Structure and function of the fourth subunit (Dpb4p) of DNA polymerase epsilon in Saccharomyces cerevisiae

DNA polymerase (Pol) of Saccharomyces cerevisiae is purified as a complex of four polypeptides with molecular masses of >250, 80, 34 (and 31) and 29 kDa as determined by SDS–PAGE. The genes POL2, DPB2 and DPB3, encoding the catalytic Pol2p, the second (Dpb2p) and the third largest subunits (Dpb3p) of the complex, respectively, were previously cloned and characterised. This paper reports the partial amino acid sequence of the fourth subunit (Dpb4p) of Pol. This protein sequence matches parts of the predicted amino acid sequence from the YDR121w open reading frame on S.cerevisiae chromosome IV. Thus, YDR121w was renamed DPB4. A deletion mutant of DPB4 (Δdpb4) is not lethal, but chromosomal DNA replication is slightly disturbed in this mutant. A double mutant haploid strain carrying the Δdpb4 deletion and either pol2-11 or dpb11-1 is lethal at all temperatures tested. Furthermore, the restrictive temperature of double mutants carrying Δdpb4 and dpb2-1, rad53-1 or rad53-21 is lower than in the corresponding single mutants. These results strongly suggest that Dpb4p plays an important role in maintaining the complex structure of Pol in S.cerevisiae, even if it is not essential for cell growth. Structural homologues of DPB4 are present in other eukaryotic genomes, suggesting that the complex structure of S.cerevisiae Pol is conserved in eukaryotes.     

The small subunits of human and mouse DNA polymerase epsilon are homologous to the second largest subunit of the yeast Saccharomyces cerevisiae DNA polymerase epsilon.

Human DNA polymerase epsilon is composed of a 261 kDa catalytic polypeptide and a 55 kDa small subunit of unknown function. cDNAs encoding the small subunit of human and mouse DNA polymerase epsilon were cloned. The predicted polypeptides have molecular masses of 59.469 and 59.319 kDa respectively and they are 90% identical. The human and mouse polypeptides show 22% identity with the 80 kDa subunit of the five subunit DNA polymerase epsilon from the yeast Saccharomyces cerevisiae. The high degree of conservation suggests that the 55 kDa subunit shares an essential function with the yeast 80 kDa subunit, which was earlier suggested to be involved in S phase cell cycle control in a pathway that is able to sense and signal incomplete replication. The small subunits of human and mouse DNA polymerase epsilon also show homology to the C-terminal domain of the second largest subunit of DNA polymerase alpha. The gene for the small subunit of human DNA polymerase epsilon (POLE2) was localized to chromosome 14q21-q22 by fluorescence in situ hybridization.     

Cell cycle-dependent phosphorylation of the DNA polymerase epsilon subunit, Dpb2, by the Cdc28 cyclin-dependent protein kinase

DNA polymerase epsilon (Polepsilon), one of the three major eukaryotic replicative polymerases, is comprised of the essential catalytic subunit, called Pol2 in budding yeast, and three accessory subunits, only one of which, Dpb2, is essential. Polepsilon is recruited to replication origins during late G(1) phase prior to activation of replication. In this work we show that the budding yeast Dpb2 is phosphorylated in a cell cycle-dependent manner during late G(1) phase. Phosphorylation results in the appearance of a lower mobility species. The appearance of that species in vivo is dependent upon the Cdc28 cyclin-dependent protein kinase (CDK), which can directly phosphorylate Dpb2 in vitro. Either G(1) cyclin (Cln) or B-type cyclin (Clb)-associated CDK is sufficient for phosphorylation. Mapping of phosphorylation sites by mass spectrometry using a novel gel-based proteolysis protocol shows that, of the three consensus CDK phosphorylation sites, at least two, Ser-144 and Ser-616, are phosphorylated in vivo. The Cdc28 CDK phosphorylates only Ser-144 in vitro. Using site-directed mutagenesis, we show that Ser-144 is sufficient for the formation of the lower mobility form of Dpb2 in vivo. In contrast, Ser-616 appears not to be phosphorylated by Cdc28. Finally, inactivation of all three CDK consensus sites in Dpb2 results in a synthetic phenotype with the pol2-11 mutation, leading to decreased spore viability, slow growth, and increased thermosensitivity. We suggest that phosphorylation of Dpb2 during late G(1) phase at CDK consensus sites facilitates the interaction with Pol2 or the activity of Polepsilon     

