A tumor necrosis factor alpha- and interleukin 6-inducible protein that interacts with the small subunit of DNA polymerase delta and proliferating cell nuclear antigen.

A cDNA encoding a protein of 36 kDa, polymerase delta-interacting protein 1 (PDIP1), that interacts with the small subunit (p50) of DNA polymerase delta (pol delta) was identified in a two-hybrid screen of a HepG2 cDNA library by using p50 as bait. The interaction of PDIP1 with p50 was confirmed by pull-down assays, and a similar assay was used to demonstrate that PDIP1 interacts directly with the proliferating cell nuclear antigen (PCNA). PCNA and p50 bound to PDIP1 simultaneously, and PDIP1 stimulated pol delta activity in vitro in the presence, but not the absence, of PCNA, suggesting that PDIP1 also interacts functionally with both p50 and PCNA. Subcellular localization studies demonstrated that PDIP1 is a nuclear protein that colocalizes with PCNA at replication foci. A putative PCNA-binding motif was identified within the C terminus of PDIP1, and a synthetic peptide containing this PCNA-binding motif was shown to bind PCNA by far-Western analysis. Northern analysis demonstrated that PDIP1 mRNA is present in a wide variety of human tissues. PDIP1 was found to be highly homologous to a previously identified protein, B12 [Wolf, F. W., Marks, R. M., Sarma. V., Byers, M. G., Katz, R. W., Shows, T. B. & Dixit, V. M. (1992) J. Biol. Chem. 267, 1317-1326], one of the early response genes induced by tumor necrosis factor alpha. PDIP1 synthesis can also be induced by tumor necrosis factor alpha and by IL-6, cytokines essential for liver regeneration after loss of hepatic tissue. It is suggested that PDIP1 provides a link between cytokine activation and DNA replication in liver as well as in other tissues.

Differential removal of thymidine nucleotide analogues from blocked DNA chains by human immunodeficiency virus reverse transcriptase in the presence of physiological concentrations of 2'-deoxynucleoside triphosphates.

Removal of 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) from a blocked DNA chain can occur through transfer of the chain-terminating residue to a nucleotide acceptor by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). ATP-dependent removal of either d4TMP or 3'-azido-3'-deoxythymidine-5'-monophosphate (AZTMP) is increased in AZT resistant HIV-1 RT (containing D67N/K70R/T215F/K219Q mutations). Removal of d4TMP is strongly inhibited by the next complementary deoxynucleoside triphosphate (50% inhibitory concentration [IC(50)] of approximately 0.5 microM), whereas removal of AZTMP is much less sensitive to this inhibition (IC(50) of >100 microM). This could explain the lack of cross-resistance by AZT-resistant HIV-1 to d4T in phenotypic drug susceptibility assays.

A mechanism of AZT resistance: an increase in nucleotide-dependent primer unblocking by mutant HIV-1 reverse transcriptase.

Mutations in HIV-1 reverse transcriptase (RT) give rise to 3'-azido-3'-deoxythymidine (AZT) resistance by a mechanism that has not been previously reproduced in vitro. We show that mutant RT has increased ability to remove AZTMP from blocked primers through a nucleotide-dependent reaction, producing dinucleoside polyphosphate and extendible primer. In the presence of physiological concentrations of ATP, mutant RT extended 12% to 15% of primers past multiple AZTMP termination sites versus less than 0.5% for wild type. Although mutant RT also unblocked ddAMP-terminated primers more efficiently than wild-type RT, the removal of ddAMP was effectively inhibited by the next complementary dNTP (IC50 approximately equal to 12 microM). In contrast, the removal of AZTMP was not inhibited by dNTPs except at nonphysiological concentrations (IC50 > 200 microM).

Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism.

HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3' end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3'-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri- or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template (Ki approximately 5 microM), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3' nucleotide from the primer to the pyrophosphate moiety in the nucleoside di- or triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the in vivo activity of many nucleoside antiretroviral drugs.

Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase.

