Differential inhibition of various mammalian DNA polymerase activities by ammonium 21-tungsto-9-antimoniate (HPA23)

The 21-tungsto-9-antimoniate ammonium salt (HPA23), known as an antiviral agent, has been shown to be a potent inhibitor of both human and murine DNA polymerase alpha and murine DNA polymerase gamma. HPA23 inhibited the activity of DNA polymerase alpha in noncompetitive fashion with respect to either deoxynucleotide substrate or nucleic acid template.primer. The Ki of murine DNA polymerase alpha for HPA23 was determined to be 24 nM. The activity of mouse DNA polymerase gamma also was strongly inhibited by HPA23 (Ki, 20 nM), and the mode of inhibition was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive to substrate, dTTP. DNA polymerase beta and terminal deoxynucleotidyltransferase, however, were relatively resistant to inhibition by HPA23. The observed inhibitions by HPA23 seem to be closely related to the polyanionic property of this drug.     

The carboxyl terminal sequence of nucleolar protein B23.1 is important in its DNA polymerase alpha-stimulatory activity

The protein B23 is a major nucleolar phosphoprotein comprising two isoforms, B23.1 and B23.2, which differ only in their carboxyl-terminal short sequences, the N-terminal 255 residues being identical in both forms. Both B23.1 and B23.2 stimulated immunoaffinity-purified calf thymus DNA polymerase alpha in a dose-dependent manner. The stimulatory effect of protein B23.1, the longer isoform, was found to be 2-fold greater than that of B23.2. Purified DNA polymerase alpha bound tightly to a protein B23.1-immobilized column, while it bound weakly to a protein B23.2-immobilized column. Surface plasmon resonance studies by BIAcore further showed that protein B23.1 bound to the DNA polymerase alpha-(dA).(dT) complex more tightly than did protein B23.2. The protein B23 isoforms appear to interact directly with the DNA polymerase alpha protein and not through the bound nucleic acid. These observations indicated that protein B23 physically bound to the DNA polymerase alpha and stimulated the enzyme activity. Product analyses showed that protein B23 greatly enhanced the reaction both in amount and length of product DNA, whereas it did not significantly alter the processivity of polymerization. In contrast, protein B23 effectively protected DNA polymerase alpha from heat inactivation. These results suggest that protein B23 stabilizes DNA polymerase alpha that is detached from product DNA, allowing the enzyme to be recruited for further elongation. Moreover, experiments using various C-terminal deletion mutants of protein B23 indicated that 12 amino acids at the C-terminal end of B23.1, which are absent in B23.2, may be essential for the full stimulation of the DNA polymerase alpha.     

Nucleolar protein B23.1 binds to retinoblastoma protein and synergistically stimulates DNA polymerase alpha activity

Phosphorylated retinoblastoma protein and nucleolar protein B23 are putative stimulatory factors for DNA polymerase alpha. We showed that these two factors interacted with each other and stimulated the activity of DNA polymerase alpha synergistically. B23 exists in two isoforms designated as B23.1 and B23.2. While B23.1 bound to a retinoblastoma protein-conjugated column, B23.2 did not. These results indicate that B23.1 can directly bind to retinoblastoma protein. It was also shown that B23 was co-immunoprecipitated with both retinoblastoma protein and DNA polymerase alpha from a HeLa cell extract by monoclonal antibodies raised against these components. These results suggest that these three proteins exist as a complex in cells, at least in part. The simultaneous addition of both B23.1 and retinoblastoma protein caused stimulation of DNA polymerase alpha activity that is much higher than the sum of the stimulation by retinoblastoma protein and B23.1 alone. The maximal stimulation was attained at the molar ratio of DNA polymerase alpha/retinoblastoma protein/B23.1 = 1:1:12. Since B23 exists as a hexamer in solution, it may act as a stimulator of DNA polymerase alpha in a form of double-hexamer, in concert with the phosphorylated retinoblastoma protein.     

Evidence of the existence of a high molecular weight form of DNA polymerase alpha in sea urchin eggs

DNA polymerase alpha combined with the endoplasmic reticulum (ER) was isolated from unfertilized sea urchin eggs. NaCl treatment of this fraction released DNA polymerase alpha from the ER. The molecular size (the S value) of the ER-free DNA polymerase alpha changed with the concentration of NaCl used; being 23 S, 11-15 S and 6-8 S in the presence of 0.05-0.12 M, 0.12-0.24 M and more than 0.24 M NaCl. DNA polymerase alpha activity decreased concomitantly with the reduction in molecular size. The 6-8 S form of DNA polymerase alpha did not aggregate by itself nor with other cellular components nonspecifically, when the 23 S form was present. These results are evidence of the presence of 6-8 S DNA polymerase alpha as a high molecular weight form (23 S-form) in sea urchin eggs.     

