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W C Copeland

Publications and source records attributed to W C Copeland.

At least 37 records · Page 2Linked to original sources
PubMed1995

Characterization of a new DNA polymerase from Schizosaccharomyces pombe: a probable homologue of the Saccharomyces cerevisiae DNA polymerase gamma.

Mitochondrial (mt) DNA replication is carried out by the nuclear-encoded DNA polymerase-gamma (Pol-gamma). We have cloned a new DNA polymerase-encoding gene from Schizosaccharomyces pombe (Sp), which we believe encodes the homologue of the Saccharomyces cerevisiae (Sc) mt DNA polymerase (MIP1). The putative Sp pol gamma gene expressed a transcript of approx. 4-kb that contained a 3-kb open reading frame encoding a polypeptide of 1018 amino acids (aa) (116 kDa). This Sp Pol-gamma is 48% identical to the Sc MIP1 and contains uniquely conserved regions not found in the bacterial PolI-type DNA polymerases. The most notable difference between these two proteins is that the MIP1 product has a 236-aa C-terminal region beyond motif C that is not found in Sp Pol-gamma. Chromosomal mapping and genomic sequencing of the Sp pol gamma places this gene on chromosome III downstream from the triose phosphate isomerase-encoding gene.

Amino Acid Sequence↗
PubMed1995

Active site mapping of the catalytic mouse primase subunit by alanine scanning mutagenesis.

In the eukaryotic cell, DNA synthesis is initiated by DNA primase associated with DNA polymerase alpha. The eukaryotic primase is composed of two subunits, p49 and p58, where the p49 subunit contains the catalytic active site. Mutagenesis of the cDNA for the p49 subunit was initiated to demonstrate a functional correlation of conserved residues among the eukaryotic primases and DNA polymerases. Fourteen invariant charged residues in the smaller catalytic mouse primase subunit, p49, were changed to alanine. These mutant proteins were expressed, purified, and enzymatically characterized for primer synthesis. Analyses of the mutant proteins indicate that residues 104-111 are most critical for primer synthesis and form part of the active site. Alanine substitution in residues Glu105, Asp109, and Asp111 produced protein with no detectable activity in direct primase assays, indicating that these residues may form part of a conserved carboxylic triad also observed in the active sites of DNA polymerases and reverse transcriptases. All other mutant proteins showed a dramatic decrease in catalysis, while mutation of two residues, Arg162 and Arg163, caused an increase in Km(NTP). Analysis of these mutant proteins in specific assays designed to separately investigate dinucleotide formation (initiation) and elongation of primer indicates that these two activities utilize the same active site within the p49 subunit. Finally, mutations in three active site codons produced protein with reduced affinity with the p58 subunit, suggesting that p58 may interact directly with active site residues.

Alanine↗
PubMed1993

Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication.

The enzymatic mechanism of primase was investigated using Escherichia coli and baculoviral overexpressed mouse primase subunits, p49 and p58. Neither of the singly purified primase subunits displayed primase activity alone, but the p49 subunit was able to extend a riboprimer, indicating that this subunit contains an RNA polymerase activity. The p58 subunit cooperated with the p49 subunit in binding the initiating purine to form the initial dinucleotide. After initiation, the p49 subunit alone was sufficient to extend the growing primer, but both the rate of p49 primer extension and its stability were influenced by the p58 subunit. The Km(ATP) in primer synthesis on poly(dT) of the p49-p58 heterodimeric primase complex was 10-fold higher than the Km(ATP) of the single p49 subunit in a ribo(A) primer extension assay. In addition, labeled ATP cross-linked to both of the individually purified subunits but with a striking difference in affinities; cross-linking was 11-fold more efficient to the p49 subunit. The interaction of the two primase subunits with polymerase alpha was also investigated. Immunoprecipitation experiments indicate that only the p58 subunit directly contacts the p180 subunit of DNA polymerase alpha. Competition experiments in the coupled primase-polymerase assay with a catalytically inactive mutant of DNA polymerase alpha and the Klenow fragment suggest that the DNA polymerase alpha-primase complex does not dissociate from the primer during the transition from RNA to DNA synthesis.

