MOLECULAR AND CELLULAR BIOLOGY,0270-7306/00/$04.0010

Nov. 2000, p. 7922–7932 Vol. 20, No. 21

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Schizosaccharomyces pombe Hsk1p Is a Potential Cds1p TargetRequired for Genome Integrity

HILARY A. SNAITH,1† GRANT W. BROWN,2 AND SUSAN L. FORSBURG1*

Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037-1099,1

and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A82

Received 15 May 2000/Returned for modification 11 July 2000/Accepted 15 August 2000

The fission yeast Hsk1p kinase is an essential activator of DNA replication. Here we report the isolation andcharacterization of a novel mutant allele of the gene. Consistent with its role in the initiation of DNA synthesis,hsk1ts genetically interacts with several S-phase mutants. At the restrictive temperature, hsk1ts cells sufferabnormal S phase and loss of nuclear integrity and are sensitive to both DNA-damaging agents and replicationarrest. Interestingly, hsk1ts mutants released to the restrictive temperature after early S-phase arrest inhydroxyurea (HU) are able to complete bulk DNA synthesis but they nevertheless undergo an abnormalmitosis. These findings indicate a second role for hsk1 subsequent to HU arrest. Consistent with a later S-phaserole, hsk1ts is synthetically lethal with Drqh1 (RecQ helicase) or rad21ts (cohesin) mutants and suppressed byDcds1 (RAD53 kinase) mutants. We demonstrate that Hsk1p undergoes Cds1p-dependent phosphorylation inresponse to HU and that it is a direct substrate of purified Cds1p kinase in vitro. These results indicate thatthe Hsk1p kinase is a potential target of Cds1p regulation and that its activity is required after replicationinitiation for normal mitosis.

Eukaryotic cells have developed elaborate regulatory mech-anisms to ensure that DNA replication is restricted to S phaseand occurs just once per cell cycle. An important component ofthis regulation is the coordination of multiple origins of repli-cation. Data from many systems have provided a model of theinitiation of DNA synthesis in which several multiprotein com-plexes interact with discrete replication origins in a specifictemporal pattern to regulate entry into S phase (24, 26, 32).Two protein kinase complexes are required to activate thereplication origins: cyclin–cyclin-dependent kinase (CDK),which acts both positively and negatively to control origin func-tion (21, 22), and the product of the hsk11 (also called CDC7)gene complex.

Schizosaccharomyces pombe hsk11 is a member of the con-served family of CDC7 protein kinases (23, 37). The hsk11

gene was originally cloned by its sequence homology to bud-ding yeast CDC7 and is essential for viability (36). Spores withhsk11 deleted are able to germinate but fail to undergo DNAreplication, demonstrating that Hsk1p is required for cell cycleprogression. Hsk1p is dependent on the transient expression ofa regulatory subunit, Dfp1p (also called Him1p), for activitytowards its substrates (6, 56). Similar to Hsk1p, Dfp1p is alsopart of a multimember family, founded by the Saccharomycescerevisiae DBF4 gene, and homologues have now been identi-fied in many other species, including Drosophila melanogasterand humans (23, 31). Dfp1p (homologous to Dbf4) is ex-pressed only during late G1 phase until mitosis, thereby re-stricting Hsk1p activity to S phase and subsequent stages of thecell cycle. Genetic and biochemical evidence indicates that theconserved MCM proteins are important Hsk1p substrates. S.pombe, S. cerevisiae, Xenopus laevis, and human CDC7 kinasesphosphorylate Mcm2 in vitro (23, 48). Also, a recessive muta-

tion in S. cerevisiae mcm5 (also called cdc46) bypasses therequirement for CDC7 entirely (20) and there are syntheticinteractions between a strain with a mutation in the Cdc7regulatory subunit, encoded by DBF4, or mcm2 mutants (33).

In addition to having a role in replication initiation, thefission yeast Dhsk1 mutant also has phenotypes suggesting arole in checkpoint responses. While most Dhsk1 spores arrestwith a predominantly 1C DNA content and fail to progress intothe cell cycle, approximately 25% of these germinated cells goon to attempt an aberrant mitosis, generating “cut” cells (36).This phenotype is typical of mutants that fail to initiate anyDNA replication; once replication is initiated, the checkpointis activated to prevent inappropriate mitosis (28).

The DNA replication and damage checkpoint responses infission yeast require two kinases acting downstream of theRad3p kinase: Cds1p (the S. pombe homologue of S. cerevisiaeRad53) and Chk1p (8, 44). rad31 encodes the homologue ofthe metazoan ATM and ATR genes and is activated by a rangeof cellular insults, including UV irradiation and nucleotidedepletion (8). The Cds1p and Chk1p kinases then act to inhibitcyclin-CDK activity, thereby preventing mitotic progression.

The replication checkpoint has been extensively investigatedusing hydroxyurea (HU), an inhibitor of ribonucleotide reduc-tase, which halts replication due to lack of nucleotides (3).Cds1p appears to respond to early replication defects such asthose induced by HU and is required for cells to recover fromS-phase arrest (35, 39). Cells lacking cds1 arrest in early Sphase upon HU treatment but do not enter mitosis. However,they are defective in return to growth, indicating that Cds1phas a role specifically in recovery from S-phase arrest, ratherthan in prevention of mitosis. Studies suggest that Dfp1p isdirectly involved in the Cds1p-mediated response to replica-tion arrest. Dfp1p is phosphorylated in vivo upon incubation ofthe cells with HU, and this phosphorylation depends on activeCds1p (6, 56). In addition, dfp1 mutants that lack a conservedmotif in the amino terminus have a mild HU sensitivity anddisplay a cut phenotype after prolonged incubation in HU (56).In budding yeast, Rad53 is required for the phosphorylation ofDbf4 in response to HU (11, 62) and may influence Cdc7

* Corresponding author. Mailing address: Molecular Biology andVirology Laboratory, The Salk Institute for Biological Studies, 10010North Torrey Pines Rd., La Jolla, CA 92037-1099. Phone: (858) 453-4100, ext. 1341. Fax: (858) 457-4765. E-mail: forsburg@salk.edu.

† Present address: Institute for Cell and Molecular Biology, Edin-burgh EH9 3JR, United Kingdom.

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activity at late origins (46, 51). Interestingly, cds11 is not anessential gene in fission yeast, in contrast to RAD53, which isrequired for viability in budding yeast (29, 39, 59), suggestingthat there may be differences between the two yeasts in theresponse to replication arrest.

To further examine the role of hsk11 in fission yeast S phase,we isolated a novel temperature-sensitive allele of the gene.We show that the mutant strain exhibits defects in both DNAreplication and checkpoint responses. Consistent with a role inthe initiation of DNA synthesis, hsk1ts cells exhibit delayedentry into S phase at the restrictive temperature. However, thecells also have an unusual phenotype for a initiation mutant:rather than showing evidence of premature mitosis, many cellsare elongated with fragmented nuclei. Upon release from HUarrest, hsk1ts cells complete bulk DNA synthesis but undergoan abnormal mitosis. This suggests a role for Hsk1p kinaseafter replication origin firing. In addition, hsk1ts cells showpronounced sensitivity to agents which trigger both the DNAreplication and damage checkpoints. Upon HU treatment,Hsk1p is phosphorylated in vivo in a Cds1p-dependent mannerand Cds1p directly phosphorylates Hsk1p in vitro. Our dataindicate that Hsk1p itself is a target of the checkpoint responseand plays a role in S phase after origin firing.

MATERIALS AND METHODS

General yeast manipulation and molecular biology. All S. pombe strains (Ta-ble 1) were maintained on yeast extract plus supplement (YES) agar plates orunder selection on Edinburgh minimal media (EMM) with appropriate supple-ments using standard techniques (38). Double mutant strains were constructed

by standard tetrad analysis. Yeast cells were transformed by electroporation asdescribed previously (54). Standard molecular biology techniques were used(45). A Dhsk1::ura41/hsk11 diploid and hsk11 genomic and cDNA clones weregifts of H. Masai. Pulsed-field gel electrophoresis was performed as describedpreviously (54). pSLF272-cds1KD contains a kinase-inactive version of cds1 witha D312E mutation (35).

