INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Cells have a complex network of mechanisms to coordinate the completion of chromosome replication and repair of damaged DNA with mitotic entry. Early cell fusion experiments demonstrated that when an S phase cell is fused with a G₂ cell, the G₂ nucleus delays its mitotic entry until the S phase nucleus finishes DNA replication. This suggests that S phase cells have a mitotic inhibitor or an inhibitory signal that prevents premature mitosis. (Rao and Johnson, 1970). Subsequent genetic studies of Saccharomyces cerevisiae and Schizosaccharomyces pombe have substantially contributed to the understanding of how cells maintain the interdependency of S phase and mitosis. In S. pombe, deletion or mutation of genes involved in the initiation of S phase (cdc18⁺, cdt1⁺, cut5⁺, cdc30⁺, and pol⁺) allow the cells to enter inappropriate mitosis (Kelly et al., 1993a,b; Saka and Yanagida, 1993; Hofmann and Beach, 1994; Saka et al., 1994; D'Urso et al., 1995; Grallert and Nurse, 1996). In contrast, cells carrying deletion of genes such as pol and pcn1 (proliferating cell nuclear antigen), which are involved in the elongation process of DNA replication, arrest with a cdc phenotype (Waseem et al., 1992; Francesconi et al., 1993). These findings suggest that it is the initiation of DNA replication that generates the signal, preventing cells from entering mitosis prematurely (Li and Deshaies, 1993; Nurse, 1994). However, it is not known whether it is the formation of the replication complex on the origin or the initiation DNA structure that is responsible for generating the S to M phase checkpoint.

Several proteins are essential for the initiation of DNA synthesis in S. pombe, including Orp1, Cdc18, and Pol. However, the roles played by each protein in this process are fundamentally distinct. A prerequisite for initiation of DNA replication is the assembly of a prereplication complex on the origin, which includes Orp1 and Cdc18 (Diffley, 1996; Aparicio et al., 1997; Donovan et al., 1997; Newlon, 1997; Tanaka et al., 1997), although neither Orp1 nor Cdc18 participates directly in the synthesis of the initiation DNA structure (Muzi and Kelly, 1995; Muzi et al., 1996; Stillman, 1996). In contrast, Pol is a component of the replication complex that directly participates in synthesis of the initiation DNA structure at the replication origin. Thus, the role of Pol in initiation is entirely different from that of Orp1 and Cdc18 (Stillman, 1996; Wang, 1996). In addition, Pol is also involved in postinitiation DNA synthesis (Wang, 1991, 1996; Campbell, 1993). Because Pol plays a dual role in both the formation of the replication complex and the synthesis of nascent DNA, Pol is the ideal replication enzyme to dissect the question of what generates the replication checkpoint signal during initiation.

Previous studies have shown that germinating spores carrying a disrupted pol⁺ gene entered mitosis when DNA synthesis was inhibited by hydroxyurea, thus implicating Pol as playing a role in the coordination of S phase with mitosis (D'Urso et al., 1995). However, this study did not resolve the question of whether the inappropriate mitotic entry was due to the physical absence of Pol, resulting in a failure to assemble the replication complex, or due to the absence of Pol catalytic activity and a subsequent inability to synthesize an initiation DNA structure. Thus the question remains as to why deletion of pol⁺ fails to bring about the appropriate replication surveillance responses in these cells.

Once DNA synthesis has initiated, cells have additional surveillance mechanisms to delay mitotic entry in the event of DNA damage or blocks to ongoing replication. Studies of S. cerevisiae and S. pombe have identified several genes involved in these mechanisms (Hartwell and Weinert, 1989; Enoch et al., 1993; Sheldrick and Carr, 1993; Nurse, 1994; Carr and Hoekstra, 1995; Carr, 1996; Elledge, 1996; Lydall and Weinert, 1996; Paulovich et al., 1997). In S. pombe, a group of six "checkpoint Rad" proteins (Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1) are thought to be involved in monitoring damaged DNA and S phase arrest caused by hydroxyurea or a cdc mutant (Al-Khodairy and Carr, 1992; Enoch et al., 1992; Rowley et al., 1992; Al-Khodairy et al., 1994). Downstream of the checkpoint Rad proteins are two effector proteins, Chk1 and Cds1. In response to DNA damage, the Chk1 protein is absolutely required for cell cycle arrest in G₂ and undergoes a checkpoint Rad-dependent phosphorylation (Walworth and Bernards, 1996), which inhibits the activation of cdc2 kinase by regulating the phosphorylation of Tyr¹⁵ (O'Connell et al., 1997; Rhind et al., 1997). Interestingly, cells arrested by a cdc mutation in a chk1 background enter mitosis inappropriately (Francesconi et al., 1995; Uchiyama et al., 1997), whereas cells arrested by the S phase inhibitor hydroxyurea at 30°C do not activate Chk1 (Walworth and Bernards, 1996). A recent study has demonstrated that the primary effector responding to hydroxyurea block is not Chk1, but Cds1 (Lindsay et al., 1998). Cds1 was originally identified as a multicopy suppressor of a DNA polymerase  thermosensitive allele, swi7-H4 (Murakami and Okayama, 1995), and has recently been shown to be required for reversible S phase arrest. It is important for maintaining the viability of cells when S phase is arrested by hydroxyurea or DNA lesions (Lindsay et al., 1998). Another protein, Rqh1, is also required for reversible S phase arrest (Murray et al., 1997; Stewart et al., 1997). Therefore, in addition to the checkpoint Rad-Chk1 pathway, cells have a checkpoint Rad-Cds1-Rqh1 subpathway for recovery of cells during S phase perturbation. Because Pol is involved in both initiation and postinitiation DNA synthesis, studies with different mutant alleles of this enzyme will help further elucidate the different cell cycle surveillance responses during S phase progression.