Adenosine Triphosphate↗
PubMed1993

Mutational studies of human DNA polymerase alpha. Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis.

Conserved site-directed mutations were introduced into the second most conserved amino acid region, region II, of the human DNA polymerase alpha catalytic subunit. These mutants were expressed in the baculovirus system and purified to near homogeneity. The mutants had polymerase activity ranging from 4 to 60% compared with the wild type polymerase alpha. Steady-state kinetic analysis of mutants G841A, D860A, D860S, D860N, Y865S, and Y865F demonstrated no significant difference in their Km values for primer-template compared with that of the wild type enzyme. In contrast, mutants D860A, Y865S, and Y865F showed a 5-10-fold increase in the Km for deoxynucleotide triphosphate (dNTP) compared with the wild type enzyme. DNA synthetic fidelity studies of these mutants showed that mutant Y865S but not Y865F had a greater than 10-fold higher misinsertion efficiency than the wild type enzyme in Mg(2+)-catalyzed reactions. However, with Mn2+ as the metal activator, Y865S and Y865F demonstrated a 2- and 9-fold higher misinsertion efficiency, respectively. These results indicate that Asp860 and Tyr865 in region II of human DNA polymerase alpha are involved in incoming dNTP substrate binding. Using three deoxynucleotide structural analogs as probes, we show that the nucleotide base is the structural requirement for dNTP binding with Tyr865. Furthermore, abolishing the hydrophobic phenyl ring side chain of Tyr865 by replacing tyrosine with serine rendered the enzyme resistant to aphidicolin. Results of these studies strongly suggest that the phenyl ring of Tyr865 directly interacts with the nucleotide base moiety of the dNTP and plays a critical role in the misinsertion fidelity of DNA synthesis. Although mutation of Gly841 to Ala did not affect the binding of primer-template, it had a significant decrease in kcat, an increase in Km for dNTP, a striking decrease of processivity, and also resistance to aphidicolin. Thus, mutation of this residue, Gly841, which is highly conserved among the alpha-like DNA polymerases, appears to affect both catalysis and substrate deoxynucleotide binding. This suggests that Gly841 is essential for the maintenance of the overall structure of the polymerase alpha catalytic site.

Amino Acid Sequence↗
PubMed1993

Mutational studies of human DNA polymerase alpha. Serine 867 in the second most conserved region among alpha-like DNA polymerases is involved in primer binding and mispair primer extension.

The second most conserved region of alpha-like DNA polymerases, region II, spans a block of 40 amino acid residues centered at the core sequence -DFNSLYPSII-. In the previous paper, we described mutational studies of 3 amino acid residues in region II which includes 2 amino acid residues in the core sequence. We showed that residues Asp860 and Tyr865 in the core sequence are involved in substrate deoxynucleotide triphosphate (dNTP) binding. We further showed that the phenyl moiety of the Tyr865 side chain interacts with the incoming dNTP and is responsible for the misinsertion fidelity of the enzyme. In this report, we investigated the function of 2 serine residues, Ser863 and Ser867, in this core sequence. Mutation of these 2 Ser residues to either Ala or Thr yielded mutant enzymes with similar Km for dNTPs, kcat, processivity, and misinsertion fidelity of DNA synthesis as the wild type enzyme. However, mutation of Ser867 to Ala demonstrated a 30-fold increase in Km for primer-template and a 5-fold higher KD for binding primer-template. DNA footprinting experiments of primer with the dideoxynucleotide terminus indicated that the structural feature of the primer recognized by Ser867 is the 3'-OH terminus. Single-stranded DNA inhibition data suggest that removal of the hydroxyl side chain of Ser867 affects the polymerase's interaction with primer and not with template. Mutation of Ser867 to Ala also decreases the mutant enzyme's Km for dNTP to extend a mispaired primer and thus enhances its capacity to extend a mispaired primer terminus. These data support the conclusion that the hydroxyl side chain of Ser867 of human DNA polymerase alpha is involved in primer interaction during DNA synthesis and plays an essential role in mispair extension fidelity of DNA synthesis.

