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A G So

Publications and source records attributed to A G So.

At least 37 records · Page 2Linked to original sources
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

Nonnucleoside reverse transcriptase inhibitors that potently and specifically block human immunodeficiency virus type 1 replication.

Certain bis(heteroaryl)piperazines (BHAPs) are potent inhibitors of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) at concentrations lower by 2-4 orders of magnitude than that which inhibits normal cellular DNA polymerase activity. Combination of a BHAP with nucleoside analog HIV-1 RT inhibitors suggested that together these compounds inhibited RT synergistically. In three human lymphocytic cell systems using several laboratory and clinical HIV-1 isolates, the BHAPs blocked HIV-1 replication with potencies nearly identical to those of 3'-azido-2',3'-dideoxythymidine or 2',3'-dideoxyadenosine; in primary cultures of human peripheral blood mononuclear cells, concentrations of these antiviral agents were lower by at least 3-4 orders of magnitude than cytotoxic levels. The BHAPs do not inhibit replication of HIV-2, the simian or feline immunodeficiency virus, or Rauscher murine leukemia virus in culture. Evaluation of a BHAP in HIV-1-infected SCID-hu mice (severe combined immunodeficient mice implanted with human fetal lymph node) showed that the compound could block HIV-1 replication in vivo. The BHAPs are readily obtained synthetically and have been extensively characterized in preclinical evaluations. These compounds hold promise for the treatment of HIV-1 infection.

Animals↗
PubMed1991

Inhibition of the RNase H activity of HIV reverse transcriptase by azidothymidylate.

The effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT.

Antiviral Agents↗
PubMed1991

Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase.

The DNA polymerase and RNase H activities of HIV reverse transcriptase are both essential for HIV replication. Although the two activities are both catalyzed by a single polypeptide, they are physically separate; i.e., the DNA polymerase resides in the N-terminal domain whereas the RNase H is localized in the C-terminal domain. The present study was undertaken to characterize the enzymatic properties of these two activities and to determine whether the two catalytic sites are also functionally distinct. We have observed that EGTA specifically stimulates, whereas CaCl2 selectively inhibits, the RNA-dependent DNA polymerase activity but that neither compound has any effect on the RNase H activity of a recombinant HIV reverse transcriptase. The stimulation of the DNA polymerase activity by EGTA is dependent on the Mg2+ concentration; the greatest stimulation is observed at low Mg2+ concentrations. Similarly, the inhibition of DNA polymerase activity by Ca2+ is influenced by Mg2+ concentration. Ca2+ inhibition can be reversed by increasing Mg2+ concentrations, suggesting the possibility that CaCl2 inhibits the reverse transcriptase activity by competing for a metal-binding site on the enzyme. The pyrophosphate analogue phosphonoformate selectively inhibits the polymerase activity but not the RNase H activity of HIV reverse transcriptase. In contrast, the RNase H activity can be selectively inhibited by deoxyadenosine 5'-monophosphate, whereas the DNA polymerase activity is not inhibited. These results suggest that the DNA polymerase and RNase activities are not only physically separate but that they are also functionally distinct.

Blotting, Western↗
PubMed1990

DNA polymerase delta: a second eukaryotic DNA replicase.

During the past few years significant progress has been made in our understanding of the structure and function of the proteins involved in eukaryotic DNA replication. Data from several laboratories suggest that, in contrast to prokaryotic DNA replication, two distinct DNA polymerases are required for eukaryotic DNA replication, i.e. DNA polymerase delta for the synthesis of the leading strand and DNA polymerase alpha for the lagging strand. Several accessory proteins analogous to prokaryotic replication factors have been identified and some of these are specific for pol delta whereas others affect both DNA replicases. The replicases and their accessory proteins appear to be highly conserved in eukaryotes, as homologous proteins have been found in species ranging from humans to yeast.

Animals↗
PubMed1989

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.

Antibodies↗
PubMed1988

Mechanisms of error discrimination by Escherichia coli DNA polymerase I.

