Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1 - PubMed (original) (raw)

Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1

Eishi Noguchi et al. Mol Cell Biol. 2003 Nov.

Abstract

The replication checkpoint is a dedicated sensor-response system activated by impeded replication forks. It stabilizes stalled forks and arrests division, thereby preserving genome integrity and promoting cell survival. In budding yeast, Tof1 is thought to act as a specific mediator of the replication checkpoint signal that activates the effector kinase Rad53. Here we report studies of fission yeast Swi1, a Tof1-related protein required for a programmed fork-pausing event necessary for mating type switching. Our studies have shown that Swi1 is vital for proficient activation of the Rad53-like checkpoint kinase Cds1. Together they are required to prevent fork collapse in the ribosomal DNA repeats, and they also prevent irreversible fork arrest at a newly identified hydroxyurea pause site. Swi1 also has Cds1-independent functions. Rad22 DNA repair foci form during S phase in swi1 mutants and to a lesser extent in cds1 mutants, indicative of fork collapse. Mus81, a DNA endonuclease required for recovery from collapsed forks, is vital in swi1 but not cds1 mutants. Swi1 is recruited to chromatin during S phase. We propose that Swi1 stabilizes replication forks in a configuration that is recognized by replication checkpoint sensors.

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Figures

FIG. 1.

Replication checkpoint function of Swi1. (A) Synergistic interactions of swi1 and chk1 mutations in UV survival assays indicate that Swi1 is required for tolerance of UV damage during DNA replication. Fivefold serial dilutions of cells were plated on YES agar medium and then exposed to the indicated dose of UV. Agar plates were incubated for 2 to 5 days at 30°C. (B) Swi1 acts independently of the proteins that promote UV damage repair. Cells of the indicated genotypes were spread on YES agar medium and exposed to the indicated dose of UV. Agar plates were incubated for 3 days at 30°C to measure UV survival. (C) Swi1 is involved in survival of HU-induced replication arrest. Synergistic interactions of swi1 with cds1 and chk1 mutations indicate that Swi1 has an HU survival function that is at least partially independent of Cds1 and Chk1. Fivefold serial dilutions of cells were incubated on YES agar medium supplemented with the indicated amount of HU for 2 to 5 days at 30°C.

FIG. 2.

Swi1 is required for Cds1 pathway of replication checkpoint arrest. The indicated strains were incubated in YES liquid medium supplemented with 0 or 12 mM HU for 6 h at 30°C and then stained with DAPI to visualize nuclear DNA. Wild-type and chk1, cds1, swi1, and swi1 cds1 mutant cells treated with HU underwent checkpoint arrest, as indicated by the appearance of elongated, uninucleate cells without septa. In contrast, rad26, cds1 chk1, and swi1 chk1 mutant cells treated with HU failed to undergo cell cycle arrest and instead displayed aberrant mitosis as indicated by a cut phenotype. The cut phenotype was also observed in ∼12% of septated swi1 chk1 mutant cells grown in the absence of HU (arrowheads). Bar, 10 μm.

FIG. 3.

Deficient Cds1 activation in swi1 mutant. (A) Cds1 activation is strongly reduced in swi1 mutant cells. Cells of the indicated genotypes were incubated in YES liquid medium supplemented with 0 or 12 mM HU for 4 h at 30°C. Kinase activity of immunoprecipitated Cds1 was measured by using myelin basic protein (MBP) substrate (upper panel). A Cds1 immunoblot confirmed that approximately equal amounts of Cds1 (absent in cds1 mutant strain) were present in the samples (lower panel). The Cds1 polyclonal antisera cross-react with nonspecific proteins that migrate faster than Cds1 (asterisk). (B) HU induction of Mik1 accumulation is deficient in swi1 mutant cells. Cells of the indicated genotypes that contained genomic _mik1_-13myc were incubated with 0 or 12 mM HU for 4 h at 30°C. Mik1 was detected with anti-Myc monoclonal antibody (upper panel). A Cdc2 immunoblot was used as a loading control (lower panel). (C) HU sensitivity of swi1 mutant cells was partially suppressed by multicopy cds1+ plasmid. Fivefold serial dilutions of swi1 mutant cells transformed with the indicated plasmids were incubated on YES agar medium supplemented with the indicated amount of HU for 2 to 5 days at 30°C. (D) Colonies of homothallic wild-type and swi1, cds1, and rad3 mutant cells were grown in sporulation medium and then stained by iodine vapor to detect spores. The swi1 mutant colonies showed a mottled phenotype, indicating a defect in mating type switching, whereas wild-type and cds1 and rad3 mutant cells stained darkly with iodine, indicating proficient mating type switching.

FIG.4.