Differentiation of lens and neural cells in chicken embryos is accompanied by simultaneous decay of DNA replication machinery

DNA polymerase alpha was detected in cells of developing chicken embryos by an immunofluorescent method using a monoclonal antibody specific for the high molecular weight polypeptide of chicken DNA polymerase alpha, and DNA polymerase beta was detected using a rabbit anti-chicken DNA polymerase beta antibody. In lens tissue of the 3- to 4-day chicken embryo, fluorescence with anti-DNA polymerase alpha antibody was detected in nuclei of lens epithelial cells but not in nuclei of lens fiber cells which had differentiated from epithelial cells. The localization of cells containing DNA polymerase alpha coincided with the distribution of cells capable of DNA replication as detected by [3H]thymidine autoradiography. Similar results were obtained during the differentiation of neural matrix cells to neuroblasts in the developing neural tube. In contrast to DNA polymerase alpha, DNA polymerase beta was detected in nuclei of both undifferentiated and differentiated cells of these tissues. Since the disappearance of DNA polymerase alpha was very rapid after the onset of differentiation, the DNA replication machinery in which DNA polymerase alpha plays a central role is thought to decay almost simultaneously with the onset of cellular differentiation in these tissues.     

Characterization of a stable, major DNA polymerase alpha species devoid of DNA primase activity.

We have purified from Xenopus laevis ovaries a major DNA polymerase alpha species that lacked DNA primase activity. This primase-devoid DNA polymerase alpha species exhibited the same sensitivity as the DNA polymerase DNA primase alpha to BuAdATP and BuPdGTP, nucleotide analogs capable of distinguishing between DNA polymerase delta and DNA polymerase DNA primase alpha. The primase-devoid DNA polymerase alpha species also lacked significant nuclease activity indicative of the alpha-like (rather than delta-like) nature of the DNA polymerase. Using a poly(dT) template, the primase-devoid DNA polymerase alpha species elongated an oligo(rA10) primer up to 51-fold more effectively than an oligo(dA10) primer. In direct contrast, the DNA polymerase DNA primase alpha complex showed only a 4.6-fold preference for oligoribonucleotide primers at the same template/primer ratio. The catalytic differences between the two DNA polymerase alpha species were most dramatic at a template/primer ratio of 300. The primase-devoid DNA polymerase alpha species was found at high levels throughout oocyte and embryonic development. This suggests that the primase-devoid DNA polymerase alpha species could play a physiological role during DNA chain elongation in vivo, even if it is chemically related to DNA polymerase DNA primase alpha.     

Stimulation of DNA polymerase alpha by hypergravity generated by centrifugal acceleration.

Gravity alteration is known to influence cell proliferation. Here we tested the effects of hypergravity on the action of DNA polymerase alpha, one of the DNA replication enzymes in eukaryotes. Hypergravity was produced by horizontal centrifugal acceleration with a hand-made rotator. The reaction rate of DNA polymerase alpha in centrifuge tubes increased along with the acceleration up to 4g, when a plateau was reached. In contrast, no stimulation was observed with primase, DNA polymerase epsilon, and the E. coli DNA polymerase I Klenow fragment. Kinetic analysis of DNA polymerase alpha reactions revealed that, under high gravity conditions, the K(m) value for template DNA decreased while the V(max) stayed constant. In contrast, the centrifugal acceleration did not affect the K(m) values for deoxyribonucleoside triphosphates. These results suggest that the hypergravity enhances the activity of DNA polymerase alpha by increasing the affinity of the enzyme for template DNA. Such enhancement was more prominent with a low concentration of DNA polymerase alpha under low ionic conditions.     

Primer utilization by DNA polymerase alpha-primase is influenced by its interaction with Mcm10p

