Two different Swi5-containing protein complexes are involved in mating-type switching and recombination repair in fission yeast

Abstract

Homologous recombination is an important biological process that occurs in all organisms and facilitates genome rearrangements and repair of DNA double-strand breaks. Eukaryotic Rad51 proteins (Rad51sp or Rhp51 in fission yeast) are functional and structural homologs of bacterial RecA protein, an evolutionarily conserved protein that plays a key role in homologous pairing and strand exchange between homologous DNA molecules in vitro. Here we show that the fission yeast swi5+ gene, which was originally identified as a gene required for normal mating-type switching, encodes a protein conserved among eukaryotes and is involved in a previously uncharacterized Rhp51 (Rad51sp)-dependent recombination repair pathway that does not require the Rhp55/57 (Rad55/57sp) function. Protein interactions with both Swi5 and Rhp51 were found to be mediated by a domain common to Swi2 and Sfr1 (Swi five-dependent recombination repair protein 1, a previously uncharacterized protein with sequence similarity to the C-terminal part of Swi2). Genetic epistasis analyses suggest that the Swi5-Sfr1-Rhp51 interactions function specifically in DNA recombination repair, whereas the Swi5-Swi2-Rhp51 interactions may function, together with chromodomain protein Swi6 (HP1 homolog), in mating-type switching.

Fig. 1.

(A) The nucleotide sequence of the swi5 region and the deduced sequence of Swi5 protein. The 5′-end sequence of the mRNA was determined by 5′-RACE, and the 3′-end sequence was determined from the cDNA. There are two introns in the swi5 gene. The swi5-39 mutation is a single G:C to A:T transition that changes Gln-38 to an ochre nonsense codon. (B) Sequence alignment of Swi5 proteins from various organisms. Multiple sequence alignment was performed by using the clustalx program (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX). Sequence data were obtained from databases. The accession numbers are XP_232740 for rat, AAH21748.1 for mouse, AAH29911.1 for human, BU480721 for chicken, and AL657116 for frog. A 102-aa form of S. cerevisiae Sae3 was predicted by V. Wood (personal communication)

Fig. 2.

Swi5 is involved in Rhp51-dependent and Rhp57-independent recombination repair. (A) Epistasis of DNA repair activity among swi5Δ, rhp51Δ, and rhp57Δ. Exponentially growing cells were irradiated with the indicated doses of γ-rays (Left) or UV light (Right), and colonies were counted. WT, wild-type YA119 (Xs); swi5Δ, YA177 (filled circles); rhp51Δ,T3(filled triangles); rhp57Δ,T5(filled squares); swi5Δ rhp51Δ, YA244 (open triangles); swi5Δ rhp57Δ, YA250 (open squares); swi5Δ rhp51Δ rhp57Δ, YA424 (crosses). (B) Multicopy plasmid expressing rhp51⁺ suppresses the DNA repair defect of swi5Δ. MMS sensitivity of the strains swi5Δ (YA177) and WT (YA119) carrying the rhp51⁺ gene on multicopy plasmid vector pSP102 was judged by performing spot tests as described (11).

Fig. 3.

Physical interactions of Swi5 with recombination repair and mating-type switching proteins. Swi5 interacts with Rhp51 and Swi6 via Swi2. (A) Two-hybrid interactions were judged by spot tests on three types of dropout (DO) plates: 4DO (SD-adenine, -histidine, -leucine, and -tryptophan; high-stringency condition), 3DO (SD-histidine, -leucine, and -tryptophan; medium-stringency condition), and the control 2DO (SD-leucine and -tryptophan). Reciprocal combinations of fusions with the GAL4-activation domain (AD) and the GAL4 DNA-binding domain (DBD) were examined, except for combinations with the DBD fusion of Swi2, because it showed strong self-activation even in the presence of empty vector pGADT7. (B) Coimmunoprecipitation among Swi2, Swi5, and Rhp51. Proteins in extracts from cells transformed with HA-Swi2 plasmid (Right) or empty vector (Left) were immunoprecipitated with antibody against HA, Rhp51, or Swi5. The immune complexes were separated by SDS/PAGE and immunoblotted with antibody against HA, Rhp51, or Swi5. (C) The two-hybrid interactions of Swi2-truncated alleles with Swi5, Rhp51, and Swi6. Full-length swi2 (encoding a 722-aa protein) or swi2-truncated alleles were subcloned in the AD plasmid pGADT7, and Swi5, Rhp51, and Swi6 were subcloned in the DBD plasmid pGBTK7. Two-hybrid interactions were expressed as follows: ++, very strong interaction (grown on 4DO plates); +, strong interaction (grown on 3 DO plates); -, no detectable interaction. a, Swi2-induced self-activation that does not depend on the AD plasmid was assayed by judging the growth of the reporter strain with the swi2 derivative on pGBKT7 and the pGADT7 empty vector on 4DO or 3DO plates and expressed in parentheses by using the same criteria as above.

Fig. 4.

Sfr1 binds to both Swi5 and Rhp51. (A) Sequence alignment of Swi2 and Sfr1. The two proteins show 23% identity and 41% similarity. (B) Sfr1 two-hybrid interaction with Swi5 and Rhp51. The interactions of the indicated proteins were examined by using the two-hybrid assay as described in Fig. 2. (C) Sfr1 coimmunoprecipitates with Swi5. Proteins in extracts from cells transformed with HA-Sfr1 plasmid (Right) or empty vector (Left) were immunoprecipitated with polyclonal antibody against HA or Swi5. The immunocomplexes were separated by SDS/PAGE and immunoblotted with anti-HA or -Swi5 antibodies. Note that HA-Sfr1 was fully active as judged by a complementation test of the DNA repair defect of an sfr1 mutant, YA431 (data not shown; see Fig. 5). (D) Coimmunoprecipitation of HA-Sfr1 with Swi5. Proteins in extracts from cells transformed with HA-Sfr1 plasmid (Right) or empty vector (Left) were immunoprecipitated with anti-HA monoclonal antibody (12CA5, Roche Applied Science). The immune complexes were separated by SDS/PAGE and immunoblotted with rat anti-Swi5 antibody.

Fig. 5.

Sfr1 protein is involved in the same repair pathway as Swi5 but not in mating-type switching. (A) The sfr1⁺ gene functions in the same repair pathway for γ-ray (Left) and UV (Right) irradiation as the swi5⁺ gene. WT, wild-type YA119 (Xs); sfr1Δ, YA431 (filled circles); swi5Δ, YA177 (filled triangles); rhp51Δ,T3(filled squares); sfr1Δ swi5Δ, YA452 (open triangles); sfr1Δ rhp51Δ, YA474 (open squares); sfr1Δ rhp57Δ, YA478 (crosses). (B) sfr1Δ is proficient in mating-type switching, as judged by the iodine staining assay. WT, wild-type, YA254; swi2Δ, YA492; and sfr1Δ, YA455.

Fig. 6.

A summary model. Three distinct pathways are involved in mitotic DNA recombination-related events, one for mating-type switching and the other two for DNA repair. Swi5 is involved in both DNA repair and mating-type switching. The Swi5/Sfr1 repair pathway is completely parallel to the Rhp55/57 pathway. Sfr1 functions specifically in repair, whereas Swi2 functions specifically in mating-type switching. Note that swi2, swi5, and swi6 belong to the same class 1b epistasis group for mating-type switching (24, 25).