2D gel analysis of replication forks in swi1 and cds1 mutants. (A) Map of the rDNA repeats as reported by Sanchez et al. (41). The ars3001 box indicates the origin region and the P box indicates a pause site mapped by Sanchez et al. (41). The HUP site is indicated, as are the probes and restriction enzyme sites (E, _Eco_RI; H, _Hin_dIII; B, _Bam_HI; K, _Kpn_I). (B) Diagram of the migration pattern of replication intermediates that can be detected by 2D gel electrophoresis. (C) Diagrams of potential Y-like arc and pause site patterns. (D) Wild-type and cds1, swi1, and swi1 cds1 mutant cells were incubated in YES liquid medium supplemented with 12 mM HU for the indicated times at 30°C. Genomic DNA prepared from cells was analyzed by 2D gel electrophoresis. The five columns on the left show 2D gels of DNA digested with _Hin_dIII and _Kpn_I and hybridized with probe A. Cells were incubated with HU for 0, 1.5, or 3 h and then HU was washed out and cells were incubated for a further 0.5 or 1.5 h. Numbers in the panels indicate fractions of RIs relative to total signal. Numbers with arrows in 1.5-h samples represent fractions of bubble structures relative to total signal. The two columns on the right show gels digested with _Hin_dIII and _Bam_HI and hybridized with probe B. Cells were incubated with HU for 0 or 3 h.

FIG. 5.

Spontaneous Rad22-YFP foci formation in swi1 mutant cells. (A) Cells that had genomic rad22-YFP were grown in EMM medium at 25°C until mid-log phase. DNA was stained with DAPI. Rad22-YFP foci formation was strikingly elevated in swi1 mutant cells. (B) Quantification of Rad22-YFP foci according to cell cycle stages. S and early G2 cells had the most Rad22-YFP foci. (C) Swi1 is not required for survival of DNA damage caused by bleomycin. Cells of the indicated genotypes were treated with the indicated concentrations of bleomycin for 2 h and then incubated on YES plates for 3 days to measure survival. (D) Swi1 is not required for survival of DNA damage caused by IR. Cells of the indicated genotypes were tested for resistance to IR. Irradiated cells were incubated on YES plates for 3 days to measure survival.

FIG. 6.

Genetic interactions involving Swi1 and Mus81. (A) Mus81 is vital for cell survival in CPT. Fivefold serial dilutions of cells were incubated for 2 to 3 days at 30°C on YES agar medium supplemented with 0 or 0.3 μM CPT. Growth of mus81 mutant cells was severely impaired by 0.3 μM CPT, whereas growth of wild-type and swi1 and checkpoint mutant strains was unaffected. The swi1 and checkpoint mutant strains were sensitive to higher concentrations of CPT (data not shown). (B) Mus81 and Rqh1 are vital in swi1 mutant background. Cells of indicated genotypes were grown in YES liquid medium and photographed. The swi1 mus81 and swi1 rqh1 double mutants showed a severe growth defect, with many elongated and vacuolated cells, whereas cds1 mus81 mutant cells and other single mutants grew relatively well.

FIG. 7.

Swi1-GFP is recruited to chromatin in S phase. (A) Cells that had genomic _swi1_-GFP were incubated in EMM liquid medium supplemented with 0 or 12 mM HU for 6 h at 25°C. Cells were modified into spheroplasts and fixed prior to microscopic analysis of Swi1-GFP fluorescence. All cells displayed nuclear Swi1-GFP. Essentially identical results were obtained with living cells (data not shown). (B) In situ chromatin binding assay of Swi1-GFP protein in wild-type and rad26 mutant backgrounds. The assay was the same as that described for panel A, except that spheroplasts were extracted with Triton X-100 to remove soluble nuclear protein and then were fixed for microscopic analysis. Representative patterns of fluorescence are shown. Swi1-GFP protein was detected predominantly in septated cells and unseptated small cells, which are in S phase or possibly early G2 phase. Representative photos of HU-treated cells are also shown. (C) Quantification of Triton X-100-resistant nuclear Swi1-GFP in wild-type and rad26 mutant cells according to cell morphology.

FIG. 8.

Proposed model of Swi1's involvement in the response to fork arrest. Swi1 is recruited to chromatin during S phase, suggesting that it might be an ancillary component of the replisome. Swi1 is required for proficient activation of Cds1 in response to replication arrest, suggesting that it stabilizes the stalled fork in a conformation that is recognized by the checkpoint sensor proteins (Rad3-Rad26 and Rad9-Rad1-Hus1 complexes) and Mrc1. Mrc1 facilities Cds1 activation by recruiting it to Rad3 and/or mediating Cds1 autophosphorylation. Cds1 enhances fork and replisome stabilization while the lesion is removed.

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Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1 - PubMed (original) (raw)