Amino Acid Sequence↗
PubMed1993

Selective action of 4'-azidothymidine triphosphate on reverse transcriptase of human immunodeficiency virus type 1 and human DNA polymerases alpha and beta.

4'-Azidothymidine (ADRT) is a novel nucleoside analogue that exhibits potent inhibitory activity against the replication of human immunodeficiency virus (HIV) in lymphocytes. The mechanisms by which ADRT inhibits HIV reverse transcriptase (HIV-RT) as ADRT 5'-triphosphate (ADRT-TP), the active intracellular metabolite of ADRT, and as the ADRT-MP molecule incorporated into DNA were examined and compared to their effects on human DNA polymerases alpha and beta. Inhibition of HIV-RT by ADRT-TP is competitive against TTP and is more potent against RNA to DNA synthesis (Ki = 0.009 microM versus Km = 3.3 microM for TTP) than it is against DNA to DNA synthesis (Ki = 0.95 microM versus Km = 16.3 microM for TTP). ADRT-TP is also a more potent inhibitor for primer elongation on RNA template than on DNA template. ADRT-TP is a poor inhibitor of human DNA polymerases alpha (Ki = 62.5 microM) and beta (Ki = 150 microM) (Chen et al., 1992). The consequences of ADRT incorporation into DNA are strikingly different for the HIV-RT and for human DNA polymerases alpha and beta. DNA polymerases alpha and beta incorporate a single ADRT-MP molecule into nascent DNA at a very slow rate and continue to elongate. They are unable to incorporate a second consecutive ADRT-MP. However, HIV-RT is able to efficiently incorporate two consecutive ADRT molecules. Incorporation of two consecutive ADRT-MP molecules by HIV-RT prevents further DNA chain elongation. Incorporation of two ADRT-MP molecules separated by one deoxyribonucleoside monophosphate (dAMP, dCMP, or dGMP) also abolishes DNA chain elongation by HIV-RT.

Antiviral Agents↗
PubMed1993

Mutational analysis of the human DNA polymerase alpha. The most conserved region in alpha-like DNA polymerases is involved in metal-specific catalysis.

Five site-directed mutations were introduced at the most conserved amino acids in region I (YGDTDS) of the human DNA polymerase alpha catalytic subunit. Mutant proteins were expressed in the baculovirus system by an improved method and purified by a rapid one-step purification in high yield and high specific activity. The Asp1004 to Asn mutation produced a protein with no detectable polymerase activity while other mutations gave activities from 1 to 20% of the wild type polymerase activity. Steady state kinetic analysis of the active mutants indicates that none of the mutations caused a change in Km(dNTP) or KD(DNA), but all active mutants showed a decrease in kcat. Thus, the effect of these conserved mutations is manifest in altered rates of catalysis. Two mutations, Asp1002 to Asn and Thr1003 to Ser, caused the enzyme to utilize Mn2+ more effectively in catalysis than Mg2+, suggesting that these amino acids are involved in metal binding. Rates of catalysis by the D1002N and T1003S mutants, as well as Y1000F mutant were improved 80-, 30-, and 70-fold, respectively, on homopolymer templates when Mn2+ replaced Mg2+ as the activator metal. The results from these mutational studies suggest that this highly conserved region binds the metal which is essential for catalysis. The Asp1002 may participate directly in chelating the metal. Results from the T1003S mutant suggest that the beta-methyl group of the threonine side chain might be locked in a hydrophobic pocket preventing free rotation around the C alpha-C beta bond, thus positioning the Thr1003 hydroxyl group to form a crucial bond with the metal ion. In addition, D1002N and T1003S displayed a 20-fold resistance to aphidicolin compared to the wild type polymerase alpha, and all of the active mutants displayed altered sensitivity to butylphenyl-dGTP. Models of the involvement of region I in catalysis and aphidicolin interaction are proposed. The mutational studies presented in this report will serve as a prototype for the functional role of region I in catalysis for all alpha-like DNA polymerases.

Amino Acid Sequence↗
PubMed1993

Fidelity studies of the human DNA polymerase alpha. The most conserved region among alpha-like DNA polymerases is responsible for metal-induced infidelity in DNA synthesis.

