DNA Polymerase ε Catalytic Domains Are Dispensable for DNA Replication, DNA Repair, and Cell Viability

Molecular Cell, Vol. 3, 679–685, May, 1999, Copyright 1999 by Cell Press

DNA Polymerase e Catalytic Domains Are Dispensable for DNA Replication, DNA Repair, and Cell Viability Tapio Kesti,*‡ Karin Flick,* Sirkka Kera¨nen,† Juhani E. Syva¨oja,‡ and Curt Wittenberg*§ * Departments of Molecular and Cell Biology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla, California 92037 † VTT Biotechnology and Food Research PO Box 1500 FIN-02044 VTT Finland ‡ Biocenter Oulu and Department of Biochemistry University of Oulu PO Box 400 FIN-90571 Oulu Finland

Summary DNA polymerase e (Pol e) is believed to play an essential catalytic role during eukaryotic DNA replication and is thought to participate in recombination and DNA repair. That Pol e is essential for progression through S phase and for viability in budding and fission yeasts is a central element of support for that view. We show that the amino-terminal portion of budding yeast Pol e (Pol2) containing all known DNA polymerase and exonuclease motifs is dispensable for DNA replication, DNA repair, and viability. However, the carboxy-terminal portion of Pol2 is both necessary and sufficient for viability. Finally, the viability of cells lacking Pol2 catalytic function does not require intact DNA replication or damage checkpoints.

Introduction DNA polymerase e has been implicated in numerous aspects of DNA metabolism, including replication, repair, recombination, and coordination of mitosis with the completion of S phase (Sugino, 1995; Wood and Shivji, 1997; Waga and Stillman, 1998). Although its precise role in these processes is unclear, Pol e has at least one essential function for viability in both budding yeast (POL2) (Morrison et al., 1990) and fission yeast (cdc201) (D’Urso and Nurse, 1997; Sugino et al., 1998). Several lines of evidence support a catalytic role for Pol2 during DNA replication. Yeast Pol e has an associated DNA polymerase activity in vitro (Morrison et al., 1990; Lee et al., 1991) that is thermolabile when prepared from cells harboring a temperature-sensitive pol2 allele. Furthermore, Pol2 resides at replication forks in cells undergoing S phase (Aparicio et al., 1997), and pol2 mutants fail to complete chromosomal DNA replication (Morrison et al., 1990; Araki et al., 1992; Budd and Campbell, 1993;

§ To whom correspondence should be addressed (e-mail: curtw@

scripps.edu).

Morrison and Sugino, 1994). Recently, 39→59 exonuclease-deficient mutants of POL2 and POL3 (Pol d) have been shown to result in strand-specific lesions in chromosomal DNA (Shcherbakova and Pavlov, 1996; Tran et al., 1997). Together, these observations have led to models for chromosomal DNA replication involving Pol e and Pol d in which each promotes replication of a specific DNA strand. Because pol3 mutants, but not pol2 mutants, accumulate lesions associated with defects in Okazaki fragment maturation (Gordenin et al., 1992; Kokoska et al., 1998), Pol2 has been proposed to be the leading strand DNA polymerase (Sugino, 1995). Support for that model in mammalian systems has been more elusive. For example, replication of SV40 DNA in vitro proceeds faithfully in the absence of Pol e (Waga and Stillman, 1994) whereas DNA polymerases a and d are both necessary and sufficient. An association has been demonstrated between Pol e and actively replicating chromosomal DNA but not replicating SV40 DNA in mammalian cells (Zlotkin et al., 1996). However, based upon both the low level of that association and its failure to be stimulated by treatment of the cells by mitogens, it was considered unlikely to reflect a central role of Pol e in DNA replication. In addition to its readily identifiable 39→59 exonuclease and DNA polymerase motifs, DNA polymerase e contains an extended carboxy-terminal domain with a conserved “zinc finger” motif. This domain is essential for viability in both the budding and fission yeast but is nonessential for the DNA polymerase activity in vitro. A number of mutations in the extreme carboxyl terminus of Pol2 render cells temperature sensitive for growth. Furthermore, those mutants are hypersensitive to the mutagen, methylmethane sulfonate (MMS), and to the DNA replication inhibitor, hydroxyurea (HU) (Dua et al., 1998). Based upon such observations, Pol2 has been suggested to be a bifunctional protein with distinct roles in DNA replication and the DNA replication checkpoint (Navas et al., 1995). We show here that the amino-terminal domain of Pol e, containing both the DNA polymerase and 39→59 exonuclease motifs, is dispensable for viability in the budding yeast. Conversely, the carboxy-terminal region of Pol2 is both necessary and sufficient for all of the essential functions of DNA polymerase e. Cells having only the carboxy-terminal region of Pol2 are proficient in DNA replication, recombination, and repair of DNA damage. Although such cells are also proficient for induction of the DNA replication checkpoint, the essential function of Pol2 is distinct from that role. We conclude that, although DNA polymerase e may play an enzymatic role during DNA replication, that function is nonessential and that the sole essential function of Pol e is, instead, provided by its noncatalytic carboxy-terminal domain. Results In a screen for suppressors of the lethality associated with the temperature-sensitive allele, pol2-18 (Araki et