The mechanism of base selection by DNA polymerase I of Escherichia coli has been investigated by kinetic analysis. The apparent KM for the insertion of the complementary nucleotide dATP into the hook polymer poly(dT)-oligo(dA) was found to be 6-fold lower than that for the noncomplementary nucleotide dGTP, whereas the Vmax for insertion of dATP was 1600-fold higher than that for dGTP. The ratio of Kcat/KM values for complementary and mismatched nucleotides of 10(4) demonstrates the extremely high specificity of base selection by DNA polymerase I and is in agreement with results obtained with a different template-primer, poly(dC)-oligo(dG) [El-Deiry, W. S., Downey, K. M., & So, A. G. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7378]. Studies on the effects of phosphate ion on the polymerase and 3'- to 5'-exonuclease activities of DNA polymerase I showed that, whereas the polymerase activity was somewhat stimulated by phosphate, the exonuclease activity was markedly inhibited, being 50% inhibited at 25 mM phosphate and greater than 90% inhibited at 80 mM phosphate. Selective inhibition of the exonuclease activity by phosphate also resulted in inhibition of template-dependent conversion of a noncomplementary dNTP to dNMP and, consequently, markedly affected the kinetic constants for insertion of noncomplementary nucleotides. The mutagenic metal ion Mn2+ was found to affect error discrimination by both the polymerase and 3'- and 5'-exonuclease activities of DNA polymerase I.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1987

Autoantibody to the proliferating cell nuclear antigen neutralizes the activity of the auxiliary protein for DNA polymerase delta.

Two murine monoclonal antibodies to the proliferating cell nuclear antigen (PCNA), a rabbit anti-N-terminal peptide antibody and human auto-antibody to PCNA reacted with the auxiliary protein for DNA polymerase delta from fetal calf thymus following SDS-polyacrylamide gel electrophoresis, confirming the identity of PCNA and the auxiliary protein. Undenatured auxiliary protein was immunoprecipitated by the human autoantibody, but not by the monoclonal antibodies, which were raised to SDS-denatured PCNA, nor by the anti-N-terminal peptide antibody, suggesting that the epitopes recognized by both the monoclonal antibodies and the anti-peptide antibody are not exposed in the native protein. The human anti-PCNA autoantibody neutralized the activity of the auxiliary protein for DNA polymerase delta, but did not inhibit the activity of pol delta itself. The ability of pol delta to utilize template/primers containing long stretches of single-stranded template was inhibited by the anti-PCNA autoantibody, whereas the activity of pol alpha on such templates was not affected, confirming the specificity of the auxiliary protein for pol delta. The ability of PCNA, a cell cycle-regulated protein, to regulate the activity of pol delta suggests a central role for pol delta in cellular DNA replication.

Animals↗
PubMed1987

Apparent stimulation of calf thymus DNA polymerase alpha by ATP.

The mechanism by which millimolar concentrations of ATP stimulate the activity and increase the processivity of calf thymus DNA polymerase alpha has been investigated with poly(dA)/oligo(dT) as template/primer to eliminate possible effects due to primer synthesis. The effect of ATP on the rate of DNA synthesis with this template/primer was found to be dependent upon whether or not the ATP was neutralized and the species of buffer used in the reaction. The present studies suggest that ATP stimulation of calf thymus DNA polymerase can be attributed to changes in the pH of the reaction mixture, a shift in the magnesium ion optimum, or both. Furthermore, effects of ATP on the processivity of DNA polymerase alpha could be mimicked by lowering the pH of the reaction mixture.

Adenosine Triphosphate↗
PubMed1986

An auxiliary protein for DNA polymerase-delta from fetal calf thymus.

An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.

Animals↗
PubMed1984

Further studies on calf thymus DNA polymerase delta purified to homogeneity by a new procedure.

DNA polymerase delta from calf thymus has been purified to apparent homogeneity by a new procedure which utilizes hydrophobic interaction chromatography with phenyl-Sepharose at an early step to separate most of the calcium-dependent protease activity from DNA polymerase delta and alpha. The purified enzyme migrates as a single protein band on polyacrylamide gel electrophoresis under nondenaturing conditions. The sedimentation coefficient of the enzyme is 7.9 S, and the Stokes radius is 53 A. A molecular weight of 173K has been calculated for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the homogeneous enzyme reveals two polypeptides of 125 and 48 kDa. This subunit structure differs from that of DNA polymerase delta prepared by our previous procedure, which was composed of subunits of 60 and 49 kDa [Lee, M. Y. W. T., Tan, C.-K., Downey , K. M., & So, A. G. (1981) Prog . Nucleic Acid Res. Mol. Biol. 26, 83-96], suggesting that the 60-kDa polypeptide may have been derived from the 125-kDa polypeptide during enzyme purification, possibly as the result of cleavage of an unusually sensitive peptide bond. DNA polymerase delta is separated from DNA polymerase alpha by hydrophobic interaction chromatography on phenyl-Sepharose; DNA polymerase delta is eluted at pH 7.2 and DNA polymerase alpha at pH 8.5. DNA polymerase delta can also be separated from DNA polymerase alpha by chromatography on hydroxylapatite; DNA polymerase alpha binds to hydroxylapatite in the presence of 0.5 M KCl, whereas DNA polymerase delta is eluted at 90 mM KCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Effect of base-pair stability of nearest-neighbor nucleotides on the fidelity of deoxyribonucleic acid synthesis.

