INTRODUCTION

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Acquired genetic instability has been proposed to be an early event in tumor evolution (24, 25). The hypothesis that cancer cells exhibit a mutator phenotype or an increased rate of mutation has been validated by the finding that mutations in mismatch repair (MMR) genes lead to microsatellite instability and are the underlying cause of both hereditary nonpolyposis colon cancer (HNPCC) and sporadic tumors (reviewed in references 18, 23, and 26). In addition to MMR genes, mutations in other genes responsible for ensuring genomic stability may also generate a mutator phenotype (25). Thus, proteins that maintain genomic stability in normal cells, including those involved in DNA replication, repair, recombination, chromosomal segregation, transcription, and cell cycle control are prime proto-mutator candidates. Mutations in any of these genes may be an early event in tumorigenesis, allowing the generation of the multiple mutations observed in cancers.

In addition to microsatellite instability, another common source of mutation in human genetic diseases is deletion of sequences flanked by short direct repeats (19). For example, this type of deletion mutation has been documented in the genes responsible for retinoblastoma (5), -, -, and -thalassemias, ataxia telangiectasia, hemophilia B, Wilm's tumor, breast cancer (BRCA-1 and BRCA-2), HNPCC (MLH-1 and MSH2), and xeroderma pigmentosa (10; also references 20 and 27 and references therein). In addition to human genetic diseases, similar spontaneous and ionizing radiation-induced deletion mutations have been characterized at the aprt locus of hamster cells (7, 14, 31).

Maintaining the integrity of genetic material during genome duplication requires normal and accurate DNA replication. In Saccharomyces cerevisiae, defects in three replication proteins, Pol3 (DNA polymerase  [Pol], Rad27 (homolog of mammalian FEN-1), and RPA (replication protein A) result in a mutator phenotype characterized by alterations of sequences flanked by short direct repeats in vivo (6, 37, 38). Strains containing thermosensitive (ts) alleles of Pol (pol3-t) or RPA (rfa1-t29, rfa1-t6, and rfa1-t33) have a mutator phenotype exhibiting an elevated rate of deletion of sequences flanked by short direct repeats (6, 38). The pol3-t allele is thought to reduce the rate of lagging strand DNA synthesis, resulting in long stretches of single-stranded DNA (ssDNA) on the lagging strand template (12). Deletions are proposed to arise from DNA polymerase slippage over a ssDNA loop formed by slip mispairing between direct repeats (12, 38).

Cells containing a null mutation of RAD27 exhibit an increased rate of duplication of sequences flanked by short direct repeats (37). Rad27 functions as either a structure-specific 5' flap endonuclease or exonuclease to process Okazaki fragments (16; see also references 2 and 21 and references therein). The observed duplications are thought to be initiated when strand displacement synthesis of Okazaki fragments occurs without the Rad27-dependent removal of the resulting 5' flap (37). A subsequent ssDNA loop formed by slip mispairing of direct repeats on the 5' flap or formation of a double-strand break (dsb) followed by mutagenic single-stranded annealing are two possible intermediates of duplication mutations (37).

These findings suggest that either the impairment or absence of a DNA replication protein could lead to a mutator phenotype that compromises genomic stability. To test this hypothesis and to further elucidate the molecular mechanisms underlying deletion and duplication mutations of sequences flanked by short direct repeats, we screened a panel of mutants of Schizosaccharomyces pombe replication proteins shown in Table 1 for a mutator phenotype. We identified specific ts alleles of pol, pol, two pol subunit genes, and a DNA ligase gene as mutators that generate deletions and a null mutant of rad2 (homolog of RAD27 and FEN-1) as a mutator that generates duplications of sequences flanked by short direct repeats. We characterized these mutators and found that the deletion and duplication mutation mechanisms are distinct but interdependent. Furthermore, the deletion and duplication mutators have differential requirements for the checkpoint Rad-mediated mechanisms to recover from aberrant replication.