Nondenaturing gel electrophoresis was used to study the nucleotide substrate-induced conformational change in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Dead-end complex was formed between HIV-1 RT, dideoxynucleotide chain-terminated primer, and DNA template in the presence of deoxynucleotide triphosphate (dNTP) complementary to the next position on the template. Complexes which form in the absence of the next complementary dNTP were disrupted by adding excess poly(rA)/oligo(dT) or heparin just prior to electrophoresis. Dead-end complex formation by noncomplementary dNTP's or ribonucleotides was at least 2000-fold less efficient than with the complementary nucleotide. When dA was the next nucleotide on the template, analogues of dTTP supported dead-end complex formation with increased apparent Kd (dTTP < dideoxy-TTP approximately alpha-thio-dTTP < dUTP < 3'-azidothymidine triphosphate). A similar relationship was observed for dGTP analogues across from dC on the template (dGTP < dideoxy-GTP < alpha-thio-dGTP << dITP < dideoxy-ITP). The optimal length of the primer/template duplex region for dead-end complex formation was between 20 and 32 base pairs. Primer-template with a mismatched primer terminus did not support dead-end complex formation, and primer terminated with 3'-azidothymidine formed dead-end complex with 25-fold elevated apparent Kd. By contrast, dead-end complex formation on primer terminated with dideoxy-IMP base paired with dC on the template was more efficient than on primer terminated with dideoxy-GMP. Implications for the mechanisms of discrimination between nucleotide analogues by HIV-1 RT are discussed.

The small subunit is required for functional interaction of DNA polymerase delta with the proliferating cell nuclear antigen.

DNA polymerase delta is usually isolated as a heterodimer composed of a 125 kDa catalytic subunit and a 50 kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA synthesis. We have recently demonstrated that the catalytic subunit of human DNA polymerase delta (p125) expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric calf thymus DNA polymerase delta, is not responsive to stimulation by PCNA. To determine whether the lack of response to PCNA of the recombinant catalytic subunit is due to the absence of the small subunit or to differences in post-translational modification in insect cells versus mammalian cells, we have co-expressed the two subunits of human DNA polymerase delta in insect cells. We have demonstrated that co-expression of the catalytic and small subunits of human DNA polymerase delta results in formation of a stable, fully functional heterodimer, that the recombinant heterodimer, similar to native heterodimer, is markedly stimulated (40- to 50-fold) by PCNA and that the increase in activity seen in the presence of PCNA is the result of an increase in processivity. These data establish that the 50 kDa subunit is essential for functional interaction of DNA polymerase delta with PCNA and for highly processive DNA synthesis.

Purification and characterization of the catalytic subunit of human DNA polymerase delta expressed in baculovirus-infected insect cells.

The catalytic subunit of human DNA polymerase delta has been overexpressed in insect cells by a recombinant baculovirus. The recombinant protein has a Mr = approximately 125,000 and is recognized by polyclonal antisera against N-terminal and C-terminal peptides of the catalytic subunit of human DNA polymerase delta. The recombinant protein was purified to near homogeneity (approximately 1200-fold) from insect cells by chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, and single-stranded DNA-cellulose. The purified protein had both DNA polymerase and 3'-5' exonuclease activities. The properties of the recombinant catalytic subunit were compared with those of the native heterodimeric DNA polymerase delta isolated from fetal calf thymus, and the enzymes were found to differ in several respects. Although the native heterodimer is equally active with either Mn2+ or Mg2+ as divalent cation activator, the recombinant catalytic subunit is approximately 5-fold more active in Mn2+ than in Mg2+. The most striking difference between the two proteins is the response to the proliferating cell nuclear antigen (PCNA). The activity and processivity of native DNA polymerase delta are markedly stimulated by PCNA whereas it has no effect on the recombinant catalytic subunit. These results suggest that the small subunit of DNA polymerase delta is essential for functional interaction with PCNA.

Schizosaccharomyces pombe proliferating cell nuclear antigen mutations affect DNA polymerase delta processivity.

