Tel2 Is Required for Activation of the Mrc1-mediated Replication Checkpoint
2006; Elsevier BV; Volume: 282; Issue: 8 Linguagem: Inglês
10.1074/jbc.m607432200
ISSN1083-351X
AutoresMiho Shikata, Fuyuki Ishikawa, Junko Kanoh,
Tópico(s)Mitochondrial Function and Pathology
ResumoProteins belonging to the Tel2/Rad-5/Clk-2 family are conserved among eukaryotes and are involved in various cellular processes, such as cell proliferation, telomere maintenance, the biological clock, and the DNA damage checkpoint. However, the molecular mechanisms underlying the functions of these molecules remain largely unclear. Here we report that in the fission yeast, Schizosaccharomyces pombe, Tel2 is required for efficient phosphorylation of Mrc1, a mediator of DNA replication checkpoint signaling, and for activation of Cds1, a replication checkpoint kinase, when DNA replication is blocked by hydroxyurea. In fact, Tel2 is required for survival of replication fork arrest and for the replication checkpoint in cells lacking Chk1, another checkpoint kinase the role of which overlaps that of Cds1 in cell cycle arrest by replication block. In addition, Tel2 plays important roles in entry into S phase and in genome stability. Tel2 is essential for vegetative cell growth, and the tel2Δ strain accumulated cells with 1C DNA content after germination. In the absence of hydroxyurea, Tel2 is vital in the mutant lacking Swi1, a component of the replication fork protection complex, and multiple Rad22 DNA repair foci were frequently observed in Tel2-repressed swi1Δ cells especially at S phase. In contrast, the cds1Δswi1Δ mutant did not show such lethality. These results indicate that S. pombe Tel2 plays important roles in the Mrc1-mediated replication checkpoint as well as in the Cds1-independent regulation of genome integrity. Proteins belonging to the Tel2/Rad-5/Clk-2 family are conserved among eukaryotes and are involved in various cellular processes, such as cell proliferation, telomere maintenance, the biological clock, and the DNA damage checkpoint. However, the molecular mechanisms underlying the functions of these molecules remain largely unclear. Here we report that in the fission yeast, Schizosaccharomyces pombe, Tel2 is required for efficient phosphorylation of Mrc1, a mediator of DNA replication checkpoint signaling, and for activation of Cds1, a replication checkpoint kinase, when DNA replication is blocked by hydroxyurea. In fact, Tel2 is required for survival of replication fork arrest and for the replication checkpoint in cells lacking Chk1, another checkpoint kinase the role of which overlaps that of Cds1 in cell cycle arrest by replication block. In addition, Tel2 plays important roles in entry into S phase and in genome stability. Tel2 is essential for vegetative cell growth, and the tel2Δ strain accumulated cells with 1C DNA content after germination. In the absence of hydroxyurea, Tel2 is vital in the mutant lacking Swi1, a component of the replication fork protection complex, and multiple Rad22 DNA repair foci were frequently observed in Tel2-repressed swi1Δ cells especially at S phase. In contrast, the cds1Δswi1Δ mutant did not show such lethality. These results indicate that S. pombe Tel2 plays important roles in the Mrc1-mediated replication checkpoint as well as in the Cds1-independent regulation of genome integrity. There are multiple safeguard systems for maintenance of genome integrity, one of which is the cell cycle checkpoint. When mammalian cells suffer DNA damage or DNA replication fork stalling, members of the phosphatidylinositol 3-kinase-related family, including ATM 3The abbreviations used are: ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3-related protein; HU, hydroxyurea; HA, hemagglutinin; ORF, open reading