Molecular Cell 680

al., 1992), plasmids encoding solely the carboxy-terminal portion of Pol2, which lacks all known enzymatic functions, were repeatedly isolated. The smallest portion of the POL2 gene that was capable of suppressing pol2-18 encoded approximately 200 carboxy-terminal amino acid residues (Figure 1). However, it was unclear which of the remaining carboxy-terminal sequences of Pol2 encoded by the suppressing plasmids were expressed or from what promoter they were transcribed since neither the wild-type translation initiation codon nor the POL2 promoter was present. Similar observations have been described previously (Navas et al., 1995). To more carefully evaluate whether POL2 lacking the amino-terminal coding sequences was sufficient to suppress the temperature-sensitive pol2-18 mutation, an in-frame deletion was constructed that lacked the sequence encoding amino acids 176–1134 (Figure 1A) of the 2222–amino acid protein. That region contains all of the conserved motifs associated with the DNA polymerase and 39→59 exonuclease functions of Pol2. The resulting mutant allele ( pol2-16), when expressed either from its own promoter (pol2-16; Figure 1B) or from the inducible GAL1 promoter (GAL1::pol2-16; Figure 1C), was capable of suppressing the lethality caused by either the pol2-18 or the pol2-11 (Budd and Campbell, 1993) temperature-sensitive mutation. In contrast, expression of only the amino-terminal portion of POL2 ( pol2DC) failed to suppress the temperature sensitivity of those mutants (Figure 1C). Cells carrying insertion mutations that disrupt the POL2 open reading frame but leave the amino- and carboxy-terminal portions intact had been found to be viable (Morrison et al., 1990). However, deletion of the carboxy-terminal coding region from that disrupted gene resulted in loss of viability (Navas et al., 1995). Since complete deletions of the POL2 open reading frame or disruption within the conserved DNA polymerase domain also resulted in lethality, it was suggested that Pol2 might be bifunctional with separable essential functions provided by the amino- and carboxy-terminal sequences (Navas et al., 1995). However, because we found the carboxyl terminus of POL2 to be sufficient to suppress two distinct temperature-sensitive alleles of POL2 having mutations in different regions of the gene, we considered the possibility that the catalytic domains of Pol2 were nonessential. To test this hypothesis, the GAL1::pol2-16 construct was introduced into a diploid strain in which the majority of the coding sequences of one copy of POL2 had been replaced by the LEU2 gene (pol2D, Figure 1A) (Morrison et al., 1990). Sporulation and tetrad analysis of that strain established that strains carrying both pol2D and GAL1::pol2-16 were frequently recovered (z40% of Leu1 segregants) whereas strains carrying only the pol2D allele were never recovered. Although slow growing, those strains were able to proliferate when the GAL1 promoter was induced by galactose but not when it was repressed by glucose (Figure 2A). The absence of the amino-terminal coding sequences and presence of the carboxy-terminal coding sequences was confirmed both by polymerase chain reaction (data not shown) and by Southern blot analysis (Figure 2B). Analysis of a tetrad having four viable segregants derived from a [POL2/pol2::LEU2 trp1/trp1:: TRP1::GALpol2-16] diploid revealed that the two Leu2

