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The many known eukaryotic DNA polymerases are classified into four families; A, B, X, and Y. Among them, DNA polymerase η, a Y family polymerase, is a low fidelity enzyme that contributes to translesional synthesis and somatic hypermutation. Although a high mutation frequency is observed in immunoglobulin genes, translesional synthesis occurs with a high accuracy. We determined whether the misincorporation rate of DNA polymerase η varies with ambient conditions. It has been reported that DNA polymerase η is unable to exclude water molecules from the active site. This finding suggests that some ions affect hydrogen bond formation at the active site. We focused on the effect of pH and evaluated the misincorporation rate of deoxyguanosine triphosphate (dGTP) opposite template T by DNA polymerase η at various pH levels with a synthetic template-primer. The misincorporation rate of dGTP by DNA polymerase η drastically increased at pH 8.0-9.0 compared with that at pH 6.5-7.5. Kinetic analysis revealed that the Km value for dGTP on the misincorporation opposite template T was markedly affected by pH. However, this drastic change was not seen with the low fidelity DNA polymerase α.

Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

DNA polymerase is one of the most important target molecules of antitumor agents, especially for antimetabolite nucleosides that include 1-beta-D-arabinofuranosylcytosine (araC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine). There are several subtypes of mammalian DNA polymerases and their localization and function have been clarified. DNA polymerase alpha, delta and epsilon have been implicated to be responsible for DNA replication, whereas DNA polymerase beta, delta and epsilon have been suggested to work in DNA repair. DNA polymerase gamma is encoded in the nucleus but localizes in the mitochondria, and is responsible for the mitochondrial DNA replication. Recently, several antiviral nucleoside analogs were reported to inhibit DNA polymerase gamma after intracellular phosphorylation and cause severe chronic toxicity. 1-(2-Deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine (4'-thio-FAC), an antimetabolite similar to araC and gemcitabine, is recently shown by us to be a very promising agent because of its potent antitumor activity by oral administration to mice. We tested for the inhibitory activities of the triphosphates of 4'-thio-FAC and gemcitabine against DNA polymerase alpha, beta and gamma. The triphosphates of 4'-thio-FAC (4'-thio-FACTP) exhibited the potent inhibitory action against DNA polymerase alpha, whereas it showed moderate inhibition against DNA polymerase beta and little inhibition against DNA polymerase gamma. The triphosphate of gemcitabine (dFdCTP) did not show potent inhibition against these three DNA polymerases. Thus, the effect on ribonucleotide reductase was suggested to be more responsible for the antitumor action of gemcitabine. The differences in the mechanisms of action against DNA polymerases between these drugs and other nucleosides were also discussed.

To elucidate the response to oxidative stress in eukaryotic cells, the effect of an oxidized nucleotide, 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), generated from dGTP with an active oxygen, on DNA synthesis was studied using a cell-free DNA replication system derived from Xenopus egg lysates with a single-stranded DNA template. Amounts of newly synthesized DNA were reduced according to the increasing concentration of 8-oxo-dGTP. Pulse labeling analysis revealed that 8-oxo-dGTP could delay DNA synthesis by reducing the rate of chain elongation. This delay was recovered by addition of a protein kinase inhibitor, staurosporine or bisindolylmaleimide I. These results indicate that a staurosporine- or bisindolylmaleimide I-sensitive protein kinase, such as a protein kinase C family member, may contribute to the delay of DNA synthesis by 8-oxo-dGTP. UV-irradiated single-stranded DNA also caused a delay of DNA synthesis on the undamaged template in the lysates. However, this delay was not recovered by staurosporine or bisindolylmaleimide I. Therefore, the mechanism of delay of DNA synthesis by 8-oxo-dGTP may be different from that by UV lesions. This is the first report that demonstrates an effect of an oxidized nucleotide on DNA replication in eukaryotes.

Sphingosine is a potent inhibitor of a mammalian DNA primase in vitro (Simbulan et al., Biochemistry 33, 9007-9012, 1994). Here we measured the inhibition of DNA primase in vitro by 9 sphingosine-analogues with respect to RNA primer synthesis and DNA primase-dependent DNA synthesis, and their potencies of inhibition in vitro were compared with their in vivo effects on human leukemic cells. Sphingosine, phytosphingosine and N, N-dimethylsphingosine strongly inhibited the activity of purified calf thymus DNA primase, and also inhibited the growth of human leukemic cell line HL-60, exerting strong cytotoxicity. Dihydrosphingosine and cis-sphingosine, which showed more subtle inhibition of DNA primase in vitro, moderately inhibited the cell growth in vivo and caused cell death. In contrast, N-acyl-, N-octyl-, and N-acetylsphingosine (ceramides) showing little inhibition of DNA primase suppressed cell growth only slightly. HL 60 cell was arrested at Go/G1 phase by exogenously added sphingosine. From these results, it is suggested that DNA primase is one of targets of sphingosine, an effector molecule in apoptosis.

