Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) was compared with dATP as a substrate for DNA synthesis by bacterial and viral DNA polymerases in vitro. Lengths of chain extension and DNA synthesis pause sites were determined by comparison with products generated by dideoxynucleotide sequencing methods on the same end-labeled primer/template duplex after high-resolution polyacrylamide gel electrophoresis. Reverse transcriptase (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) incorporated CldATP efficiently. DNA strand elongation continued past most chloroadenine (ClA) insertion sites but resulted in shorter chains than when dATP was inserted. Phage T4 DNA polymerase incorporated CldATP least efficiently; Klenow fragment of Escherichia coli DNA polymerase I and modified T7 DNA polymerase (Sequenase) showed intermediate ability to utilize the analogue. Incorporation of several consecutive ClA residues into the replicating strand dramatically reduced the ability of Sequenase, Klenow fragment, and T4 DNA polymerases to continue strand elongation. In the absence of the corresponding normal deoxyribonucleoside triphosphate during DNA synthesis, ClA was frequently misincorporated as thymine, cytosine, or guanine by both AMV RT and HIV-1 RT but rarely, if at all, by Klenow fragment, Sequenase, and T4 DNA polymerase. Except T4, for most DNA polymerases, CldATP at 10-20-fold molar excess over dATP was not a strong competitive inhibitor of dATP, as judged by the amount of strand extension and polymerase pause sites during DNA synthetic reactions. Our results indicate that the degree of strand extension in the presence of CldATP, the number and location of polymerase pause sites, and the amount of misincorporation of the analogue are both polymerase- and sequence-dependent.
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PMID:Effects of 2-chloro-2'-deoxyadenosine 5'-triphosphate on DNA synthesis in vitro by purified bacterial and viral DNA polymerases. 170 19

We investigated the inhibitory effects of aurochloric acid (AuCl4H) on reverse transcriptase (RT) derived from avian myeloblastosis virus and DNA polymerase alpha (pol. alpha) purified from HeLa S3 cells. The activities of RT, pol. alpha and E. coli DNA polymerase I (pol. I) with dTTP as the substrate were inhibited 50% at AuCl4H concentrations of 18 microM, 43 microM and 230 microM, respectively. AuCl4H inhibited RT activity competitively with respect to the substrate, dTTP, and uncompetitively with the template/primer, (rA)n(dT)12-18. In assays with dGTP as the substrate, 50% inhibitions of RT, pol. alpha and pol. I activities were observed at AuCl4H concentrations of 100 microM, 450 microM and 580 microM, respectively. AuCl4H inhibited RT activity uncompetitively with respect to the substrate, dGTP, and noncompetitively with the template/primer, (rC)n(dG)12-18. AuCl4H at concentrations causing more than 50% inhibition of RT activity had little inhibitory effect on the colony-forming ability of HeLa cells or their syntheses of DNA, RNA and protein.
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PMID:Inhibition of avian myeloblastosis virus reverse transcriptase by aurochloric acid. 170 21

We have examined the properties of reverse transcriptases (RTs) required for strand transfer synthesis on poly(rA). In this process, a primer is elongated on one template and then switches to other templates for additional elongation until it is much longer than the templates on which it was made. Models of retrovirus replication require the RT to catalyze two distinct strand transfers. Additionally, they propose that the RT ribonuclease H (RNase H) activity is involved in both transfers. RTs from human immunodeficiency virus (HIV), avian myeloblastosis virus, and murine leukemia virus differ in molecular mass and subunit composition. However, they all catalyzed strand transfer synthesis on (rA)300, generating characteristically long products. An RNase H-deficient enzyme, HIV-RTRD, catalyzed strand transfer synthesis to the same degree as native HIV-RT, indicating that a functional RNase H activity is not required. Additionally, N-ethylmaleimide, which inhibits RNase H but not polymerase activity of HIV-RT, did not diminish strand transfer synthesis. Highly processive DNA synthesis by each RT was found to be required for the strand transfer reaction. RNase H- murine leukemic virus RT has a structural modification that not only eradicates RNase H, but also makes the polymerase much less processive for DNA synthesis. However, conditions that allow this modified enzyme to bind repeatedly to the same primer during synthesis, i.e. conditions that simulate higher processivity, allow strand transfer synthesis. Catalysis of strand transfer synthesis is not a property of all DNA polymerases, since the Klenow fragment of Escherichia coli DNA polymerase I is unable to catalyze this reaction even if high processivity is simulated. These results suggest that strand transfer synthesis relies on an unidentified functional activity present in RTs.
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PMID:Requirements for the catalysis of strand transfer synthesis by retroviral DNA polymerases. 171 74

