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PubMed1998

DNA polymerase II (epsilon) of Saccharomyces cerevisiae dissociates from the DNA template by sensing single-stranded DNA.

Two forms of DNA polymerase II (epsilon) of Saccharomyces cerevisiae, Pol II* and Pol II, were purified to near homogeneity from yeast cells. Pol II* is a four-subunit complex containing a 256-kDa catalytic polypeptide, whereas Pol II consists solely of a 145-kDa polypeptide derived from the N-terminal half of the 256-kDa polypeptide of Pol II*. We show that Pol II* and Pol II are indistinguishable with respect to the processivity and rate of DNA-chain elongation. The equilibrium dissociation constants of the complexes of Pol II* and Pol II with the DNA template showed that the stability of these complexes is almost the same. However, when the rates of dissociation of the Pol II* and Pol II from the DNA template were measured using single-stranded DNA as a trap for the dissociated polymerase, Pol II* dissociated 75-fold faster than Pol II. Furthermore, the rate of dissociation of Pol II* from the DNA template became faster as the concentration of the single-stranded DNA was increased. These results indicate that the rapid dissociation of Pol II* from the DNA template is actively promoted by single-stranded DNA. The dissociation of Pol II from the DNA template was also shown to be promoted by single-stranded DNA, although at a much slower rate. These results suggest that the site for sensing single-stranded DNA resides within the 145-kDa N-terminal portion of the catalytic subunit and that the efficiency for sensing single-stranded DNA by this site is positively modulated by either the C-terminal half of the catalytic subunit and/or the other subunits.

Amino Acid Sequence↗
PubMed1971

Analysis of DNA polymerases II and 3 in mutants of Escherichia coli thermosensitive for DNA synthesis.

A series of double mutants carrying one of the thermosensitive mutations for DNA synthesis (dnaA, B, C, D, E, F, and G) and the polA1 mutation of DeLucia and Cairns, were constructed. Enzyme activities of DNA Polymerases II and III were measured in each mutant. DNA Polymerase II activity was normal in all strains tested. DNA Polymerase III activity is thermosensitive specifically in those strains having thermoscnsitive mutations at the dnaE locus. From these results we conclude that DNA Polymerases II and III are independent enzymes and that DNA Polymerase III is an enzyme required for DNA replication in Escherichia coli.

Chromatography↗
PubMed1999

DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross-links in Escherichia coli.

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be approximately 0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in a polB+ ( approximately 0. 6) than in a DeltapolB (approximately 0.1) strain. Complementation with a polB+-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type < polB < recA < polB recA approximately uvrA. Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing, E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.

DNA Polymerase II↗
PubMed1990

Purification and characterization of DNA polymerase II from the yeast Saccharomyces cerevisiae. Identification of the catalytic core and a possible holoenzyme form of the enzyme.

We have purified yeast DNA polymerase II to near homogeneity as a 145-kDa polypeptide. During the course of this purification we have detected and purified a novel form of DNA polymerase II that we designate as DNA polymerase II. The most highly purified preparations of DNA polymerase II are composed of polypeptides with molecular masses of 200, 80, 34, 30, and 29 kDa. Immunological analysis and peptide mapping of DNA polymerase II and the 200-kDa subunit of DNA polymerase II indicate that the 145-kDa DNA polymerase II polypeptide is derived from the 200-kDa polypeptide of DNA polymerase II. Activity gel analysis shows that the 145- and the 200-kDa polypeptides have catalytic function. The polypeptides present in the DNA polymerase II preparation copurify with the polymerase activity with a constant relative stoichiometry during chromatography over five columns and co-sediment with the activity during glycerol gradient centrifugation, suggesting that this complex may be a holoenzyme form of DNA polymerase II. Both forms of DNA polymerase II possess a 3'-5' exonuclease activity that remains tightly associated with the polymerase activity during purification. DNA polymerase II is similar to the proliferating cell nuclear antigen (PCNA)-independent form of mammalian DNA polymerase delta in its resistance to butylpheny-dGTP, template specificity, stimulation of polymerase and exonuclease activity by KCl, and high processivity. Although calf thymus PCNA does not stimulate the activity of DNA polymerase II on poly(dA):oligo(dT), possibly due to the limited length of the template, the high processivity of yeast DNA polymerase II on this template can be further increased by the addition of PCNA, suggesting that conditions may exist for interactions between PCNA and yeast DNA polymerase II.