	MATERIALS AND METHODS

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Genetic and cell biology techniques. S. pombe 972 h was used as the wild-type strain. All the fission yeast strains used in this study contain the wild-type ura4⁺ gene either at their endogenous locus or in the place of a deleted gene in the null mutants. Double mutants were constructed by standard genetic techniques (15) and were ensured to contain only one wild-type copy of the ura4⁺ gene in the strain. The rich medium (YE) and the minimal medium (PM) were prepared as described previously (15, 28). Unless otherwise stated, cells were grown in YES medium which is YE medium supplemented with 0.2 mg of adenine per ml and 0.5 mg (each) of uracil, leucine, and histidine per ml. Strains were generally propagated at 25°C unless otherwise stated. Conjugation and sporulation were also performed at 25°C.

Determination of permissive, semipermissive, and nonpermissive temperatures. The permissive, semipermissive, and nonpermissive temperatures of each of the strains tested were determined by a variety of methods in rich medium (YES). The permissive temperature was defined as the condition under which cells grew at the same rate as the wild-type cells did and displayed very few, if any, elongated cells. The semipermissive condition was defined as the temperature at which a strain exhibited a lower growth rate and slightly elongated cells but still maintained a plating efficiency comparable to that observed at the permissive temperature. The plating efficiency was determined by counting the number of colonies resulting from plating 10³ cells/plate on rich medium (YES). The nonpermissive temperature was determined to be the lowest temperature at which the plating efficiency of a strain was compromised (to <40%) relative to the colony-forming ability at the permissive temperature, thereby preventing the measurement of a mutation rate.

Mutator analysis. Yeast strains shown in Table 1 were first grown on PM plates without uracil to select for cells harboring wild-type ura4⁺. After 4 days, whole colonies of ura⁺ cells were suspended in nonselective YES medium and grown overnight for approximately 6.5 generations. Log-phase cells were plated onto YES plates containing 1 mg of 5-fluoroorotic acid (5-FOA) per ml at a concentration of 10⁶ cells/plate. After ~5 days of growth, the number of FOA^r colonies was assessed. Mutation rates per generation were calculated by using the formula adapted from the formula in reference 35: rate of mutation = 1 e^{(1/n)lnR_n/R₀}, where R₀ and R_n are the proportions of ura⁺ cells 0 and n generations after removal from selection, respectively. R₀ was taken as 1, since the progenitor cells for each experiment were ura⁺. The overall mutation rate was taken as the median value from ~10 to 30 independent experiments per strain. The relative mutation rate was expressed as the fold induction compared to the wild-type value, which was 2.89 × 10⁸ per generation. Unless otherwise mentioned, all strains were designated as having a mutator phenotype within 95% confidence intervals by a two-tailed t test ( = 0.05,  = 0.20).

	RESULTS

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Identification of replication mutators. It has been shown in S. cerevisiae that mutations of POL3, RPA, and RAD27 result in genomic instability. pol3-t, rfa1-t29, rfa1-t6, and rfa1-t33 generate deletions and a rad27 null strain generates duplications of sequences flanked by short direct repeats (6, 37, 38). To determine whether aberrant replication caused by mutations in other replication proteins could also lead to a mutator phenotype, we screened a bank of S. pombe mutants involved in DNA replication (Table 1) for an increased mutation rate. We used a forward mutation rate assay that detects mutations inactivating the ura4⁺ gene (FOA^r mutations). Strains that had a mutation rate fivefold higher than that of the wild-type cells, which have a mutation rate of 2.89 × 10⁸ per generation, were considered to have a mutator phenotype and will hereafter be referred to as mutators.

Mutation spectra of the replication mutators. We analyzed the nature of the mutations generated in the ura4 gene of the FOA^r mutant cells. The size of the ura4 gene from the FOA^r cells was determined by PCR amplification and agarose gel analysis as described in Materials and Methods. The wild-type ura4⁺ PCR product is 1,406 bp long. The mutators had either smaller, larger, or similar-sized PCR fragments compared to the wild-type ura4⁺ PCR product (designated Del, Ins, or NDSC in Table 2, respectively; see also Fig. 1). ura4 PCR products that were smaller than wild-type products were found in FOA^r colonies derived from ts replication mutants pol1-1, polts11, polts13, polts1, cdc1-7, cdc27-K3, and cdc17-K42, indicating that mutations at these specific alleles of these replication proteins caused deletions in the ura4⁺ gene. ura4 PCR products that were larger than wild-type products, indicative of insertion mutation events, were rarely found in those ts replication mutators that generated deletions. These mutants will hereafter be referred to as the ts deletion mutators (Table 2). The percentages of deletions versus total types of mutations generated by a ts replication mutator ranged from 24 in cdc27-K3 to 94 in polts1. The observation that insertions, but very few deletions, were detected in FOA^r rad2 mutant cells (Table 2) is similar to the mutation spectrum described for S. cerevisiae rad27 mutants (37).

