A role for DNA polymerase delta in gene conversion and crossing over during meiosis in Saccharomyces cerevisiae

A screen for mutants of budding yeast defective in meiotic gene conversion identified a novel allele of the POL3 gene. POL3 encodes the catalytic subunit of DNA polymerase delta, an essential DNA polymerase involved in genomic DNA replication. The new allele, pol3-ct, specifies a protein missing the last four amino acids. pol3-ct shows little or no defect in DNA replication, but displays a reduction in the length of meiotic gene conversion tracts and a decrease in crossing over. We propose a model in which DNA synthesis determines the length of strand exchange intermediates and influences their resolution toward crossing over.     

Characterization of genetic interactions with RFA1: the role of RPA in DNA replication and telomere maintenance

Replication protein A (RPA) is a heterotrimeric single-stranded DNA binding protein whose role in DNA replication, recombination and repair has been mainly elucidated through in vitro biochemical studies utilizing the mammalian complex. However, the identification of homologs of all three subunits in Saccharomyces cerevisiae offers the opportunity of examining the in vivo role of RPA. In our laboratory, we have previously isolated a missense allele of the RFA1 gene, encoding the p70 subunit of the RPA complex. Strains containing this mutant allele, rfa1-D228Y, display increased levels of direct-repeat recombination, decreased levels of heteroallelic recombination, UV sensitivity and a S-phase delay. In this study, we have characterized further the role of RPA by screening other replication and repair mutants for a synthetic lethal phenotype in combination with the rfa1-D228Y allele. Among the replication mutants examined, only one displayed a synthetic lethal phenotype, pol12-100, a conditional allele of the B subunit of pol alpha-primase. In addition, a delayed senescence phenotype was observed in raf1-D228Y strains containing a null mutation of HDF1, the S. cerevisiae homolog of the 70 kDa subunit of Ku. Interestingly, a synergistic reduction in telomere length observed in the double mutants suggests that the shortening of telomeres may be the cause of the decreased viability in these strains. Furthermore, this result represents the first evidence of a role for RPA in telomere maintenance.     

Fission yeast with DNA polymerase delta temperature-sensitive alleles exhibits cell division cycle phenotype.

DNA polymerases alpha and delta are essential enzymes believed to play critical roles in initiation and replication of chromosome DNA. In this study, we show that the genes for Schizosaccharomyces pombe (S.pombe) DNA polymerase alpha and delta (pol alpha+ and pol delta+) are essential for cell viability. Disruption of either the pol alpha+ or pol delta+ gene results in distinct terminal phenotypes. The S.pombe pol delta+ gene is able to complement the thermosensitive cdc2-2 allele of Saccharomyces cerevisiae (S.cerevisiae) at the restrictive temperature. By random mutagenesis in vitro, we generated three pol delta conditional lethal alleles. We replaced the wild type chromosomal copy of pol delta+ gene with the mutagenized sequence and characterized the thermosensitive alleles in vivo. All three thermosensitive mutants exhibit a typical cell division cycle (cdc) terminal phenotype similar to that of the disrupted pol delta+ gene. Flow cytometric analysis showed that at the nonpermissive temperature all three mutants were arrested in S phase of the cell cycle. The three S.pombe conditional pol delta alleles were recovered and sequenced. The mutations causing the thermosensitive phenotype are missense mutations. The altered amino acid residues are uniquely conserved among the known polymerase delta sequences.     

Analysis of the stimulation of DNA polymerase V of Escherichia coli by processivity proteins

Bypass of replication-blocking lesions in Escherichia coli is carried out by DNA polymerase V (UmuC) in a reaction that requires UmuD', RecA, and single-strand DNA-binding protein (SSB). The activity of this four-component basic bypass system is a low-fidelity and low-processivity activity. Addition of the processivity subunits of pol III, the beta subunit sliding DNA clamp, and the five-subunit gamma complex clamp loader increased the rate of translesion replication approximately 3-fold. This stimulation was specific to the lesion bypass step, with no effect on the initiation of synthesis by pol V. The beta subunit and gamma complex increased the processivity of pol V from 3 to approximately 14-18 nucleotides, providing a mechanistic basis for their stimulatory effect. Stimulation of bypass was observed over a range of RecA and SSB concentrations. ATPgammaS, which strongly inhibits translesion replication by pol V, primarily via inhibition of the initiation stage, caused the same inhibition also in the presence of the processivity proteins. The in vivo role of the processivity proteins in translesion replication was examined by assaying UV mutagenesis. This was done in a strain carrying the dnaN59 allele, encoding a temperature-sensitive beta subunit. When assayed in an excision repair-defective background, the dnaN59 mutant exhibited a level of UV mutagenesis reduced up to 3-fold compared to that of the isogenic dnaN(+) strain. This suggests that like in the in vitro system, the beta subunit stimulates lesion bypass in vivo.     