Figure 1. The Pol2 Carboxyl Terminus Lacking Catalytic Domains Is Sufficient to Complement Temperature-Sensitive Mutants of POL2 (A) Structure of the Pol2 protein, point mutants, and deletion mutants. The numbering refers to the amino acid residues in Pol2 polypeptide. The positions of conserved 39→59 exonuclease and DNA polymerase motifs are shown. The most conserved polymerase motif I is indicated by I. pol2-3 (referred to here as pol2D), pol2-18, and pol2-11 mutations have been described previously (Morrison et al., 1990; Araki et al., 1992; Budd and Campbell, 1993; Navas et al., 1995). pol2-16 lacks the 39→59 exonuclease and DNA polymerase motifs, while pol2DC is devoid of the entire carboxy-terminal portion of Pol2. The relevant restriction sites for the Southern analysis presented in Figure 3 are presented at the bottom of the diagram. (B) The carboxyl terminus of Pol2 (pol2-16) is sufficient to complement temperature-sensitive mutants of POL2. pol2-16 can complement both pol2-11 (C-terminal) and pol2-18 (N-terminal) temperature-sensitive mutants. Yeast cells were transformed with the indicated plasmids, streaked onto SC-uracil plates (Glc), and incubated for 3–4 days at 258C or 368C. (C) GAL-pol2-16, but not GAL-pol2DC, can complement both pol2 ts mutants. Yeast cells were streaked onto SC-uracil plates containing galactose (Gal) to induce the GAL promoter and incubated for 3–4 days at 258C or 368C. The pol2-18 allele is present in a centromeric plasmid over a pol2 disruption since pol2-18 is insufficient for viability when present in the chromosome in a single copy (our observations; H. Araki and A. Sugino, personal communication).

Trp2 spores had solely the wild-type POL2 allele whereas the two Leu1 Trp1 spores that carried pol2::LEU2 also carried the plasmid bearing pol2-16. Using a similar approach, we established that a chromosomal allele of pol2-16 expressed under its own promoter was also

Pol e Catalytic Activity Is Dispensable 681

Figure 2. The Carboxyl Terminus of Pol2 (pol2-16) Is Sufficient for DNA Replication and Cell Proliferation (A) pol2-16 can complement a complete disruption of POL2. Cells containing a pol2 disruption (pol2-3) were transformed with either a plasmid with wild-type POL2, or pol2-16 expressed from the plasmid indicated. pol216 and pol2-11 were expressed from the endogenous chromosomal locus. Yeast cells were streaked onto YEPD (Glc) or YPGal (Gal) plates and incubated at 258C or 368C. (B) Southern analysis was performed using SalI and SphI-digested genomic DNA isolated from the indicated strain probed with a 4.2 kb PstI fragment of POL2. The expected fragments are indicated (see Figure 1). Segregants A–D (lanes 4–7) represent the four viable products dissected from a single tetrad obtained by sporulation of a POL2/pol2D trp1/ trp1::TRP1::GAL1::pol2-16 diploid (TAY120, lane 1). The Leu and Trp phenotypes of each spore and the parent are indicated below each lane and are consistent with the genotype demonstrated by Southern analysis. (C) Reduced rate of chromosomal DNA replication and delay in cell cycle progression in pol2-16 mutants. rho0 derivatives of POL2 and pol2-16 strains were synchronized with mating pheromone a factor and released into fresh medium lacking mating pheromone. Samples were taken at the times indicated, and their DNA content was determined by flow cytometry.

sufficient for viability (Figure 2A). Spores having only the genomic pol2-16 allele often germinated but grew into colonies only infrequently (z10% of expected segregants). Since the viability of vegetative pol2-16 cells is not significantly different than that of POL2 cells, this suggests that the Pol2 catalytic domains may be especially important during the first cell cycles following spore germination. Again, the absence of the wild-type POL2 coding region and the presence of the genomic pol2-16 allele in viable segregants were confirmed by Southern blot analysis (Figure 2B). Similar strains have been constructed in two distinct genetic backgrounds with essentially the same results (data not shown). Based upon these observations, we conclude that the carboxy-terminal domain of Pol2 is both necessary and sufficient for viability even when present in its chromosomal locus expressed under the wild-type POL2 promoter. In contrast, the allele of POL2 having only the amino-terminal region (YCpGAL::pol2DC) is insufficient to complement the inviability of the pol2::LEU2 deletion (Navas et al., 1995; data not shown). Cells carrying either pol2-16 or GAL1::pol2-16 as their only source of Pol2 exhibited a decrease in the rate of cell proliferation. This is most likely a consequence of the increase in the duration of S phase in the pol2-16 mutants. The duration of S phase can be observed by flow cytometric analysis of DNA content in cells synchronously traversing the cell cycle following release from mating pheromone–induced G1 phase arrest (Figure 2C and data not shown). Whereas wild-type cells take less than 30 min to progress from a 1C to a 2C DNA content, that same process takes approximately 1 hr in the pol216 mutants.