Mutational studies in the highly conserved region I domain of the human DNA polymerase alpha enzyme demonstrated a change in metal cation-specific catalysis. Here, we extend the investigation to include the fidelity of DNA synthesis by these mutants, studying misinsertion, mispair extension, and the nucleotide analog utilization. The fidelity of region I mutants and wild type human DNA polymerase alpha enzyme were analyzed with either Mg2+ or Mn2+ as the metal activator. Despite the known mutagenic effect of Mn2+ in causing polymerases to misinsert nucleotides and to utilize dideoxynucleotides, we have found that two region I mutants, D1002N and T1003S, which utilize Mn2+ in catalysis more effectively than Mg2+, actually have a 70- and 40-fold higher misinsertion fidelity, respectively, in Mn(2+)-catalyzed reactions than that of the wild type enzyme. The enhanced misinsertion fidelity of these two mutants in Mn(2+)-catalyzed reactions is due to Km discrimination of the incorrect nucleotide where the D1002N and T1003S had a 850- and 62-fold higher Km for insertion of incorrect than correct nucleotide, respectively. In Mg(2+)-catalyzed reactions, all of the region I mutants exhibited similar misinsertion efficiencies as the wild type polymerase. Study of mispair extension showed that in Mn(2+)-catalyzed ractions, the wild type polymerase alpha enzyme readily extended mispair termini. In contrast, the two region I mutants, D1002N and T1003S, were unable to extend the mispaired termini in either Mg(2+)- or Mn(2+)-catalyzed reactions. These results suggest that the side chains of region I amino acids play an essential role in the Mn(2+)-induced infidelity during DNA synthesis by human DNA polymerase alpha. The effects of the metal activator on the utilization of two nucleotide analogs, 3'-azido-3'-deoxythymidine triphosphate and ddCTP, by the region I mutants were also investigated.

Base Sequence↗
PubMed1993

In vivo species specificity of DNA polymerase alpha.

The DNA polymerase alpha enzymes from human, and budding (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are homologous proteins involved in initiation and replication of chromosomal DNA. Sequence comparison of human DNA polymerase alpha with that of S. cerevisiae and S. pombe shows overall levels of amino acid sequence identity of 32% and 34%, respectively. We report here that, despite the sequence conservation among these three enzymes, functionally active human DNA polymerase alpha fails to rescue several different conditional lethal alleles of the budding yeast POL1 gene at nonpermissive temperature. Furthermore, human DNA polymerase alpha cannot complement a null allele of budding yeast POL1 either in germinating spores or in vegetatively growing cells. In fission yeast, functionally active human DNA polymerase alpha is also unable to complement the disrupted pol alpha::ura4+ allele in germinating spores. Thus, in vivo, DNA polymerase alpha has stringent species specificity for initiation and replication of chromosomal DNA.

Alleles↗
PubMed1993

Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen.

Initiation of cell-free simian virus 40 (SV40) DNA replication requires the interaction of DNA polymerase alpha/primase with a preinitiation complex containing the viral T antigen and cellular proteins, replication protein A, and topoisomerase I or II. To further understand the molecular mechanisms of the transition from preinitiation to initiation, the intermolecular interaction between human DNA polymerase alpha and T antigen was investigated. We have demonstrated that the human DNA polymerase alpha catalytic polypeptide is able to associate with SV40 large T antigen directly under physiological conditions. A physical association between these two proteins was detected by coimmunoprecipitation with monoclonal antibodies from insect cells coinfected with recombinant baculoviruses. A domain of human polymerase alpha physically interacting with T antigen was identified within the amino-terminal region from residues 195 to 313. This domain of human polymerase alpha was able to form a nonproductive complex with T antigen, causing inhibition of the SV40 DNA replication in vitro. Kinetics of the inhibition indicated that this polymerase domain can inhibit viral replication only during the preinitiation stage. Extra molecules of T antigen could partially overcome the inhibition only prior to initiation complex formation. The data support the conclusion that initiation of SV40 DNA replication requires the physical interaction of T antigen in the preinitiation complex with the amino-terminal domain of human polymerase alpha from amino acid residues 195 to 313.