In this study using a pol strain as well as a strain carrying a structurally intact but catalytically dead pol mutant, we demonstrate that the initiation DNA structure is required to generate the S phase to mitosis checkpoint signal. In addition, using polts mutants, we clearly demonstrate that the different extents of perturbation and disruption of DNA replication caused by these mutations induce differential downstream cell cycle kinase responses.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Strains, Media, and Genetic and Molecular Methods

S. pombe strains used in this study are listed in Table 1. Rich medium (yeast extract) and Edinburgh minimal medium (EMM) were as described by Moreno et al. (1991). All standard genetic methods were as described by Gutz et al. (1974). Standard molecular biology techniques were carried out as described by Maniatis et al. (1982). The plasmid pDblet (Brun et al., 1995) was modified by replacing the ura4⁺ marker with Leu2⁺, and the modified plasmid is named pDblet(leu). Transformation of fission yeast was performed by using the lithium acetate method described by Griffiths et al. (1995). For growth analysis of mutant strains, cells were first grown at 25°C to exponential phase and then shifted to 36°C. At the indicated time, cell number was determined by hemocytometer count. Cell viability measured at the restrictive temperature was performed by removal of a fixed number of cells at defined time intervals after shift to 36°C. Cells were diluted and plated onto yeast extract plates and incubated at 25°C for 3 d. Colonies were scored, and viability was expressed as a percentage of the colonies formed on cell samples plated immediately before shifting to 36°C.

Construction of pol Strains

Heterozygous diploid strain DB23 (Table 1) carrying a full deletion of the pol gene was constructed by a one-step gene replacement method. A 1.2-kb his3⁺ gene flanked by the pol⁺ genomic sequences (486-bp upstream sequence and 700-bp downstream sequence) was transformed into the diploid strain KG23 (Burke and Gould, 1994). Histidine prototrophic transformants were selected. The replacement of pol⁺ coding sequence by his3⁺ was confirmed by two methods: 1) genomic Southern analysis of the stable his3⁺ prototrophs, and 2) sporulation followed by tetrad dissection, which yielded two viable, histidine auxotrophic spores. For analysis of cells containing pol, histidine prototrophic spores derived from DB23 (pol⁺/pol) were selected to germinate at 30°C.

A haploid strain was constructed by transforming the heterozygous diploid DB23 (pol⁺/pol) with pREP82-pol⁺ containing the ura4⁺-selectable marker (Maundrell, 1993). Histidine and uracil prototrophic transformants were selected, followed by sporulation and tetrad dissection. Haploid cells derived from the histidine and uracil prototrophic spores were designated DB3, which contains pol::his3⁺[pREP82-pol⁺].

Another diploid strain, DB24, heterogeneous for pol, was constructed by crossing DB3 with the thermosensitive haploid strain DBts13 (polts13). After 5-fluoro-orotic acid (FOA) selection, the diploid was sporulated in EMM and inoculated in media minus leucine for selective germination of spores carrying polts13 at 36°C. Diploid strains DB25, DB26, and DB27 were constructed by transforming the diploid strain DB23 (pol⁺/pol) with pDblet(leu)pol⁺, pDblet(leu)polts13, and pDblet(leu)pol(D984N), respectively. The diploids were sporulated and germinated at 25°C in EMM containing adenine and uracil for selective germination of spores containing pol::his3⁺ [pDblet(leu)pol].

Isolation of Temperature-sensitive pol Mutants

The pol⁺ gene on plasmid pREP81 (Maundrell, 1993) was mutagenized using hydroxylamine as described (Rose et al., 1990). After mutagenesis, the DNA was transformed into Escherichia coli strain CJ236 (ung) (Kunkel et al., 1987). Mutagenized plasmid DNAs were prepared from 1 × 10⁵ ampicillin-resistant colonies.

Thermosensitive pol mutants were isolated by two different approaches. 1) Mutagenized plasmid pREP81-pol DNAs were transformed into the haploid strain DB3 containing pol::his3⁺ [pREP82-pol⁺] followed by plasmid shuffling (Boeke et al., 1987). Transformants were replica plated onto EMM plates lacking histidine and leucine but containing FOA and incubated at 25°C for 4 d. Colonies that survived FOA selection were then replica plated onto selective medium containing phloxin B at 36°C for 24 h. Red colonies were selected as putative pol thermosensitive mutants and confirmed by several rounds of temperature selection. 2) Mutagenized pREP81-pol plasmid DNAs were transformed into the heterozygous diploid strain DB23 (pol/pol⁺). Transformants were pooled, sporulated, and germinated in selective EMM medium. The haploid cells derived from histidine and leucine prototrophic spores were replica plated at 36°C onto selective EMM medium containing phloxin B. Red colonies were selected as potential thermosensitive mutant clones and confirmed as described above. After screening ~5 × 10⁴ colonies, 18 thermosensitive pol mutants were isolated. Four representative thermosensitive mutant alleles were identified by sequence analysis (Table 2).