Genetic mapping of the Saccharomyces cerevisiae DNA polymerase I gene and characterization of a pol1 temperature-sensitive mutant altered in DNA primase-polymerase complex stability

Summary The cloned DNA polymerase I gene has been used to map the POL1 locus on the left arm of chromosome XIV, between MET4 and TOP2. Temperature-sensitive mutants in POL1 have been obtained by in vitro mutagenesis of the cloned gene and in vivo replacement of the wild-type allele with the mutated copy. Physiological and biochemical characterization of one temperature-sensitive mutant (pol1-1) shows that cells shifted to the non-permissive temperature can complete one round of cell division and DNA replication before they arrest. Analysis of DNA polymerase I in crude extracts and in partially purified preparations indicates that the pol1-1 mutation results in a conformational change and affects the stability of the DNA primase-polymerase complex.     

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.     

In Vivo Analysis Reveals That the Interdomain Region of the Yeast Proliferating Cell Nuclear Antigen Is Important for DNA Replication and DNA Repair

To identify the regions of the proliferating cell nuclear antigen (PCNA) that are important for function in vivo, we used random mutagenesis to isolate 10 cold-sensitive (Cs(-)) and 31 methyl methanesulfonate-sensitive (Mms(s)) mutations of the PCNA gene (POL30) in Saccharomyces cerevisiae. Unlike the Mms(s) mutations, the Cs(-) mutations are strikingly clustered in the interdomain region of the three-dimensional PCNA monomer structure. At the restrictive temperature, the Cs(-) pol30 mutants undergo a RAD9-dependent arrest as large-budded cells with a 2c DNA content. Defects in DNA synthesis are suggested by a significant delay in the progression of synchronized pol30 cells through S phase at the restrictive temperature. DNA repair defects are revealed by the observation that Cs(-) pol30 mutants are very sensitive to the alkylating agent MMS and mildly sensitive to ultraviolet radiation, although they are not sensitive to gamma radiation. Finally, analysis of the chromosomal DNA in pol30 cells by velocity sedimentation gradients shows that pol30 cells accumulate single-stranded DNA breaks at the restrictive temperature. Thus, our results show that PCNA plays an essential role in both DNA replication and DNA repair in vivo.     

In vivo species specificity of DNA polymerase α

The DNA polymerase a enzymes from human, and budding (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are homologous proteins involved in initiation and replication of chromosomal DNA. Sequence comparision of human DNA polymerase α with that of S. cerevisiae and S. pombe shows overall levels of amino acid sequence identity of 32% and 34%, respectively. We report here that, despite the sequence conservation among these three enzymes, functionally active human DNA polymerase a fails to rescue several different conditional lethal alleles of the budding yeast POL1 gene at nonpermissive temperature. Furthermore, human DNA polymerase α cannot complement a null allele of budding yeast POL1 either in germinating spores or in vegetatively growing cells. In fission yeast, functionally active human DNA polymerase α is also unable to complement the disrupted polα::ura4 ⁺ allele in germinating spores. Thus, in vivo, DNA polymerase α has stringent species specificity for initiation and replication of chromosomal DNA.     

Proliferating cell nuclear antigen (pol30) mutations suppress cdc44 mutations and identify potential regions of interaction between the two encoded proteins.

In addition to its role as a processivity factor in DNA replication, proliferating cell nuclear antigen (PCNA) may function in the regulation of cell cycle progression. We present genetic evidence that PCNA interacts with the gene product of CDC44, an essential nucleotide-binding protein that encodes the large subunit of yeast replication factor C (K. Fien and B. Stillman, personal communication). Mutations in POL30 (PCNA) suppress cold-sensitive alleles of cdc44 that contain mutations in or near nucleotide-binding consensus domains, but they do not suppress a null allele. Thus, it appears that PCNA interacts with Cdc44p but cannot substitute for its function. pol30 mutations suppress additional phenotypes of cdc44 mutations, including the cold sensitivity that they were selected to suppress. This observation suggests an intimate association between PCNA and Cdc44p. Each of five independent pol30 mutants contains a unique single mutation that maps to a localized region on one face of the predicted three-dimensional structure of PCNA. This face identifies a region likely to be important for functional interaction between the CDC44 and POL30 gene products.     