Antigens, Viral, Tumor↗
PubMed1992

Human DNA polymerases alpha and beta are able to incorporate anti-HIV deoxynucleotides into DNA.

Deoxynucleoside analogs, AZT and/or ddN, are the therapeutic agents currently utilized to inhibit the human immunodeficiency virus (HIV) reverse transcriptase. The effects of their anabolic products, AZT-triphosphate (AZT-TP) and ddCTP on human cellular DNA metabolic processes were studied using highly purified, structurally and enzymatically defined forms of the two major human host DNA polymerases, alpha and beta, and compared to those of the reverse transcriptase purified from HIV viron. Human DNA polymerase alpha during processive DNA synthesis is able to incorporate AZT-monophosphate (AZT-MP) but not ddCMP into DNA, causing chain termination. During its initial encounter with a primer terminus, polymerase alpha is able to incorporate both AZT-MP and ddCMP into DNA chains. Polymerase beta is able to incorporate AZT-MP and ddCMP into DNA, causing chain termination in both modes of DNA synthesis. Steady state kinetic analyses demonstrate that polymerase alpha inserts one AZT-MP molecule into DNA for every 2500 dTMP molecules incorporated. Polymerase beta incorporates ddCMP with efficiency nearly equal to that of dCMP. HIV reverse transcriptase prefers to incorporate AZT-MP and ddCMP rather than dTMP and dCMP, respectively. The findings described here raise the concern that the capability of the two major host DNA polymerases to incorporate AZT-MP or ddCMP into DNA might cause adverse side effects on human DNA metabolism and mutation in the genomes of patients under long term continuous treatment with AZT and ddC.

Antiviral Agents↗
PubMed1991

Catalytic subunit of human DNA polymerase alpha overproduced from baculovirus-infected insect cells. Structural and enzymological characterization.

The human DNA polymerase alpha catalytic polypeptide has been functionally overexpressed by a recombinant baculovirus in insect cells at greater than 1000-fold higher levels than that found in cultured normal human cells. The recombinant polymerase alpha protein is translated from its natural translation start codon under the control of the baculovirus polyhedron promoter producing a protein of 180 kDa, identical in size to that isolated from cultured human cells. This recombinant polymerase alpha is phosphorylated and reactive to a panel of monoclonal antibodies directed against the native polymerase alpha-primase complex and to polyclonal antisera against N- and C-terminal peptides of the polymerase alpha catalytic polypeptide. The recombinant enzyme was immunopurified from insect cells as a single polypeptide. The single subunit recombinant polymerase alpha has no detectable 3'-5' exonuclease activity. The Km for primer-template and dNTP, reactivity to inhibitors, N2-(p-n-butylphenyl)-dGTP (BuPdGTP) and aphidicolin, thermosensitivity, and DNA synthetic processivity and fidelity of the recombinant polymerase alpha are identical to that observed with the four-subunit polymerase alpha-primase complex immunopurified from cultured human cells. These results strongly suggest that the presence of the other subunits, (the p70 and the two primase subunits, p48 and p58), does not influence kinetic parameters of polymerase alpha catalysis, sensitivity to inhibitors, or DNA synthetic fidelity and processivity.

Animals↗
PubMed1991

Site-directed alteration of four active-site residues of a pyruvoyl-dependent histidine decarboxylase.

Site-directed mutagenesis has been used to examine the chemical roles of four active-site residues in histidine decarboxylase (HDC) from Lactobacillus 30a. This protein is known to undergo an autoactivation in which chain cleavage between serines-81 and -82 leads to cofactor (pyruvoyl) formation at position 82. Conversion of Ser-81 to Ala virtually eliminates productive cleavage. It is proposed that the residue plays a key role in stabilizing the transition state of the chain cleavage reaction. Conversion of Phe-83 to Met renders the proenzyme thermally less stable than wild type and appears to slightly increase the rate of autoactivation. The Km value for histidine is increased about 8-fold, confirming crystallographic evidence that Phe-83 is involved in substrate binding. Both wild-type and F83M enzymes show constant Km and steadily increasing kcat values as a function of temperature. Lys-155 and Tyr-262, by virtue of their positions in the active site of HDC, have been proposed to possibly play specific roles in either autoactivation or catalysis by active HDC. Conversion to Gln and Phe respectively suggests that these residues have real but minor roles in those processes.