Models of DNA replication in yeast and Xenopus suggest that Mcm10p is required to generate the pre-initiation complex as well as progression of the replication fork during the elongation of DNA chains. In this report, we show that the Schizosaccharomyces pombe Mcm10p/Cdc23p binds to the S. pombe DNA polymerase (pol) alpha-primase complex in vitro by interacting specifically with the catalytic p180 subunit and stimulates DNA synthesis catalyzed by the pol alpha-primase complex with various primed DNA templates. We investigated the mechanism by which Mcm10p activates the polymerase activity of the pol alpha-primase complex by generating truncated derivatives of the full-length 593-amino acid Mcm10p. Their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA and to pol alpha were compared. Concomitant with increased deletion of the N-terminal region (from amino acids 95 to 415), Mcm10p derivatives lost their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA. Truncated derivatives of Mcm10p containing amino acids 1-416 retained the pol alpha binding activity, whereas the C terminus, amino acids 496-593, did not. These results demonstrate that both the single-stranded DNA binding and the pol alpha binding properties of Mcm10p play important roles in the activation. In accord with these findings, Mcm10p facilitated the binding of pol alpha-primase complex to primed DNA and formed a stable complex with pol alpha-primase on primed templates. A mutant that failed to activate or bind to DNA and pol alpha, was not observed in this complex. We suggest that the interaction of Mcm10p with the pol alpha-primase complex, its binding to single-stranded DNA, and its activation of the polymerase complex together contribute to its role in the elongation phase of DNA replication.     

A unique subpopulation of murine DNA polymerase alpha/primase specifically interacts with polyomavirus T antigen and stimulates DNA replication.

Murine cells or cell extracts support the replication of plasmids containing the replication origin (ori-DNA) of polyomavirus (Py) but not that of simian virus 40 (SV40), whereas human cells or cell extracts support the replication of SV40 ori-DNA but not that of Py ori-DNA. It was shown previously that fractions containing DNA polymerase alpha/primase from permissive cells allow viral ori-DNA replication to proceed in extracts of nonpermissive cells. To extend these observations, the binding of Py T antigen to both the permissive and nonpermissive DNA polymerase alpha/primase was examined. Py T antigen was retained by a murine DNA polymerase alpha/primase but not by a human DNA polymerase alpha/primase affinity column. Likewise, a Py T antigen affinity column retained DNA polymerase alpha/primase activity from murine cells but not from human cells. The murine fraction which bound to the Py T antigen column was able to stimulate Py ori-DNA replication in the nonpermissive extract. However, the DNA polymerase alpha/primase activity in this murine fraction constituted only a relatively small proportion (approximately 20 to 40%) of the total murine DNA polymerase alpha/primase that had been applied to the column. The DNA polymerase alpha/primase purified from the nonbound murine fraction, although far more replete in this activity, was incapable of supporting Py DNA replication. The two forms of murine DNA polymerase alpha/primase also differed in their interactions with Py T antigen. Our data thus demonstrate that there are two distinct populations of DNA polymerase alpha/primase in murine cells and that species-specific interactions between T antigen and DNA polymerases can be identified. They may also provide the basis for initiating a novel means of characterizing unique subpopulations of DNA polymerase alpha/primase.     

DNA polymerases alpha and delta are immunologically and structurally distinct

The relationship between DNA polymerases alpha and delta are evaluated immunologically by monoclonal antibody specifically against DNA polymerase alpha and murine polyclonal antiserum against calf thymus DNA polymerase delta. DNA polymerases alpha and delta are found to be immunologically distinct. The structural relationship between the proliferating cell nuclear antigen (PCNA)-dependent calf DNA polymerase delta and DNA polymerase alpha from human and calf was analyzed by two-dimensional tryptic peptide mapping of the catalytic polypeptides. The results demonstrate that the catalytic polypeptides of the PCNA-dependent calf polymerase delta and DNA polymerase alpha are distinct, unrelated, and do not share any common structural determinants. The immunological and structural relationship between a recently identified PCNA-independent form of DNA polymerase delta from HeLa cells was also assessed. This PCNA-independent human polymerase delta was found to be immunologically unrelated to human polymerase alpha but to share some immunological and structural determinants with the PCNA-dependent calf thymus polymerase delta.     

DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination.

DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the discontinuous DNA synthesis was attributable to DNA polymerase alpha. Additionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase alpha activity. To test the relative efficiency of each nuclear DNA polymerase for discontinuous synthesis, equal amounts (as measured by DNA polymerase activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and straightepsilon (+/- PCNA) were used in the discontinuous DNA synthesis assay. DNA polymerase alpha showed the most discontinuous DNA synthesis activity, although small but detectable levels were seen for DNA polymerases delta (+PCNA) and straightepsilon (- PCNA). Klenow fragment and DNA polymerase beta showed no discontinuous DNA synthesis, although at much higher amounts of each enzyme, discontinuous synthesis was seen for both. Discontinuous DNA synthesis by DNA polymerase alpha was seen with substrates containing 3 and 4 bp single-strand stretches of complementarity; however, little synthesis was seen with blunt substrates or with 1 bp stretches. The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryotic cells. These data suggest that DNA polymerase alpha may be able to rejoin double-strand breaks in vivo during replication.     

Studies of Schizosaccharomyces pombe DNA polymerase alpha at different stages of the cell cycle.