frame; ssDNA, single-stranded DNA. 3The abbreviations used are: ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3-related protein; HU, hydroxyurea; HA, hemagglutinin; ORF, open reading frame; ssDNA, single-stranded DNA. and ATR, are activated and phosphorylate their downstream targets to cause cell cycle arrest until the DNA is fully repaired or until replication can resume (1Rhind N. Russell P. J. Cell Sci. 2000; 113: 3889-3896Crossref PubMed Google Scholar, 2Boddy M.N. Russell P. Curr. Biol. 2001; 11: R953-R956Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 3Branzei D. Foiani M. Curr. Opin. 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Chk1 and Cds1 have overlapping roles in both the DNA damage checkpoint and the DNA replication checkpoint; therefore, the chk1 cds1 double mutant is highly sensitive to various forms of DNA damage and to replication block, comparable with rad3 or rad26 mutants, whereas chk1 or cds1 single mutants show only moderate or weak sensitivity (16Boddy M.N. Furnari B. Mondesert O. Russell P. Science. 1998; 280: 909-912Crossref PubMed Scopus (279) Google Scholar, 17Lindsay H.D. Griffiths D.J. Edwards R.J. Christensen P.U. Murray J.M. Osman F. Walworth N. Carr A.M. Genes Dev. 1998; 12: 382-395Crossref PubMed Scopus (327) Google Scholar, 18Zeng Y. Forbes K.C. Wu Z. Moreno S. Piwnica-Worms H. Enoch T. Nature. 1998; 395: 507-510Crossref PubMed Scopus (305) Google Scholar). Chk1 and Cds1 phosphorylate and inactivate the tyrosine phosphatase Cdc25, and activation of Chk1 and Cds1 leads to accumulation of the tyrosine kinase Mik1, in response to DNA damage or replication block (16Boddy M.N. Furnari B. 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Cds1 also plays a role in stabilization of replication forks. The frequencies of replication fork collapse and arrest are significantly increased in the cds1Δ strain (25Noguchi E. Noguchi C. Du L.-L. Russell P. Mol. Cell. Biol. 2003; 23: 7861-7874Crossref PubMed Scopus (140) Google Scholar). Another important system for maintaining genome integrity is protection of replication forks. In S. pombe, the Swi1-Swi3 complex is localized at and protects stalled replication forks (25Noguchi E. Noguchi C. Du L.-L. Russell P. Mol. Cell. Biol. 2003; 23: 7861-7874Crossref PubMed Scopus (140) Google Scholar, 26Dalgaard J.Z. Klar A.J.S. Cell. 2000; 102: 745-751Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 27Krings G. Bastia D. Proc. Natl. Acad. Sc. U. S. A. 2004; 101: 14085-14090Crossref PubMed Scopus (91) Google Scholar, 28Noguchi E. Noguchi C. McDonald W.H. Yates J.R. II I Russell P. Mol. Cell. 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Thus, the presence of the Swi1-Swi3 complex at replication forks is conserved at least in the two yeasts. The Tel2/Rad-5/Clk-2 family proteins are highly conserved among eukaryotic species. The tel2 mutant in S. cerevisiae has short telomeric DNA (34Lustig A.J. Petes T.D. Proc. Natl. Acad. Sc. U. S. A. 1986; 83: 1398-1402Crossref PubMed Scopus (203) Google Scholar). S. cerevisiae Tel2 is essential for cell viability and binds to both single-stranded and double-stranded telomeric DNA in vitro, suggesting that it regulates telomere length by binding to the ends of telomeres (35Runge K.W. Zakian V.A. Mol. Cell. Biol. 1996; 16: 3094-3105Crossref PubMed Scopus (84) Google Scholar, 36Kota R.S. Runge K.W. Nucleic Acids Res. 1998; 26: 1528-1535Crossref PubMed Scopus (28) Google Scholar, 37Kota R.S. Runge K.W. Chromosoma (Berl.). 1999; 108: 278-290Crossref PubMed Scopus (22) Google Scholar). 