Purification and characterization of the Schizosaccharomyces pombe origin recognition complex: interaction with origin DNA and Cdc18 protein

The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombe ORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro. Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins.     

Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis.

A series of adenovirus type 5 precursor terminal protein (pTP) and DNA polymerase (Ad pol) genes with linker insertion mutations were separately introduced into the vaccinia virus genome under the control of a late vaccinia virus promoter. The recombinant viruses were used for overexpression of the mutant genes in HeLa cells. In total, 22 different mutant pTP and 10 different Ad pol vaccinia virus recombinants were constructed, including some that expressed carboxyl-terminus-truncated forms of both proteins and one that produced the mutant H5ts149 Ad pol. To investigate the structure-function relationships of both proteins, extracts from cells infected with the recombinant viruses were tested for in vitro complementation of the initiation and elongation steps in adenovirus DNA replication. The results were in accordance with those of earlier in vivo experiments with these insertion mutants and indicate that multiple regions of both proteins are essential for adenovirus DNA replication. The carboxyl termini of both pTP and Ad pol were shown to be essential for proper functioning of these proteins during initiation of adenovirus DNA replication. Three different DNA replication-negative pTP mutants were shown to have residual activity in the initiation assay, suggesting not only that pTP is required for initiation but also that it may play a role in DNA replication after the deoxycytidylation step.     

Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.     

DNA polymerase alpha in the fission yeast Schizosaccharomyces pombe: identification and tracing of the catalytic subunit during the cell cycle.

A recombinant protein was obtained in Escherichia coli by subcloning part of the Schizosaccharomyces pombe POL1 gene at the 3'-end of lacZ. Antibodies raised against this protein were used to identify the POL1 gene product in extracts of exponentially growing S. pombe cells. A major 170-kDa protein, whose structure and properties were typical of the catalytic subunit of eukaryotic DNA polymerases alpha (pol alpha), was detected. The same antibodies were used to trace pol alpha and to quantify its level during the S. pombe cell cycle. We found that pol alpha was present at all stages of the cycle and that its cellular pool was subject to limited (three-fold) increase in G1 and S phases, with a decline to the initial level soon after. In addition, we found that a second form of pol alpha with slightly lower molecular weight (165 kDa) existed only during late G1 and S phases. Moreover, absence of initiation or perturbations in the course of DNA replication induced overproduction of the 165-kDa form.     

The Pol32 subunit of DNA polymerase delta contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding

We have carried out a domain analysis of POL32, the third subunit of Saccharomyces cerevisiae DNA polymerase delta (Pol delta). Interactions with POL31, the second subunit of Pol delta, are specified by the amino-terminal 92 amino acids, whereas interactions with the replication clamp proliferating cell nuclear antigen (PCNA, POL30) reside at the extreme carboxyl-terminal region. Pol32 binding, in vivo and in vitro, to the large subunit of DNA polymerase alpha, POL1, requires the carboxyl-proximal region of Pol32. The amino-terminal region of Pol32 is essential for damage-induced mutagenesis. However, the presence of its carboxyl-terminal PCNA-binding domain enhances the efficiency of mutagenesis, particularly at high loads of DNA damage. In vitro, in the absence of effector DNA, the PCNA-binding domain of Pol32 is essential for PCNA-Pol delta interactions. However, this domain has minimal importance for processive DNA synthesis by the ternary DNA-PCNA-Pol delta complex. Rather, processivity is determined by PCNA-binding domains located in the Pol3 and/or Pol31 subunits. Using diagnostic PCNA mutants, we show that during DNA synthesis the carboxyl-terminal domain of Pol32 interacts with the carboxyl-terminal region of PCNA, whereas interactions of the other subunit(s) of Pol delta localize largely to a hydrophobic pocket at the interdomain connector loop region of PCNA.     

The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication.