Despite the increase in the duration of S phase, pol216 mutants are quite unremarkable in terms of their capacity to complete DNA replication, recombination, and excision repair. This is best illustrated by their behavior in response to mutagens and inhibitors of DNA replication (summarized in Table 1). Two representative experiments are presented in more detail (Figure 3). First, cells carrying either mutant or wild-type alleles of POL2 were evaluated for their sensitivity to ultraviolet irradiation. Each of the mutant strains exhibited comparable sensitivity to wild-type cells whereas cells deficient in MEC1, an established element of the DNA damage checkpoint pathway, were highly sensitive (Figure 3A). Next, the ability of cells to successfully repair an HO endonuclease–induced double-stranded DNA break at the MAT locus was evaluated by analyzing the plating efficiency of cells expressing a galactose-inducible HO gene under inducing (galactose) versus repressing (glucose) conditions (Figure 3B). Although pol2-16 mutants of each mating type were noticeably more sensitive than wild-type cells of the same mating type, that difference was modest relative to rad52 mutants that are known to be deficient in their capacity to repair doublestranded DNA breaks. We have observed similarly small differences in the sensitivity of pol2-16 mutants and wild-type cells to the DNA alkylating agent methylmethane sulfonate and to ionizing radiation (Table 1). We conclude that cells lacking Pol2 catalytic domains are either unaffected or only mildly deficient in their response to DNA damage and, therefore, that DNA polymerase e catalytic function is either unimportant for those repair processes or is functionally redundant with another DNA polymerase for its role in those processes.

Molecular Cell 682

Table 1. Comparison of the Phenotypes of pol2-16 to Those of Other pol2 Mutants Functiona

POL2

pol2-16

pol2-18

pol2-11

mec1b

Growth at 258C Growth at 378C Resistance to HU (Transient) (Constitutive) Resistance to MMS Resistance to UV Resistance to g Resistance to ds breaks in DNA

111 111

11 1

111 2

111 2

NA

111 111 111 111 111 111

1 1 11 111 11 11

11 1 11 11 111 NA

11 1/2 11 11 11 NA

2 2 2 2 2 NA

Congenic strains carrying the pol2 allele shown were subjected to the treatment indicated and their response was analyzed. Plus signs or minus signs reflect the phenotype of the strain relative to wild type. a NA, not analyzed. b A strain with a checkpoint-defective mec1-1 allele (Vallen and Cross, 1995) was used as a control.

Budding yeast Pol2 has been implicated in transduction of the DNA replication checkpoint signal. The extreme sensitivity of pol2-11 and pol2-12 mutants to continuous exposure to the ribonucleotide reductase inhibitor, hydroxyurea, provides one line of evidence supporting a checkpoint function (Navas et al., 1995; Table 1). Because both of those alleles contain nonsense mutations and, therefore, express protein products lacking the extreme carboxyl terminus (Figure 1A), the checkpoint function has been assigned to that portion of the molecule. The pol2-16 allele retains that portion

of the open reading frame. Consequently, it was important to evaluate the behavior of those mutants in response to HU. Cells expressing pol2-16 under either the endogenous POL2 promoter or the inducible GAL1 promoter were only slightly more sensitive than either wild-type or pol2-18 mutant strains to continuous exposure to subarresting concentrations (10–50 mM) of HU (Table 1, Figure 4, and data not shown). Although pol211 mutants were quite sensitive to the same concentrations of HU, neither those mutants (Table 1 and data not shown) nor pol2-16 mutants (Figure 4) exhibit HU sensitivity comparable to mec1 mutants. Finally, pol2-16 mutants exhibit only a modest defect in their ability to recover from exposure to arresting doses of HU (200 mM). It is unclear whether the modest HU sensitivity exhibited by pol2-16 mutants represents a significant defect in the DNA replication checkpoint. However, an intact replication checkpoint is not required for the viability of those mutants. Although their plating efficiency is somewhat diminished, the majority of pol2-16 mutant cells remain viable in a mec1-1 background (Figure 4 and data not shown). The decrease in viability that is observed relative to wild type is likely a result of the increased duration of S phase in pol2-16 mutants. Because pol2-16 mec1 mutants are easily recovered, we can conclude that the ability of cells to survive in the