The quaternary structure of DNA polymerase epsilon from Saccharomyces cerevisiae

DNA polymerase epsilon (Pol epsilon) from Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication. Biochemical characterizations of Pol epsilon have been cumbersome due to protease sensitivity and the limited amounts of Pol epsilon in cells. We have developed a protocol for overexpression and purification of Pol epsilon from S. cerevisiae. The native four-subunit complex was purified to homogeneity by conventional chromatography. Pol epsilon was characterized biochemically by sedimentation velocity experiments and gel filtration experiments. The stoichiometry of the four subunits was estimated to be 1:1:1:1 from colloidal Coomassie-stained gels. Based on the sedimentation coefficient (11.9 S) and the Stokes radius (74.5 A), a molecular mass for Pol epsilon of 371 kDa was calculated, in good agreement with the calculated molecular mass of 379 kDa for a heterotetramer. Furthermore, analytical equilibrium ultracentrifugation experiments support the proposed heterotetrameric structure of Pol epsilon. Thus, both DNA polymerase delta and Pol epsilon are purified as monomeric complexes, in agreement with accumulating evidence that Pol delta and Pol epsilon are located on opposite strands of the eukaryotic replication fork.     

Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron microscopy

The structure of the multisubunit yeast DNA polymerase epsilon (Pol epsilon) was determined to 20-A resolution using cryo-EM and single-particle image analysis. A globular domain comprising the catalytic Pol2 subunit is flexibly connected to an extended structure formed by subunits Dpb2, Dpb3 and Dpb4. Consistent with the reported involvement of the latter in interaction with nucleic acids, the Dpb portion of the structure directly faces a single cleft in the Pol2 subunit that seems wide enough to accommodate double-stranded DNA. Primer-extension experiments reveal that Pol epsilon processivity requires a minimum length of primer-template duplex that corresponds to the dimensions of the extended Dpb structure. Together, these observations suggest a mechanism for interaction of Pol epsilon with DNA that might explain how the structure of the enzyme contributes to its intrinsic processivity.     

Saccharomyces cerevisiae DNA polymerase epsilon and polymerase sigma interact physically and functionally,suggesting a role for polymerase epsilon in sister chromatid cohesion

The large subunit of Saccharomyces cerevisiae DNA polymerase epsilon, Pol2, comprises two essential functions. The N terminus has essential DNA polymerase activity. The C terminus is also essential, but its function is unknown. We report here that the C-terminal domain of Pol2 interacts with polymerase sigma (Pol sigma), a recently identified, essential nuclear nucleotidyl transferase encoded by two redundant genes, TRF4 and TRF5. This interaction is functional, since Pol sigma stimulates the polymerase activity of the Pol epsilon holoenzyme significantly. Since Trf4 is required for sister chromatid cohesion as well as for completion of S phase and repair, the interaction suggested that Pol epsilon, like Pol sigma, might form a link between the replication apparatus and sister chromatid cohesion and/or repair machinery. We present evidence that pol2 mutants are defective in sister chromatid cohesion. In addition, Pol2 interacts with SMC1, a subunit of the cohesin complex, and with ECO1/CTF7, required for establishing sister chromatid cohesion; and pol2 mutations act synergistically with smc1 and scc1. We also show that trf5 Delta mutants, like trf4 Delta mutants, are defective in DNA repair and sister chromatid cohesion.     