View this table: [in this window] [in a new window]   Table 2.  Representative thermosensitive mutant alleles of pol+

Integration of Wild-Type and Mutant pol

Wild-type pol⁺ and the mutant polts13 gene under its endogenous chromosomal promoter and terminator sequences in tandem with the S. pombe leu1 sequence was cloned into the plasmid pJK148 (Keeney and Boeke, 1994). Plasmid pJK148 containing the pol sequence was linearized at an unique PstI site in the pol⁺ upstream region to facilitate recombination at the pol chromosomal locus. Linearized plasmid DNA was transformed into the heterozygous diploid strain DB23 containing pol::his3⁺ followed by sporulation and germination. Haploid leucine and histidine prototrophs were selected. Stable integrants DB10 (pol⁺), DBts11 (polts11), and DBts13 (polts13) were identified by several rounds of selection on nonselective media and further confirmed by genomic Southern analysis. DBts13 (polts13) was further crossed with wild-type SP808 to remove the leu1⁺ marker, and the resulting strain was named DBts131 (polts13/leu). Strains DBts13 and DBts131 yielded identical results in all studies. Thus, DBts13 (polts13) was used as the representative thermosensitive mutant for most of the studies in this paper.

Generation and Purification of Cds1 Antibody

Cds1 protein expressed in S. pombe as a GST fusion protein was affinity purified on a glutathione-agarose column followed by a Hitrap Q column (Pharmacia). The purified GST-Cds1 protein (300 µg) was used as antigen to immunize rabbits. The crude sera was affinity purified on a tandem GST column and GST Cds1 column. The affinity-purified antibody was used to test cross-reactivity against the purified protein and crude extracts from S. pombe wild-type cells and cds1 null mutant cells. The antibody recognized a single Cds1-specific band in the crude extract from wild-type cells, and this band was not present in extracts derived from the cds1 strain.

Cds1 Kinase Assay

Cds1 kinase assay was performed as described by Lindsay et al. (1998) with modification. Cells were grown to midlog phase, washed in PBS, and then washed in lysis buffer (150 mM HEPES, pH 7.9, 250 mM KCl, 50 mM NaF, 60 mM -glycerol phosphate, 15 mM p-nitrophenyl phosphate, 1 mM DTT, 1 mM EDTA, supplemented with a mixture of protease inhibitors). Cells suspended in lysis buffer were disrupted by vortexing with glass beads. The protein extracts were spun at 15,000 rpm for 15 min at 4°C to remove the glass beads and cell debris. Protein concentrations of the supernatant were determined, and 300 µg of the protein extract in 500 µl of lysis buffer were incubated with a 1:400 dilution of the affinity-purified Cds1 antibody at 4°C for 2 h. Immunocomplexes were further incubated with 30 µl of protein A beads (50% slurry) at 4°C for an additional 1 h. The protein A beads were precipitated and washed three times with lysis buffer and three times with kinase buffer (10 mM HEPES, pH 7.5, 75 mM KCl, 5 mM MgCl₂, 0.5 mM EDTA, 1 mM DTT). The immunocomplex-protein A pellet was incubated in a 20-µl reaction containing 100 µM ATP, 5 µg of myelin basic protein (MBP), 5 µCi of [-³²P]ATP at 30°C for 10 min. The reaction was terminated by the addition of 5 µl of 5× SDS sample buffer. After boiling for 3 min, the samples were run on 15% gels, fixed in 40% methanol and 10% acetic acid, and dried before exposure to films. Equal amounts of Cds1-immunoprecipitate used in the kinase assay were quantitated by gel analysis. The extent of phosphorylated MBP was quantitated by using an IS-1000 digital imaging system (Alpha Innotech, San Leandro, CA).

Reciprocal Shift Experiments Using Hydroxyurea Block and Release

Reciprocal shift experiments using a hydroxyurea block and release were performed with either single mutant DBts13 (polts13) or double mutants harboring polts13 and cdc20 or cdc25 mutant alleles as described by Nasmyth and Nurse (1981). Hydroxyurea was added to a final concentration of 12 mM to each cell culture at 25°C and incubated in YES. After 4 h in hydroxyurea, cells were washed extensively with prewarmed (36°C) YES and then resuspended and grown in YES at 36°C. Cell samples were removed at indicated time intervals for analysis of growth rate, viability, DNA content, and nuclear and cell morphology.

Cytological Analysis

Cells were fixed in 70% ethanol and stained by addition of DAPI followed by calcofluor, processed, examined, and photographed as described (Uchiyama et al., 1997).

Flow Cytometry Analysis

Cells were collected, washed in water, and fixed in 70% ethanol before staining with propidium iodide as described by Paulovich and Hartwell (1995). DNA contents was measured by a Coulter Electronics (Hialeah, FL) fluorescence-activated cell sorter.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Cells with pol Enter Mitosis with a 1C DNA Content

We have previously shown that cells carrying a disrupted pol gene display heterogeneous cell morphology (Francesconi et al., 1993). D'Urso et al. (1995) have shown that cells harboring a disrupted pol gene arrested with variable amounts of DNA and entered aberrant mitosis. The variable amounts of DNA synthesis observed in spores carrying a disrupted pol gene were thought to be due to residual Pol being carried over from the original diploid after sporulation (D'Urso et al., 1995). To definitively discern the DNA content of cells in the absence of pol⁺, we constructed a diploid strain (DB23) that is heterozygous for a complete deletion of the pol⁺ coding sequence and pol⁺. pol spores, derived from the diploid DB23 (pol/pol⁺), were selected for germination. Sixteen hours after inoculation, no DNA synthesis was observed in spores deleted of pol⁺. After 12 h, ~60% of the cells were either anucleated or had missegregated nuclear material across the septum (Figure 1). The phenotype of the pol spores is similar to that shown by Francesconi et al. (1993) and D'Urso et al. (1995) and identical to that of cdc18 and cdc30 germinating spores (Kelly et al., 1993a; Grallert and Nurse, 1996). To further substantiate this observation, we constructed a heterozygous diploid DB24 (pol/polts13) carrying a complete deletion of pol⁺ and a copy of the pol gene containing a thermosensitive polts allele in tandem with the leu⁺ gene (see description of polts alleles below and Table 1 for strain description). Spores derived from DB24 (pol/polts13) were germinated in a leucine-minus medium at 36°C for selective germination of spores carrying polts13. After 10 h at 36°C, these spores displayed aberrant nuclear phenotypes identical to those of pol spores. These results demonstrate that Pol plays a critical role in coordinating S phase with mitosis.