View larger version (63K): [in this window] [in a new window]   FIG. 1.   Analysis of ura4 PCR products from FOAr cells. Genomic DNA isolated from FOAr cells was amplified by PCR and analyzed by 1.5% agarose gel electrophoresis as described in Materials and Methods. The wild-type (WT) ura4 PCR product is 1,406 bp long and marked as having 0-bp alteration. The difference in size between the wild-type and other ura4 PCR products (+/ bp) is indicated to the right of the gel, with + indicating larger and  indicating smaller than the wild-type product.

Deletion mutator phenotype is dependent upon semipermissive growth conditions. We further characterized the replication mutators to elucidate the deletion and duplication mechanisms. Studies of an S. cerevisiae pol3-t mutant have shown an eightfold increase of the mutation rate at 30°C over that at 21°C, suggesting that Pol has an increased mutation rate at the semipermissive temperature (38). We thus tested the effects of temperature on the mutation rates of the ts deletion replication mutators (polts11, polts13, polts1, cdc1-7, cdc27-K3, and cdc17-K42), a ts replication nondeletion mutator (polts3), and a ts replication nonmutator (polts2) at 21, 25, or 30°C. Mutation rates of wild-type cells were similar at 25, 30, and 37°C (data not shown). The ts replication mutators polts11, polts13, polts1, and cdc1-7 that displayed an elevated mutation rate relative to that of wild-type cells at 25°C had a lower mutation rate at 21°C. At 21°C, the mutation rates in polts1 and cdc1-7 were reduced ca. five- to ninefold to near-wild-type levels, and the mutation rates in polts11 and polts13 were reduced from ~180- to ~50-fold higher than that of wild-type cells (compare Tables 2 and 6). At 25°C, cells harboring the polts11, polts13, polts1, or cdc1-7 allele were slightly elongated, which is indicative of a mild replication perturbation and 25°C being the semipermissive condition for these mutants (3; also data not shown). At 21°C, cells harboring the polts11 or polts13 allele displayed a higher mutation rate than the wild-type cells did, indicating that 21°C is still a semipermissive temperature for these two mutants. Analogously, regardless of their ability to generate deletions, mutants such as cdc17-K42, cdc27-K3, polts3, and cdc6-121 mutants that exhibited a mutator phenotype at 30°C had decreased mutation rates to near-wild-type levels at 25°C (compare Tables 2 and 6). Viability analysis indicated that 30°C is the semipermissive temperature of these replication mutators (Table 1). The mutation rate of a ts nonmutator (polts2) remained at the wild-type rates at 21, 25, and 28°C (Table 2 and data not shown). Cells harboring rad2 are not temperature-sensitive (30), and thus, the associated mutation rate did not change in a temperature-dependent manner (data not shown). These results indicate that the observed increased mutation rate in all the ts replication mutators is dependent upon semipermissive growth conditions and confirms that the mutated replication protein is the source of the mutator phenotype.

