Mapping and Analysis of Swi5 and Sfr1 Phosphorylation Sites

The evolutionarily conserved Swi5-Sfr1 complex plays an important role in homologous recombination, a process crucial for the maintenance of genomic integrity. Here, we purified Schizosaccharomyces pombe Swi5-Sfr1 complex from meiotic cells and analyzed it by mass spectrometry. Our analysis revealed new phosphorylation sites on Swi5 and Sfr1. We found that mutations that prevent phosphorylation of Swi5 and Sfr1 do not impair their function but swi5 and sfr1 mutants encoding phosphomimetic aspartate at the identified phosphorylation sites are only partially functional. We conclude that during meiosis, Swi5 associates with Sfr1 and both Swi5 and Sfr1 proteins are phosphorylated. However, the functional relevance of Swi5 and Sfr1 phosphorylation remains to be determined.


Introduction
Homologous recombination is a conserved process for repairing several types of lesions, including DNA double-strand breaks [1]. It is a precise DNA repair pathway, during which homologous DNA sequence is copied from an intact donor template. The ability to identify and exchange the strands of two homologous DNA molecules is mediated by RecA-family recombinases such as Rad51 and meiosis specific Dmc1, eukaryotic orthologs of the prokaryotic recombinase RecA. Rad51 and Dmc1 require several auxiliary factors in order to function properly. The Swi5-Sfr1 complex in the fission yeast Schizosaccharomyces pombe (Mei5-Sae3 in Saccharomyces cerevisiae) has been characterized as an auxiliary factor that stimulates Rad51 and Dmc1 activity. The Swi5-Sfr1 complex stabilizes Rad51 and Dmc1 filaments and stimulates their ATPase activity [2][3][4][5][6][7]. Swi5 in complex with Sfr1C (180 residue N-terminal deletion mutant of Sfr1) was successfully crystallized. Structural studies demonstrated that Swi5 and Sfr1C form a complex with a parallel coiled-coil heterodimer joined together via two leucine-zipper motifs and a bundle. Swi5-Sfr1C forms a kinked structure that is able to stimulate Rad51-mediated strand exchange. Docking of the atomic model of the Swi5-Sfr1C complex into the Rad51 filament model showed that the kinked structure fits well into the helical groove of Rad51 filament [8]. The N-terminal part of Sfr1 (Sfr1N) is intrinsically disordered and contains two sites that cooperatively bind Rad51, indicating that the primary function of Sfr1N is to mediate the interaction between Swi5-Sfr1 and Rad51 [6].
Swi5 and Sfr1 play an important role during meiosis, a specialized cell division that generates gametes with a haploid set of chromosomes from a diploid precursor [9]. This reduction in chromosome number results from one round of DNA replication followed by two nuclear divisions, meiosis I and meiosis II. During meiotic prophase I, homologous chromosomes pair and crossovers are created as one outcome of the repair of programmed DNA double-strand breaks via homologous recombination. Crossovers provide a physical connection between homologs, which is essential for their faithful segregation during the first meiotic division [10]. Mutation of either Swi5 or Sfr1 results in reduced homologous recombination in both mitotic and meiotic cells and elevated sensitivity to a number of DNA damaging agents, including ionizing radiation and methyl-methanesulfonate (MMS) [11][12][13][14][15].
The importance of homologous recombination is emphasized by its critical role during development and for tumor suppression. Defects in homologous recombination are associated with sensitivity to DNA damage, loss of genomic integrity and various diseases including Fanconi anemia and Bloom syndrome [16].
In this study, we investigated the role of post-translational modification by phosphorylation in the regulation of Swi5-Sfr1 function. We purified S. pombe Swi5-Sfr1 from meiotic cells and identified phosphorylation sites by mass spectrometry. We found that although the identified phosphorylation sites are not essential for the function of Swi5 and Sfr1, phosphomimetic swi5 and sfr1 mutants are only partially functional.  [17][18][19]. Deletions of swi5 and sfr1 genes were performed as described in Gregan et al. [20]. TAP-tagging was performed as described in Cipak et al. [21]. S. pombe strains were constructed by standard meiotic crosses, genotypes of strains used are listed in Table S3. Transformants were confirmed by PCR-based analysis and mutations of phosphorylation sites by nucleotide sequencing.

Microscopy
The immunofluorescence and microscopy techniques used to analyze chromosome segregation were performed as described in Rabitsch et al. [22].
To monitor the progression of meiosis, 1 ml aliquots of cultures were collected and fixed in 70% (v/v) ethanol. To visualize DNA, mounting medium with DAPI (Vectashield, Vector Laboratories, Burlingame, CA, USA) was used for microscopy (Zeiss Axio Imager Z2; Carl Zeiss AG, Oberkochen, Germany).

Spot Tests
YES plates containing methyl methanesulfonate (YES+MMS) were freshly prepared two days before the experiment. Cells were grown on YES plates for one day at 32 • C, resuspended in sterile water and cell concentration was determined using a Burker chamber. Cells were diluted in sterile water in 10-fold steps and 3-5 microliters of suspension was spotted onto standard YES and YES+MMS plates. The plates were incubated for 3-5 days at 30 • C.