Isolation of an hsk1-1312 temperature-sensitive strain. pREP1-hsk1* plasmidswere mutagenized in 1 M hydroxylamine solution, pH 6.7, for 20 h at 37°C (43).The mutant library was transformed into a Dhsk1::ura41/hsk11 diploid (FY818),and leu1 transformants were recovered (Fig. 1Ai). Each diploid transformant(3,000 in total) was picked and sporulated, and Dhsk1/p(nmt-hsk1) haploids wereisolated on selective minimal media (Fig. 1Aii). Each haploid was replica platedonto YES agar containing phloxin B, and temperature-sensitive strains wereidentified. Plasmids from temperature-sensitive strains were recovered (16 intotal), and the entire mutagenized hsk1 sequence was integrated into FY818 atthe Dhsk1 locus (Fig. 1Aiii). leu11 diploids were sporulated, and temperature-sensitive Dhsk1::p(hsk1ts-leu11)::ura41 haploids were identified. Counterselec-tion on 5-fluoro-orotic acid was used to isolate strains containing the restoredgenomic locus, now with a mutant copy of hsk1 (Fig. 1Aiv). hsk1-1312 is mutatedat codon 314 from TCT to ATT, resulting in a serine-to-isoleucine substitution.

Construction of hemagglutinin (HA) and myc-tagged Hsk1p and Hsk1tsp. The1,707-bp hsk11 sequence was amplified by PCR from FY254 wild-type or FY945hsk1ts genomic DNA and cloned into pSLF172 (15), generating pHS60 (wildtype) and pHS69 (temperature sensitive). A version of this plasmid with a LEU2marker was constructed, generating pHS139. The 39 hsk13HA sequence wascloned into pJK210 (25) and used to integrate Hsk1pHA or Hsk1tspHA into thegenome as a partial tandem duplication (FY982 or FY1035, respectively). C-terminally tagged hsk1-myc (pHS136) was generated by subcloning hsk1 alonefrom pHS60 into pDS672, which contains two myc tags (D. A. Sherman, S. G.Pasion, and S. L. Forsburg, unpublished data).

HU, bleomycin, and UV treatment. Cells were treated with 20 mM HU for 4 hat either 25 or 32°C in minimal media or grown on EMM agar plus 5 mM HU.For block and release experiments, 10 to 15 mM HU was added for 4 h, the cellswere washed extensively in HU-free EMM, and the cells were reinoculated intoEMM. Cells were treated with 5 mU of bleomycin sulfate (Sigma-Aldrich, St.

TABLE 1. Strain list

Strain Genotypea Source or reference

FY254 h2 urad4-D18 leu1-32 ade6-M210 can1-1 Our stockFY440 h1 rad4-116 ura4-D18 leu1-32 15FY818 h1/h2 Dhsk::ura41/hsk11 ura4-D18/ura4-D18 leu1-32/leu1-32 ade6-M210/ade6-M216 36FY945 h2 hsk1-1312 ura4-D18 leu1-32 ade6-M210 This studyFY961 h1 mis5-268 (mcm6) ura4-D18 leu1-32 ade6-M210 can1-1 M. YanagidaFY980 h2 hsk1-1312 mcm5HA::leu11 leu1-32 ura4-D18 leu1-32 ade6-M216 This studyFY981 h2 hsk1-1312 Dmcm2::(mcm2HA-leu11) leu1-32 ura4-D18 leu1-32 ade6-M210 This studyFY982 h2 hsk1::p(hsk1HA-ura41) ura4-D18 leu1-32 ade6-M210 can1-1 This studyFY983 h1 hsk1-1312 mis5-268 ura4-D18 leu1-32 ade6-M210 This studyFY984 h1 hsk1-1312 cdc19-P1 ura4-D18 leu1-21 ade6-M210 This studyFY998 h2 hsk1-1312 cdc21-M68 ura4-D18 leu1-32 ade6-M210 This studyFY999 h1 hsk1-1312 Dcds1::ura41 ura4-D18 leu1-32 This studyFY1000 h2 cdc10-V50 hsk1::p(hsk1HA-ura41) ura4-D18 leu1-32 ade6-M210 This studyFY1002 h2 cdc21-M68 hsk1::p(hsk1HA-ura41) ura4-D18 leu-32 ade6-M210 This studyFY1011 h2 cdc22-M45 hsk1::p(hsk1HA-ura41) ura4-D18 leu-32 ade6-M210 This studyFY1035 h2 hsk1::p(hsk1-1312HA-ura41) ura4-D18 leu1-32 ade6-M210 can1-1 This studyFY1072 h1 hsk1-1312 cdc25-22 ura4-D18 leu1-32 ade6-M216 This studyFY1082 h1 Dcds1::ura41 hsk1::p(hsk1HA-ura41) ura4-D18 leu1-32 ade6-M210 This studyFY1085 h1 Dchk1::ura41 hsk1::p(hsk1HA-ura41) ura4-D18 leu1-32 ade6-704 This studyFY1089 h1 cdc23-M36 ura4-D1 leu1-32 ade6-M210 This studyFY1105 h2 Drad3::ura41 ura4-D18 leu1-32 ade6-704 P. RussellFY1124 h1 hsk1-1312 rad4-116 ura4-D18 leu1-32 This studyFY1125 h1 hsk1-1312 cdc23-M36 ura4-D18 leu1-32 ade6-M210 This studyFY1127 mat2-102 hsk1-1312 ura4-D18 leu1-32 ade6-M216 This studyFY1132 h1 hsk1-1312 orp1-1 ura4-D18 leu1-32 ade6-M210 This studyFY1135 h2 hsk1-1312 cdc24-G1 ura4-D18 leu1-32 ade6-M210 This studyFY1137 h1 hsk1-1312 pol1-1 ura4-D18 leu1-32 ade6-M210 This studyFY1159 h2 rad21-K1::ura41 ura4-D18 leu1-32 ade6-M216 his7-366 57FY1163 h2 rad12::ura41 ura4-D18 leu1-32 ade6-M210 10FY1176 h1 chk1:ep leu1-32 ade6-M216 59FY1181 h2 hsk1-1312 chk1::ep ura4-D18 leu1-32 ade6-M210 This studyFY1282 h2 cdc10-V50 hsk1-1312 ura4 leu1 ade6-M210 This studyHSY1 h2/mat2-102 ura4-D18/1 leu1-32/1 ade6-M210/ade6-M216 This studyHSY2 h2/mat2-102 hsk1-1312/hsk1-1312 ura4-D18/ura4-D18 leu1-32/leu1-32 ade6-M210/ade6-M216 This study

a All strains are isogenic with h2 972. We have described previously other strains used in this work (34, 53).

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Louis, Mo.) per ml in minimal media as described previously (16). Cultures ofstrains to be tested for UV sensitivity were grown to mid-exponential phase inminimal media. One thousand cells of each strain were plated onto two YES agarplates and exposed to a range of UV doses in a Stratalinker (Stratagene, La Jolla,Calif.). Viability was calculated as a percentage of cells surviving to form colo-nies. Viability was determined by serial dilutions, plating on YES agar at 25°C,and comparing the efficiencies of colony formation of treated and untreated cells.

Cell biology. Immunofluorescence and DAPI (49,69-diamidino-2-phenylin-dole) staining were performed as described previously (10, 54). For all immu-nofluorescence experiments, Hsk1pHA and Hsk1tsp were integrated into thechromosome under the native promoter (see tagging of Hsk1p and Hsk1tspabove). Cells were examined using a Leitz Laborlux S microscope, and images

were acquired directly by Adobe Photoshop for Macintosh using a Spot IIchange-coupled-device digital camera (Diagnostic Instruments, Inc., SterlingHeights, Mich.). Samples for flow cytometry were prepared as described previ-ously (54). Chromosome loss rates were determined as described previously (34).