View larger version (38K): [in this window] [in a new window]   Figure 1.   pol germinating spores undergo mitosis with 1C DNA content. FACS profile and phenotype of pol+ (A) and pol (B) germinating spores at 30°C. Shown here are germinating spores 12 h after inoculation into selective medium.

The Catalytic Function, Not the Physical Presence of Pol, Is Required to Generate the S Phase to Mitosis Checkpoint

To distinguish whether it is only the physical presence of Pol in the replication complex or whether the catalytic function of Pol for synthesis of an initiation DNA structure is necessary for bringing about the replication checkpoint, we constructed a catalytically dead but structurally intact Pol mutant. Asp⁹⁸⁴ of S. pombe Pol is a critical residue in region I, the most conserved region of the -like DNA polymerases (Figure 2A) (Delarue et al., 1990; Ito and Braithwaite, 1991; Wang, 1991, 1996). Previous mutational studies have shown that conservative mutation of the second Asp residue of human Pol Asp¹⁰⁰⁴ to Asn completely abolishes the catalytic activity of Pol. This mutation, however, does not alter either the protein structure of Pol or the ability of the mutant Pol protein to assemble into the Pol-primase complex (Copeland and Wang, 1993a,b). We therefore introduced an identical mutation into the S. pombe Pol by changing Asp⁹⁸⁴ to Asn and investigated the effect of the physically intact but catalytically dead Pol mutant, pol(D984N), on the S phase to mitosis checkpoint.

View larger version (49K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   Figure 2.   Germinating spores lacking Pol catalytic activity undergo inappropriate mitosis. (A) Primary sequence conservation of the region I of -like DNA polymerases (Delarue et al., 1990; Ito and Braithwaite, 1991; Wang, 1991, 1996). Asp984 of S.pombe DNA polymerase  was mutagenized to Asn. (B) Phenotype of germinating spores containing a chromosomal pol and plasmids pDblet(leu)pol+, pDblet(leu)pol(D984N), and pDblet(leu)polts13. Diploid DB23 with one copy of the chromosomal pol+ deleted was transformed with pDblet(leu)pol+ or pDblet(leu)pol(D984N) and inoculated into selective media for germination of spores containing pol/pDblet(leu)pol+ and pol/pDblet(leu)pol+ at 30°C. The phenotype of the cells shown here is 14 h after inoculation. Diploid DB23 cells transformed with pDblet(leu)polts13 were inoculated into selective media for germination of spores containing pol/pDblet(leu)polts13 at 36°C, and the phenotype shown is 10 h after inoculation. Bar, 4 µm. (C) Dominant negative effect of overexpressing Pol(D984N) mutant. Cell number increase after induction by removal of thiamine or repression by addition of thiamine was measured by counting cells starting from 10 h using a hemocytometer. After 16-h removal of thiamine from the media (Maundrell, 1993), the overexpression of catalytically dead Pol(D984N) caused a significant slowdown of cell growth.

Mutant pol(D984N) was cloned into the vector pDblet(leu) and transformed into the diploid DB23 (pol/pol⁺). As controls, plasmids pDblet(leu)pol⁺ and pDblet(leu)polts13 (see description of polts mutations below) were also constructed and transformed into the diploid DB23 (pol/pol⁺). The diploid cells carrying each of the three individual pDblet(leu)pol constructs were sporulated and selectively germinated for the pol/pDblet(leu)pol. Fourteen hours after inoculation of the spores at 30°C, the spores sustained with plasmid pDblet(leu)pol⁺ had germinated into normal cells (Figure 2B). In contrast, spores containing the plasmid pDblet(leu)pol(D984N) entered mitosis in the absence of DNA synthesis. Approximately 50% of these germinating spores displayed an aberrant mitotic nuclear phenotype, with either anucleated cells or cells with missegregated nuclear material across the septum (Figure 2B). Furthermore, none of these cells arrested with a cdc phenotype. An identical phenotype was observed when pol spores harboring the plasmid pDblet(leu)polts13 were germinated at the restrictive temperature. To further ensure that the observed aberrant mitotic phenotype is caused by the catalytically dead mutant, pol(D984N) was constructed into an inducible vector (Maundrell, 1990). Cells harboring the pRep4 pol(D984N) plasmid under uninduced conditions displayed a similar growth rate as the cells harboring the wild-type pol⁺ plasmid, with a doubling time of 3 h. In contrast, induced cells with overexpressed Pol(D984N) had a doubling time of 6 h, showing that expression of the catalytically dead Pol(D984N) mutant has a dominant negative effect on cell growth (Figure 2C). Furthermore, 24 h after induction, ~20% of the cells had an elongated phenotype. Dominant negative effects are usually attributed to assembly of the defective protein into complexes with other cellular components, rendering a population of nonfunctional complex. Thus, our results indicate that the Pol(D984N) is competent to assemble into the replication complex, disabling the replication complex, and causing the observed slower cell growth rate. This result strongly supports the notion that the aberrant nuclear morphology observed in cells containing the Pol(D984N) (Figure 2B) is caused by the presence of a catalytically nonfunctional mutant Pol in the replication complexes. Our results thus indicate that it is the catalytic function of Pol, essential for the synthesis of an initiation DNA structure, and not the physical presence of Pol in the replication complex, that is required for generating the signal that prevents cells from entering inappropriate mitosis.