In order to elucidate the biological activities of cordycepin (3'-deoxyadenosine) and related 3'-deoxyribonucleosides on eukaryotic DNA replication, the inhibitory effects of triphosphate derivatives of 3'-deoxyadenosine(3'-dATP), 8-azido-3'-deoxyadenosine(8-N3-3'-dATP), 3'-deoxyguanosine(3'-dGTP), 3'deoxyuridine(3'dUTP), 5-fluoro-3'deoxyuridine(5-F-3'-dUTP), 3'-deoxycytidine(3'-dcTP), and 5-fluoro-3'-deoxycytidine(5-F-3'dCTP) on DNA primase and replicative DNA polymerases alpha, delta, and epsilon purified from cherry salmon (Oncorhynchus masou) testes or calf thymus were examined. All analogs, except 8-N3-3'-dATP, showed strong inhibitory effects on DNA primase, but none of them inhibited DNA polymerases alpha, delta, or epsilon. Kinetic analysis revealed that the inhibition modes of them were competitive with respect to the incorporation of natural substrate that had the corresponding base moiety and non-competitive with respect to other substrates. Based on the kinetic data, we compared the affinities of 3'-dNTPs between DNA primase and RNA polymerases I and II, since 3'-dNTPs also inhibit eukaryotic RNA polymerases. Although the Ki values for DNA primase were much larger than those for RNA polymerases, the Ki/K(m) values, which indicate the affinity of the analog to the enzyme, were very similar.

Oxidative metabolism is known to generate mutagenic compounds within cells, among which is 8-oxodeoxyguanosine. Here the mutagenic potential of the triphosphate form of this base analog (8-O-dGTP) is investigated during replication in vitro of the lacZ alpha-complementation sequence in M13mp2 DNA. Adding 8-O-dGTP at equimolar concentration with the normal dNTPs to polymerization reactions decreases the fidelity of DNA synthesis by exonuclease-deficient Klenow, T4, and Thermus thermophilus DNA polymerases. Sequence analysis of mutants suggests that 8-O-dGMP is misincorporated opposite template adenines, yielding A-->C transversions. The degree of polymerase selectivity against this error is enzyme-dependent, with rates varying by > 25-fold. To determine if the A.8-O-dGMP mispair is proofread, a direct comparison of the fidelity of proofreading-proficient and proofreading-deficient Klenow and T4 DNA polymerases was made. Although the exonuclease activity of Klenow polymerase did not substantially reduce overall misincorporation of 8-O-dGMP, misincorporation was lower for the proofreading-proficient T4 enzyme as compared to its proofreading-deficient derivative. These data suggest that the A.8-O-dGMP mispair can be proofread. The mutagenic potential of 8-O-dGTP with eukaryotic systems was also examined. Misincorporation of 8-O-dGTP opposite adenine was observed during SV40 origin-dependent replication of double-stranded DNA in HeLa cell extracts. When present during replication at a concentration equal to the four normal dNTPs, 8-O-dGTP was at least 13-fold more mutagenic for A.T-->C.G transversions than was a 100-fold excess of normal dGTP.(ABSTRACT TRUNCATED AT 250 WORDS)

We have measured the fidelity of leading and lagging strand DNA replication in HeLa cell extracts. Providing an excess of one dNTP in reactions induces replication errors consistent with misincorporation of that dNTP. With excess dTTP, both substitutions and single-nucleotide frameshifts are induced. Error distribution is nonrandom; reproducible hot spots for a substitution and a frameshift error are observed. Measurements with two vectors having the origin of replication on opposite sides of the mutational target demonstrate that error rates for G.dTTP and C.dTTP mispairs depend on whether the strand is replicated as the leading or lagging strand. Also, the two hot spots are only observed in one origin-target orientation. Replication reactions reconstituted from two fractions derived from extracts are 3-fold less accurate, but the error specificity with excess dTTP is similar to that with extracts. This suggests that the processes responsible for the nonrandom error rates are not lost as a result of fractionation. Furthermore, the reconstituted system is devoid of mismatch repair activity. Thus, mismatch repair is not responsible for the mispair-, site-, and strand-specific differences observed.