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Shoubridge E.A. Hekimi S. J. Biol. Chem. 2003; 278: 21678-21684Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). However, the molecular mechanisms of action of Rad-5/Clk-2 or hCLK2 in DNA replication remain unknown. Here we report the identification and characterization of tel2+, the gene encoding the fission yeast TEL2/Rad-5/Clk-2-related protein. We found that Tel2 is required for HU-induced activation of the Mrc1-Cds1 DNA replication checkpoint. In addition, the genetic interaction between tel2 and swi1 indicates that Tel2 also plays an important role in maintenance of genome integrity, especially at S phase. Strains and General Techniques—The S. pombe strains used in this study are listed in Table 1. Yeast extract media YES, SD, MEA, and EMM were used to grow cells. Growth media, basic genetics, and biochemical techniques for fission yeast were described previously (46Alfa C. Fantes P. Hyams J. McLeod M. Warbrick E. Experiments with Fission Yeast: A Laboratory Course Manual. 1993; (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar, 47Moreno S. Klar A. Nurse P. Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3125) Google Scholar). UV sensitivity assays and 4′,6-diamino-2-phenylindole staining of nuclear DNA were performed as described previously (28Noguchi E. Noguchi C. McDonald W.H. Yates J.R. II I Russell P. Mol. Cell. Biol. 2004; 24: 8342-8355Crossref PubMed Scopus (171) Google Scholar). EMM was supplemented with 14 μm thiamine to repress tel2+ gene expression regulated by the nmt81 promoter (48Basi G. Schmid E. Maundrell K. Gene (Amst.). 1993; 123: 131-136Crossref PubMed Scopus (565) Google Scholar, 49Maundrell K. Gene (Amst.). 1993; 123: 127-130Crossref PubMed Scopus (927) Google Scholar).TABLE 1Yeast strains used in this studyStrainGenotypeJK317h- leu1-32 ura4-D18TN360h+ leu1-32CS903h+/h- tel2+/tel2::ura4+ ade6-M216/ade6-M210 leu1-32/leu1-32 ura4-D18/ura4-D18CS904h+/h- tel2+/tel2::ura4+ ade6-M216/ade6-M210 leu1-32/leu1-32 ura4-D18/ura4-D18CS1091h- tel2-3HA:ura4+ cdc25-22 ade6-M216 ura4-D18CS1526h+ tel2-12myc:Kanr leu1-32 ura4-D18CS1529h- nmt81-tel2-12myc:LEU2+:Kanr leu1-32 ura4-D18CS1531h+ nmt81-tel2-12myc:LEU2+:Kanr leu1-32 ura4-D18CS1539h- nmt81-tel2-12myc:LEU2+:Kanr cds1::ura4+ leu1-32 ura4-D18TM1667h- rad22-YFP:Kanr swi1::LEU2+ leu1-32 ura4-D18CS1717h- nmt81-tel2-12myc:LEU2+:Kanr chk1::ura4+ leu1-32 ura4-D18CS1725h- tel2-12myc:Kanr cds1::ura4+ ura4-D18CS1728h- tel2-12myc:Kanr chk1::ura4+ ura4-D18CS1732h- nmt81-tel2:12myc:LEU2+:Kanr leu1-32YT1738h+/h- ade6-M216/ade6-M210 leu1-32/leu1-32 ura4-D18/ura4-D18CS1742h- tel2-12myc:Kanr mrc1-3HA:ura4+ leu1-32 ura4-D18CS1745h- nmt81-tel2-12myc:LEU2+:Kanr mrc1-3HA:ura4+ leu1-32 ura4-D18CS1746h- nmt81-tel2-12myc:LEU2+:Kanr chk1-3HA leu1-32 ura4-D18CS1761h- tel2-12myc:Kanr chk1::ura4+ cds1::ura4+ ura4-D18CS1773h- tel2-12myc:Kanr chk1-3HA leu1-32 ura4-D18CS1785h- tel2-12myc:KanrCS1789h- tel2-12myc:Kanr rad3::ura4+ ura4-D18CS1830h- nmt81-tel2-12myc:LEU2+:Kanr rad22-YFP:ura4+ leu1-32 ura4-D18CS1833h- tel2-12myc:Kanr rad22-YFP:ura4+ ura4-D18CS1837h- nmt81-tel2-12myc:LEU2+:Kanr swi1::LEU2+ leu1-32 ura4-D18CS1847h- tel2-12myc:Kanr swi1::LEU2+ leu1-32 ura4-D18CS2045h- tel2-12myc:Kanr mrc1-3HA:ura4+ cds1::ura4+ leu1-32 ura4-D18CS2278h- tel2-12myc:Kanr swi1::LEU2+ cds1::ura4+ leu1-32 ura4-D18CS2298h- nmt81-tel2-12myc:LEU2+:Kanr swi1::LEU2+ rad22-YFP:ura4 leu1-32 ura4-D18 Open table in a new tab Gene Disruption—For disruption of the tel2+ gene, the tel2+ ORF was amplified by PCR with primers cs11 (5′-GCTTACCGATTAGCGGAGC-3′) and cs12 (5′-CAGCGGACCCAACAAGCTG-3′) using wild-type genomic DNA as a template and cloned into pBlueScript SK– (Stratagene). The resultant plasmid was digested with HincII and PstI, and a ura4+ cassette was inserted. The resultant plasmid was digested with SpeI and XhoI, and the tel2+::ura4+ fragment was used for transformation. Flow Cytometric Analysis of Germinated Spores—Diploid cells were induced for sporulation on MEA medium for 2 days at 25 °C and were treated with 0.5% glusulase for 12 h at 30 °C. Cells were washed with water and treated with 5 mg/ml Lysing Enzymes (Sigma) for 1 h at 37°C. Spores were washed twice with water and stored at 4 °C. Spores were germinated in SD medium at 32 °C. Flow cytometric analysis was performed as described previously (50Hatanaka M. Shimoda C. Yeast. 