Immunoblotting and immunoprecipitation. Protein lysates were prepared byglass bead lysis, and immunoprecipitation was carried out essentially as describedpreviously (50, 54). The phosphatase inhibitors 50 mM sodium fluoride, 50 mMb-glycerophosphate, and 20 mM sodium vanadate were included as necessary.Enhanced resolution of the phosphorylated forms of Hsk1p and Chk1p wasobtained using a 50:1 ratio of acrylamide-piperazine diacrylamide. Western blot-ting was performed as described previously (54), and the blots were digitallyscanned using a Hewlett-Packard ScanJet IIcx scanner and analyzed using Can-vas 6 software for Macintosh (Deneba, Miami, Fla.). Hsk1pHA and Dfp1p-mycwere specifically immunoprecipitated using anti-HA 12CA5 (gift of TonyHunter) and anti-myc 9E10 (BABCO) antibodies as described previously (54).

l phosphatase assays. Immunoprecipitates of Hsk1pHA from lysates treatedin the presence or absence of HU were washed into l phosphatase buffer (NewEngland Biolabs, Beverley, Mass.) plus 2 mM manganese chloride. The pelletswere resuspended in 50 ml of assay buffer, 400 U of l phosphatase (New EnglandBiolabs) was added to each sample, and the reaction mixture was incubated at30°C for 30 min. The reaction was halted by the addition of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boil-ing for 3 min. Proteins were separated by SDS-PAGE and analyzed by immu-noblotting as described above.

Kinase assays. For Hsk1p autokinase assays, Hsk1pHA or Hsk1tspHA wasimmunoprecipitated from protein lysates as described above. The immunopre-cipitates were resuspended in 40 ml of assay buffer (25 mM MOPS [morpho-linepropanesulfonic acid, pH 7.2], 15 mM MgCl2, 15 mM EGTA, 1% TritonX-100, 1 mM dithiotreitol, 60 mM b-glycerophosphate, 15 mM r-nitrophenolphosphate, 0.1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 20 mgof leupeptin per ml, 40 mg of aprotinin per ml). Twenty microliters of this slurrywas incubated with 200 mM [g-32P]ATP (NEN, Boston, Mass.) for 30 min at32°C. The reaction was halted by boiling the slurry in SDS-PAGE sample buffer.Immunoprecipitates were separated on SDS–10% PAGE gels, the gels wereexposed to a PhosphorImager screen, and the images were analyzed using Im-ageQuant software (Molecular Dynamics, Sunnyvale, Calif.). Cds1p was purifiedfrom extracts of 2 g of GBY438 following growth for 20 h in the absence ofthiamine, with 15 mM HU being added during the last 4 h of growth. Purificationto apparent homogeneity was achieved in two steps, using Talon-Sepharose andanti-HA monoclonal antibody 12CA5–protein A-Sepharose, as described previ-ously (7). Cds1p kinase assays were performed as previously described (6) exceptthat 100 mM ATP was used and bovine serum albumin was omitted. Assaymixtures contained the Hsk1pHA immunoprecipitate from approximately 4 mgof extract and 50 ng of Cds1p.

RESULTS

Isolation of a novel temperature-sensitive allele of hsk1. Toallow genetic analysis of the fission yeast hsk11, we isolated thefirst conditional allele of this gene, using a plasmid shufflescreen (Fig. 1A) (52). From a total of 3,000 plasmids initiallyscreened, we isolated a single integrated allele, hsk1-1312.hsk1-1312 supports growth at 25°C, is semipermissive forgrowth at 29°C, but is insufficient for growth at 32°C and above(Fig. 1B). This phenotype can be completely complemented byexpression of hsk11 from a plasmid (data not shown). Thetemperature-sensitive lesion in hsk1-1312 is a 2-bp mutationthat changes serine 314 in kinase subdomain IX to isoleucine(Fig. 1C). This serine residue, which is completely conserved inS. cerevisiae Cdc7 and other metazoan CDC7 homologues, islocated 3 residues from a similarly conserved aspartate residuethat stabilizes the catalytic loop in kinase subdomain VIB (Fig.1D) (19).

We constructed a panel of double mutant strains with hsk1-1312, hereinafter denoted hsk1ts, and other components in-volved in the early stages of DNA replication (Table 2). hsk1ts

interacts genetically with two of the mcm mutants, the mcm2and mcm6 mutants, but not with the third, the mcm4 mutant.The double mutant hsk1ts strains with the rad4 initiator proteinor the MCM10 homologue cdc23 also have a lower maximumpermissive temperature than that of either of the single mu-tants alone. These genetic interactions with putative initiatorsare consistent with the expected role of hsk11 in replicationinitiation.

FIG. 1. Isolation of a temperature-sensitive allele of hsk1. (A) hsk1-1312 wasconstructed as described in Materials and Methods. (B) Wild-type (WT)(FY254) and hsk1-1312 (FY945) cells were streaked onto YES agar and incu-bated at 25, 29, and 32°C for 4 days. (C) Hsk1p structure showing the location ofthe temperature-sensitive lesion. The kinase domains are shown in light-grayboxes, and the kinase insert domains are shown in dark-gray boxes. (D) Align-ment of the amino acid sequences in kinase subdomain IX in the CDC7 familyof hsk1-1312, showing the temperature-sensitive S314I lesion, and wild-type S.pombe (S.p., GenBank accession no. D50493), S. cerevisiae (S.c., accession no.M12624), Homo sapiens (H.s., accession no. AF015592), X. laevis (X.l., accessionno. AB003699), and M. musculus (M.m., accession no. AB018574) hsk11/CDC7homologues. Residues conserved among all the kinases are shown in black boxes,and residues conserved between most of the proteins are shown in gray boxes.

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Effects of hsk1ts on protein function. We investigated theeffect of the hsk1ts mutation on the stability and protein inter-actions of the Hsk1ts protein. The temperature-sensitive Hsk1protein (Hsk1tsp) is slightly less abundant than the wild-typeversion in asynchronously growing cells (data not shown). Todetermine if this reduction in protein level is due to increasedprotein turnover, we compared the stabilities of Hsk1p andHsk1tsp at the restrictive temperature. Hsk1p and Hsk1tspwere expressed from the thiamine-repressible nmt1 promoter.Following repression of expression, Hsk1p was degradedwithin 8 h, whereas temperature-sensitive Hsk1tsp was unde-tectable after only 4 h at 36°C (Fig. 2A), demonstrating thatHsk1tsp is indeed less stable than the wild-type protein. It hasrecently been reported that Cdc7 in S. cerevisiae is able tohomo-oligomerize (49). We found that this interaction is con-served with Hsk1p in S. pombe and that Hsk1tsp is still capableof multimerizing with the wild-type Hsk1 protein (data notshown).

However, other aspects of Hsk1tsp activity are aberrant.Hsk1p requires its regulatory subunit Dfp1p for activity againstthe Mcm2p substrate (6). We found that even at the permissivetemperature, Hsk1tsp displays a much lower affinity for Dfp1pthan that observed with wild-type Hsk1p (Fig. 2B). Further-more, Hsk1tsp has severely reduced autophosphorylation ac-tivity at the restrictive temperature (Fig. 2C). It has been ob-served that certain cdc7 mutants of S. cerevisiae can becomplemented by overexpression of the Dfp1p homologueDbf4 (30). However, we did not observe a similar rescue ofhsk1ts by high levels of Dpf1p (data not shown). Thus, thetemperature-sensitive lesion in Hsk1tsp clearly disrupts severalfeatures which are necessary for the proper activity of theprotein.