Thermosensitive Mutant Alleles of pol

To further investigate how mutations of Pol affect cell cycle events during S phase progression, we isolated 18 thermosensitive pol mutants by two approaches described in MATERIALS AND METHODS. Four mutants carrying polts11, polts13, polts16, and polts17 alleles display aberrant mitotic nuclear morphology at the restrictive temperature of 36°C. We identified and sequenced these four mutant alleles (Table 2). Because polts13 contains a deletion of three contiguous amino acid residues, we further tested whether mutation of each of the individual amino acid residues of polts13 would cause temperature-sensitive cell growth. Ser⁴⁷⁰, Leu⁴⁷¹, and Arg⁴⁷² were individually mutagenized to Ala and found to have no effect on cell growth at 36°C. This indicates that the observed thermosensitivity of DBts13 (polts13) is caused by the deletion of more than one amino acid residue. In this study, we characterized two mutants, DBts11 (polts11) and DBts13 (polts13), and investigated the effects of these two polts alleles on different cell cycle events.

Characterization of pol Thermosensitive Mutants

At the permissive temperature, the mutants DBts11 (polts11) and DBts13 (polts13) exhibit a slightly elongated cell morphology with normal nuclear morphology (Figure 3F). The growth rate is comparable to the wild-type DB10 (pol⁺) cells (our unpublished observations). When midlog phase cultures of DBts11 (polts11) and DBts13 (polts13) were shifted to 36°C, they doubled their cell number once and then arrested cell growth after 3 h. In contrast, wild-type DB10 (pol⁺) cells continued to double every 2 h (Figure 3A). Viability analysis showed that the mutant cells could be recovered 2 h after shift to 36°C, but there was an overt decrease in their ability to recover after 3 h (Figure 2B). Both DBts11 (polts11) and DBts13 (polts13) began to display aberrant nuclear morphology 3 h after shift to 36°C. After 6-8 h, ~40% of the mutant cells exhibited heterogeneous cell sizes and aberrant nuclear morphology with a mixed population of anucleated cells, cells with unevenly distributed nuclear material, or small cells with condensed nucleus localized at one end of the cell (Figure 3, C and inset, G, and H). We further investigated the aberrant phenotypes of these two mutants in cells synchronized in a lactose gradient. The kinetics of the appearance of the aberrant phenotypes at 36°C of the synchronized mutant cells were found to be identical to that of the asynchronous culture (our unpublished observations). Flow cytometry analysis of mutant cells 4 h after shift to 36°C indicated that both mutants arrested in early to mid S phase (Figure 3D). As a comparison, the polts mutant pol1-1, isolated by D'Urso et al. (1995), was analyzed in parallel. After 4 h at the restrictive temperature, pol1-1 displayed a cdc phenotype and 2C DNA flow cytometry profile as described by D'Urso et al. (1995). This indicates that the polts alleles isolated in this study induce different cell cycle responses than the pol1-1 allele previously isolated by D'Urso et al. (1995). Furthermore, the four polts mutant alleles shown in Table 2 were not substantially sensitive to either UV irradiation or hydroxyurea at the permissive temperature (our unpublished results).

View larger version (44K): [in this window] [in a new window]   Figure 3.   Characterization of temperature-sensitive mutants of pol+. Cell number increase and viability were determined as described in MATERIALS AND METHODS. (A) Cell number increase of wild-type DB10 (pol+) and mutants upon shift to 36°C. DBts11 (polts11) and DBts13 (polts13) arrested at 36°C after one cell division. (B) Viability of wild type and thermosensitive mutants. (C) Percentage of cells displaying aberrant nuclear phenotype. Aberrant phenotype described as cut was scored by microscopic examination of DAPI- and calcofluor-stained cells. The inset shows the three types of aberrant phenotypes observed. (D) Flow cytometry analysis of DB10 (pol+), DBts11 (polts11), and DBts13 (polts13) 4 h after shift to the restrictive temperature. 1C and 2C standards are arrested cdc10 cells and exponentially growing haploid wild-type cells, respectively. (E-H). Photomicrographs of DB10 (pol+) at 36°C; DBts13 (polts13) 4 and 6 h after shift to 36°C. Bar, 5.8 µm.