We introduced nine site-directed mutations into seven conserved fission yeast proliferative cell nuclear antigen (PCNA) residues, Leu2, Asp63, Arg64, Gly69, Gln201, Glu259, and Glu260, either as single or as double mutants. Both the recombinant wild type and mutant PCNAs were able to form homotrimers in solution and to sustain growth of a null pcna strain (Deltapcna). Wild type Schizosaccharomyces pombe PCNA and PCNA proteins with mutations in Asp63, Gln201, Glu259, or Glu260 to Ala were able to stimulate DNA synthetic activity and to enhance the processivity of calf thymus DNA polymerase delta holoenzyme similar to calf thymus PCNA. Mutations of Leu2 to Val or Arg64 to Ala, either singly or as a double mutant, yielded PCNA mutant proteins that had reduced capacity in enhancing the processivity of DNA polymerase delta but showed no deficiency in stimulation of the ATPase activity of replication factor C. S. pombe Deltapcna strains sustained by these two mutant-pcna alleles had moderate defects in growth and displayed elongated phenotypes. These cells, however, were not sensitive to UV irradiation. Together, these in vitro and in vivo studies suggest that the side chains of Leu2 and Arg64 in one face of the PCNA trimer ring structure are two of the several sites involved in tethering DNA polymerase delta for processive DNA synthesis during DNA replication.

The benzylthio-pyrimidine U-31,355, a potent inhibitor of HIV-1 reverse transcriptase.

U-31,355, or 4-amino-2-(benzylthio)-6-chloropyrimidine is an inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound acts as a specific inhibitor of the RNA-directed DNA polymerase function of HIV-1RT and does not impair the functions of the DNA-catalyzed DNA polymerase or the Rnase H of the enzyme. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-31,355. The data were analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP, and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived that allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates, and the inhibitor. The results obtained indicate that U-31,355 acts as a mixed inhibitor with respect to the template:primer and dNTP binding sites associated with the RNA-directed DNA polymerase domain of the enzyme. The inhibitor possessed a significantly higher binding affinity for the enzyme-substrate complexes, than for the free enzyme and consequently did not directly affect the functions of the substrate binding sites. Therefore, U-31,355 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond. Moreover, the potency of U-31,355 depends on the base composition of the template:primer in that the inhibitor showed a much higher binding affinity for the enzyme-poly (rC):(dG)10 complexes than for the poly (rA):(dT)10 complexes.

Cloning of the cDNAs for the small subunits of bovine and human DNA polymerase delta and chromosomal location of the human gene (POLD2).

cDNAs encoding the small subunit of bovine and human DNA polymerase delta have been cloned and sequenced. The predicted polypeptides, 50,885 and 51,289 Daltons, respectively, are 94% identical, similar to the catalytic subunits. The high degree of conservation of the polypeptides suggests an essential function for the small subunit in the heterodimeric core enzyme. Although the catalytic subunit of DNA polymerase delta shares significant homology with those of the herpes virus family of DNA polymerases, the small subunit of mammalian DNA polymerase delta is not homologous to the small subunit of either herpes simplex virus type 1 DNA polymerase (UL42 protein) or the Epstein-Barr virus DNA polymerase (BMRF1 protein). Searches of the protein databases failed to detect significant homology with any protein sequenced thus far. PCR analysis of DNA from a panel of human-hamster hybrid cell lines localized the gene (POLD2) for the small subunit of DNA polymerase delta to human chromosome 7.

Catalytically distinct conformations of the ribonuclease H of HIV-1 reverse transcriptase by substrate cleavage patterns and inhibition by azidothymidylate and N-ethylmaleimide.

The RNase H activity of recombinant HIV-1 reverse transcriptase (RT) has been characterized with respect to inhibition by azidothymidylate (AZTMP) and N-ethylmaleimide (NEM) and to cleavage patterns using either poly(rA)/poly(dT) or poly(rG)/poly(dC) as model substrate and either Mg2+ or Mn2+ as divalent cation activator. The inhibitory potency of AZTMP and other nucleotide analogues was found to be dependent on both the composition of the substrate and the divalent cation. The enzyme was significantly more sensitive to AZTMP inhibition with poly(rG)/poly(dC) than with poly(rA)/poly(dT) as substrate and in Mn2+ than in Mg2+ with either substrate. Kinetic studies indicated that AZTMP is a competitive inhibitor with respect to the substrate in Mn2+ whereas it behaves as an uncompetitive inhibitor in Mg2+. These results suggest that the enzyme may exist in two distinct forms depending on whether Mg2+ or Mn2+ is the divalent cation activator. Consistent with this suggestion is the alteration in the mode of cleavage of the substrate upon substitution of Mg2+ with Mn2+. In Mg2+, hydrolysis of poly(rA)/poly(dT) appears to be solely endonucleolytic, whereas in Mn2+, hydrolysis is both endonucleolytic and exonucleolytic. With poly(rG)/poly(dC) as substrate, hydrolysis is both endonucleolytic and exonucleolytic in either Mg2+ or Mn2+. There is a positive correlation between sensitivity to AZTMP and production of mononucleotides, suggesting that the exonuclease activity of RNase H is preferentially inhibited by AZTMP. The sensitivity of RNase H to inhibition by N-ethylmaleimide was also found to be markedly influenced by the substrate composition and the divalent cation activator, being most sensitive under conditions in which endonucleolytic activity predominates.(ABSTRACT TRUNCATED AT 250 WORDS)