A bovine genomic DNA library was surveyed with respect to the template activity for RNA-primed DNA synthesis by calf thymus DNA polymerase alpha-primase complex. About 7% of the single-stranded DNA clones contained distinct initiation sites consisting of pyrimidine clusters of pyrimidine-rich sequences. The initiation sites were located at or near the 3'-end of the pyrimidine clusters. One of these sequences, containing a 10-mer pyrimidine cluster with major initiation sites, was analyzed in detail. By the successive substitutions of pyrimidines in the cluster with oligodeoxydenylate [(dA)n] in the 5' to 3' direction, the minimum length of the initiation sequence was estimated to be as long as the 7-mer. In contrast, when one or three pyrimidines at the 3'-end of the cluster were replaced with (dA)n, the priming activity was largely lost, indicating that these pyrimidine residues were indispensable for priming. Furthermore, base substitutions of upstream or downstream sequences outside the pyrimidine cluster also decreased the total priming frequencies. Interestingly, the base substitutions inside or outside of the pyrimidine cluster sometimes caused a shift in the major priming sites. These results indicate that the minimum priming unit of the CTPPS1 template for RNA-primed DNA synthesis consists of a pyrimidine cluster (6-mer) with one purine at its 3'-border and that both the 3'-downstream 6-bases and the 5'-upstream 17-bases modulate the priming by enhancing the priming frequency and/or slightly shifting the sites of initiation of primer synthesis. It was also revealed that the lengths of the product RNA primers became shorter as the length of pyrimidine cluster was shortened by substitution with (dA)n. The gel retardation assay further showed that the complex formation between DNA polymerase alpha-primase and the DNA templates was strongly in competition with poly(dC), poly(dG), and poly(dT) but not with poly(dA). Furthermore, template activities as well as the pyrimidine contents of a series of base-substituted DNA correlated well with their affinities to the enzyme, as measured by both gel retardation assay and their Km values for the priming reaction. Apparently, DNA polymerase alpha-primase primarily recognizes the minimum priming unit consisting of a pyrimidine cluster with a purine at the 3'-boundary of purine, but the initiation of primer RNA synthesis can be modified by pyrimidine residues outside of the minimum priming unit.

We examined the mechanism of the inhibition of DNA synthesis by a new platinum compound, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato+ ++)-2-platinum(II) monohydrate (DWA-2114R), a derivative of the antitumor drug cis-diamminedichloroplatinum(II) (CDDP), using prokaryotic and eukaryotic DNA polymerases. Preincubating activated DNA with CDDP or DWA-2114R reduced its template activity for prokaryotic and eukaryotic DNA polymerases in a dose-dependent manner. DWA2114R required six times greater drug concentration and two times longer incubation time to show the same decrease of the template activity compared to CDDP. Treatment of primed pUC118 ssDNA templates with the two drugs followed by second-strand synthesis by prokaryotic and eukaryotic DNA polymerases revealed that DWA2114R bound to DNA in a similar manner to CDDP and these adducts blocked DNA elongation by DNA polymerases of eukaryotes as well as of prokaryotes. With these two drugs, the elongations by E. coli DNA polymerase I (Klenow fragment), T7 DNA polymerase and calf thymus DNA polymerase alpha were strongly arrested at guanine-guanine sequences (GG). Stop bands were also observed at adenine-guanine sequences (AG) guanine-adenine-guanine sequences (GAG) and mono-guanine sequence (G). Calf testis DNA polymerase beta was also arrested efficiently at AG, GAG and G, but much more weakly at GG. This pattern was common to DWA2114R and CDDP.

To search a novel factor possibly involved in the regulation of DNA replication for eukaryotic cells, we fractionated the Xenopus egg extract frequently used as a cell free DNA replication system, and obtained a single polypeptide of 50 KDa on SDS-polyacrylamide gel electrophoresis. Data of the amino acid sequence reduced from cloned gene of this protein revealed that this might be a Xenopus homolog of human PA2G4 that was known as a proliferation-associated protein or a cell cycle-regulated protein. Recombinant mouse PA2G4 protein also showed the inhibitory effect on the DNA replication reaction in Xenopus egg extract with the single-stranded DNA template. These results strongly suggest that PA2G4 may have an important role on the regulation of DNA replication in eukaryotic cells.