Formation of deletion mutations requires Rad2. The differential mutation spectra associated with the ts replication mutators and rad2 suggest that the processes of generating deletions and duplications are distinct. However, the two mechanisms may be related, since proteins mutated in both pathways are directly involved in DNA replication and the sequence that is altered is always flanked by short direct repeats. To test whether the underlying mechanisms responsible for generating deletions and duplications by these replication mutators are interdependent, we constructed double mutants of each of the deletion-generating ts replication mutators in a rad2 background. Deletion of rad2 had no effect on the viability or restrictive temperature of all the double mutants tested (data not shown). We then compared the mutation rate of the ts replication mutators in a rad2⁺ or rad2 background at the semipermissive temperature of each respective ts replication mutator (Table 4). Surprisingly, the mutation rates of polts11 and polts13 decreased 4.6- to 7.1-fold, respectively, in a rad2 background. It was difficult to evaluate the effect of rad2 on the mutation rate of the other ts replication mutators within 95% confidence levels, since these ts replication mutators and rad2 had similar mutation rates which were 9- to 22-fold higher than that of wild-type cells (Table 4). However, agarose gel analysis of the FOA^r PCR products revealed that all the ts deletion mutators in a rad2 background had reduced percentages of deletion mutations (Table 4). For example, in a rad2⁺ background, mutations generated by polts1 were 94% deletions and 6% NDSCs (Tables 2 and 4), whereas in a rad2 background, deletions in polts1 were no longer detectable (Table 4). Finding decreases in both the mutation rates in polts11 and polts13 and in the percentages of deletion mutations formed in all the ts deletion mutators in a rad2 background indicates that Rad2 is required to generate the observed deletion mutations in the ts replication mutators.

View this table: [in this window] [in a new window]   TABLE 4.   Mutation rates and types of mutations of ts replication mutators in rad2+ and rad2 backgroundsa

Formation of duplication mutations in rad2 requires normal DNA polymerization, but not DNA ligation. We also measured the duplication mutation rate of rad2 in all the ts replication mutator backgrounds. With the exception of the ligase mutator (cdc17-K42), the duplication mutation rate of rad2 was reduced in a ts replication mutator background. The percentages of duplication mutations decreased from 37 in the rad2 single mutant to 0 to 6 in a polts11, polts13, cdc1-7, or cdc27-K3 background and to 14 in a polts1 background (Table 4). These results suggest that duplication mutations caused by rad2 are dependent upon normal DNA polymerization, but not upon DNA ligation. Together, these results indicate that the deletion and duplication mutation mechanisms are distinct but interdependent.

Cds1 is required for growth and viability of the ts replication mutators, but not the duplication mutator rad2. All the replication mutators described in this study involve proteins that directly function at the replication fork, suggesting that these replication mutators generate aberrant replication structures at the semipermissive temperature. We thus investigated how the cell responds to prevent cell death and mutation formation from aberrant replication(s). To this end, we analyzed these replication mutators in a cds1 background (Table 5). In response to hydroxyurea (HU) or DNA damage, the protein kinase Cds1 enables cells to arrest S phase reversibly through a checkpoint-Rad mediated S-phase recovery pathway (22). Cds1 is phosphorylated and activated by S-phase arrest (22). Our previous studies have shown that Cds1 protein kinase is activated in polts11 and polts13 mutants at the semipermissive temperature (25°C), suggesting that these mutants have aberrant replication structures that activate Cds1 (3). Furthermore, Cds1 function is required for viability of mutant cells containing polts alleles, since cells with polts11 or polts13 in a cds1 background at 25°C are either inviable or grow poorly (3). We therefore tested the effect of deleting cds1⁺ in the replication mutators.

View this table: [in this window] [in a new window]   TABLE 5.   Growth of replication mutants in rad26 and cds1 backgrounds

Absence of Cds1 exacerbates the deletion, but not duplication, mutator phenotype. We next analyzed the mutation rate of the ts replication mutators in a cds1 background. Since cds1 did not have a mutator phenotype (Table 6) and deletion of cds1⁺ reduced the semipermissive temperature of all the ts replication mutators (Table 5), analyses were performed at the permissive temperature of the respective ts replication mutators. The polts11 cds1 double mutant was inviable, and the polts13 cds1 double mutant grew too poorly to be assessed at 21°C. In a cds1 background, the mutation rates of ts deletion replication mutators, polts1, cdc1-7, cdc27-K3, and cdc17-K42, increased 3.6- to 18.5-fold over that in a cds1⁺ background (Table 6). Thus, at the permissive temperature, Cds1 plays a role in preventing formation of mutations in these ts replication mutators. Similar to the effect of Cds1 on viability, deletion of cds1⁺ also did not have any effect on the mutation rate in the duplication mutator rad2 or ts nonmutator polts2 (Table 6).