Figure 3. Cells Lacking Pol2 Catalytic Domains Are Proficient in Excision Repair and Double-Strand Break Repair (A) Plating efficiency of pol2 mutants following exposure to UV irradiation. Dilutions of cells were spread on YEPD plates and irradiated with UV light at 254 nm followed by incubation for 3–5 days and counting of colonies. The relevant genotypes of the strains are indicated. (B) Sensitivity of pol2-16 mutants to HO-induced double-strand DNA breaks. The relative plating efficiency of cells containing the YCp50GAL-HO (URA3) plasmid (open bars) was determined by plating on SC-uracil containing glucose or galactose. Cells containing a YCpGAL (URA3) plasmid (solid bars) were used as a control. rad52 strains, known to be defective in the double-strand break repair, were included for comparison.

Figure 4. Cells Lacking Pol2 Catalytic Domains Are Viable in the Absence of the MEC1-Dependent Replication and Damage Checkpoints Equal numbers of wild-type, mec1, pol2-16, and pol2-16 mec1 cells from fresh cultures were streaked onto YPGAL plates with or without 20 mM HU. The plates were incubated at 308C for 3 days. All strains are cln1D cln2D, which is required for viability of the mec1-1 mutant in the 15Dau background (Vallen and Cross, 1999).

Pol e Catalytic Activity Is Dispensable 683

absence of DNA polymerase e catalytic activity does not require intact DNA replication or DNA damage checkpoints and, conversely, that the essential role of the Pol2 carboxyl terminus does not depend upon induction of either of those checkpoints. Discussion The observation that the POL2 gene of Saccharomyces cerevisiae is essential for viabilty and that temperaturesensitive mutants are defective in cell cycle progression is central to the argument that DNA polymerase e is important for the replication of chromosomal DNA in eukaryotes. However, experiments reported here establish that the catalytic function of S. cerevisiae Pol2 is not required for cell cycle progression or viability. Furthermore, we demonstrate that the essential function of Pol2 can be provided by the carboxyl terminus. Although we do not dispute the ample evidence that Pol2 can participate in chromosomal replication in eukaryotes, we argue that it need not participate directly in the polymerization of deoxyribonucleotides. Whether that is because it plays no significant role in that process in wildtype cells or whether another polymerase, for instance DNA polymerase d, is sufficient in its absence is unclear from our data. Nevertheless, it is clear that Pol2 plays no requisite role. Previous analysis of pol2 mutants led to the proposal that Pol2 was a bifunctional enzyme with essential but separable DNA polymerase and DNA replication checkpoint domains (Navas et al., 1995). However, it now appears that the requirement for the carboxyl terminus is sufficient to explain most of the observations that led to that conclusion. For example, that function is sufficient to explain the temperature-sensitive defect of pol2-11 and pol2-18 mutants since both can be complemented by pol2-16. Although this could be explained by intragenic complementation in the case of pol2-11, the fact that pol2-16 is sufficient on its own renders that explanation unnecessary. Intragenic complementation is insufficient to explain the complementation of pol218 because the lesion in pol2-18 is in sequences that are absent in the pol2-16 allele. In fact, cells arrested by incubation of pol2-18 at the restrictive temperature can be rescued by induction of GAL-pol2-16 (unpublished data). Conversely, introduction of the mutation that is responsible for the temperature sensitivity of pol2-11 into pol2-16 results in loss of its essential function. These observations argue that the lesions in those pol2 mutants that give rise to their lethality result from inactivation of the essential carboxy-terminal function of Pol2. Recent evidence demonstrates that Pol e plays an important role in DNA replication associated with double-strand break-induced DNA repair (Holmes and Haber, 1999). That study found that the process of mating type switching in pol2-18 mutants at the restrictive temperature was strongly delayed but not completely compromised. This led the authors to conclude that, although Pol3 is sufficient, Pol2 plays a primary role in that process. We present evidence that pol2-16 mutants are reasonably proficient in repair of HO-induced double-strand breaks at the MAT locus, raising the possibility that the requirement for POL2 in that process is also