2001; 18: 207-217Crossref PubMed Scopus (48) Google Scholar). Chromosomal Integration of tel2-12myc, tel2-3HA, and mrc1-3HA—To tag genomic tel2+ with a sequence encoding 12 copies of the Myc epitope at the C terminus, the tel2+ ORF was amplified by PCR with primers cs26 (5′-TATAAAGGATCCTCCCAGCCAGTTACTTCCAG-3′; the BamHI site is underlined) and cs33 (5′-TATAAAGGTACCTTAAGTCCGGCTAATCCAAATC-3′; the KpnI site is underlined), cloned into pJK202 (51Kanoh J. Ishikawa F. Curr. Biol. 2001; 11: 1624-1630Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar), and the ura4+ marker was replaced with the Kanr marker. The resultant plasmid was partially digested with HincII and used for transformation. To tag genomic tel2+ with a sequence encoding three copies of the HA epitope at the C terminus, the tel2+ ORF was amplified by PCR with primers cs13 (5′-TATAAACTCGAGTCCCAGCCAGTTACTTCCAG-3′; the XhoI site is underlined) and cs14 (5′-AAATATGCGGCCGCAAAGTCCGGCTAATCCAAATC-3′; NotI site is underlined) and cloned into pTN151 (51Kanoh J. Ishikawa F. Curr. Biol. 2001; 11: 1624-1630Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). The resultant plasmid was partially digested with HincII and used for transformation. To tag genomic mrc1+ with a sequence encoding three copies of the HA epitope at the C terminus, the mrc1+ ORF was amplified by PCR using primers cs70 (5′-TATAAACTCGAGAGCTTTGGACGATAGTGACG-3′; the XhoI site is underlined) and cs71 (5′-TATAAAGCGGCCGCAGTCAAAGTCCGAGTAATTATTC-3′; the NotI site is underlined) and cloned into pTN151 (51Kanoh J. Ishikawa F. Curr. Biol. 2001; 11: 1624-1630Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). The resultant plasmid was digested with SpeI and used for transformation. Construction of the nmt81-tel2+ Strain—To insert the nmt81 promoter upstream of the tel2+ ORF, the tel2+ ORF was amplified by PCR using primers cs31 (5′-TATAAACATATGAAATCCTTTGCTAGCGAAC-3′; the NdeI site is underlined) and cs32 (5′-TATAAAGGATCCTTAAAGTCCGGCTAATCCAAATC-3′; the BamHI site is underlined) and cloned into pRIP81 (49Maundrell K. Gene (Amst.). 1993; 123: 127-130Crossref PubMed Scopus (927) Google Scholar). The upstream region of the chromosomal tel2+ ORF was amplified by PCR using primers cs11 (5′-GCTTACCGATTAGCGGAGC-3′) and cs34 (5′-TATAAAAAGCTTATCCTTGGAGTTGAAAAATAACC-3′; the HindIII site is underlined) and cloned into pRIP81-tel2+. The resultant plasmid was digested with SpeI and SacI and used for transformation of the strain CS1526, yielding the strain CS1531. CS1531 was crossed with JK317 to obtain CS1529, and CS1529 was crossed with TN360 to obtain CS1732. Cds1 Kinase Assay and Immunoblotting of Chk1 and Mrc1—Cds1 kinase assays were performed as described previously (16Boddy M.N. Furnari B. Mondesert O. Russell P. Science. 1998; 280: 909-912Crossref PubMed Scopus (279) Google Scholar). Chk1 immunoblotting was performed as described previously (52Walworth N.C. Bernards R. Science. 