The influence of the stability of base pairs formed by nearest-neighbor nucleotides on misincorporation frequency has been studied with the large fragment of DNA polymerase I, the alternating DNA copolymers, poly(dI-dC) and poly-(dG-dC), as template-primers, and dGTP, dITP, and dCTP as substrates. We have utilized the difference in thermodynamic stability between the doubly H-bonded I X C base pair and triply H-bonded G X C base pair to examine the effects of base-pair stability of both the "preceding" and the "following" nucleotides on the frequency of insertion of a mismatched nucleotide, as well as on its stable incorporation into polynucleotide. The present studies demonstrate that the stability of the base pairs formed by nearest-neighbor nucleotides affects the frequency of incorporation of noncomplementary nucleotides. Misincorporation frequency is increased when the nearest-neighbor nucleotides form more stable base pairs with the corresponding nucleotides in the template and is decreased when they form less stable base pairs. The stability of the base pair formed by a nucleotide either preceding (5' to) or following (3' to) a misincorporated nucleotide influences misincorporation frequency, but by different mechanisms. The stability of base pairs formed by preceding nucleotides affects the rate of insertion of mismatched nucleotide but does not protect the mismatched nucleotide from removal by the 3' to 5' exonuclease activity. In contrast, the stability of a base pair formed by a following nucleotide determines whether a misincorporated nucleotide is extended or excised by affecting the ability of the enzyme to edit errors of incorporation.

Base Composition↗
PubMed1984

Molecular mechanisms of manganese mutagenesis.

The mechanism by which DNA polymerase discriminates between complementary and noncomplementary nucleotides for insertion into a primer terminus has been investigated. Apparent kinetic constants for the insertion of dGTP and dATP into the hook polymer d(C)194-d(G)12 with Escherichia coli DNA polymerase I (large fragment) were determined. The results suggest that the high specificity of base selection by DNA polymerase I is achieved by utilization of both Km and Vmax differences between complementary and noncomplementary nucleotides. The molecular basis for the increased error frequency observed with DNA polymerase I in the presence of Mn2+ has also been investigated. Our studies demonstrate that when Mn2+ is substituted for Mg2+, there is a higher ratio of insertion of incorrect to correct dNTP by the polymerase activity, accompanied by a decreased hydrolysis of a mismatched dNMP relative to a matched dNMP at the primer terminus by the 3',5' exonuclease activity. Kinetic analysis revealed that in the presence of Mn2+, the kcat for insertion of a complementary dNTP is reduced, whereas the catalytic rate for the insertion of a mismatched nucleotide is increased. The apparent Km values for either complementary or noncomplementary nucleotide substrates are not significantly altered when Mg2+ is replaced by Mn2+. The rate of hydrolysis of a mismatched dNMP at the primer terminus is greater in the presence of Mg2+ vs. Mn2+, whereas the rate of hydrolysis of a properly base-paired terminal nucleotide is greater in Mn2+ vs. Mg2+. These studies demonstrate that both the accuracy of base selection by the polymerase activity and the specificity of hydrolysis by the 3',5' exonuclease activity are altered by the substitution of Mn2+ for Mg2+.

DNA Polymerase I↗
PubMed1983

Danazol for the treatment of idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura is an autoimmune disorder, most common in young women. We treated 22 patients with this disorder (12 of whom were women) with danazol, an androgen with reduced virilizing capability, for two months or longer. Fifteen had undergone splenectomy, all were receiving glucocorticoids, and 18 had also been given other treatments. Fifteen of the patients were benefited, 11 with sustained normalization of their platelet counts. Six of eight patients tested had initial increases in circulating platelet-reactive IgG; in all six there was a marked decrease concomitant with danazol therapy. Danazol was effective in both men and women, irrespective of previous treatments. The duration of remissions ranged from 2 to 13 months. The drug was well tolerated and appears to be better suited than glucocorticoids for long-term management of idiopathic thrombocytopenic purpura, but the exact indications for the use of danazol in this disorder remain to be determined.

Adult↗
PubMed1983

Treatment of idiopathic thrombocytopenic purpura.

ITP is a common disease that is sorely in need of better management. Treatment strategy requires consideration of both long-term benefits and long-term hazards of each available therapeutic option. This discussion reviews conventional therapy as well as newer approaches to refractory ITP, including immunosuppressants, vinca alkaloids, colchicine, androgens, tamoxifen, and plasmapheresis.

Adult↗