Fidelity of DNA polymerase epsilon holoenzyme from budding yeast Saccharomyces cerevisiae

DNA polymerases delta and epsilon (pol delta and epsilon) are the major replicative polymerases and possess 3'-5' proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of the holoenzyme of wild-type pol epsilon, the 3'-5' exonuclease-deficient pol2-4, a +1 frameshift mutator for homonucleotide runs, pol2C1089Y, and pol2C1089Y pol2-4 enzymes using a synthetic 30-mer primer/100-mer template. The nucleotide substitution rate for wild-type pol epsilon was 0.47 x 10(-5) for G:G mismatches, 0.15 x 10(-5) for T:G mismatches, and less than 0.01 x 10(-5) for A:G mismatches. The accuracy for A opposite G was not altered in the exonuclease-deficient pol2-4 pol epsilon; however, G:G and T:G misincorporation rates increased 40- and 73-fold, respectively. The pol2C1089Y pol epsilon mutant also exhibited increased G:G and T:G misincorporation rates, 22- and 10-fold, respectively, whereas A:G misincorporation did not differ from that of wild type. Since the fidelity of the double mutant pol2-4 pol2C1089Y was not greatly decreased, these results suggest that the proofreading 3'-5' exonuclease activity of pol2C1089Y pol epsilon is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.     

The noncatalytic C-terminus of AtPOLK Y-family DNA polymerase affects synthesis fidelity, mismatch extension and translesion replication

Cell survival depends not only on the ability to repair damaged DNA but also on the capability to perform DNA replication on unrepaired or imperfect templates. Crucial to this process are specialized DNA polymerases belonging to the Y family. These enzymes share a similar catalytic fold in their N-terminal region, and most of them have a less-well-conserved C-terminus which is not required for catalytic activity. Although this region is essential for appropriate localization and recruitment in vivo, its precise role during DNA synthesis remains unclear. Here we have compared the catalytic properties of AtPOLK, an Arabidopsis orthologue of mammalian pol kappa, and a truncated version lacking 193 amino acids from its C-terminus. We found that C-terminally truncated AtPOLK is a high-efficiency mutant protein, the DNA-binding capacity of which is not affected but it has higher catalytic efficiency and fidelity than the full-length enzyme. The truncated protein shows increased propensity to extend mispaired primer termini through misalignment and enhanced error-free bypass activity on DNA templates containing 7,8-dihydro-8-oxoGuanine. These results suggest that, in addition to facilitating recruitment to the replication fork, the C-terminus of Y-family DNA polymerases may also play a role in the kinetic control of their enzymatic activity.     

Subunit interactions within the Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon ) complex. Demonstration of a dimeric pol epsilon

Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon) is essential for chromosomal replication. A major form of pol epsilon purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay. The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits. We propose that Pol2p.Dpb2p and Dpb3p.Dpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes. We present biochemical evidence to support the proposal that pol epsilon may be dimeric in vivo. Gel filtration of the Pol2p.Dpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2p.Dpb2p. Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p. This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication.     

Double-stranded DNA binding, an unusual property of DNA polymerase epsilon, promotes epigenetic silencing in Saccharomyces cerevisiae

We have previously shown that DNA polymerase epsilon (Pol epsilon)of Saccharomyces cerevisiae binds stably to double-stranded DNA (dsDNA), a property not generally associated with DNA polymerases. Here, by reconstituting Pol epsilon activity from Pol2p-Dpb2p and Dpb3p-Dpb4p, its two component subassemblies, we report that Dpb3p-Dpb4p, a heterodimer of histone-fold motif-containing subunits, is responsible for the dsDNA binding. Substitution of specific lysine residues in Dpb3p, highlighted by homology modeling of Dpb3p-Dpb4p based on the structure of the histone H2A-H2B dimer, indicated that they play roles in binding of dsDNA by Dpb3p-Dpb4p, in a manner similar to the histone-DNA interaction. The lysine-substituted dpb3 mutants also displayed reduced telomeric silencing, whose degree paralleled that of the dsDNA-binding activity of Pol epsilon in the corresponding dpb3 mutants. Furthermore, additional amino acid substitutions to lysines in Dpb4p, to compensate for the loss of positive charges in the Dpb3p mutants, resulted in simultaneous restoration of dsDNA-binding activity by Pol epsilon and telomeric silencing. We conclude that the dsDNA-binding property of Pol epsilon is required for epigenetic silencing at telomeres.     