Binding Sites↗
PubMed1990

Human DNA polymerase alpha catalytic polypeptide binds ConA and RCA and contains a specific labile site in the N-terminus.

The catalytic polypeptide of DNA polymerase alpha is often observed in vitro as a family of phosphopolypeptides predominantly of 180 and 165 kDa derived from a single primary structure. The estimated Mr of this polypeptide deduced from the full-length cDNA is 165 kDa. Immunoblot analysis with polyclonal antibodies against peptides of the N- and C-termini of the deduced primary sequence indicates that the observed family of polypeptides from 180 kDa to lower molecular weight results from proteolytic cleavage from the N-terminus. Antibodies against the N-terminal peptide detect only the 180 kDa species suggesting that this higher molecular weight polypeptide may be the result of posttranslational modification of the 165 kDa primary translation product. The catalytic polypeptide is not only phosphorylated but is also found to react with lectins ConA and RCA. N-terminal sequencing of the isolated catalytic polypeptide from human cells and of the recombinant fusion proteins indicates that the often observed 165 kDa polypeptide is the in vitro proteolytic cleavage product of the modified 180 kDa protein at the specific site between lys123 and lys124 within the sequence -RNVKKLAVTKPNN-.

Amino Acid Sequence↗
PubMed1989

The molecular cloning, sequence and expression of the hdcB gene from Lactobacillus 30A.

We previously cloned the structural gene hdcA, which encodes the enzyme histidine decarboxylase (HDC; EC 4.1.1.22), from Lactobacillus 30a and found what appeared to be the start of a second gene 59 nucleotide (nt) downstream from the hdcA stop codon [Vanderslice et al., J. Biol. Chem. 32 (1986) 15186-15191]. Here we report the complete nt sequence of this second gene, which we have named hdcB, and show that it encodes a 20-kDa protein, HDCB, which was purified from Escherichia coli. The hdcA and hdcB genes together comprise an operon, the transcription from which is shown to be increased threefold by the presence of histidine in the growth medium. Western blots were used to quantitate the rise in concentrations of both gene products during histidine induction of the hdc operon. This increase was found to be proportional to the observed threefold increase in the concentration of the respective mRNAs. Transcription of the hdc operon in the mutant-3 strain of Lactobacillus 30a [Recsei and Snell, Biochemistry 12 (1973) 365-371] was shown to be constitutively 15-fold greater than in uninduced wild type cells and was unaffected by histidine. The transcription start point was defined as a guanine 73 nt 5' to the start codon of the hdcA gene. Of the transcripts initiated at this promoter, 15% include both hdcA and hdcB sequences, the remainder terminate in the intergenic region and thus encode only hdcA.

Amino Acid Sequence↗
PubMed1988

Site-directed alteration of serine 82 causes nonproductive chain cleavage in prohistidine decarboxylase.

Prohistidine decarboxylase from Lactobacillus 30a normally autoactivates by cleavage of the Ser-81-Ser-82 peptide bond, converting Ser-82 to a pyruvoyl moiety which serves as the enzymatic cofactor. We have used site-directed methods to make two conservative mutations, converting Ser-82 to cysteine (S82C) and threonine (S82T). Both mutant proteins autoactivate, although dramatically (20- to 80-fold), more slowly than wild type. Roughly 55% of the mutant protein in each case undergoes a nonproductive chain cleavage which does not result in cofactor production. This finding suggests that an important feature in the enzyme's evolution has been development of an activation scheme which minimizes nonproductive side reactions. Catalytic constants are also affected by the mutations, particularly kcat which drops 8-fold in S82C and 450-fold in S82T. In addition, the S82T protein activates to produce a novel alpha-ketobutyroyl cofactor.

Carboxy-Lyases↗