largely a consequence of a requirement for its noncatalytic carboxy-terminal domain. What, then, is the essential function of the Pol2 carboxyl terminus? Two lines of evidence argue against that function being its proposed role as a sensor of the DNA replication checkpoint signal. First, neither the DNA replication nor DNA damage checkpoints are essential for viability (reviewed by Elledge, 1996; Weinert, 1998). In addition, rescue of a deficiency of POL2 by pol216 is independent of MEC1, an essential downstream element of the replication checkpoint machinery. Another possibility is that the carboxyl terminus of Pol2 is important for the integrity of the replication complex or recruits essential factors to that complex. Notably, the essential protein Dpb2 interacts with Pol2 through this region (Morrison et al., 1990; Araki et al., 1991; Dua et al., 1998). Perhaps the carboxy-terminal domain of Pol2 is required to properly localize or regulate the activity of Dpb2. Although it is intriguing to speculate, the precise role of the Pol2 carboxyl terminus awaits further investigation. Our findings raise significant questions concerning the importance of the catalytic function of DNA polymerase e in replication of chromosomal DNA in eukaryotes. Whereas a mechanism involving Pol e catalytic activity may be the most efficient, a simpler system that utilizes Pol d for both strands, such as that demonstrated for in vitro replication of the SV40 genome (Waga and Stillman, 1994), may be sufficient. Clearly, a mechanism for DNA replication that necessitates the involvement of Pol e in the replication of one strand must be revised. Experimental Procedures Strains All yeast strains used in this study were congenic with 15Daub, a bar1D ura3Dns derivative of BF264-15D (MATa ade1 his2 leu2-3,112 trp1-1), and their relevant genotypes are as follows: TAY237 (MATa pol2-16), TAY 238 (MATa pol2-16), TAY205 (MATa pol2-3::LEU2 YCppol2-18), TAY245 (MATa pol2-11), TAY209 (MATa pol2-3::LEU2 YEppol2-16), TAY228 (MATa pol2-3::LEU2 trp1::TRP1::GAL1-pol216), TAY201 (MATa pol2-3::LEU2 YCpPOL2), TAY120 (MATa/MATa POL2/pol2-3::LEU2 trp1/trp1::TRP1::GAL1-pol2-16), TAY352 (MATa mec1-1 cln1 cln2 pol2-3::LEU2 trp1::TRP1::GAL1-pol2-16), TAY 285 (MATa rad52::LEU2), TAY295 (MATa rad52::LEU2), 26858A (MATa mec1-1 cln1D cln2D) (Vallen and Cross, 1995), CWY869 (MATa cln1 cln2), and TAY523 (MATa cln1::KANR cln2::URA3 pol2-3::LEU2 trp1::TRP1::GAL1-pol2-16). The pol2-3::LEU2 disruption allele is referred to as pol2D throughout. The cln1 cln2 mutation in all mec1-1 strains is required for maintenance of viability (Vallen and Cross, 1999). Experiments using these strains utilize controls having cln1 cln2 mutations. Standard genetic methods were used in strain construction (Guthrie and Fink, 1991). Mutant Alleles of POL2 The structures of POL2 alleles are outlined in Figure 1. The pol2-3::LEU2 fragment for disruption of the POL2 gene was amplified by PCR from genomic DNA of strain YHA302 (Araki et al., 1992). YEppol2-16 was generated by partial BglII digestion of YEpPOL2 (Araki et al., 1992). A 6.4 kb NgoMI-EagI fragment was subcloned into pRS406 integrating vector, and the construct was used to introduce pol2-16 into POL2 locus by transplacement. YCpGALpol2-16 was created by introducing a SacI site upstream of pol2-16 translation initiation codon and subcloning a SacI-SphI fragment into pYCS1, a single-copy derivative of pYES2. The YCpGALpol2DC plasmid contains a SacI-ClaI fragment of POL2 encoding the amino acid residues 1–1142 in pYCS1. A pRS306pol2-11 plasmid (Budd and