Purification of DNA polymerase II, a distinct DNA polymerase, from Saccharomyces cerevisiae

Yeast DNA polymerases I and III have been well characterized physically, biochemically, genetically and immunologically. DNA polymerase II is present in very small amounts, and only partially purified preparations have been available for characterization, making comparison with DNA polymerases I and III difficult. Recently, we have shown that DNA polymerases II and III are genetically distinct (Sitney et al., 1989). In this work, we show that polymerase II is also genetically distinct from polymerase I, since polymerase II can be purified in equal amounts from wild-type and mutant strains completely lacking DNA polymerase I activity. Thus, yeast contains three major nuclear DNA polymerases. The core catalytic subunit of DNA polymerase II was purified to near homogeneity using a reconstitution assay. Two factors that stimulate the core polymerase were identified and used to monitor activity during purification and analysis. The predominant species of the most highly purified preparation of polymerase II is 132,000 Da. However, polymerase activity gels suggest that the 132,000-Da form of DNA polymerase II is probably an active proteolytic fragment derived from a 170,000-Da protein. The highly purified polymerase fractions contain a 3'----5'-exonuclease activity that purifies at a constant ratio with polymerase during the final two purification steps. However, DNA polymerase II does not copurify with a DNA primase activity.     

DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.     

Sld2, Which Interacts with Dpb11 in Saccharomyces cerevisiae, Is Required for Chromosomal DNA Replication

The DPB11 gene, which genetically interacts with DNA polymerase II (), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint in Saccharomyces cerevisiae. To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal with dpb11-1) mutations which fall into six complementation groups (sld1 to -6). In this study, we characterized SLD2, encoding an essential 52-kDa protein. High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1. Conversely, high-copy DPB11 suppressed the temperature-sensitive growth defect caused by sld2-6. The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay. This interaction was evident at 25°C but not at 34°C when Sld2-6 or Dpb11-1 replaced its wild-type protein. No interaction between Sld2-6 and Dpb11-1 could be detected even at 25°C. Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2. sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1 cells. Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift. These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication.     

Saccharomyces cerevisiae Est3p dimerizes in vitro and dimerization contributes to efficient telomere replication in vivo

In Saccharomyces cerevisiae at least five genes, EST1, EST2, EST3, TLC1 and CDC13, are required for telomerase activity in vivo. The telomerase catalytic subunit Est2p and telomerase RNA subunit Tlc1 constitute the telomerase core enzyme. Est1p and Est3p are the other subunits of telomerase holoenzyme. In order to dissect the function of Est3p in telomere replication, we over-expressed and purified recombinant wild-type and mutant Est3 proteins. The wild-type protein, as well as the K71A, E104A and T115A mutants were able to dimerize in vitro, while the Est3p-D49A, -K68A or -D166A mutant showed reduced ability to dimerize. Mutations in Est3p that decreased dimerization also appeared to cause telomere shortening in vivo. Double point mutation of Est3p-D49A-K68A and single point mutation of Est3p-K68A showed similar telomere shortening, suggesting that the K68 residue might be more important for telomerase activity. The ectopic co-expression of K71A or T115A mutant with wild-type Est3p using centromere plasmids caused telomere shortening, while co-expression of the D49A, K68A, D86A or F103A mutants with wild-type Est3p had no effect on telomere length regulation. These data suggested that dimerization is important for Est3p function in vivo.     

The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication.

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.     

DNA replication defect in Salmonella typhimurium mutants lacking the editing (epsilon) subunit of DNA polymerase III.