Protein Purification and Analysis
Induction of meiosis and monitoring of progression of meiosis were performed as described in Cipak et al. [23]. Meiotic cultures expressing Sfr1-TAP or Sfr1-5A-TAP were harvested around 3 hours after induction of meiosis and cells from fifteen-liter cultures were collected by centrifugation. The tagged proteins were isolated and analyzed by mass spectrometry as described previously in Cipak et al. [21].
Mass spectrometry analysis of proteins co-purifying with Sfr1-TAP revealed that, similarly as in cycling cells, Sfr1-TAP associated with high levels of Swi5 (Table S1 and  Table S2. Mass spectrometry analysis of proteins co-purifying with Sfr1-TAP revealed that, similarly as in cycling cells, Sfr1-TAP associated with high levels of Swi5 (Tables S1 and S2).
Our identification of S84 phosphorylation on Swi5 that co-purified with Sfr1-TAP suggests that phosphorylated Swi5 is part of the Swi5-Sfr1 complex. We also found that Sfr1-5A-TAP but not Sfr1-TAP co-purified with small amounts of Dmc1, a meiosis specific member of the RecA-family of recombinases, suggesting that the interaction between the Swi5-Sfr1 complex and Dmc1 is transient, substoichiometric or easily disrupted (Tables S1 and S2). We conclude that during meiosis, Swi5 associates with Sfr1-TAP and both Swi5 and Sfr1 proteins are phosphorylated.
The observed phenotype of phosphomimetic mutants swi5-2D and sfr1-13D is not due to lack of expression, as all four mutant proteins (Swi5-2A-TAP, Swi5-2D-TAP, Sfr1-13A-TAP and Sfr1-13D-TAP) were expressed, as detected by Western blot analysis of TAP-tagged proteins (Figure 3, Figures S3 and S4). The Western blot analysis showed that protein levels of both Swi5-TAP and Sfr1-TAP increase during meiosis, which is consistent with previous observation that swi5 and sfr1 mRNAs are upregulated during meiosis [34]. Swi5 and Sfr1 are also important for proper segregation of chromosomes during meiosis [5,31]. In order to analyze chromosome segregation, we introduced swi5 mutations into a homothallic h 90 strain where chromosome I was marked with GFP (lys1-GFP) [32]. In this strain, GFP-tagged LacI molecules bind to lacO repeats inserted within the lys1 locus located near the centromere. This strain generates cells of both mating types and undergoes mating and meiosis on a sporulation medium. Similarly, we introduced sfr1 mutations into h 90 strain where chromosome II was marked with GFP (cen2-GFP) [33]. During anaphase I, homologous centromeres in wild-type cells segregated to opposite poles. However, we frequently observed homolog non-disjunction in swi5Δ and sfr1Δ anaphase I cells ( Figure 2B). While expression of a wild-type Swi5 (swi5Δ swi5-wt) and Swi5-2A mutant (swi5Δ swi5-2A) rescued the missegregation phenotype of the swi5Δ mutant, Swi5-2D mutant protein only partially rescued the phenotype of the swi5Δ mu- Taken together, we showed that Swi5 co-purifies with Sfr1-TAP isolated from meiotic cells. Our mass spectrometry analysis identified new phosphorylation sites on Swi5 and Sfr1 proteins. However, we found that the identified phosphorylation sites are dispensable for proper segregation of chromosomes during meiosis I and for repair of damaged DNA, as assessed by an MMS sensitivity test. While our results show that the absence of phosphorylation on the analyzed residues is not essential for the function of Swi5 and Sfr1, the phenotype of phosphomimetic mutations is difficult to interpret. Although phosphomimetic mutations are often used to mimic the constitutively phosphorylated state, it is known that the chemical environment created by phosphorylation is different from that of negatively charged amino acids [35]. Therefore, the potential functional relevance of Swi5 and Sfr1 phosphorylation remains to be determined. Cells were harvested at the indicated time points (hours). Proteins extracted from meiotic cells were analyzed by gel electrophoresis and Western blotting using anti-tubulin antibodies. The TAP epitope was detected using PAP antibodies (rabbit antiperoxidase antibody linked to peroxidase). As a positive control (PC), protein extracts were prepared from a pool of cells expressing TAP tag alone, harvested at 4-6 hours after meiosis induction [36]. Full original images of Western blots are shown in Figure S4. (B) Progression of pat1-induced meiosis. Cells as described in (A) were harvested at the indicated time points (hours). Fixed cells were stained with DAPI and nuclei were counted in 100 cells per time point. Shown are the fractions of cells that contained one nucleus (1n), two nuclei (2n) or more than two nuclei (>3n) at the indicated time points.  Proteins extracted from meiotic cells were analyzed by gel electrophoresis and Western blotting using anti-tubulin antibodies. The TAP epitope was detected using PAP antibodies (rabbit antiperoxidase antibody linked to peroxidase). As a positive control (PC), protein extracts were prepared from a pool of cells expressing TAP tag alone, harvested at 4-6 hours after meiosis induction [36]. Full original images of Western blots are shown in Figure S4. (B) Progression of pat1-induced meiosis. Cells as described in (A) were harvested at the indicated time points (hours). Fixed cells were stained with DAPI and nuclei were counted in 100 cells per time point. Shown are the fractions of cells that contained one nucleus (1n), two nuclei (2n) or more than two nuclei (>3n) at the indicated time points.