to that of the canonical DSB repair mechanism (23,49,50). Following the fate of newly synthesized 13 …”

Regulation of Histone H3 Lysine 56 Acetylation in Schizosaccharomyces pombe

“…Homothallic fission yeast cells switch their mating type by making use of a site-specific single-stranded lesion that is transformed into a double strand break during the passage of a unidirectional replication fork. This initiates a gene conversion event that replaces the mating type cassette at the active mating type locus (42)(43)(44)(45). Therefore, we analyzed the effect of the absence of H3 Lys-56 acetylation in mating-type switching efficiency in S. pombe.…”

Comparative Genomics and Molecular Dynamics of DNA Repeats in Eukaryotes

“…In Schizosaccharomyces pombe, mating-type switching is induced by the replication fork encountering a single-strand nick at the mat1 locus, transforming this nick into a DSB, thus initiating homologous recombination with one of the two homologous mat cassettes (references 13, 14, 237, and 237a). This is a highly regulated process under the control of several proteins, including the conserved fork protection Swi1/Swi3 complex (Tim/Tipin in mammals), responsible for the temporary replication fork pausing near mat1 (200,237). The possible presence of single-strand nicks at fragile sites in vivo, during or after replication, is an open question.…”