Hsk1tsp disrupts nuclear localization. We next examinedthe localization of Hsk1p in wild-type cells and in mutantsblocked at different stages of the cell cycle by indirect immu-nofluorescence. In wild-type cells, Hsk1p exhibits punctate nu-clear staining throughout the cell cycle (Fig. 3A and B). Inmitotic cells just undergoing nuclear division, the localizationof Hsk1p is less well defined (Fig. 3A and B), although theprotein clearly remains in the nucleus even in cells arrested inmitosis by various cell cycle mutants (data not shown). SinceDfp1p is quite unstable and present only during S and G2phases (6, 56), this indicates that Hsk1p nuclear localization isindependent of Dfp1p.

Although the Hsk1p substrate Mcm2 (also called Cdc19p) is

located in the nucleus throughout the cell cycle (41), in mcmmutant strains MCM proteins are redistributed to the cyto-plasm (42). However, these same mutations have no effect onthe localization of Hsk1p, which remains nuclear (Fig. 3C to Dand data not shown), demonstrating that proper localization ofHsk1p is independent of its substrate. Hsk1p localization wasalso unaffected by mutation in a subunit of the origin recogni-tion complex, orp1, or treatment with HU (data not shown).

In contrast, Hsk1tsp is redistributed to the cytoplasm at therestrictive temperature (Fig. 3E to H). Even at 25°C, there isan increase in cytoplasmic background staining in comparisonwith that of wild-type cells (Fig. 3A and B). At the restrictivetemperature, specific nuclear localization of Hsk1tsp is all butlost and staining is seen throughout the cell body (Fig. 3H),

TABLE 2. Summary of genetic interactions of hsk1ts mutants

Mutation Function Genetic interactiona

orp1 ORC subunitb 2 max. perm. temp.rad4 Initiator protein 2 max. perm. temp.cdc19 MCM2 2 max. perm. temp.cdc21 MCM4 No interactionmis5 MCM6 2 max. perm. temp.cdc23 MCM10/DNA43 2 max. perm. temp.cdc24 S phase? 2 max. perm. temp.pol1 DNA polymerase a No interactionDrqh1 DNA helicase Synthetically lethalcdc25 Cdc2p phosphatase 2 max. perm. temp.rad21 Mitotic cohesin Synthetically lethalDcds1 Checkpoint kinase SuppressionDchk1 Checkpoint kinase Synthetically lethalDrad3 Checkpoint kinase Synthetically lethal

a Downward-pointing arrows indicate reduced growth temperature. max.perm. temp., maximum permissive temperature.

b ORC, origin recognition complex.

FIG. 2. Hsk1p protein analysis. (A) Hsk1tsp has lower stability at 36°C thanHsk1p. Wild-type cells (FY254) were transformed with plasmids bearing genesexpressing either Hsk1pHA (nmt-hsk11, pHS60) or Hsk1tspHA (nmt-hsk1ts,pHS69). Hsk1pHA expression was induced for 16 h at 25°C, 15 mM thiamine wasadded back to the media, and the cultures were shifted to 36°C for 8 h. Aliquotswere removed at 216 h (before induction, lanes 1 and 7), at 0 h (after induction,lanes 2 and 8), and at 2 h (lanes 3 and 9), 4 h (lanes 4 and 10), 6 h (lanes 5 and11), and 8 h (lanes 6 and 12) after the addition of thiamine. Each lysate (10 mg)was immunoblotted for Hsk1pHA (lanes 1 to 6), Hsk1tspHA (lanes 7 to 12), andtubulin (lanes 1 to 12). (B) Hsk1tsp fails to interact with Dfp1p. Expression ofDfp1p-myc (pSLF272-dfp11myc [6, 15]) was induced in wild-type (WT)(FY982) or hsk1ts (FY1035) cells. The culture of hsk1ts was split, with half beingkept at 25°C and the other half being incubated at 36°C for 6 h. Proteins wereimmunoprecipitated from each lysate with either anti-HA (H), anti-myc (M), oranti-tubulin (T) antibodies, and the immunoprecipitates (IP) were immunoblot-ted with anti-HA antibodies. Lanes 1 to 3, wild type; lanes 4 to 6, hsk1ts cells at25°C; lanes 7 to 9, hsk1ts cells at 36°C. The lower gel indicates the loading control.Crude extracts used for immunoprecipitation were blotted for Hsk1-HA andDfp1-myc. Lane 10, wild type; lane 11, hsk1ts cells at 25°C; lane 12, hsk1ts cellsat 36°C. (C) Kinase activity is abrogated in hsk1ts cells. Kinase assays were carriedout as described in Materials and Methods. HA-tagged Hsk1p (lanes 1 and 3) orHsk1tsp (lanes 2 and 4) was immunoprecipitated with an anti-HA antibody(a-HA). Half the immunoprecipitate was subjected to kinase assay (lanes 1 and2), and the other half was subjected to immunoblotting (lanes 3 and 4). autorad,autoradiograph.

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suggesting that the protein is either being specifically exportedfrom the nucleus or failing to localize correctly.

hsk1ts causes G1 phase delay and defects in nuclear integ-rity. We examined the phenotype of the hsk1ts mutant. Upon ashift to the restrictive temperature for 4 h, hsk1ts cells displayeda transient G1 delay (Fig. 4A). Most cells finally arrested withan approximately 2C DNA content, but there were increasingnumbers of cut cells containing less than 1C DNA (data notshown). Furthermore, cells with fragmented nuclei were alsoapparent when the culture was examined microscopically (Fig.4B). This fragmentation suggests a defective checkpoint and issimilar to the phenotype reported for the Dhsk1 allele (36).Several initiation mutants undergo premature mitosis in theabsence of replication. However, in contrast to those mutantswith typical initiation checkpoint defects, hsk1ts cells were no-ticeably elongated, indicating a cell cycle delay, and cells with,1C DNA content appeared only 4 h following the shift to thenonpermissive temperature. This timing and morphologystrongly argue against premature entry into mitosis.

We determined the hsk1ts execution point for both the rep-lication and DNA fragmentation phenotypes by using an HUblock and release experiment (Fig. 4C). hsk1ts and wild-typecells were arrested in early S phase by treatment with HU atthe permissive temperature of 25°C and then shifted to 36°C toinactivate Hsk1tsp. hsk1ts cells in HU became elongated withsingle nuclei, as did wild-type cells (data not shown). After HUwas removed, both wild-type and hsk1ts cells completed DNAsynthesis in about 30 min, indicating that hsk1ts was not re-quired for DNA replication after the HU arrest point. How-ever, the flow cytometry profile of hsk1ts revealed kinetics ofDNA accumulation different from those of the wild type. Thisfinding is consistent with the failure of late origins to fire inhsk1ts cells at the restrictive temperature, as seen in analogousexperiments with S. cerevisiae cdc7 mutants (12).

Strikingly, the nuclear structure of hsk1ts cells released fromHU to the restrictive temperature became highly disorderedafter the completion of DNA replication (Fig. 4E and data notshown). A large fraction of the hsk1ts cells were elongated, andthe DNA fragmented into several visible pieces of DAPI-stained material. This suggests that the Hsk1p kinase has asecond execution point, after replication initiation, which isrequired to maintain chromosome integrity. Significantly, thisphenotype resembled that observed in many of the cells fol-lowing the shift of the hsk1ts strain to the restrictive tempera-ture (Fig. 4A). Therefore, the nuclear fragmentation pheno-type is not simply a consequence of HU arrest but indicates arole for Hsk1p after the initiation of bulk DNA synthesis inorder to maintain chromosome integrity during mitosis. Thisfunction is apparently separable from its role in replicationinitiation, which has an execution point prior to the HU arrest.

Establishment of chromosome cohesion depends upon suc-cessful passage through S phase (53, 58). In fission yeast, atemperature-sensitive mutant in the rad21 cohesin exhibits ab-normal mitosis at the restrictive temperature, with aberrantlycondensed chromosomes and unequally divided nuclei (57).These phenotypes are similar to those we observed in hsk1ts

cells (Fig. 4B). We found that the rad21ts cohesin mutant issynthetically lethal with hsk1ts (Table 2), which may indicate arole for Hsk1p in ensuring correct chromosome cohesion as Sphase progresses.