1996; 271: 353-356Crossref PubMed Scopus (348) Google Scholar). For analyses of Mrc1, cell extracts were prepared, and SDS-PAGE and immunoblotting were performed as described previously (15Zhao H. Tanaka K. Noguchi E. Noguchi C. Russell P. Mol. Cell. Biol. 2003; 23: 8395-8403Crossref PubMed Scopus (45) Google Scholar). S. pombe tel2+ Is Essential for Vegetative Cell Growth—To investigate the molecular mechanisms underlying the function of Tel2/Rad-5/Clk-2 family proteins in DNA replication, we searched for a tel2/rad-5/clk-2-related gene in the Sanger Centre S. pombe genome data base. We identified the ORF SPAC458.03 (hereafter referred to as tel2+) that encodes a protein showing 14.9% identity to S. cerevisiae Tel2, 13.8% to C. elegans Rad-5/Clk-2, and 15.7% to Homo sapiens CLK2 throughout the entire sequence. We found three short and unique amino acid sequences highly conserved among Tel2/Rad-5/Clk-2 family proteins (Figs. 1A and supplemental Fig. S1). The Tel2 protein was expressed throughout the cell cycle (Fig. 1B), and its expression showed no significant changes after UV or HU treatment (data not shown). The function of Tel2 was investigated by generating a tel2 null mutation. One copy of tel2+ in a ura4– diploid strain was replaced with the ura4+ marker gene. Two independent gene disruption transformants (CS903 and CS904) were confirmed by Southern blotting analysis (data not shown). Tetrad analysis of the tel2+/tel2::ura4+ diploids revealed that no more than two spores could form colonies in each set of tetrads and that all the viable cells were Ura–. The terminal morphology of tel2– spores on germination plates was examined. Fifteen percent of the spores ceased growth immediately after germination (Fig. 1C, left), whereas 85% formed microcolonies of 2–50 cells (Fig. 1C, middle and right). To exclude the possibility that the lethality observed in the tel2 null mutant was because of unexpected effects on neighboring genes, tel2+/tel2::ura4+ diploid cells were transformed with pREP1 (control vector) or pREP1-tel2+ (expression plasmid containing the tel2+ gene), and their spores were incubated on plates lacking uracil to select tel2::ura4+ haploid mutant cells. The growth of the tel2 haploid mutant was dependent on pREP1-tel2+ (Fig. 1D). Therefore, we concluded that the tel2 gene function is essential for vegetative cell growth. The function of tel2+ in DNA replication was investigated by germinating spores from a tel2+/tel2::ura4+ diploid (CS903) in SD medium lacking uracil. The tel2Δ cells completed the first cycle of DNA replication in 8 h and started to form a germ projection at 8 h, as observed in wild-type spores (Fig. 2A and data not shown). The tel2Δ cells progressed to the second cycle of DNA replication (∼8–16 h in Fig. 2C), possibly using inherited Tel2 protein. This and the variety of terminal cell morphology shown in Fig. 1C probably reflect a requirement of only a low level of Tel2 protein for cell growth (see also Fig. 3). At 20 h, a population with 1C DNA content appeared, which was not observed in the wild-type cells, indicating that the entry into S phase was inhibited or delayed in tel2Δ. These observations suggested that Tel2 is required for the normal entry into S phase.FIGURE 3Characterization of the nmt81-tel2+ strain. A, construction of the nmt81-tel2+ strain. A thiamine-inducible nmt81 promoter was inserted upstream of the chromosomal tel2+ locus with the LEU2+ marker gene, and 12 copies of the Myc tag were added at the C terminus of the Tel2 ORF. The activity of the nmt81 promoter is repressed in the presence of thiamine, and it is induced in the absence of thiamine. B, expression of Tel2-Myc protein after the addition of thiamine. Wild-type (CS1785) and nmt81-tel2+ (CS1732) cells were grown in EMM medium (without thiamine) at 30 °C to log phase, and then 14 μm thiamine was added. Whole-cell extracts were prepared, and immunoblotting was performed with anti-Myc antibodies for Tel2-Myc protein and with anti-PSTAIRE antibodies for Cdc2 protein (control). C, viability of the wild-type (CS1785) and nmt81-tel2+ (CS1732) strains. Cells were grown in EMM (without thiamine) to mid-log phase, and then thiamine was added at time 0. The viability of each strain was calculated by dividing the