Checkpoint-dependent and -independent roles of Swi3 in replication fork recovery and sister chromatid cohesion in fission yeast

Abstract

Multiple genome maintenance processes are coordinated at the replication fork to preserve genomic integrity. How eukaryotic cells accomplish such a coordination is unknown. Swi1 and Swi3 form the replication fork protection complex and are involved in various processes including stabilization of replication forks, activation of the Cds1 checkpoint kinase and establishment of sister chromatid cohesion in fission yeast. However, the mechanisms by which the Swi1-Swi3 complex achieves and coordinates these tasks are not well understood. Here, we describe the identification of separation-of-function mutants of Swi3, aimed at dissecting the molecular pathways that require Swi1-Swi3. Unlike swi3 deletion mutants, the separation-of-function mutants were not sensitive to agents that stall replication forks. However, they were highly sensitive to camptothecin that induces replication fork breakage. In addition, these mutants were defective in replication fork regeneration and sister chromatid cohesion. Interestingly, unlike swi3-deleted cell, the separation-of-functions mutants were proficient in the activation of the replication checkpoint, but their fork regeneration defects were more severe than those of checkpoint mutants including cds1Δ, chk1Δ and rad3Δ. These results suggest that, while Swi3 mediates full activation of the replication checkpoint in response to stalled replication forks, Swi3 activates a checkpoint-independent pathway to facilitate recovery of collapsed replication forks and the establishment of sister chromatid cohesion. Thus, our separation-of-function alleles provide new insight into understanding the multiple roles of Swi1-Swi3 in fork protection during DNA replication, and into understanding how replication forks are maintained in response to different genotoxic agents.

Figure 1. Sensitivity of swi3 mutants to S-phase stressing agents.

(A, B) Five-fold serial dilutions of cells of the indicated genotypes were incubated on YES agar medium supplemented with the indicated amounts of HU (top panels), MMS (middle panels) and CPT (bottom panels) for 3 to 5 days at 32°C. In A, classes (C I to C IV) of swi3 mutants are indicated in parentheses. In B, original swi3 alleles from which the single point mutations were derived are also indicated in parentheses. Representative images of repeat experiments are shown.

Figure 2. Effects of swi3 mutations on the formation of the Swi1-Swi3 complex.

(A) Protein extracts were prepared from cells expressing the indicated fusion proteins. Swi3-FLAG (Swi3-FL) was precipitated, and associated proteins were probed with the anti-Myc 9E10 and anti-FLAG M2 antibodies. Classes (C I to C IV) of Swi3 mutants are indicated in parentheses. The appearance of two to three bands in Swi1-Myc Western blots is due to degradation of the fusion protein , . The Swi3-E40 mutant protein showed slower mobility, which is possibly due to mutational effects. Although only small amount of the Swi3-E10 protein was recovered by immunoprecipitation, Swi1-13Myc was efficiently co-precipitated with Swi3-E10. Western blotting of tubulin was performed as a loading control. (B) Protein extracts from the indicated strains were subjected to Swi3-TAP precipitation experiments, and associated proteins were probed with the anti-FLAG M2 and PAP antibodies. Original swi3 alleles from which the single point mutations were derived are also indicated in parentheses. Although reduced amount of Swi3 were recovered by immunoprecipitation in swi3-D84H, L112R and R124R, they were all readily detected. Asterisk indicates non-specific bands. Representative results of repeat experiments are shown. IP, immunoprecipitation; WB, Western blotting; WCE, whole cell extract.

Figure 3. Structure of Swi3 related proteins.

(A) Schematic drawing of Swi3 homologs from S. pombe (Sp Swi3), S. cerevisiae (Sc Csm3) and humans (Hs Tipin). Gray boxes indicate regions of amino acid sequences that are highly conserved throughout evolution. This region in each protein is called the Swi3 domain. The RPA-binding motif is found only in human Tipin. Mutation sites found in swi3 alleles are indicated. aa, amino acid. (B) The Swi3 polypeptide was divided into 4 putative functional sub-domains. The dark gray box (Domain II) indicates the region with amino acid sequences that are conserved throughout evolution. This region contains a putative NLS (42–49 aa) and the Swi3 domain (52–116 aa), the latter of which includes three conserved α-helices: h1 (63–69 aa), h2 (81–97), and h3 (105–114 aa). The light gray box (Domain III) has amino acid sequences that are weakly conserved among species and contain a conserved α-helix (h4, 119–131 aa). Swi3 also has a stretch of acidic amino acids at 26–32 within Domain I. The positions of mutations that abolish Swi1–Swi3 complex formation are indicated. aa, amino acid. (C) Multiple sequence alignment of Swi3 homologs from S. pombe (Sp Swi3), humans (Hs Tipin), C. elegans (Ce Swi3), Drosophila melanogaster (Dm Swi3) and S. cerevisiae (Sc Csm3). Locations of the putative NLS, the conserved acidic region, the conserved α-helices, and mutations found in our swi3 mutant collection are shown.