Replication protein A (RAP) is a eukaryotic single-stranded DNA binding protein involved in DNA replication, repair, and recombination. Recent studies indicate that RPA preferentially binds the damaged sites rather than the undamaged sites. Therefore, RPA is thought to be a member ofrepair factories or a sensor of lesion on DNA. To obtain further information of behavior of RPA against the oxidized lesion, we studied the binding affinity of RPA for the single-stranded DNA containing 5-formyluracil, a major lesion of thymine base yielded by the oxidation, using several synthetic oligonucleotides. The affinity of RPA for oligonucleotides was determined by gel shift assay. Results suggest that the surrounding sequence of 5-formyluracil may affect the affinity for RPA, and that the 5-formyluracil on the purine stretch but not the pyrimidine stretch increases the affinity for RPA. Results of affinity labeling experiment of RPA with the oligonucleotides containing 5-formyluracil indicate that RPA1 subunit may directly recognize and bind to the 5-formyluracil on the single-stranded DNA.

To obtain the information for the exact role of replication protein A (RPA) on both eukaryotic DNA replication and repair, the binding preference of RPA purified from Xenopus egg extract against the undamaged and UV-damaged single-stranded DNA was studied by the gel shift assay. Chemically synthesized oligonucleotide containing the pyrimidine(6-4)pyrimidone photoproduct at one site was used as a model of UV-damaged DNA. Results of competition assay and Scatchard plots indicate that RPA preferentially binds to the 6-4 photoproduct oligonucleotide than the undamaged DNA.

Productive infection and replication of herpesviruses usually occurs in growth-arrested cells, but there has been no direct evidence in the case of Epstein-Barr virus (EBV), since an efficient lytic replication system without external stimuli does not exist for the virus. Expression of the EBV lytic-switch transactivator BZLF1 protein in EBV-negative epithelial tumor cell lines, however, is known to arrest the cell cycle in G 0 /G 1 by induction of the tumor suppressor protein p53 and the cyclin-dependent kinase (CDK) inhibitors p21 WAF-1/CIP-1 and p27 KIP-1 , followed by the accumulation of a hypophosphorylated form of the Rb protein. In order to determine the effect of the onset of lytic viral replication on cellular events in latently EBV-infected B LCLs, a tightly controlled induction system of the EBV lytic-replication program by inducible BZLF1 protein expression was established in B95-8 cells. The induction of lytic replication completely arrested cell cycle progression and cellular DNA replication. Surprisingly, the levels of p53, p21 WAF-1/CIP-1 , and p27 KIP-1 were constant before and after induction of the lytic program, indicating that the cell cycle arrest induced by the lytic program is not mediated through p53 and the CDK inhibitors. Furthermore, although cellular DNA replication was blocked, elevation of cyclin E/A expression and accumulation of hyperphosphorylated forms of Rb protein were observed, a post-G 1 /S phase characteristic of cells. Thus, while the EBV lytic program promoted specific cell cycle-associated activities involved in the progression from G 1 to S phase, it inhibited cellular DNA synthesis. Such cellular conditions appear to especially favor viral lytic replication.

Human retinoblastoma (Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10-100-fold the activity of DNA polymerase alpha from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and under-phosphorylated forms. Dephosphorylation of Rb protein by protein phosphatase 2A largely diminished its stimulatory effect. On the other hand, a hyperphosphorylated Rb protein, obtained from insect cells overexpressing Rb protein, cyclin E and cyclin-dependent kinase 2 simultaneously, stimulated DNA polymerase alpha more strongly than the singly-expressed Rb protein. These results indicate that the phosphorylation is crucial for the stimulation. Rb protein isolated from human Burkitt lymphoma Raji cells also stimulated DNA polymerase alpha. In contrast, Rb protein did not affect eukaryotic DNA primase or Klenow fragment of Escherichia coli DNA polymerase I. By immunoprecipitation using anti-DNA polymerase alpha antibody, Rb protein in nuclear extract of Raji cells was co-precipitated with DNA polymerase alpha. This result indicates that DNA polymerase alpha exists as a complex containing phosphorylated Rb protein in cells. DNA polymerase alpha specifically bound to a purified Rb protein-immobilized Sepharose column. Rb protein also bound to DNA polymerase alpha trapped to anti-DNA polymerase alpha antibody-Sepharose column, suggesting the direct association of these two proteins. These observations suggest a new function of phosphorylated Rb protein in the regulation of DNA replication.