Despite the fragmentation phenotypes we observed, hsk1mutant cells maintained high viability following 4 h at therestrictive temperature or after 4 h in HU, although colony sizewas heterogeneous (data not shown). This result suggests thatthe cells can recover from the damage if exposure to restrictiveconditions is not prolonged.

To determine whether DNA fragmentation in the hsk1ts

cells depends upon passage through mitosis, we repeated theHU block and release experiment with cdc10, hsk1ts cdc10, andhsk1ts cdc25 mutant strains. First, we monitored the appear-ance of normal mitotic cells and cells with fragmented nuclei inthe two cdc10 strains. Following HU release, the cdc10 mutantarrested in the G1 phase of the following cell cycle, ensuringthat we were observing events in a single mitosis. As seen inFig. 4D, the cdc10 mutant underwent mitosis, with a peak at2.5 h. The hsk1ts cdc10 strain accumulated cells with frag-mented nuclei with similar timing, which suggests that frag-mentation occurs as a consequence of mitotic entry. This resultalso demonstrates that entry into mitosis in the double mutantstrain occurs with normal timing; this phenotype was identicalto that observed in the hsk1ts mutant alone. When the hsk1ts

cdc25 cells were released to 36°C, they completed S phase andarrested at the G2/M phase transition due to the lack of activeCdc25p (data not shown). As shown in Fig. 4E, no chromo-some fragmentation was detected in the hsk1ts cdc25 strain.

FIG. 3. Localization of Hsk1p. Hsk1pHA or Hsk1tspHA was expressed fromthe endogenous promoter in the genome. Cells were prepared for indirectimmunofluorescence as described in Materials and Methods and stained withDAPI to detect the DNA (A, C, E, and G) or with the anti-HA antibody 16B12to visualize Hsk1p (B, D, F, and H). Wild-type cells were grown at 32°C inminimal media prior to being stained. Temperature-sensitive strains were grownto mid-log phase at 25°C and then shifted to 36°C for 4 h prior to being stained.In all cases, HA-tagged hsk11 was integrated into the genome. (A to D)Hsk1pHA localizes to discrete spots in the nucleus independently of MCMproteins. Shown are the localizations of Hsk1p in wild-type cells (FY982) (A andB) and in cdc21-M38 cells (FY1002) (C and D). The arrows in panels A and Bindicate mitotic cells. (E to H) Hsk1tsp is redistributed from the nucleus at 36°C.hsk1ts cells (FY1035) were grown in minimal media to mid-log phase at 25°C (Eand F) or shifted to 36°C for 6 h (G and H). In all cases, the scale bar represents10 mm.

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Thus, the abnormal morphology in the hsk1ts strain reflectspassage through mitosis of cells with apparently replicatedDNA but does not reflect premature mitosis, since timing ofentry appeared normal.

Since the hsk1ts mutant showed abnormal, fragmented nu-clei and Hsk1tsp was lost from the nucleus, we next askedwhether other nuclear proteins were also redistributed. Asshown in Fig. 5A to D, the typical nuclear localization ofMcm2p or Mcm5p was unaffected by the relocalization ofHsk1tsp, suggesting that MCM protein localization is indepen-dent of Hsk1p function and showing that overall nuclear in-tegrity is maintained in hsk1ts cells. In cells where chromo-somal fragmentation was observed, there was little associationof the MCM proteins with the smaller DAPI staining material.

We examined the chromosome structure in the hsk1ts mu-tant strain using pulsed-field gel electrophoresis, which allowsresolution of the three individual chromosomes of S. pombe.Chromosomes that are undergoing DNA replication or haveaberrant structures fail to enter the gel (27, 34). As seen in Fig.5E, the three chromosomes from hsk1ts cells did not enter thegel very efficiently compared to the wild type, even at thepermissive temperature, and this effect was exacerbated inchromosomes isolated from hsk1ts cells at the restrictive tem-perature. We also observed that the mobility of chromosome 3in the mutant was higher than in the wild type, suggesting thatthis chromosome may be smaller than normal. A similar effecthas been reported for mcm2 mutants (34). There is no obvioussmear of chromosomal fragments, such as that observed incdc24 mutants (17); thus, if hsk1ts chromosomes are frag-mented, they must have sufficient structural abnormalities toprevent migration into the gel.

Finally, we investigated the genome stability of hsk1ts cells bycomparing rates of chromosome loss as assessed by hap-loidization. Diploid fission yeast cells that lose one chromo-some rapidly are reduced to the haploid state. Hence, the rateof chromosome loss can be inferred by haploidization of non-sporulating diploids (4). As shown in Fig. 5F, hsk1ts/hsk1ts

diploids display a much higher rate of chromosome loss thanthat of the wild type, even at the permissive temperature, andthis is further elevated by shifting the culture to the restrictivetemperature. These results clearly demonstrate that hsk1ts

causes a significant decrease in genome stability at all temper-atures.

Hsk1p is a target of the replication checkpoint kinaseCds1p. Strains with defects in genome stability are often sen-sitive to agents that challenge chromosomal integrity. We ex-amined the HU response of the hsk1ts mutant more closely.Arrest in HU triggers the so-called replication checkpoint, andrecovery from HU arrest requires the activities of the Cds1 andRqh1 proteins (3). cds11 encodes a nonessential checkpointkinase (39), and rqh11, also known as rad121 or hus21, en-codes a RecQ-type helicase (9, 40, 55). Mutation of theseproteins does not cause premature mitosis but rather affectsthe ability of arrested cells to recover and reenter the cell cycle

FIG. 4. hsk1ts exhibits a G1 delay and loss of nuclear integrity at the restric-tive temperature. (A) hsk1ts exhibits G1 delay. The DNA profiles of wild-type(FY254) (WT) and hsk1ts (FY945) cells were examined by flow cytometry. Cellswere grown at 25°C in minimal media to mid-log phase before being shifted to36°C for 4 h. Aliquots were removed at 0 and 4 h and prepared for flow cytometryas described in Materials and Methods. The positions of 1C and 2C DNAcontents are indicated. (B) hsk1ts has abnormal chromosomes. Shown is DAPIstaining to visualize nuclei of wild-type (FY254) (top) and hsk1ts (FY945) (bot-tom) cells after 6 h at 36°C. Arrows indicate chromosome fragments. (C) Bothwild-type and hsk1ts cells complete DNA synthesis after HU arrest and release to36°C. Wild-type (FY254) and hsk1ts (FY945) cells were arrested in HU at 25°Cand released to 36°C. Aliquots were removed for analysis by flow cytometry priorto the addition of HU and at 0, 10, 20, and 30 min after release from HU. Fewerthan 5% of the cells showed a cut phenotype (data not shown). (D) Timing of

mitosis and fragmentation in hsk1ts cdc10 and cdc10 cells. Cells were arrested inHU for 4 h and released to 36°C without HU. Cell morphology was determinedby DAPI staining and examination by fluorescence microscopy. The percentagesof cells displaying the indicated phenotypes were plotted against time. Opensymbols, normal mitotic cells in cdc10 (E) or cdc10 hsk1 (h) cells; closedsymbols, fragmented nuclei in cdc10 (F) or cdc10 hsk1 (■) cells. (E) Fragmen-tation in hsk1 strains requires entry into mitosis. Samples from the experimentwhose results are shown in panel D were photographed after 4 h at the restrictivetemperature. Left, cdc10 cells; middle, hsk1ts cdc10 cells (arrows indicate frag-mented nuclei); right, hsk1ts cdc25 cells. hsk1ts cdc10 and hsk1ts cells have iden-tical phenotypes (data not shown).