All replication mutators require checkpoint Rad proteins for viability. Incomplete replication is monitored by the checkpoint Rad proteins either to activate Cds1 to ensure a reversible S-phase arrest (intra-S checkpoint) or to activate Chk1 and Cds1 to establish a G₂ arrest (S-to-M checkpoint) (4, 22, 40). Finding that deletion of cds1⁺ decreased the restrictive temperature (Table 5) and enhanced the mutation rate of the ts replication mutators (Table 6) led us to test whether deletion of a Rad protein, Rad26, would similarly affect these replication mutators. Six checkpoint Rad proteins, Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1, form a "guardian" complex that establishes the DNA damage and replication checkpoints, and loss of any single component results in a nonfunctional complex (1). As expected, all the ts replication mutators were either inviable or grew poorly in a rad26 background at the semipermissive temperature (Table 5). Similar to cds1⁺, deletion of rad26⁺ also exacerbated the mutator phenotype of two ts replication deletion mutators tested. The mutation rates of cdc27-K3 and cdc17-K42 increased ca. fourfold in a rad26 background at the permissive temperature (25°C) over those of the single mutants (data not shown). This suggests that aberrant replication in these two ts replication mutants is monitored through the checkpoint Rad proteins to activate Cds1 to prevent cell death and mutation formation at the semipermissive and permissive temperatures, respectively. However, the requirement for Rad26 is not restricted to the ts replication mutators. Two nonmutator Pol mutants, polts2 and cdc6-23, were also inviable in a rad26 background at their semipermissive temperatures (Table 5). This indicates that many types of replication defects are monitored through the checkpoint Rad proteins.

	DISCUSSION

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In this study, we have identified mutant alleles of S. pombe replication proteins that confer a mutator phenotype. Characterization of these mutators reveal the following interesting and novel findings. (i) ts alleles of pol, pol, two pol subunit genes, cdc1 and cdc27, and a DNA ligase gene cause aberrant replication that induces a mutator phenotype characterized by deletions of sequences flanked by short direct repeats. (ii) Generation of deletion mutations in these ts replication mutators requires a functional Rad2. (iii) Deletion of rad2⁺ results in an increased rate of duplication of sequences flanked by short direct repeats that requires normal DNA polymerization, but not DNA ligation. (iv) The checkpoint Rad proteins are required to prevent cell death from aberrant replication generated in the deletion and duplication mutators. (v) Cds1 is required to maintain cell viability and prevent deletion mutation formation specifically in the ts replication mutators, but not in the rad2 duplication mutator.

ssDNA gap initiates formation of deletion mutations in a Rad2-dependent manner. Studies of S. cerevisiae pol3-t have suggested that the observed deletion of sequences flanked by short direct repeats depends on their orientation relative to the origin of replication and are specific to either the leading or lagging strand, but not both (38). Simian virus 40 reconstituted replication studies have shown that Pol, ligase, and FEN-1 (mammalian homolog of Rad2) specifically function in lagging strand DNA synthesis, while Pol functions in both leading and lagging strand synthesis (39). Our finding that mutant alleles of pol, pol, two pol subunit genes, a ligase gene and deletion of rad2 destabilize sequences flanked by short direct repeats indicates that the observed sequence alterations in these replication mutators occur predominantly during lagging strand synthesis. These mutant alleles could cause a decreased rate of lagging strand synthesis or a stalled replication fork, yielding a ssDNA gap on the template similar to the proposed mutational mechanism in S. cerevisiae pol3-t (12, 38) (Fig. 2A). The absence of ligase function results in a nick that may be resected into a ssDNA gap (Fig. 2A). Deletions are proposed to arise from DNA polymerase slippage over a ssDNA loop formed by slip mispairing of short direct repeats on the template strand (12, 38).