The quinoline U-78036 is a potent inhibitor of HIV-1 reverse transcriptase.

The quinoline U-78036 represents a new class of non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitors. The agent possesses excellent antiviral activity at nontoxic doses in HIV-1-infected lymphocytes grown in tissue culture. Enzymatic kinetic studies of the HIV-1 reverse transcriptase (RT)-catalyzed RNA-directed DNA polymerase function were carried out in order to determine whether the inhibitor interacts with the template-primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The data were analyzed using steady-state or Briggs-Haldane kinetics assuming that the template-primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The calculated rate constants are in agreement with this model. The results show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template-primer and the dNTP binding sites of the enzyme. Hence, U-78036 inhibits the RNA-directed DNA polymerase activity of RT by interacting with a site distinct from the template-primer and dNTP binding sites. Moreover, the potency of U-78036 is dependent on the base composition of the template-primer. The equilibrium constants for various enzyme-substrate-inhibitor complexes were at least seven times lower for the poly(rC).(dG)10-catalyzed system than the one catalyzed by poly(rA).(dT)10. In addition, the inhibitor does not impair the DNA-dependent DNA polymerase activity and the RNase H function of HIV-1 RT nor does it inhibit the RNA-directed DNA polymerase activity of the HIV-2, avian myoblastoma virus, and murine leukemia virus RT enzymes.

U-90152, a potent inhibitor of human immunodeficiency virus type 1 replication.

Bisheteroarylpiperazines are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). We describe a novel bisheteroarylpiperazine, U-90152 [1-(5-methanesulfonamido-1H-indol-2-yl-carbonyl)-4-[3-(1-methyl eth yl-amino)pyridinyl]piperazine], which inhibited recombinant HIV-1 RT at a 50% inhibitory concentration (IC50) of 0.26 microM (compared with IC50s of > 440 microM for DNA polymerases alpha and delta). U-90152 blocked the replication in peripheral blood lymphocytes of 25 primary HIV-1 isolates, including variants that were highly resistant to 3'-azido-2',3'-dideoxythymidine (AZT) or 2',3'-dideoxyinosine, with a mean 50% effective dose of 0.066 +/- 0.137 microM. U-90152 had low cellular cytotoxicity, causing less than 8% reduction in peripheral blood lymphocyte viability at 100 microM. In experiments assessing inhibition of the spread of HIV-1IIIB in cell cultures, U-90152 was much more effective than AZT. When approximately 500 HIV-1IIIB-infected MT-4 cells were mixed 1:1,000 with uninfected cells, 3 microM AZT delayed the evidence of rapid viral growth for 7 days. In contrast, 3 microM U-90152 totally prevented the spread of HIV-1, and death and/or dilution of the original inoculum of infected cells prevented renewed viral growth after U-90152 was removed at day 24. The combination of U-90152 and AZT, each at 0.5 microM, also totally prevented viral spread. Finally, although the RT amino acid substitutions K103N (lysine 103 to asparagine) and Y181C (tyrosine 181 to cysteine), which confer cross-resistance to several nonnucleoside inhibitors, also decrease the potency of U-90152, this drug retains significant activity against these mutant RTs in vitro (IC50s, approximately 8 microgramM).

Purification and characterization of delta helicase from fetal calf thymus.