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after HU is removed. We analyzed the genetic interactionsbetween hsk1ts, Dcds1, and Drqh1 mutants. Strikingly hsk1ts andDrqh1 mutants were synthetically lethal, since it was not pos-sible to recover an hsk1ts Drqh1 double mutant (Table 2; datanot shown). In contrast, deletion of cds11 appeared to partiallysuppress hsk1ts, since the double mutant formed small coloniesat 32°C, a temperature restrictive for hsk1ts growth (Table 2;Fig. 6A). Moreover hsk1ts was hypersensitive to overexpressionof active, but not kinase-inactive, Cds1p (Fig. 6B). However,the fluorescence-activated cell sorter profiles were not signifi-cantly different (data not shown).

If Hsk1p activity is required for cells to recover from repli-cation arrest, we would expect hsk1ts strains to display pheno-types similar to those of Dcds1 and Drqh1 mutants following

prolonged exposure to sublethal doses of HU. We comparedthe responses of wild-type, hsk1ts, Dcds1, Drqh1, and hsk1ts

Dcds1 cells to low levels of HU at the permissive temperature(Fig. 6C). Although the wild-type strain formed colonies effi-ciently in the presence of 5 mM HU, hsk1ts, Dcds1, Drqh1, andhsk1ts Dcds1 cells were unable to grow. Since hsk1ts cells ar-rested normally in response to HU treatment, as did Dcds1 and

FIG. 5. hsk1ts has increased genome instability. (A to D) MCM proteinsremain nuclear in hsk1ts cells. The hsk1ts allele was combined with a straincarrying an integrated HA-tagged copy of either Mcm2p (FY981) (A and B) orMcm5p (FY980) (C and D). The strains were grown in minimal media for 6 h at36°C. The arrows in panels A and B indicate cells with highly abnormal chro-mosomes which have reduced MCM protein localization. The scale bar repre-sents 10 mm. (E) hsk1ts cells have structurally abnormal chromosomes as deter-mined by pulsed-field gel electrophoresis. Wild-type (FY254) (WT) and hsk1ts

(FY945) cells were grown at either 25 or 36°C for 6 h. Whole chromosomes wereprepared in agarose plugs and run in a 0.6% agarose gel as described in the text.The positions of chromosomes 1 to 3 are indicated. (F) hsk1ts homozygousdiploids display elevated rates of chromosome loss. Wild-type (HSY1) or hsk1ts

(HSY2) diploids were scored for chromosome loss as described in the text.Results of an experiment representative of three separate experiments whichgave similar results are shown.

FIG. 6. hsk1ts is required for recovery from replication arrest. (A) hsk1ts canbe partially suppressed by deletion of cds1. hsk1ts (FY945) or hsk1ts Dcds1(FY999) double mutant cells were streaked onto YES agar at 25 or 32°C. (B)Moderate overexpression of Cds1p is toxic in hsk1ts cells. Wild-type (WT) andhsk1ts cells were transformed with either pREP4X (V), pSLF272-cds11 (cds11),or pSLF272-cds1KD (cds1KD), and expression was induced at 25°C. (C) hsk1ts issensitive to HU. Wild-type (FY254), Dcds1 (FY866), hsk1ts (FY945), Drqh1(FY1163), and hsk1ts Dcds1 (FY999) cells were grown on minimal media orminimal media plus 5 mM HU at 25°C. (D) Abnormal cell morphologies werescored after treatment with HU. Wild-type, hsk1ts, Drqh1, Dcds1, and hsk1ts

Dcds1 cells were treated with 10 mM HU for 11 h at 25°C (left), or 4 h at 25°Cfollowed by release to HU-free media at 36°C for 4 h (right). Cells were stainedwith DAPI and examined microscopically. Abnormal cells were classified asthose displaying a cut morphology (black), unequal segregation (white), or frag-mented or irregular nuclei (gray). Two hundred cells were counted for eachstrain.

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Drqh1 cells (Fig. 4C and data not shown), we conclude fromthis assay that hsk1ts cells are similarly defective in the recoveryfrom S-phase arrest. Microscopic examination of hsk1ts, Dcds1,Drqh1, and hsk1ts Dcds1 cells following prolonged incubation inHU at the permissive temperature revealed that all strainsdeveloped increasing numbers of cut cells and other aberrantnuclear morphologies (Fig. 6D) (55). In particular, both hsk1ts

and Drqh1 cells exhibited fragmentation similar to that seen inthe hsk1ts mutant at the restrictive temperature (Fig. 4A anddata not shown) (55). Interestingly, in the hsk1ts Dcds1 mutant,the fraction of abnormal cells was higher than that observed ineither single mutant (Fig. 6D, left graph). Upon examinationof hsk1ts and Drqh1 cells after release from HU arrest to therestrictive temperature for hsk1ts, we found that both strainsaccumulated large numbers of cells with aberrant chromosomestructures and that both were more severely affected than theDcds1 mutant alone (Fig. 6D, right graph). Thus, the chromo-some integrity of all these mutants appeared to be affectedupon prolonged arrest in HU. However, the double mutantphenotypes and complex genetic interactions suggest thatthese effects are mediated through separable pathways, indi-cating that recovery from HU requires multiple components ofthe replication and repair machinery.

The genetic suppression of hsk1ts by deletion of cds11 andits sensitivity to Cds1p overproduction suggest that Cds1p an-tagonizes some aspect of Hsk1p activity. To determine whetherHsk1p is itself a target of Cds1p, we examined Hsk1p from cellextracts grown in the presence or absence of HU by gel elec-trophoresis (Fig. 7A). Hsk1p from HU-treated cells migratedwith a reduced mobility compared to that from untreated cells.This modest mobility shift could be reversed by treatment withl phosphatase, indicating that the modification reflects phos-phorylation. The same modification of Hsk1p is seen in a strainwith a mutation in the catalytic subunit of the ribonucleotidereductase cdc22 (Fig. 7B), which phenocopies HU treatment(47). Since the hsk1ts strain is sensitive to HU, we speculatedthat the mutant protein might not respond to HU treatment.Indeed, we found that temperature-sensitive Hsk1tsp does notdisplay a detectable phosphorylated form following treatmentwith HU at either the permissive or restrictive temperature(Fig. 7B).

We investigated which checkpoint kinases were required forthe observed Hsk1p phosphorylation. We treated wild-type,Dcds1, and Dchk1 cells with HU and examined Hsk1p modifi-cation. HU-induced phosphorylation of Hsk1p was not de-tected in Dcds1 cells (Fig. 7C) but did occur in Dchk1 mutants.We also treated cells with the radiation mimetic drug bleomy-cin, which activates the damage response pathway (16), but weobserved no alteration in the mobility of Hsk1p (Fig. 7C).Thus, these data suggest that Hsk1p is a specific target of theDNA replication checkpoint rather than the DNA damageresponse pathway.

To determine whether Hsk1p is a direct target of Cds1p, weexamined the ability of the purified Cds1p kinase to phosphor-ylate immunoprecipitated Hsk1p (Fig. 7D). In order to elimi-nate any background due to autophosphorylation, we usedheat-inactivated Hsk1p immunoprecipitated from appropriatestrains as a substrate (Fig. 7D) (6). Both Hsk1p and Hsk1tspwere phosphorylated by Cds1p in vitro (Fig. 7D). This resultprovides the first evidence that the catalytic subunit of theHsk1p kinase itself can be phosphorylated by Cds1p kinase.

hsk1ts requires the damage checkpoint for viability. hsk1ts issensitive to several agents that induce DNA damage. As can beseen in Fig. 8A, hsk1ts cells were considerably more sensitive toUV light than wild-type cells but were not as severely affectedas Drad3 cells. We also tested the sensitivity of hsk1ts to the

drug methyl methane sulfonate (MMS) and found that hsk1ts

cells are much more sensitive to MMS than wild-type and rad4cells (Fig. 8B). Interestingly, we were unable to construct dou-ble mutants with hsk1ts and the Dchk1 or Drad3 checkpointkinases (Table 2). This inability of cells to grow in the absenceof chk1 or rad3 when there were mutations in hsk1 suggeststhat hsk1ts may cause chromosomal lesions even at the permis-sive temperature which require the presence of a fully func-tional DNA damage checkpoint pathway to delay mitosis foradequate repair. This requirement would be consistent witheither defects in initiation or defects in overall chromosomestability. We tested this theory by examining the phosphoryla-tion state of the Chk1 protein, since it has previously beenshown that the phosphorylation of Chk1p correlates with ac-tivation of the DNA damage checkpoint (60). In wild-typecells, Chk1p was activated only upon treatment with bleomy-cin. In contrast, Chk1p phosphorylation was observed in hsk1ts

cells even in the absence of bleomycin, at both 25 and 36°C,indicating partial activation of the DNA damage checkpoint inthese mutant cells (Fig. 8C).