Molecular Cell 684

Campbell, 1993) was used to make a pol2-11 strain by transplacement. Southern Analysis (Figure 2B) was achieved using genomic DNA digested with SalI and SphI and by probing with the 4.2 kb PstI fragment of POL2 (Figure 1). This probe detects fragments of 2.0 kb and 4.7 kb from the wild-type POL2 gene, a 3.8 kb fragment from pol2-16 and GALpol2-16, and a 3.0 kb fragment from the pol2-3 deletion construct. The latter fragment is only weakly detected because it has only 100 bp of overlap with the probe. Flow Cytometric Analysis rho0 derivatives of POL2 and pol2-16 strains were prepared by growth on ethidium bromide (Fox et al., 1991) and then grown at 308C in YEPD medium to an optical density at 600 nm of 0.3 and synchronized with 100 ng/ml mating pheromone a factor for 3.5 and 4.5 hr, respectively. a factor was removed by centrifugation, and cells were suspended in fresh medium. Cells were stained with propidium iodide as described (Haase and Lew, 1997), and they were analyzed by a FACScan analyzer (Becton-Dickinson). Analysis of Sensitivity to Mutagens and Inhibitors Survival after exposure to 0.2 M HU was assayed as described (Araki et al., 1995). The ability to form colonies on YEPD plates containing 0–200 mM HU was scored. Sensitivity to MMS was assayed by a transient exposure to 0.15% MMS (Schar et al., 1997). UV mutagenesis was performed by spreading appropriate dilutions of cells onto YEPD plates, irradiating with Stratalinker (Stratagene) at 254 nm, followed by incubation for 3–5 days and counting of colonies. Gamma irradiation was performed by diluting log phase cultures to 104 cells/ml in YEPD, irradiating with 0–200 Gy from a 137Cs source, and spreading onto YEPD plates. The plates were incubated for 3–5 days and the colonies were scored. To evaluate resistance to HO-induced double-strand breaks, strains containing the YCp50-GAL-HO plasmid were plated on CMUra Gal medium, and the relative capacity to form colonies was determined (Herskowitz and Jensen, 1991). Acknowledgments

cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint. Proc. Natl. Acad. Sci. USA 92, 11791–11795. Budd, M.E., and Campbell, J.L. (1993). DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 496–505. Dua, R., Levy, D.L., and Campbell, J.L. (1998). Role of the putative zinc finger domain of Saccharomyces cerevisiae DNA polymerase e in DNA replication and the S/M checkpoint pathway. J. Biol. Chem. 273, 30046–30055. D’Urso, G., and Nurse, P. (1997). Schizosaccharomyces pombe cdc201 encodes DNA polymerase epsilon and is required for chromosomal replication but not for the S phase checkpoint. Proc. Natl. Acad. Sci. USA 94, 12491–12496. Elledge, S.J. (1996). Cell cycle checkpoints: preventing an identity crisis. Science 274, 1664–1672. Fox, T.D., Folley, L.S., Mulero, J.J., MuMullin, T.W., Thorsness, P.E., Hedin, L.O., and Costanzo, M.C. (1991). Analysis and manipulation of yeast mitochondrial genes. Methods Enzymol. 194, 149–165. Gordenin, D.A., Malkova, A.L., Peterzen, A., Kulikov, V.N., Pavlov, Y.I., Perkins, E., and Resnick, M.A. (1992). Transposon Tn5 excision in yeast: influence of DNA polymerases alpha, delta, and epsilon and repair genes. Proc. Natl. Acad. Sci. USA 89, 3785–3789. Guthrie, C., and Fink, G.R. (1991). A guide to yeast genetics and molecular biology. In Methods in Enzymology (San Diego, CA: Academic Press) pp. 1–933. Haase, S.B., and Lew, D.J. (1997). Flow cytometric analysis of DNA content in budding yeast. Methods Enzymol. 283, 322–332. Herskowitz, I., and Jensen, R.E. (1991). Putting the HO gene to work: practical uses for mating-type switching. Methods Enzymol. 194, 132–146. Holmes, A.M., and Haber, J.E. (1999). Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases. Cell 96, 415–424. Kokoska, R.J., Stefanovic, L., Tran, H.T., Resnick, M.A., Gordenin, D.A., and Petes, T.D. (1998). Destabilization of yeast micro- and minisatellite DNA sequences by mutations affecting a nuclease involved in Okazaki fragment processing (rad27) and DNA polymerase delta (pol3-t). Mol. Cell. Biol. 18, 2779–2788. Lee, S.H., Pan, Z.Q., Kwong, A.D., Burgers, P.M., and Hurwitz, J. (1991). Synthesis of DNA by DNA polymerase epsilon in vitro. J. Biol. Chem. 266, 22707–22717.