A DNA helicase (delta helicase) which partially copurifies with DNA polymerase delta has been highly purified from fetal calf thymus. delta helicase differs in physical and enzymatic properties from other eukaryotic DNA helicases described thus far. The enzyme has an apparent mass of 57 kDa by gel filtration and is associated with polypeptides of 56 and 52 kDa by SDS-polyacrylamide gel electrophoresis. Photo-cross-linking of the purified enzyme with [alpha-32P]ATP resulted in labeling of a polypeptide of approximately 58 kDa, suggesting that the active site is present on the larger polypeptide. Unwinding of a partial duplex requires a nucleoside triphosphate which can be either ATP or dATP but not a nonhydrolyzable analogue of ATP. Other ribo- and deoxyribonucleoside triphosphates have little or no activity as cofactors. delta helicase also has DNA-dependent ATPase activity which has a relatively low Km for ATP (40 microM). delta helicase binds to single-stranded DNA but has little or no affinity for double-stranded DNA or single-stranded RNA. Similar to replicative DNA helicases from prokaryotes and the herpes simplex virus type 1 helicase-primase, delta helicase translocates in the 5'-3' direction along the strand to which it is bound and preferentially unwinds DNA substrates with a forklike structure.

Eukaryotic DNA replication.

The past decade has witnessed an exciting evolution in our understanding of eukaryotic DNA replication at the molecular level. Progress has been particularly rapid within the last few years due to the convergence of research on a variety of cell types, from yeast to human, encompassing disciplines ranging from clinical immunology to the molecular biology of viruses. New eukaryotic DNA replicases and accessory proteins have been purified and characterized, and some have been cloned and sequenced. In vitro systems for the replication of viral DNA have been developed, allowing the identification and purification of several mammalian replication proteins. In this review we focus on DNA polymerases alpha and delta and the polymerase accessory proteins, their physical and functional properties, as well as their roles in eukaryotic DNA replication.

Primary structure of the catalytic subunit of calf thymus DNA polymerase delta: sequence similarities with other DNA polymerases.

The 125- and 48-kDa subunits of bovine DNA polymerase delta have been isolated by SDS-polyacrylamide gel electrophoresis and demonstrated to be unrelated by partial peptide mapping with N-chlorosuccinimide. A 116-kDa polypeptide, usually present in DNA polymerase delta preparations, was shown to be a degraded form of the 125-kDa catalytic subunit. Amino acid sequence data from Staphylococcus aureus V8 protease, cyanogen bromide, and trypsin digestion of the 125- and 116-kDa polypeptides were used to design primers for the polymerase chain reaction to determine the nucleotide sequence of a full-length cDNA encoding the catalytic subunit of bovine DNA polymerase delta. The predicted polypeptide is 1106 amino acids in length with a calculated molecular weight of 123,707. This is in agreement with the molecular weight of 125,000 estimated from SDS-polyacrylamide gel electrophoresis. Comparison of the deduced amino acid sequence of the catalytic subunit of bovine DNA polymerase delta with that of its counterpart from Saccharomyces cerevisiae showed that the proteins are 44% identical. The catalytic subunit of bovine DNA polymerase delta contains the seven conserved regions found in a number of bacterial, viral, and eukaryotic DNA polymerases. It also contains five additional regions that are highly conserved between bovine and yeast DNA polymerase delta, but these regions share little or no homology with the alpha polymerases. Four of these additional regions are also highly homologous to the herpes virus family of DNA polymerases, whereas one region is not homologous to any other DNA polymerase that has been sequenced thus far.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure of the catalytic subunit of human DNA polymerase delta and chromosomal location of the gene.

The catalytic subunit (Mr approximately 124,000) of human DNA polymerase delta has been cloned by PCR using poly(A)+ RNA from HepG2 cells and primers designed from the amino acid sequence of regions highly conserved between bovine and yeast DNA polymerase delta. The human cDNA was 3443 nucleotides in length and coded for a polypeptide of 1107 amino acids. The enzyme was 94% identical to bovine DNA polymerase delta and contained the numerous highly conserved regions previously observed in the bovine and yeast enzymes. The human enzyme also contained two putative zinc-finger domains in the carboxyl end of the molecule, as well as a putative nuclear localization signal at the amino-terminal end. The gene coding for human DNA polymerase delta was localized to chromosome 19.

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)