DISCUSSION

In this report we have described the isolation and charac-terization of the first temperature-sensitive allele of S. pombehsk11. The mutant allele, hsk1-1312, changes serine 314 inkinase subdomain IX, which is conserved in all CDC7 familymembers, into an isoleucine. This mutation causes a decreasein the stability of Hsk1tsp, as well as reduced autophosphory-lation activity and decreased association with the Dfp1p sub-unit. The wild-type protein is found constitutively in the nu-cleus, and its localization is unaffected by treatment with HUor the absence of its substrate Mcm2p. In contrast, the mutantprotein fails to localize appropriately, with reduced nuclearlocalization even under permissive conditions. This redistribu-tion of Hsk1tsp may reflect abnormal protein structure or theinability of the protein to interact with a factor required fornuclear retention. However, it is unlikely that the redistribu-tion of Hsk1tsp reflects its failure to interact correctly withDfp1p, since wild-type Hsk1p remains nuclear throughout thecell cycle even though Dfp1p is present only transiently (thiswork and references 6 and 56).

A postinitiation role for Hsk1p. Under restrictive condi-tions, hsk1ts mutant cells are severely delayed in S-phase pro-gression and some cells show abnormal entry into mitosis,suggesting that they suffer a defect in the checkpoint thatmonitors DNA replication. At first glance, this possibility isconsistent with observations of other mutants that block thecells prior to initiation of DNA synthesis, including rad4 (alsocalled cut5) (14), orp1 (18), cdc18 (27), and pol1 (13) mutants.The failure of these mutants to restrain M phase is thought toindicate defects in assembly of replication structures that signalto the checkpoint apparatus, causing the cells to enter mitosisprematurely, without entering S phase (28). However, in con-trast to these other mutants, some hsk1ts cells at the restrictivetemperature show a single elongated cell body, with multipleDAPI-stained dots. This phenotype is distinct from the small,aneuploid cells of a typical cut mutant. Importantly, cellularelongation suggests that a cell cycle delay occurs prior to ab-normal mitosis.

To probe the role of Hsk1p in later stages of DNA replica-tion, we blocked hsk1ts cells in HU and then released them tothe restrictive temperature. The cells completed DNA synthe-sis and underwent mitosis with apparently normal timing. Thiswas expected; data from budding yeast show that replicationforks originating from early-firing replication origins are suffi-

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cient to complete genome duplication in cdc7 mutants, even iflate origins do not fire (5, 12). This result indicates that theinitiation function of Hsk1p at early origins occurs prior to theHU block. However, although hsk1ts cells released from HU tothe restrictive temperature completed DNA synthesis and en-tered mitosis with normal timing, that mitosis was severelydisrupted, with a high fraction of cells exhibiting fragmentednuclei. Thus, when Hsk1p activity is lost during replicationelongation, it affects mitotic progression, a phenotype not ob-served in budding yeast (5, 12). The execution point for thiseffect is subsequent to the HU block, which suggests two pos-sibilities: either there is a second role for Hsk1p during normalS phase or there is a role for Hsk1p in recovery from the DNAreplication block caused by HU treatment. These possibilitiesare not mutually exclusive.

Importantly, the phenotype upon HU treatment is similar tothat observed in a fraction of cells when they were shifted tothe restrictive temperature without a prior HU block. Thisresult implies that the mitotic defect observed following HUtreatment is not solely an effect of S-phase arrest but rather

FIG. 7. Hsk1p is phosphorylated by Cds1p in response to HU. (A) Hsk1p isphosphorylated in the presence of HU. Wild-type cells were or were not treatedwith HU. Lysates were prepared from wild-type cells (FY982) in the presence ofphosphatase inhibitors, and HA-tagged Hsk1p was immunoprecipitated from thelysates using an anti-HA antibody. Samples were run on 6% acrylamide–pipera-zine diacrylamide gels, as described in Materials and Methods. Lanes: 1, Hsk1pminus HU; 2, Hsk1p plus HU; 3, Hsk1p plus HU treated with l phosphatase (lp’ase) as described in Materials and Methods. (P), phosphorylated. (B) Hsk1p ismodified in cdc22 cells, but Hsk1tsp is not phosphorylated in the presence of HUat any temperature. Lysates were prepared from wild-type (FY982) (WT), hsk1ts

(FY1035), and cdc22 (FY1011) strains carrying HA-tagged integrated Hsk1p,grown in the presence (H) or absence (2) of 20 mM HU. Protein lysate (10 mg)was analyzed by immunoblotting following electrophoresis as described for panelA above. Lanes: 1, wild type, 32°C; 2, wild type plus HU for 4 h at 32°C; 3, hsk1ts

cells at 25°C; 4, hsk1ts cells plus HU at 25°C; 5, hsk1ts cells for 4 h at 36°C; 6,hsk1ts cells plus HU for 4 h at 36°C; 7, cdc22 cells for 4 h at 36°C; 8, cdc22 cellsplus HU for 4 h at 36°C. (C) Hsk1p phosphorylation requires Cds1p. Whole-celllysates were prepared from wild-type (FY982), Dcds1 (FY1082), and Dchk1(FY1085) cells, and cells expressing Hsk1pHA were grown in the absence (2) orpresence of HU (H) or bleomycin (B). Protein lysate (10 mg) was analyzed byimmunoblotting following electrophoresis as described for panel A above. Lanes:1, wild-type untreated cells; 2, wild type cells plus HU; 3, wild-type cells plusbleomycin; 4, Dcds1 untreated cells; 5, Dcds1 cells plus HU; 6, Dcds1 cells plusbleomycin; 7, Dchk1 untreated cells; 8, Dchk1 cells plus HU; 9, Dchk1 cells plusbleomycin. (D) Hsk1p and Hsk1tsp are Cds1p kinase substrates in vitro. (Uppergel) Hsk1p was immunoprecipitated from extracts of fission yeast strains con-taining pHS60 (Materials and Methods). The Hsk1p immunoprecipitates werepreincubated (pre-inc) with cold ATP (lanes 3 and 4) or heat inactivated at 70°C(lanes 5 and 6) (6) and incubated with Cds1p kinase and [g-32P]ATP. Reactionproducts were fractionated by SDS-PAGE and exposed to a PhosphorImagerscreen. Lanes: 1, no protein; 2, Cds1p only; 3, Hsk1p preincubated with coldATP; 4, Hsk1p preincubated with cold ATP plus Cds1p; 5, Hsk1p preincubatedat 70°C; 6, Hsk1p preincubated at 70°C plus Cds1p. (Lower gel) Hsk1p andHsk1tsp were immunoprecipitated from extracts of fission yeast strains contain-ing pHS60 and pHS69, respectively. The immunoprecipitates were heat inacti-vated at 70°C and incubated in the absence or presence of Cds1p. The relativelevels of incorporation of 32P into Hsk1p and Hsk1tsp were determined usingImageQuant software and are given in arbitrary units. Lanes: 7, Hsk1p only; 8,Hsk1p plus Cds1p; 9, Hsk1tsp only; 10, Hsk1tsp plus Cds1p.