We are grateful to Martin Budd, Judy Campbell, Akio Sugino, Hiroyuki Araki, and Elizabeth Vallen for strains and plasmids. We also thank A. Sugino, H. Araki, and J. Campbell for enthusiastic and informative discussions and to Jim Haber for his thoughtful comments on the manuscript. We thank members the TSRI Cell Cycle Group for many helpful discussions. This work was supported by The National Research Council for the Environment and Natural Resources, Academy of Finland (T. K. and J. E. S), a fellowship from the Emil Aaltonen Foundation (T. K.), and National Institutes of Health, USA (GM43487 and GM46006 to C. W.).

Morrison, A., Araki, H., Clark, A.B., Hamatake, R.K., and Sugino, A. (1990). A third essential DNA polymerase in S. cerevisiae. Cell 62, 1143–1151.

Received February 17, 1999; revised March 15, 1999.

Schar, P., Herrmann, G., Daly, G., and Lindahl, T. (1997). A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks. Genes Dev. 11, 1912–1924.

References

Morrison, A., and Sugino, A. (1994). The 39→59 exonucleases of both DNA polymerases delta and epsilon participate in correcting errors of DNA replication in Saccharomyces cerevisiae. Mol. Gen. Genet. 242, 289–296. Navas, T.A., Zhou, Z., and Elledge, S.J. (1995). DNA polymerase epsilon links the DNA replication machinery to the S phase checkpoint. Cell 80, 29–39.

Aparicio, O.M., Weinstein, D.M., and Bell, S.P. (1997). Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91, 59–69.

Shcherbakova, P.V., and Pavlov, Y.I. (1996). 39→59 exonucleases of DNA polymerases epsilon and delta correct base analog induced DNA replication errors on opposite DNA strands in Saccharomyces cerevisiae. Genetics 142, 717–726.

Araki, H., Hamatake, R.K., Johnston, L.H., and Sugino, A. (1991). DPB2, the gene encoding DNA polymerase II subunit B, is required for chromosome replication in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 88, 4601–4605.

Sugino, A. (1995). Yeast DNA polymerases and their role at the replication fork. Trends Biochem. Sci. 20, 319–323.

Araki, H., Ropp, P.A., Johnson, A.L., Johnston, L.H., Morrison, A., and Sugino, A. (1992). DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae. EMBO J. 11, 733–740. Araki, H., Leem, S.H., Phongdara, A., and Sugino, A. (1995). Dpb11, which interacts with DNA polymerase II (epsilon) in Saccharomyces

Sugino, A., Ohara, T., Sebastian, J., Nakashima, N., and Araki, H. (1998). DNA polymerase epsilon encoded by cdc201 is required for chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe. Genes Cells 3, 99–110. Tran, H.T., Keen, J.D., Kricker, M., Resnick, M.A., and Gordenin, D.A. (1997). Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants. Mol. Cell. Biol. 17, 2859–2865.

Pol e Catalytic Activity Is Dispensable 685

Vallen, E.A., and Cross, F.R. (1999). Interaction between the MEC1dependent DNA synthesis checkpoint and G1 cyclin function in Saccharomyces cerevisiae. Genetics 151, 459–471. Vallen, E.A., and Cross, F.R. (1995). Mutations in RAD27 define a potential link between G1 cyclins and DNA replication. Mol. Cell. Biol. 15, 4291–4302. Waga, S., and Stillman, B. (1994). Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nature 369, 207–212. Waga, S., and Stillman, B. (1998). The DNA replication fork in eukaryotic cells. Annu. Rev. Biochem. 67, 721–751. Weinert, T. (1998). DNA damage and checkpoint pathways: molecular anatomy and interactions with repair. Cell 94, 555–558. Wood, R.D., and Shivji, M.K. (1997). Which DNA polymerases are used for DNA-repair in eukaryotes? Carcinogenesis 18, 605–610. Zlotkin, T., Kaufmann, G., Jiang, Y., Lee, M.Y., Uitto, L., Syva¨oja, J., Dornreiter, I., Fanning, E., and Nethanel, T. (1996). DNA polymerase epsilon may be dispensable for SV40- but not cellular- DNA replication. EMBO J. 15, 2298–2305.