In Salmonella typhimurium, dnaQ null mutants (encoding the epsilon editing subunit of DNA polymerase III [Pol III]) exhibit a severe growth defect when the genetic background is otherwise wild type. Suppression of the growth defect requires both a mutation affecting the alpha (polymerase) subunit of DNA polymerase III and adequate levels of DNA polymerase I. In the present paper, we report on studies that clarify the nature of the physiological defect imposed by the loss of epsilon and the mechanism of its suppression. Unsuppressed dnaQ mutants exhibited chronic SOS induction, indicating exposure of single-stranded DNA in vivo, most likely as gaps in double-stranded DNA. Suppression of the growth defect was associated with suppression of SOS induction. Thus, Pol I and the mutant Pol III combined to reduce the formation of single-stranded DNA or accelerate its maturation to double-stranded DNA. Studies with mutants in major DNA repair pathways supported the view that the defect in DNA metabolism in dnaQ mutants was at the level of DNA replication rather than of repair. The requirement for Pol I was satisfied by alleles of the gene for Pol I encoding polymerase activity or by rat DNA polymerase beta (which exhibits polymerase activity only). Consequently, normal growth is restored to dnaQ mutants when sufficient polymerase activity is provided and this compensatory polymerase activity can function independently of Pol III. The high level of Pol I polymerase activity may be required to satisfy the increased demand for residual DNA synthesis at regions of single-stranded DNA generated by epsilon-minus pol III. The emphasis on adequate polymerase activity in dnaQ mutants is also observed in the purified alpha subunit containing the suppressor mutation, which exhibits a modestly elevated intrinsic polymerase activity relative to that of wild-type alpha.     

DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair

High accuracy (fidelity) of DNA replication is important for cells to preserve the genetic identity and to prevent the accumulation of deleterious mutations. The error rate during DNA replication is as low as 10(-9) to 10(-11) errors per base pair. How this low level is achieved is an issue of major interest. This review is concerned with the mechanisms underlying the fidelity of the chromosomal replication in the model system Escherichia coli by DNA polymerase III holoenzyme, with further emphasis on participation of the other, accessory DNA polymerases, of which E.?coli contains four (Pols I, II, IV, and V). Detailed genetic analysis of mutation rates revealed that (1) Pol II has an important role as a back-up proofreader for Pol III, (2) Pols IV and V do not normally contribute significantly to replication fidelity, but can readily do so under conditions of elevated expression, (3) participation of Pols IV and V, in contrast to that of Pol II, is specific to the lagging strand, and (4) Pol I also makes a lagging-strand-specific fidelity contribution, limited, however, to the faithful filling of the Okazaki fragment gaps. The fidelity role of the Pol III τ subunit is also reviewed.     

A nuclear mutant of Saccharomyces cerevisiae deficient in mitochondrial DNA replication and polymerase activity

We have isolated a thermosensitive mutant which is transformed into a population of cells devoid of mitochondrial DNA (rho 0 cells) at 35 degrees C and is deficient in mitochondrial (mt) DNA polymerase activity. A single recessive nuclear mutation (mip1) is responsible for rho 0 phenotype and mtDNA polymerase deficiency in vitro. At 25 degrees C (or 30 degrees C) a dominant suppressor mutation (SUP) masks the deficiency in vivo. The meiotic segregants (mip1 sup) which do not harbor the suppressor have a rho 0 phenotype both at 25 and 35 degrees C. They have no mtDNA polymerase activity, in contrast with MIP rho 0 mutants of mitochondrial inheritance which do exhibit mtDNA polymerase activity. In the thermosensitive mutant (mip1 SUP), the replication of mtDNA observed in vivo at 30 degrees C is completely abolished at 35 degrees C. In the meiotic segregants (mip1 sup), no mtDNA replication takes place at 30 and 35 degrees C. The synthesis of nuclear DNA is not affected. DNA polymerases may have replicative and/or repair activity. There is no evidence that mip mutants are deficient in mtDNA repair. In contrast the MIP gene product is strictly required for the replication of mtDNA and for the expression of the mtDNA polymerase activity. This enzyme might be the replicase of mtDNA.