FIG. 8. hsk1ts is sensitive to DNA-damaging agents. (A) hsk1ts cells aresensitive to UV irradiation. Wild-type (FY254) (WT), hsk1ts (FY945), and Drad3(FY1105) cells were treated with UV irradiation as described in Materials andMethods. The graph represents relative levels of viability following treatmentwith the indicated doses. The results of one representative experiment are pre-sented. The experiment was repeated three times with similar results. (B) hsk1ts

cells are sensitive to MMS. Wild-type (FY254), hsk1ts (FY945), rad4 (FY440),and hsk1ts rad4 (FY1124) cells were streaked onto YES agar plates with orwithout 0.025% MMS at 25°C. (C) The DNA damage checkpoint is activated inhsk1ts cells. Wild-type (FY1176) or hsk1ts (FY1181) cells were grown at 25 or36°C for 6 h in the presence or absence of 5 mU of bleomycin per ml. Lysateswere prepared, and 20 mg of total protein was separated on 8% acrylamide–piperazine diacrylamide gels, as described in Materials and Methods. The mo-bility of Chk1p was determined by Western blotting with anti-HA antibodies.Lanes: 1, wild type at 25°C; 2, wild type at 25°C plus bleomycin; 3, wild type at36°C; 4, wild type at 36°C plus bleomycin; 5, hsk1ts cells at 25°C; 6, hsk1ts cells at25°C plus bleomycin; 7, hsk1ts cells at 36°C. (P), phosphorylated.

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that it is enhanced due to the synchrony imposed by the HUblock.

We posit that the nuclear fragmentation may reflect a defectin chromosome cohesion, which is established during S phase(53, 58). In support of this, we observed synthetic lethalitybetween hsk1ts and the mitotic rad21ts cohesin. If the hsk1ts

cells are unable to establish correct cohesion during S phase,this might prevent proper chromosome segregation at mitosis,resulting in the observed loss in nuclear integrity. Intriguingly,the cohesin is phosphorylated during S phase (2). Very re-cently, a novel DNA polymerase was shown to be required forboth replication and cohesion in budding yeast, indicating afurther connection between DNA replication and sister chro-matid cohesion (61). Hsk1p may contribute directly to theestablishment of cohesion, or it may carry out a more generalfunction during DNA synthesis that affects chromosome seg-regation.

This evidence indicates a role for Hsk1p in maintaininggenome integrity that may be separate from its role in repli-cation initiation. Interestingly, we found that Chk1p check-point kinase is phosphorylated in hsk1ts cells even at the per-missive temperature, indicating that the damage checkpoint ischronically activated in this strain. This chronic activation fur-ther suggests that defects in hsk1 cause genome damage evenwhen the cells are able to divide and explains why the hsk1ts

mutant requires the checkpoint kinases Rad3p and Chk1p forviability.

Hsk1p is a potential target of Cds1p. The phenotypes thatwe observed in hsk1ts cells following prolonged exposure toHU are similar to the recovery defects observed for Dcds1 andDrqh1 cells upon treatment with HU, which reflect the inabilityof the cells to restart S phase and complete a normal cell cycleafter replication arrest. Moreover, the synthetic lethality be-tween hsk1ts and Drqh1 mutants indicates that Hsk1p is re-quired for recovery from replication arrest in a pathway that isat least partially separate from that requiring Rqh1p.

Our experiments reveal interactions between Hsk1p andCds1p both during a normal cell cycle and during replicationarrest. We found that the temperature sensitivity of hsk1ts ispartially suppressed by Dcds1 cells and, conversely, that hsk1ts

cells are hypersensitive to overexpression of active, but notkinase-dead, Cds1p. Several replication mutants, such as DNApolymerase a, require cds11 for viability (1), which makes thesuppression of hsk1ts particularly unusual. This observationmight indicate that Cds1p is modestly activated in the hsk1ts

mutant because of its replication initiation defect, resulting innegative feedback which then further down-regulates Hsk1pactivity. Alternatively, there may be a nonessential role forCds1p in normal S phase in which it negatively regulatesHsk1p. Relief of Hsk1pts inhibition by the deletion of Cds1pwould be permissive for growth at higher temperatures.

The Dcds1 hsk1 double mutant has noticeably more abnor-mal cells following HU treatment than those of either singlemutant, indicating that while both genes operate in the re-sponse to HU, they are likely to affect at least partially separatepathways. We suggest that the defect in HU in the doublemutant is due to two overlapping effects: the absence of aproper Cds1p response and the inability of the hsk1 mutantcells to carry out their late-S-phase function. These possibilitiessuggest that release from HU elicits multiple responses re-quired to restart replication and maintain genome integrity.

The Hsk1p subunit in HU-treated wild-type cells undergoesphosphorylation that requires Cds1p. Using purified proteins,we determined that both wild-type and mutant Hsk1 proteinsare direct substrates of Cds1p in vitro, even if the Hsk1pproteins are heat inactivated. However, the mutant Hsk1tsp is

not detectably phosphorylated in vivo. The reason for thisdiscrepancy remains unclear, although a number of mecha-nisms, such as defects in mutant Hsk1p localization or defec-tive interactions with other proteins, may account for the in-ability of Cds1p to mediate phosphorylation of Hsk1tsp in vivo.It is also formally possible that both Cds1p phosphorylationand Hsk1p autophosphorylation are required in order to de-tect a shift in Hsk1p mobility. Previous studies have shown thatthe Hsk1p regulatory subunit Dfp1p is also phosphorylated ina Cds1p-dependent manner in response to HU treatment (6,56). Mutations in dfp1 confer sensitivity to HU and causeaberrant mitoses upon prolonged exposure to HU (56). There-fore, both subunits of the Hsk1 kinase may be direct substratesof Cds1 kinase and are elements of the response to S-phasearrest. These observations are consistent with models of bud-ding yeast suggesting that RAD53 kinases prevent activation oflate origins by regulating CDC7 and DBF4 kinases (29, 46, 51,62).

Our investigation provides the first evidence that Hsk1pkinase is itself a potential Cds1p substrate, indicating thatthere are levels of regulation of Hsk1p not necessarily medi-ated by its association with Dfp1p. The requirement for Hsk1pactivity after HU arrest and genetic interactions with Drqh1and rad21ts mutants in the absence of HU suggest that Hsk1pmay function during S phase subsequent to origin firing. Thissecond function may occur normally during replication and isrequired for successful recovery from replication arrest. Ourdata also provide evidence for the functional separation of thecheckpoint kinases Chk1p and Cds1p, since deletion of eachgene has opposite effects in hsk1ts cells. Additional geneticanalysis under way should allow us to forge further ties be-tween hsk1 and other components of the replication and repairpathways in fission yeast and to determine the role of hsk1 inthe maintenance of genome integrity.

ACKNOWLEDGMENTS

We are grateful to Hisao Masai for strains and plasmids and discus-sion of unpublished work. We thank Tamar Enoch, Greg Freyer, HisaoIkeda, Paul Russell, Shelley Sazer, Nancy Walworth, and MitsuhiroYanagida for strains and plasmids; Sally Pasion and Dan Sherman forplasmids; Tony Hunter for antibodies; and Jeff Hodson, John Marlett,and Andrew Waight for excellent technical assistance. We are alsomuch indebted to Magdalena Bezanilla, Mike Catlett, Eliana Gomez,Joel Leverson, Debbie Liang, and Sally Pasion for their critical read-ings of the manuscript.

This work was supported by American Cancer Society grant RPG-00-132-01-CCG (S.L.F.) and Medical Research Council of Canadagrant MOP-36360 (G.W.B.). H.A.S. was partly supported by The SalkInstitute President’s Club and the Ralph M. Parsons Foundation. Weacknowledge the Dreyfus Foundation for their support. S.L.F. is ascholar of the Leukemia and Lymphoma Society. G.W.B. is a specialfellow of the Leukemia and Lymphoma Society.

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