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Proposed mechanisms for formation of deletion and duplication mutations and their interdependency in replication mutators. Two adjoining Okazaki fragments are shown, with thick arrows representing short direct repeats. (A) A ssDNA gap generated by a slow or stalled replication fork in ts DNA polymerases or by a resected nick in ts ligase mutant yields a potentially mutagenic structure that can form deletions. The ssDNA gap may initiate deletion mutation formation via a dsb or upon replication slippage over a ssDNA loop formed by slip mispairing of short direct repeats on the template strand. Following are two proposed roles (21) for Rad2 in processing a deletion mutation intermediate: (i) Rad2 may play a role in homology-dependent DNA end joining in dsb repair. After DNA ends are aligned by terminal microhomology, DNA on each strand beyond the short stretches of homology would be 3' and 5' flaps that would have to be cleaved off. Rad2 is the only known 5' flap endonuclease and the only known 5'3'-specific double-stranded DNA exonuclease activity in higher eukaryotes and thus, may be involved in resolving these structures (reviewed in reference 21). (ii) Deletion formation may involve a ssDNA loop as an intermediate and is shown in panel A. Initial nicking of the ssDNA loop by an unknown enzyme would result in 3' and 5' flaps that would have to be cleaved off. Rad2 may be required to remove the 5' flaps to generate deletion mutations. (B) Duplication mutations are initiated when strand displacement synthesis of Okazaki fragments occur without Rad2-dependent removal of the resulting 5' flap. The 5' flap yields duplication mutations via a dsb or upon replication slippage over a ssDNA loop formed by slip mispairing of short direct repeats on the nascent strand as previously proposed (37) and described in the Discussion. (C) In a ts ligase mutant, displacement of the downstream Okazaki fragment occurs to generate a potentially mutagenic 5' flap. The absence of Rad2 to process the displaced 5' flap structure initiates duplication mutations as described above for panel B. (D) In a double mutant of ts polymerase and rad2, a semidisabled polymerase has a reduced potential to displace a 5' flap in the downstream Okazaki fragment. Decreased levels of displaced 5' flap structures result in a reduced duplication mutation rate as described in the Discussion.

Duplication mutations in rad2 are dependent upon normal DNA polymerization, but not DNA ligation. The duplication mutations observed in S. cerevisiae rad27 mutants are proposed to be initiated by strand displacement of a 5' flap on a downstream Okazaki fragment by synthesis of the upstream Okazaki fragment. The similarity of the duplication mutator phenotypes of S. pombe rad2 and S. cerevisiae rad27 mutants suggests that the source of the duplication mutations observed in rad2 is likely to also be initiated by a 5' flap (Fig. 2B).

Cds1 responds specifically to aberrant replication induced in the ts replication mutators. Our finding that in addition to DNA polymerases, mutations in DNA ligase could result in a mutator phenotype suggests that the observed deletion mutations are induced by aberrant replication rather than solely by polymerase slippage. The aberrant replication in rad2 cells that induces duplication mutations is distinct from that of the ts replication mutators. However, cells with either type of aberrant replication in a rad26 background are inviable (Table 5). This suggests that the checkpoint Rad proteins respond to both types of aberrant replication in the deletion and duplication mutators. The loss of viability in a rqh1 background indicates that the checkpoint Rad-mediated Rqh1-dependent pathway is required to prevent inappropriate recombination (35) and maintain viability from aberrant replication in the deletion and duplication mutators. However, Rqh1 does not seem to be involved in deletion mutation production because the absence of Rqh1 does not affect the mutation rate of the ts replication mutators (data not shown) and the requirement for Rqh1 is not specific to the replication mutators (data not shown).

View larger version (46K): [in this window] [in a new window]   FIG. 3.   How checkpoint Rad proteins and Cds1 respond to aberrant replication in the deletion and duplication mutators. ts replication proteins at the semipermissive temperature and deletion of Rad2 generate different types of aberrant replication structures that are monitored by checkpoint Rad proteins. Checkpoint Rad proteins activate Rqh1 to prevent inappropriate recombination and cell death from both types of aberrant replication structures. In response to prevalent levels of aberrant replication specifically in ts replication mutators at the semipermissive temperature, checkpoint Rad proteins activate the Cds1-dependent recovery subpathway to prevent cell death and formation of deletion mutations. Some aberrant replication structures escape the Cds1-dependent recovery mechanism and thus result in deletion mutations. In the presence of lower levels of aberrant replication at the permissive temperature, Cds1 prevents formation of deletion mutations in the ts replication mutators. Details of these checkpoint responses are described in the Discussion.