Figure 4. Effects of swi3 mutations on cell growth and length.

(A) Cells of the indicated genotypes were grown in YES media at 30°C and measured for OD_{600 nm} values at the indicated times. (B, C) Cells of the indicated genotypes were grown in YES supplemented with 0 (B) or 30 µM (C) CPT for 7 h at 25°C, and cell length at septation was measured. At least 25 septated cells were measured for each strain. Error bars correspond to standard deviations. * P-values (<0.01) determined by paired Student's t-test indicate that these mutants show statistically significant elongation phenotype compared to wild-type cells.

Figure 5. Effects of swi3 mutations on the recovery of replication forks.

(A, B, C) Chromosome samples from cells of the indicated genotypes were examined by PFGE. Cells were grown until mid-log phase and then incubated in the presence of 30 µM CPT (A), 20 mM HU (B) or 15 µM CPT (C) for 3 h at 30°C. Cells were then washed and released into fresh medium. Chromosomal DNA samples were prepared at the indicated times. swi3 (except for swi3-E39) and cds1 mutants appeared to harbor a shorter chromosome III, which is probably due to recombination at rDNA repeats , , . Representative results from repeat experiments are shown. (D) Five-fold serial dilutions of cells of the indicated genotypes were incubated on YES agar medium supplemented with the indicated amounts of CPT for 3 days at 32°C.

Figure 6. Effects of swi3 mutations on Cds1 kinase activity and DNA repair foci formation.

(A) Cells of the indicated genotypes were incubated in YES medium supplemented with 12 mM HU for 0 (open bars) and 2 h (closed bars) at 30°C. Kinase activity of immunoprecipitated Cds1 was measured using myelin basic protein (MBP) as a substrate. MBP was separated on 15% polyacrylamide gels and detected by Coomassie Brilliant Blue staining. The gel was dried, and radioactivity levels (cpm) of MBP were determined in a liquid scintillation counter. Relative radioactivity levels of Cds1 were calculated by setting the radioactivity of MBP from the HU-treated wild type sample to 100%. Error bars correspond to standard deviations obtained from three independent experiments. (B) Cells of indicated swi3 mutants were engineered to express Rad22-YFP and grown in YES medium at 25°C until mid-log phase. The percentages of nuclei with at least one focus are shown. At least 200 cells were counted for each strain. Error bars correspond to standard deviations obtained from at least three independent experiments. This analysis shows that a large increase in Rad22-YFP foci accumulation was observed in swi3 mutants that have a defect in Swi1–Swi3 complex formation.

Figure 7. Effects of swi3 mutations on sister chromatid cohesion.

(A) Cells of the indicated genotypes were grown to mid-log phase and incubated at 20°C for 3 and 5 h to obtain prophase/metaphase cells. All cells contain the nda3-KM311 mutation and LacO repeats near centromere 1 and express LacI-GFP-NLS. Representative images at 5 h are shown for cells of indicated genotypes. (B) Quantification of prophase/metaphase cells that had two GFP foci shown in A. At least 200 cells were counted for each strain. Error bars correspond to the standard deviations obtained from at least three independent experiments. (C) Five-fold serial dilutions of cells of the indicated genotypes were incubated on YES agar medium supplemented with the indicated amounts of HU, CPT, and TBZ for 3 to 5 days at 32°C. swi3-E31 and swi3-E39 has synergistic genetic interaction with ctf18Δ in CPT and TBZ sensitivities. However, swi3-E31 but not swi3-E39 had additive genetic effect with ctf18Δ in HU sensitivity, strengthening the idea that swi3-E39 is proficient in the Cds1-dependent replication checkpoint. Representative images of repeat experiments are shown.

Figure 8. Models for Swi1–Swi3 dependent preservation of genomic integrity in S. pombe.

Swi1–Swi3 complex is involved in both checkpoint-dependent and -independent pathways to maintain genomic integrity. Swi1–Swi3 regulates Mrc1 and Cds1 to promote checkpoint activation and fork stabilization in response to HU-dependent fork arrest. Swi1–Swi3 uses a checkpoint-independent mechanism to regenerate broken replication forks when cells are treated with CPT. Swi1–Swi3 may regulate Chl1 to promote efficient establishment of sister chromatid cohesion, which might also be involved in fork regeneration.