number of colonies formed on EMM (without thiamine) plates by the number of cells plated. Error bars indicate the standard error from three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Characterization of the nmt81-tel2+ Mutant—To further investigate the function of Tel2, we generated a strain in which the thiamine-repressible nmt81 promoter was inserted upstream of the chromosomal tel2+ locus, and the Myc tag was added at the C terminus of the Tel2 ORF (Fig. 3A). In the absence of thiamine, the nmt81 promoter induces expression of the gene located downstream, whereas it "partially" represses the expression in the presence of thiamine (48Basi G. Schmid E. Maundrell K. Gene (Amst.). 1993; 123: 131-136Crossref PubMed Scopus (565) Google Scholar). The resultant nmt81-tel2+ strain was grown to exponential phase in the absence of thiamine. Before addition of thiamine (time 0), the level of Tel2-myc expression in nmt81-tel2+ cells was approximately double that in wild-type cells (Fig. 3B). After addition of thiamine, the expression level of Tel2-myc in the nmt81-tel2+ strain decreased gradually (Fig. 3B). This strain showed lower viability than the wild-type strain, although it did not die completely and kept growing slowly even after 60 h of thiamine treatment, indicating that a very small amount of Tel2 protein is sufficient for cell viability (Fig. 3C). Thirty six hours after addition of thiamine, Tel2-myc was hardly detectable by Western blotting, and the viability started to decrease (Fig. 3, B and C). Therefore, we used cells that had been treated for at least 36 h with thiamine in subsequent experiments to investigate the effects of Tel2 repression. Tel2 Is Required for Survival of Replication Fork Arrest in chk1Δ—C. elegans rad-5/clk-2 mutants are hypersensitive to HU, IR, or UV treatment (38Hartman P.S. Herman R.K. Genetics. 1982; 102: 159-178Crossref PubMed Google Scholar, 41Ahmed S. Alpi A. Hengartner M.O. Gartner A. Curr. Biol. 2001; 11: 1934-1944Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 53Gartner A. Milsten S. Ahmed S. Hodgkin J. Hengartner M.O. Mol. Cell. 2000; 5: 435-443Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar), and overexpression of human CLK2 causes hypersensitivity to HU (45Jiang N. Benard C.Y. Kebir H. Shoubridge E.A. Hekimi S. J. Biol. Chem. 2003; 278: 21678-21684Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Therefore, we examined whether Tel2 is involved in tolerance to DNA damage in fission yeast. Cells were grown in the presence of thiamine for 36 h to repress tel2+ expression and then spotted onto plates containing HU and thiamine (Fig. 4A). Wild-type cells readily formed colonies on plates containing 5 mm HU, whereas the nmt81-tel2+ strain formed a smaller number (∼1/20) of colonies, indicating that Tel2 is involved in tolerance to replication fork arrest induced by HU treatment. The nmt81-tel2+ chk1Δ double mutant showed much stronger sensitivity to HU than each single mutant, which was similar to rad3Δ. In contrast, the nmt81-tel2+ cds1Δ double mutant showed only slightly stronger sensitivity than each single mutant. These observations indicated that Tel2 functions in HU tolerance and that this is at least partially independent of Chk1. Next, we examined whether Tel2 is involved in UV tolerance (Fig. 4A). Exposure of DNA to UV irradiation causes the formation of DNA lesions that block replication forks. The Tel2-repressed nmt81-tel2+ strain and chk1Δ single mutant showed moderate sensitivity to UV irradiation. However, the nmt81-tel2+ chk1Δ double mutant showed acute sensitivity similar to rad3Δ or chk1Δ cds1Δ. In contrast, the nmt81-tel2+ cds1Δ double mutant showed sensitivity similar to that of the nmt81-tel2+ sin
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