Genetic Interactions of polts Mutant with Other Cell Cycle Mutants

Studies of budding yeast have shown that S phase mutants have an extensive network of synthetic interactions with other cell cycle genes (Hennessy et al., 1991; Yan et al., 1991a,b; Li and Herskowitz, 1993). We thus explored potential genetic interactions of polts mutants with cdc mutants. We used polts13 as a representative for this study and constructed double mutants of polts13 and several cdc mutants (Table 3). As expected, polts13 cdc10 and polts13 cdc25 arrested with the elongated cdc10 and cdc25 phenotype, respectively, not the polts13-like phenotype. polts13 in cdc18, cdc19 (MCM protein), or cdc21 (MCM protein) backgrounds arrested with a mid S-phase flow cytometry profile and polts13-like phenotype. Although cdc18⁺ and the MCM proteins are involved in initiation of S phase, the alleles used in this study, cdc18-K46, cdc19-P1, and cdc21-M68, all arrest the cell cycle with a G₂ DNA content (Kelly et al., 1993a; Forsburg and Nurse, 1994; Forsburg, 1996; Maiorano et al., 1996). Thus double mutants of these genes with polts13 arrest with a polts13 phenotype. cdc2-3w is a semidominant mutant (Enoch and Nurse, 1990). The double mutant polts13 cdc2-3w arrested with a cdc2-3w-like phenotype. Double mutant polts13 cdc22 (cdc22 encodes the large subunit of ribonucleotide reductase) arrested with a cdc22-like phenotype with a very low percent of abnormal nuclear morphology. In agreement with the known biochemical functions of Pol, Pol, and DNA ligase, polts13 arrested the cell cycle in either polts03 or cdc17 (DNA ligase) background with a polts-like phenotype. The recovery of both double mutants polts13 polts03 and polts13 cdc17 was lower than that of the single mutant DBts13 (polts13), indicating that cells with two essential replication enzymes impaired have lower viability. The double mutant polts13 cdc20 (cdc20⁺ is Pol in S. pombe) arrested with a G₁-S flow cytometry profile, cdc20-like elongated phenotype, and a very low percent of abnormal nuclear morphology. After 4 h at the restrictive temperature, in contrast to the single mutant polts13, the double mutant polts13 cdc20 recovered with full viability. This was surprising, because Pol is thought to be the first DNA polymerase that functions at the replication fork; polts13 cdc20 is expected to arrest with a polts-like phenotype, not a cdc20 phenotype.

View this table: [in this window] [in a new window]   Table 3.  Analysis of polts13 in cdc mutant backgrounds

polts13 cdc20 Double Mutant Arrests Early in S Phase with a cdc Phenotype

To confirm the cell cycle arrest point of polts13 relative to cdc20, we carried out reciprocal shift experiments using hydroxyurea (see MATERIALS AND METHODS). The single mutant DBts13 (polts13) and the double mutant polts13 cdc20 were used for the experiment, and the double mutant polts13 cdc25 was used as a control. Mutants were first arrested in S phase by hydroxyurea at 25°C for 4 h. The hydroxyurea was then removed, and cells were shifted to the restrictive temperature. As the cells proceed through the cell cycle, they are expected to arrest with either a cdc phenotype or a polts13 phenotype, depending on their point of execution in the cell cycle with respect to hydroxyurea. Mutants that arrest the cell cycle after the hydroxyurea block will not increase their cell number at the restrictive temperature, whereas mutants that arrest before the hydroxyurea block will double their cell number once, before arrest in the cell cycle.

After a 4-h block in hydroxyurea at the permissive temperature, both DBts13 (polts13) and the double mutant polts13 cdc20 had a 1C DNA profile (Figure 4A). Four hours after shifting to the restrictive temperature, the single mutant DBts13 (polts13) arrested with 1.5 C DNA (Figure 4A), and 40% of the cells displayed aberrant nuclear phenotypes (Figure 4B). However, the cell number of DBts13 (polts13) only increased 1.5-fold (our unpublished observation), suggesting that polts13 arrests the cell cycle very near the hydroxyurea block point. It has been reported that cdc20 arrests the cell cycle before the hydroxyurea block point (Nasmyth and Nurse, 1981) and with 1C DNA content (D'Urso and Nurse, 1997). Double mutant polts13 cdc20 arrested the cell cycle with a DNA content slightly greater than 1C (Figure 4A), doubled in cell number, and displayed a cdc phenotype with no abnormal nuclear morphology, similar to the cdc20 single mutant (Figure 4B). As expected, double mutant polts13 cdc25 had no increase in cell number after 4 h at 36°C and arrested with a phenotype and DNA content identical to the single mutant cdc25 (Figure 4, A and B).

View larger version (38K): [in this window] [in a new window]   Figure 4.   polts13 cdc20 double mutant arrested with cdc phenotype. (A) FACS analysis of single mutant DBts13 (polts13) and double mutants polts13 cdc20 and polts13 cdc25 in a hydroxyurea reciprocal shift experiment at 0, 2, and 4 h after shift to the restrictive temperature. The hydroxyurea reciprocal shift experiments were performed as described in MATERIALS AND METHODS. Double mutant polts13 cdc25 at 25°C is moderately elongated, resulting in a >1C profile at 0 h. (B) Phenotypes of single and double mutants of polts13 strains. Cells were stained with DAPI and calcofluor after shift to the restrictive temperature for 4 h. Bar, 5.8 µm.

Previous study has shown that cdc20 arrests the cell cycle in late G₁ or early S phase with 1C DNA content (D'Urso and Nurse, 1997). In addition, p25^rum1, a specific inhibitor of the p34^cdc2/p56^cdc13 mitotic kinase, accumulates only in preSTART cells, not in postSTART cells. It has been reported that p25^rum1 is not present in cdc20-arrested cells (Correa-Bordes and Nurse, 1995). This indicates that cdc20 arrests the cell cycle postSTART. In addition, our reciprocal shift experiments clearly showed that polts13 cdc20 doubles its cell numbers and arrests with a slightly greater than 1C DNA content (Figure 4A). Together, this indicates that the double mutant polts13 cdc20 arrests postSTART and the cdc phenotype of the double mutant is not caused by cells arresting at preSTART.