We have shown that calf thymus DNA polymerase alpha-DNA primase complex (pol alpha-primase) preferentially binds to pyrimidine-rich sequences and initiates RNA primer synthesis [Suzuki, M. et al. (1993) Biochemistry 32, 12782-12792]. Here we tested the association of pol alpha-primase with a guanine-rich DNA fragment (SVG, 30-mer) containing in vivo initiation sites of simian virus 40 DNA replication. While pyrimidine-rich fragment (CTPPS 1, 30-mer), that is a preferred sequence for calf thymus DNA primase, was well co-precipitated with pol alpha-primase using anti-pol alpha antibody, SVG was hardly precipitated under the same conditions. Competition studies in either gel-retardation assay or during de novo DNA synthesis by pol alpha-primase demonstrated that the interaction of pol alpha-primase with SVG was much weaker than that with CTPPS-1. On the other hand, replication protein-A (RP-A) could bind SVG, although less efficiently than CTPPS 1. After preincubation with RP-A, SVG could bind pol alpha-primase that was immobilized on Sepharose beads. The simian virus 40 large T antigen also enhanced association of SVG to pol alpha-primase, while Escherichia coli single-stranded DNA-binding protein did not. However, pol alpha-primase, bound to SVG in the presence of RP-A, failed to synthesize RNA primers. When SVG was extended 10 nucleotides at its 5'-end, pol alpha-primase synthesized trace amounts of RNA primers, and this activity was stimulated more than 10-fold by adding RP-A. These results suggest a new role for RP-A, i.e., as a molecular tether that allows pol alpha-primase to bind guanine-rich regions of DNA in order to initiate RNA primer synthesis.

We have determined the fidelity of DNA replication by human cell extracts in reactions containing excess dGTP. Replication errors were scored using two M13 DNA substrates having the replication origin on opposite sides of the lacZ alpha-complementation gene. The data suggest that the average rates for replication errors resulting from G(template), T.dGTP, and A.dGTP mispairs are 25 x 10(-6), 12 x 10(-6), and 3 x 10(-6), respectively. The data also suggest that error rates for both the (+) and (-) strands differ by less than 2-fold when they are replicated either as the leading or lagging strand. This is in contrast to the 33- and 8-fold differences observed earlier for G.dTTP and C.dTTP mispairs on the (+) strand when replicated by the leading or lagging strand complex (Roberts, J. D., Izuta, S., Thomas, D. C., and Kunkel, T. A. (1994) J. Biol. Chem. 269, 1711-1717). Thus, the relative fidelity of the leading and lagging strand replication proteins varies with the mispair and sequence considered. Misincorporation of dGTP preferentially occurs at template positions where dGTP is the next correct nucleotide to be incorporated. This "next nucleotide" effect is characteristic of reduced exonucleolytic proofreading and suggests that these replication errors are normally proofread efficiently. Fidelity measurements performed in the absence or presence of dGMP, an inhibitor of proofreading exonuclease activity, suggest that the leading strand replication complex proofreads some mispairs more efficiently than does the lagging strand replication complex.

Eukaryotic DNA polymerase alpha pauses at some sites on the natural DNA template of M13mp2. Terminal misincorporations of dA or dG, in place of dT, by DNA polymerase alpha have been reported to be within one of the pause sites, pause site II (positions 6269 and 6270 (Fry, M., and Loeb, L.A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 763-767)). The DNA products arrested within pause site II (position 6270) were separated, annealed with synthetic templates, and further elongated by DNA polymerase alpha. It was confirmed that a considerable amount of terminal misincorporation of dG in place of dT occurred at this position. When M13mp2 DNA was coated with various amounts of replication protein-A (RP-A), however, DNA polymerase alpha was able to overcome the pause site II, whereas pause bands at other sites barely decreased. In contrast, Escherichia coli single-stranded DNA-binding protein did not specifically abolish the arrested band at pause site II, though it generally suppressed the reaction. Since RP-A hardly increased the elongation frequency from the primer carrying a 3‘-mismatched terminal deoxynucleotide, the reduction of arrested products by RP-A may be attributed to the change in the incorporation mode from noncomplementary to complementary deoxynucleotides within pause site II and may not be due to the reinitiation from the mismatched 3‘-terminal deoxynucleotide. To confirm this, we amplified the reaction products at pause site III by means of a polymerase chain reaction method and showed that the complementary strand to pause site II, which was elongated in the presence of RP-A, did not carry any detectable misinsertion. Therefore, the errorprone step of the DNA synthesis catalyzed by DNA polymerase alpha may be readily avoided by RP-A.