Base Composition↗
PubMed1995

Effects of gypenosides on mouse splenic lymphocyte transformation and DNA polymerase II activity in vitro.

AIM: To study the effects of gypenosides (Gyp) on lymphocyte transformation and DNA polymerase II activity. METHODS: Lymphocyte transformation response was induced by concanavalin A and lipopolysaccharides respectively. The activity of DNA polymerase II and DNA synthesis were assayed with TTP and [3H]TdR incorporation respectively in mixed lymphocyte culture test. RESULTS: Gyp 2.5-20 mg L-1 enhanced splenic T- and B- cell transformation, increased the DNA synthesis and potentiated the activity of DNA polymerase II. However, Gyp > 40 mg L-1 showed contrary effects. CONCLUSION: Gyp regulated lymphocyte transformation and DNA synthesis by regulating DNA polymerase II activity.

Animals↗
PubMed2001

Resolving a fidelity paradox: why Escherichia coli DNA polymerase II makes more base substitution errors in AT- compared with GC-rich DNA.

The activity of DNA polymerase-associated proofreading 3'-exonucleases is generally enhanced in less stable DNA regions leading to a reduction in base substitution error frequencies in AT- versus GC-rich sequences. Unexpectedly, however, the opposite result was found for Escherichia coli DNA polymerase II (pol II). Nucleotide misincorporation frequencies for pol II were found to be 3-5-fold higher in AT- compared with GC-rich DNA, both in the presence and absence of polymerase processivity subunits, beta dimer and gamma complex. In contrast, E. coli pol III holoenzyme, behaving "as expected," exhibited 3-5-fold lower misincorporation frequencies in AT-rich DNA. A reduction in fidelity in AT-rich regions occurred for pol II despite having an associated 3'-exonuclease proofreading activity that preferentially degrades AT-rich compared with GC-rich DNA primer-template in the absence of DNA synthesis. Concomitant with a reduction in fidelity, pol II polymerization efficiencies were 2-6-fold higher in AT-rich DNA, depending on sequence context. Pol II paradoxical fidelity behavior can be accounted for by the enzyme's preference for forward polymerization in AT-rich sequences. The more efficient polymerization suppresses proofreading thereby causing a significant increase in base substitution error rates in AT-rich regions.

Base Sequence↗
PubMed1990

Purification of DNA polymerase II stimulatory factor I, a yeast single-stranded DNA-binding protein.

Incidental to the purification of yeast DNA polymerase II was the observation that various chromatographic fractions contained activities that stimulated synthesis by this polymerase. In this paper we report the purification and initial characterization of one such factor, stimulatory factor I (SFI). SFI, which is associated with an apparent complex of three polypeptides of 66, 37, and 13.5 kDa, binds preferentially to single-stranded DNA, possibly explaining its ability to stimulate DNA polymerase II. Single-stranded DNA-binding activity is associated with the 66-kDa polypeptide.

Centrifugation, Density Gradient↗
PubMed1971

Purification and DNA synthesis in cell-free extracts: properties of DNA polymerase II.

The major DNA-synthesizing enzyme present in Pol A(1) (-)Escherichia coli (DNA polymerase II) has been purified to homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme requires all four deoxynucleoside triphosphates, Mg(++), NH(4) (+), and native DNA for maximal activity. The enzyme activity is sensitive to sulfhydryl reagents and is insensitive to anti-DNA polymerase I antiserum. A second DNA-synthesizing enzyme, present in low amounts, has been identified in Pol A(1) (-) extracts. The relationship of this enzyme to DNA polymerases I and II is discussed.

Cell-Free System↗
PubMed2004

Pyrophosphorolysis by Type II DNA polymerases: implications for pyrophosphorolysis-activated polymerization.

We find that Type II DNA polymerases can catalyze pyrophosphorolysis, the reverse reaction of DNA polymerization. This property is applied utilizing pyrophosphorolysis-activated polymerization (PAP), a method of nucleic acid amplification using serial coupling of pyrophosphorolysis and polymerization. PAP can be used for ultrarare allele detection (detection of minimal residual disease and cancer risk assessment through measurement of mutation load) and for microarray-based scanning for unknown mutations. Herein, we show that Type II DNA polymerases efficiently catalyze template-dependent pyrophosphorolysis to activate oligonucleotides blocked at their 3' termini with acyclonucleotides in which a 2-hydroxyethoxymethyl group substitutes for the 2'-deoxyribofuranosyl sugar. Type II archeon DNA polymerases Vent (exo-) and Pfu (exo-) can be utilized for PAP or a bidirectional form of PAP with acyclonucleotide-blocked oligonucleotides, but not with dideoxynucleotide-blocked oligonucleotides. In contrast, a Type I DNA polymerase, TaqFS, can utilize either acyclonucleotide-blocked or dideoxynucleotide-blocked oligonucleotides. These findings expand the potential of nascent PAP technology.