Replication Perturbation Caused by polts Alleles Activates Cds1 Kinase and Requires the Checkpoint Rads, Cds1, and Rqh1, but Not Chk1, for Maintenance of Cell Viability

Our observation that polts mutants have a slightly elongated cell morphology at the permissive temperature compared with the wild-type cells (Figure 3, E and F) suggests that polts11 and polts13 cause mild replication perturbations even at the permissive temperature. We thus investigated the cell cycle surveillance responses that could be induced by polts alleles at 25°C. We found that cells carrying either of the polts11 or polts13 mutant alleles are synthetic lethal in all checkpoint rad gene deletion backgrounds (Table 4). Thus, the replication perturbation caused by these two polts alleles requires the function of checkpoint Rads for viability of the cells at 25°C.

View this table: [in this window] [in a new window]   Table 4.  Genetic interactions of polts mutants with cell cycle response genes

Because Cds1 is thought to be involved in a checkpoint Rad-dependent "S-phase recovery" subpathway to maintain cell viability in the event of S phase perturbation (Lindsay et al., 1998), attempts were made to construct double mutants of polts11 and polts13 in a cds1 background. The double mutant polts11 cds1 was found to be synthetic lethal at either 22 or 25°C (Table 4). The double mutant polts13 cds1 formed microcolonies at 22°C (Table 4). At 25°C, polts13 cds1 had a severely reduced growth rate in comparison with either of the single mutants cds1 or polts13 (Figure 5A) and displayed elongated cell morphology but normal nuclear morphology (Figure 5B).

View larger version (30K): [in this window] [in a new window]   Figure 5.   At 25°C, Cds1 is required to maintain normal growth of polts13 mutant and is activated. (A) Double mutant polts13 cds1 has reduced growth rate at 25°C compared with the respective single mutant cds1 and polts13. Serial dilutions of exponentially growing cells at 25°C by 10-fold were spotted on YES plates. Plates were incubated at 25°C for 3 d. (B) Double mutant polts13 cds1 displays an elongated phenotype compared with the respective single mutant. Shown are phenotypes of single mutant polts13 and double mutant polts13 cds1 at 25°C. Bar, 3.5 µm. (C) Cds1 kinase is activated in polts mutants at 25°C. Cds1 protein was immunoprecipitated from logarithmically growing wild-type cells, (wt), wild-type cells treated with 20 mM hydroxyurea [wt(HU)], polts11 and polts13, and cds1 cells. The immunoprecipitated Cds1 proteins were used to assay for kinase activity using MBP as the substrate as described in MATERIALS AND METHODS. Shown here is the phosphorylation of MBP by Cds1 kinase derived from different strains. (D) The histogram shows that Cds1 kinase activity is fourfold higher in polts11 and polts13 as compared with the wild-type pol+ integrant DB10 cells. In DB10 cells treated with hydroxyurea, the Cds1 kinase activity is 25-fold higher than in untreated cells. The Cds1 kinase activity from polts13 is defined as the 100% maximum activity.

Finding that Cds1 is required to maintain the viability of polts mutants at the permissive temperature prompted us to assay the levels of Cds1 kinase activity in these polts mutants. The Cds1 kinase activity in both polts mutants was fourfold higher than that of the wild-type cells at 25°C (Figure 5, C and D). Cells treated with hydroxyurea was used as a control for the kinase assay, and cells containing a cds1 were used as a kinase-negative control. Similar to previous observations (Lindsay et al., 1998) the Cds1 kinase activity was activated ~25-fold in wild-type cells treated with hydroxyurea, whereas no detectable MBP phosphorylation was observed in cds1 cell lysates (Figure 5C), similar to the Cds1 kinase dead mutant described by Lindsay et al. (1998).

In addition to Cds1, Rqh1 is also thought to be involved in the checkpoint Rad-dependent recovery subpathway to prevent inappropriate recombination or to bypass lesions during S phase arrest or DNA damage (Murray et al., 1997; Stewart et al., 1997). Attempts to generate double mutants of polts11 or polts13 in an rqh1 background indicated that spores carrying the double mutants either did not germinate or formed microcolonies with reduced growth rates (Table 4). Thus, the replication perturbation caused by polts11 or polts13 at the permissive temperature requires both Cds1 and Rqh1 for maintaining normal growth and cell viability.

Previous studies have shown that S phase arrest or S phase delay of cells caused by a polts mutation requires the checkpoint Rad-Chk1 pathway to prevent inappropriate mitotic entry (Francesconi et al., 1995, 1997; Uchiyama et al., 1997). To test the requirement of Chk1 in polts mutants at 25°C, double mutants of polts in a chk1 background were analyzed. The double mutants polts11 chk1 and polts13 chk1 at 25°C had the same growth rate and identical cell size as those of the single polts mutants (Table 4). This suggests that at the permissive temperature, Chk1, unlike Cds1 and Rqh1, does not play a role in maintaining the viability of the polts mutants. Furthermore, at 25°C Chk1 was not phosphorylated in the polts13 mutant (Figure 6B, lane 4).