The status of Schizosaccharomyces pombe (fission yeast) DNA polymerase alpha was investigated at different stages of the cell cycle. S.pombe DNA polymerase alpha is a phosphoprotein, with serine being the exclusive phosphoamino acid. By in vivo pulse labeling experiments DNA polymerase alpha was found to be phosphorylated to a 3-fold higher level in late S phase cells compared with cells in the G2 and M phases, but the steady-state level of phosphorylation did not vary significantly during the cell cycle. Tryptic phosphopeptide mapping demonstrated that the phosphorylation sites of DNA polymerase alpha from late S phase cells were not the same as that from G2/M phase cells. DNA polymerase alpha partially purified from G1/S cells had a different mobility in native gels from that from G2/M phase cells. The partially purified polymerase alpha from G1/S phase cells had a higher affinity for single-stranded DNA than that from G2/M phase cells. Despite the apparent differences in cell cycle-dependent phosphorylation, mobility in native gels and affinity for DNA, the in vitro enzymatic activity of the partially purified DNA polymerase alpha did not appear to vary during the cell cycle. The possible biological significance of these cell cycle-dependent characteristics of DNA polymerase alpha is discussed.     

DNA polymerase alpha and beta in the California urchin.

DNA polymerase alpha and beta were identified in the urchin, Strongylocentrotus purpuratus. The DNA polymerase beta sedimented at 3.4 S, constituted 5% of total DNA polymerase activity, and was resistant to N-ethylmaleimide and high ionic strength. The polymerase alpha sedimented at 6--8 S, was inhibited by N-ethylmalemide or 0.1 M (NH4)2SO4, and was dependent upon glycerol for preservation of activity. Both the polymerases alpha and beta were nuclear associated in embryos. The DNA polymerase alpha was markedly heterogeneous on DEAE-Sephadex ion exchange and showed three modal polymerase species. These polymerase alpha species were indistinguishable by template activity assays but the DNA polymerase associated ribonucleotidyl transferase (Biochemistry 75 : 3106-3113, 1976) was found predominantly with only one of the DNA polymerase alpha species.     

Inhibition of eukaryotic dna polymerase alpha by persimmon (Diospyros kaki) extract and related polyphenols.

The effects of persimmon extract (Diospyros kaki) and related polyphenols on eukaryotic DNA polymerase alpha were examined. It was found that persimmon extract, epigallocatechin gallate, and epicatechin gallate strongly inhibited the activity of DNA polymerase alpha purified from calf thymus. Among these polyphenols, persimmon extract had the most potent effect on DNA polymerase alpha activity and the concentration of persimmon extract producing 50% inhibition of the activity was 0.191 microM. Persimmon extract showed a weaker effect on DNA polymerase beta and slightly inhibited primase and DNA polymerase I. The inhibition of DNA polymerase alpha by persimmon extract was competitive with the template-primer and noncompetitive with dTTP substrate. The Ki value of DNA polymerase alpha for persimmon extract was estimated to be 70 nM. Moreover, persimmon extract inhibited [3H]thymidine incorporation of human peripheral lymphocyte cells stimulated by PHA.     

Resolution and purification of free primase activity from the DNA primase-polymerase alpha complex of HeLa cells.

DNA primase activity has been resolved from a purified DNA primase-polymerase alpha complex of HeLa cells by hydrophobic affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase alpha. The free DNA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velocity sedimentation has an S20'W of 5. DNA primase synthesizes RNA oligomers with single-stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase alpha in a manner that has already been described for several purified eukaryotic DNA primase-polymerase alpha complexes. The purified free DNA primase activity is resistant to neutralizing anti-human DNA polymerase alpha antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase alpha and also DNA polymerase alpha complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase alpha. Taken together these results indicate that the DNA primase and polymerase alpha activities of the DNA primase-polymerase alpha complex reside on separate polypeptides that associate tightly through hydrophobic interactions.     

Murine DNA polymerase alpha fills gaps to completion in a direct assay. Altered kinetics of de novo DNA synthesis at single nucleotide gaps