DNA polymerase alpha is the only enzyme in eukaryotic cells capable of starting DNA chains de novo and is required for the initiation of SV40 DNA replication in vitro. We have cloned the 70 kDa subunit of human DNA polymerase alpha (hereafter referred to as the B subunit) and expressed it as a fusion protein in bacteria. The purified fusion protein forms a stable complex with SV40 T antigen, both in solution and when T antigen is bound to the SV40 origin of DNA replication. Analysis of mutant forms of the B subunit indicates that the N-terminal 240 amino acids are sufficient to mediate complex formation. The B subunit fusion protein promotes formation of a complex containing T antigen and the catalytic subunit (subunit A) of DNA polymerase alpha, suggesting that it serves to tether the two proteins. These physical interactions are functionally significant, since the ability of T antigen to stimulate the activity of the catalytic subunit of DNA polymerase alpha is highly dependent upon the B subunit. We suggest that the interactions mediated by the B subunit play an important role in SV40 DNA replication by promoting DNA chain initiation at the origin and/or facilitating the subsequent priming and synthesis of DNA chains on the lagging strand template. The protein may play similar roles in cellular DNA replication.     

Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene.

The budding yeast Saccharomyces cerevisiae is proving to be an useful and accurate model for eukaryotic DNA replication. It contains both DNA polymerase alpha (I) and delta (III). Recently, proliferating cell nuclear antigen (PCNA), which in mammalian cells is an auxiliary subunit of DNA polymerase delta and is essential for in vitro leading strand SV40 DNA replication, was purified from yeast. We have now cloned the gene for yeast PCNA (POL30). The gene codes for an essential protein of 29 kDa, which shows 35% homology with human PCNA. Cell cycle expression studies, using synchronized cells, show that expression of both the PCNA (POL30) and the DNA polymerase delta (POL3, or CDC2) genes of yeast are regulated in an identical fashion to that of the DNA polymerase alpha (POL1) gene. Thus, steady state mRNA levels increase 10-100-fold in late G1 phase, peak in early S-phase, and decrease to low levels in late S-phase. In addition, in meiosis mRNA levels increase prior to initiation of premeiotic DNA synthesis.     

Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta

The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus. Specifically, we show that deletion of PIF1 suppresses the lethality of a DNA2-null mutant. The pif1delta dna2delta strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol delta, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32delta strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol delta and/or by an as yet unidentified helicase. Thus, suppression of dna2delta can be rationalized if deletion of POL32 and/or PIF1 results in a reduction in long flaps that require Dna2 for processing. We further show that deletion of DNA2 suppresses the long-telomere phenotype and the high rate of formation of gross chromosomal rearrangements in pif1Delta mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.     

Isolation and characterization of new proliferating cell nuclear antigen (POL30) mutator mutants that are defective in DNA mismatch repair

A number of studies have suggested a role for proliferating cell nuclear antigen (PCNA) in DNA mismatch repair (MMR). However, the majority of mutations in the POL30 gene encoding PCNA that cause MMR defects also cause replication and other repair defects that contribute to the increased mutation rate caused by these mutations. Here, 20 new pol30 mutants were identified and screened for MMR and other defects, resulting in the identification of two mutations, pol30-201 and pol30-204, that appear to cause MMR defects but little if any other defects. The pol30-204 mutation altered an amino acid (C81R) in the monomer-monomer interface region and resulted in a partial general MMR defect and a defect in MSH2-MSH6 binding in vitro. The pol30-201 mutation altered an amino acid (C22Y) located on the surface of the PCNA trimer that slides over the DNA but did not cause a defect in MSH2-MSH6 binding in vitro. The pol30-201 mutation caused an intermediate mutator phenotype. However, the pol30-201 mutation caused almost a complete defect in the repair of AC and GT mispairs and only a small defect in the repair of a "+T" insertion, an effect similar to that caused by an msh6Delta mutation, indicating that pol30-201 primarily effects MSH6-dependent MMR. The chromosomal double mutant msh3-FF>AA msh6-FF>AA eliminating the conserved FF residues of the PCNA interacting motif of these proteins caused a small (<10%) defect in MMR but showed synergistic interactions with mutations in POL30, indicating that the FF>AA substitution may not eliminate PCNA interactions in vivo. These results indicate that the interaction between PCNA and MMR proteins is more complex than was previously appreciated.