View larger version (47K): [in this window] [in a new window]   Figure 6.   Cds1 and Chk1 are both activated in polts mutants at 36°C. (A) Phenotype of double mutant DB242 (polts13 chk1) at the restrictive temperature. Midlog phase double mutant DB242 (polts13 chk1) grown at 25°C was shifted to 36°C for 6 h. Cells were stained with DAPI and calcofluor. (B) Activation of p56chk1:ep in DB232. Thirty micrograms of protein from cell lysates of DB232 carrying polts13 and epitope-tagged chk1+, DBts13 (polts13), and NW222 containing epitope-tagged chk1+ were fractionated on 8% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and probed with the 12CA5 antibody, and p56chk1:ep was detected using the ECL system (Amersham, Arlington Heights, IL). The unphosphorylated p56chk1:ep is marked by arrow a, and the phosphorylated p56chk1:ep is marked by arrow b. Lanes 1 and 3, Strains NW222 and DB232 (polts13 p56chk1:ep) treated with MMS; lane 2, DBts13 (polts13) containing no epitope-tagged chk1+ as a control; lanes 4-7, lysates from DB232 (polts13 p56chk1:ep) after 0, 2, 4, and 6 h at 36°C; lane 8, phosphatase-treated lysates from DB232 (polts13 p56chk1:ep) grown at 36°C for 6 h. (C) Activation of Cds1 kinase activity at 36°C. Cells were grown to midlog phase at 25°C and then shifted to 36°C for 3 h. Cds1 kinase activity was measured in wild-type cells, (wt), polts11, and polts13 as described in MATERIALS AND METHODS.

Disruption of Replication by polts Mutants at the Restrictive Temperature Induces Phosphorylation of Chk1 Protein

Previous studies have shown that cells arrested by a cdc mutation in a chk1 background enter mitosis inappropriately (Francesconi et al., 1995; Uchiyama et al., 1997). We thus investigated the cell cycle checkpoint responses of polts mutants at the restrictive temperature. At 36°C, nearly all of the polts11 chk1 and polts13 chk1 double mutants died with a small cell size and classic cut nuclear morphology (Table 4 and Figure 6A). This suggests that at 36°C, severe disruption of replication caused by these two Polts enzymes requires a functional Chk1 kinase to prevent cells from proceeding to inappropriate mitosis.

We then analyzed the phosphorylation status of Chk1 in the polts mutant cells at the restrictive temperature. Strains containing either polts11 or polts13 and chk1⁺ tagged with three copies of hemagglutinin epitope (Walworth and Bernards, 1996) were constructed. We tested the phosphorylation status of p56^chk1:ep in these polts strains at the permissive and the restrictive temperatures, using the phosphorylation of p56^chk1:ep in MMS-treated cells as a reference for the phosphorylated protein band shift (Figure 6B). As expected, at the permissive temperature, there was no discernible p56^chk1:ep phosphorylation in the polts mutant (Figure 6B, lane 4, and Table 4). In contrast, 2 h after shifting to 36°C, phosphorylation of p56^chk1:ep was observed in both polts11 and polts13 strains. The levels of p56^chk1:ep phosphorylation increased after 4 h and were maintained up to 6 h (Figure 6B, lanes 5-7). It is not yet known whether the phosphorylation of Chk1 protein correlates to an induction of Chk1 kinase activity. Attempts to discern whether the Chk1 kinase activity positively correlated to the phosphorylation of p56^chk1:ep by assaying the kinase activity of the anti-hemagglutinin immunoprecipitates were not successful. Immunoprecipitates of DBts13 (polts13) with no epitope-tagged Chk1 yielded a high background level of nonspecific kinase activity, and this precluded resolution of this question. Efforts to differentiate between the phosphorylation status of p56^chk1:ep in the polts13 strain at the restrictive temperature versus the MMS-treated polts13 strain using electrofocusing followed by SDS gel electrophoresis also did not yield any informative information.

To further clarify the roles played by Chk1 and Cds1, we also investigated the Cds1 kinase response in polts mutants at 36°C. The Cds1 kinase activity of DBts11 (polts11) and DBts13 (polts13) at the restrictive temperature was induced eightfold higher than in the wild-type integrant DB10 (pol⁺) cells (Figure 6C). This is not significantly higher than the induction observed at 25°C (Figure 5C). In addition, the double mutant polts13 cds1 at the restrictive temperature has a similar phenotype as the DBts13 (polts13) single mutant.

Thus, at the restrictive temperature, Chk1 and not Cds1 plays a major role in preventing the cells from entering inappropriate mitosis. Interestingly, despite the phosphorylation of Chk1 protein in these mutant cells at the restrictive temperature, a population of the cells still enter inappropriate mitosis after 4 h (Figure 3, C and H). The possible reasons for these phenotypes are discussed below.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In this study we investigated cell cycle responses induced by mutations in Pol. We report 1) the initiation DNA structure synthesized by Pol is required to bring about the S-M phase checkpoint; 2) polts mutants in cdc20 (pol) background arrest the cell cycle with a cdc phenotype, not a polts-like phenotype; and 3) during S phase progression, different degrees of replication defects caused by Pol mutations induce different downstream cell cycle surveillance kinases.

The Catalytic Function of Pol Is Required to Generate the Signal to Bring about the Replication Checkpoint

Genetic evidence has indicated that initiation of S phase generates a signal activating the S phase to mitosis checkpoint (Kelly et al., 1993b; Li and Deshaies, 1993). In this study, we generated a catalytically dead but structurally intact Pol mutant, Pol(D984N), to dissect the nature of the signal that is generated at the initiation of S phase. Previous mutational studies indicate that mutation of Asp⁹⁸⁴ to Asn completely abolishes the catalytic function of Pol without affecting the mutant protein's structure and stability or its ability to assemble into the Pol-primase complex (Copeland and Wang, 1993a,b). Our results showed that the catalytically dead Pol mutant when overexpressed had a dominant negative effect on vegetative cell growth (Figure 2C). This further indicates