Alleles↗
PubMed1976

Escherichia coli DNA polymerases II and III: activation by magnesium or by manganous ions.

Escherichia coli DNA polymerases II and III have been extensively studied in vitro when activated with Mg2+. The Mn2+-activated polymerization reactions are considered here, and shown to differ from the Mg2+-activated reactions. The Mn2+-activated DNA polymerase II reaction requires K+ or spermidine, and the effects of monovalent cation and polyamine are additive. In contrast, the Mg2+-activated reaction does not require, but is stimulated by, K+ or spermidine, in a non-additive manner. Under optimal conditions, DNA polymerase II is activated better with Mn2+ than it is with Mg2+, suggesting a physiological role for the Mn2+-activated enzyme. The observed preference for Mn2+ over Mg2+ in reaction kinetics and at high DNA template concentrations suggest that Mg2+ may preferentially activate the associated exonuclease activity. At 29 degrees C, the Mn2+-activated DNA polymerase III reaction is stimulated by K+ and inhibited by ethanol or phosphatidylethanolamine. In contrast, the latter compounds and Triton X-100 increase the initial rate of the Mg2+-activated reaction, whereas K+ inhibits this reaction at all concentrations. The K+ inhibition is reduced at low Mg concentrations when Mn2+ is also present. After stimulating the initial reaction rate, ethanol causes a rapid decrease in the rate of the Mg2+-activated reaction during incubation at 20 degrees C. At 27 degrees C, all surface-active compounds inhibit the Mg2+-activated reaction. Preincubation of the enzyme at 30 degrees C or below with DNA template and divalent cation increases the initial reaction rate, suggesting that formation of an enzyme-divalent cation-DNA template complex occurs as the first step in DNA polymerase III catalysis. The apparent Km at 21 degrees C for gapped calf thymus DNA was 25 muM with Mn2+ and 125 muM with Mg2+ for DNA polymerase III, and 18 muM at 30 degrees C for DNA polymerase II with either Mn2+ or Mg2+. Reactions with poly[d(A-T)] were enhanced by Mn2+ relative to Mg2+, and activity with poly(rA)-poly(dT) was Mn2+ dependent for both enzymes.

DNA Polymerase II↗
PubMed1993

Absence of a role for DNA polymerase II in SOS-induced translesion bypass of phi X174.

In order to examine the possible role of Escherichia coli DNA polymerase II in SOS-induced translesion bypass, Weigle reactivation and mutation induction were measured with single-stranded phi X174 transfecting DNA containing individual lesions. No decrease in bypass of thymine glycol or cyclobutane pyrimidine dimers in the absence of DNA polymerase II was observed. Furthermore, DNA polymerase II did not affect bypass of abasic sites when either survival or mutagenesis was the endpoint. Lastly, repair of gapped DNA molecules, intermediates in methyl-directed mismatch repair, was also unaffected by the presence or absence of DNA polymerase II.

Bacteriophage phi X 174↗
PubMed1972

Isolation and partial characterization of a mutant of Escherichia coli deficient in DNA polymerase II.

A mutant of Escherichia coli deficient in DNA polymerase II has been isolated from E. coli polA1 by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and assay of polymerase activity in extracts of survivors. The polA1 mutation was suppressed during mutagenesis by introduction of the suppressor, su7(+), into the parental strain. The mutant, HMS83 polA1 polB1, contains less than 0.5% of the normal levels of DNA polymerase II. The only polymerase activity detected in the mutant is DNA polymerase III. E. coli HMS83 grows normally at both 25 and 42 degrees , and supports the growth of bacteriophages T4, T7, lambda, varphiX174, 186, P2, and fd. The polB mutation does not affect sensitivity to ultraviolet irradiation or recombination frequencies.