DNA polymerase alpha was studied in a direct gap-filling assay. Using a defined template, DNA synthesis was primed from the M13 17-mer universal primer and blocked by an oligonucleotide hybridized 56 nucleotides downstream of the primer. DNA polymerase alpha filled this gap to completion. A time course of the reaction showed that in 50% of the substrate molecules, gaps were filled to completion within 10 min. In another 35% of the molecules the final nucleotide was lacking after 10 min. This nucleotide was added at a reduced rate, and was not incorporated into all of the molecules even after 6 h. The reduced rate of incorporation of the final nucleotide is reflected in an increased Km for de novo incorporation of one nucleotide at a single nucleotide gap (0.7 microM), as opposed to the Km for de novo incorporation of one nucleotide into singly primed M13 DNA (0.18 microM). DNA polymerase alpha purified from murine cells infected with the parvovirus minute virus of mice, and HeLa cell DNA polymerase alpha 2, exhibited the same kinetics of gap filling as did DNA polymerase alpha purified from uninfected Ehrlich ascites murine tumor cells. T4 DNA polymerase filled gaps to completion in this assay. Escherichia coli DNA polymerase I Klenow fragment quantitatively displaced the downstream oligonucleotide, and extended nascent DNA chains for an additional 100 nucleotides. Nicks and single-nucleotide gaps produced in gap-filling reactions by murine DNA polymerase alpha and T4 DNA polymerase were sealed by T4 DNA ligase.     

Purification and properties of an accessory protein for DNA polymerase alpha/primase

A protein that stimulates DNA polymerase alpha/primase many-fold on unprimed poly(dT) was purified to homogeneity from extracts of cultured mouse cells. The protein contains polypeptides of approximately 132 and 44 kDa, and the total molecular mass of 150 kDa calculated from Stokes radius (54 A) and sedimentation coefficient (6.7 S) indicates that it contains one each of the two subunits. The purified "alpha accessory factor" (AAF) also stimulates DNA polymerase alpha/primase in the self-primed reaction with unprimed single-stranded DNA. In addition to these effects on the coordinate activities of DNA polymerase alpha and DNA primase, stimulatory effects were also demonstrated separately on both the polymerase and primase activities of the enzyme complex. However, there was no stimulation with DNase-treated ("activated") DNA under normal conditions for assay of DNA polymerase alpha. The stimulatory activity of mouse AAF is highly specific for DNA polymerase alpha/primase; no effect was observed with mouse DNA polymerases beta, gamma, or delta, nor with retroviral, bacteriophage, or bacterial DNA polymerases. Mouse AAF stimulated human DNA polymerase alpha/primase with several different templates, similar to results with the mouse enzyme. However, it had very little effect on the DNA polymerase/primase from either Drosophila embryo or from yeast.     

Isolation and partial characterization of a high-molecular-weight DNA polymerase from Leishmania mexicana.

This paper describes for the first time the isolation and characterization of a high-molecular-weight predominant DNA polymerase from the genus Leishmania, which are parasitic flagellated protozoa. Like mammalian DNA polymerase alpha, the leishmanial DNA polymerase, designated DNA polymerase A, is of high-molecular-weight, is sensitive to N-ethylmaleimide and is inhibited by high ionic strength. Unlike mammalian DNA polymerase alpha, but similar to the predominant DNA polymerase isolated from the related lower eukaryotic organisms, Trypanosoma cruzi and Crithidia fasciculata, the leishmanial DNA polymerase A is resistant to inhibition by aphidicolin, a potent inhibitor of DNA replication in mammalian cells and of DNA polymerase alpha. The DNA polymerase A was purified 28,000-fold and properties such as pH optimum, salt sensitivity, template requirements and response to DNA polymerase inhibitors were determined. A low-molecular-weight DNA polymerase was detected during the isolation procedures, but was separated from the polymerase A activity. Differences in responses to specific antisera and specific mammalian DNA polymerase alpha inhibitors suggest that the leishmanial high-molecular-weight A enzyme is sufficiently different to suggest this enzyme as a chemotherapeutic target.     

Monoclonal antibody that blocks phosphoinositide-dependent activation of mouse tumor DNA polymerase alpha

A monoclonal antibody (MaB) against mouse sarcoma DNA polymerase alpha was isolated from the culture medium of an IgG-secreting hybridoma. The MaB demonstrated reactivity against two murine DNA polymerase alpha preparations and a calf thymus DNA polymerase alpha. Murine sarcoma polymerase was activated in vitro by phosphatidylinositol-4-monophosphate (PIP) showing increased deoxynucleotidyltransferase activity and enhanced binding affinity to activated DNA template. The MaB did not neutralize polymerase activity, but blocked further activation of the enzyme by PIP. Treatment of polymerase with MaB prior to treatment with PIP inhibited both increased enzyme activation and increased binding of the enzyme to DNA template. Treatment of polymerase with MaB subsequent to treatment with PIP did not block enzyme activation or increased DNA template binding. The data suggest that this anti-DNA polymerase alpha IgG is directed against a regulatory subunit of the polymerase rather than the catalytic subunit. The antibody may serve to distinguish between DNA polymerase alpha preparations with distinctly different regulatory subunits.