Cell Fractionation↗
PubMed1991

DPB2, the gene encoding DNA polymerase II subunit B, is required for chromosome replication in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DNA polymerase II holoenzyme consists of five polypeptides. The largest is the catalytic subunit, whose gene (POL2) has been cloned and sequenced. Herein we describe the cloning and sequencing of DPB2, the gene for the second largest subunit of DNA polymerase II, and the isolation of temperature-sensitive dpb2 mutations. The DNA sequence revealed an open reading frame encoding a protein of Mr 79,461 and lacking significant sequence similarity to any protein in data bases. Disruption of DPB2 was lethal for the cell and the temperature-sensitive dpb2-1 mutant was partially defective in DNA synthesis at the restrictive temperature, indicating that the DPB2 protein is required for normal yeast chromosomal replication. Furthermore, the DNA polymerase II complex was difficult to obtain from dpb2-1 mutant cells, suggesting that a stable DNA polymerase II complex requires DPB2 and is essential for chromosomal replication. The DPB2 transcript periodically fluctuated during the cell cycle and, like those of other genes encoding DNA replication proteins, peaked at the G1/S phase boundary.

Amino Acid Sequence↗
PubMed1994

Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation.

DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity (polB delta 1 mutants) exhibited 5- to 10-fold-greater sensitivity to mode 1 killing by H2O2 compared with isogenic polB+ cells. Survival decreased by about 15-fold when polB mutants containing defective superoxide dismutase genes, sodA and sodB, were compared with polB+ sodA sodB mutants. Resistance to peroxide killing was restored following P1 transduction of polB cells to polB+ or by conjugation of polB cells with an F' plasmid carrying a copy of polB+. The rate at which Lac+ mutations arose in Lac- cells subjected to selection for lactose utilization, a phenomenon known as adaptive mutation, was increased threefold in polB backgrounds and returned to wild-type rates when polB cells were transduced to polB+. Following multiple passages of polB cells or prolonged starvation, a progressive loss of sensitivity to killing by peroxide was observed, suggesting that second-site suppressor mutations may be occurring with relatively high frequencies. The presence of suppressor mutations may account for the apparent lack of a mutant phenotype in earlier studies. A well-established polB strain, a dinA Mu d(Apr lac) fusion (GW1010), exhibited wild-type (Pol II+) sensitivity to killing by peroxide, consistent with the accumulation of second-site suppressor mutations. A high titer anti-Pol II polyclonal antibody was used to screen for the presence of Pol II in other bacteria and in the yeast Saccharomyces cerevisiae. Cross-reacting material was found in all gram-negative strains tested but was not detected in gram-positive strains or in S. cerevisiae. Induction of Pol II by nalidixic acid was observed in E. coli K-12, B, and C, in Shigella flexneri, and in Salmonella typhimurium.

Adaptation, Physiological↗
PubMed2001

DNA polymerases II and V mediate respectively mutagenic (-2 frameshift) and error-free bypass of a single N-2-acetylaminofluorene adduct.

The NarI sequence represents a strong mutation hot spot for -2 frameshift mutations induced by N-2-acetylaminofluorene (AAF), a strong chemical carcinogen. Only when bound to the third (underlined) guanine (5'-GGCGCC-->GGCC) can AAF trigger frameshift mutations, suggesting the involvement of a slipped replication intermediate with a two-nucleotide bulge. While base substitutions induced by UV light or abasic sites require DNA polymerase V (Pol V; umuDC), the AAF-induced -2 frameshift pathway requires DNA polymerase II, the polB gene product. Interestingly, error-free bypass of the G-AAF adduct requires Pol V. The genes encoding both Pol II and Pol V are induced by the SOS regulon, a co-ordinated cellular response to environmental stress. A given lesion, G-AAF, can thus be bypassed by two SOS-controlled DNA polymerases (II and V), generating mutagenic (-2 frameshifts) and error-free replication products respectively. Therefore both Pol II and Pol V can compete for the blocked replication intermediate in the vicinity of the lesion and engage in replication by transiently replacing the replicative DNA Pol III. Our data suggest that, in order to cope with the large diversity of existing DNA lesions, cells use a single or a combination of translesional DNA polymerases to achieve translesion synthesis.

2-Acetylaminofluorene↗
PubMedDate not supplied

[Molecular mechanisms of stabilization and preparation of metabolic breaks in DNA strands in vivo. I. Tandem action of exonuclease V and DNA polymerase II].

Investigation of the formation of metabolic imbalance breaks in the DNA of thy- cells of E. coli during thymine starvation is described. The results of experiments indicate that two enzymes--exonuclease V and 3' exonuclease activity of DNA polymerase II take part in the formation of metabolic imbalance gaps. The manifestation of activity of the enzymes has a tandem character.

DNA Polymerase II↗