Psychotrine dihydrogen oxalate and O-methylpsychotrine sulfate heptahydrate (MP), the salts of isoquinoline alkaloids from ipecac, were found to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT). We currently report the results of additional studies designed to characterize the mechanism of inhibition facilitated by MP. The inhibition was noncompetitive with respect to TTP and uncompetitive with respect to poly(rA) and oligo(dT)12-18 (4:1) at low template-primer concentrations but competitive at high concentrations (greater than 200 microM). Identical non-Michaelis-type kinetics were observed when activated DNA was used as the template. The biphasic nature of the double-reciprocal plots and Hill coefficients of less than 1 indicate that MP functions as an allosteric inhibitor of the enzyme which appears to possess multiple active sites that interact in a cooperative (negative) fashion in the presence of the inhibitor. MP was selective for the recombinant HIV-1 RT (p66) utilizing poly(rA) and oligo(dT)12-18 (4:1) as template-primer. Greater inhibition was observed with this template primer as compared with other natural and synthetic template-primers tested. MP had significantly less effect on avian myeloblastosis virus RT as well as mammalian or bacterial DNA and RNA polymerases. Other members of the ipecac class of alkaloids, e.g. emetine hydrochloride, were inactive against all of these enzymes, including HIV-1 RT. Conversely, MP did not inhibit in vitro protein synthesis, a property manifested by all the other ipecac alkaloids tested. Studies conducted with structural analogs revealed that the imine functionality at positions 1' and 2' of MP is the key structural requirement for HIV-1 RT inhibitory activity. Therefore, MP appears to possess unique structural properties that enable interaction with HIV-1 RT in a manner that can be differentiated from other polymerases. Use of these alkaloids for the definition of this viral enzyme-specific topology may lead to the development of therapeutically useful chemotherapeutic agents.
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PMID:Psychotrine and its O-methyl ether are selective inhibitors of human immunodeficiency virus-1 reverse transcriptase. 172 Oct 50

3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta, gamma-diphosphate (I) and 2'-deoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma-diphosphate (II) were synthesised. Reverse transcriptases of HIV and avian myeloblastosis virus, rat liver DNA polymerase beta, calf thymus terminal deoxynucleotidyl transferase and E. coli DNA polymerase I KF incorporated both compounds into the growing DNA chain, KF being the least effective. Compound I revealed termination substrate properties, but II was repeatedly incorporated into the DNA chain, for example, by HIV reverse transcriptase - up to 8 residues. Human placenta DNA polymerases alpha and epsilon incorporated neither I nor II into the DNA chain, although DNA synthesis, catalyzed by all the investigated enzymes, was inhibited in the presence of I or II and compound II was a more effective inhibitor then I. The DNA fragments containing alpha-phosphonomethyl groups were hydrolyzed by 3'----5' exonuclease of DNA polymerase I and not hydrolyzed by ExoIII from E. coli.
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PMID:[Formation of phosphonoester bonds, catalyzed by DNA polymerases]. 172 22

A quantitative and efficient assay was developed to measure the 3'-OH terminal DNA endonuclease activity of the avian myeloblastosis virus (AMV) integrase protein. A retroviral-like linearized plasmid containing long terminal repeat (LTR) sequences at its recessed 3'-OH termini was filled in and labeled with the Escherichia coli Klenow DNA polymerase fragment. The 32P-labeled nucleotide was located at the penultimate position. The labeled linearized plasmid or restriction fragments derived from it were incubated with AMV IN and release of the label was quantitated by conversion to acid-soluble counts. The structure of the released product was characterized on 23% sequencing gels. Results indicate that AMV integration protein is functioning as an endonuclease releasing a dinucleotide and that the activity is stoichiometric with a preference for the cleavage of the U3 LTR terminus over that of the U5 LTR terminus.
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PMID:Development of an acid-soluble assay for measuring retrovirus integrase 3'-OH terminal nuclease activity. 188 32

2',3'-dideoxy-2',3'-dehydrothymidine 5'-triphosphate (dddTTP) shows termination substrate properties in the DNA synthesis catalyzed by E. coli DNA polymerase I KF, rat liver DNA polymerase beta, reverse transcriptases of avian myeloblastosis virus and Raus sarcoma virus and calf thymus terminal deoxynucleotidyl transferase. This implies that the mononucleotide residue of dddTTP incorporates into 3'-termini of newly synthesized DNA chains. However, dddTTP has no influence on the DNA synthesis catalyzed by calf thymus DNA polymerase alpha. In the case of some DNA polymerases dddTTP was one order of magnitude more effective in comparison with the other known termination substrates.
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PMID:Properties of 2',3'-dideoxy-2',3'-dehydrothymidine 5'-triphosphate in terminating DNA synthesis catalyzed by several different DNA polymerases. 243 82

A synthetic procedure has been developed by which stable abasic sites are introduced into oligodeoxynucleotides at any desired position in the sequence. A modified tetrahydrofuran moiety, isosteric with 2'-deoxyribofuranose, serves as a structural analog of the natural apurinic/apyrimidinic site. We have also prepared oligodeoxynucleotides that lack cyclic structure at the abasic site but retain the carbon atoms of the phosphodiester backbone. These synthetic oligodeoxynucleotides are cleaved on the 5' side of the abasic site by endonuclease IV and by exonuclease III; they serve also as templates for avian myeloblastosis virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow fragment), and calf thymus DNA polymerase-alpha. Extension of primed templates by these DNA polymerases is blocked initially at the position immediately 3' to the abasic site; nucleoside monophosphates are subsequently incorporated opposite the lesion. The nucleotide most frequently incorporated opposite all abasic sites, regardless of structure, is dAMP. Significant "readthrough" at the abasic site was observed in experiments using avian myeloblastosis virus reverse transcriptase and DNA polymerase-alpha and, to a much lesser degree, with DNA polymerase I. We conclude that a modified tetrahydrofuran group can serve as a stable structural analog of 2'-deoxyribose in the apurinic/apyrimidinic site. These modified oligodeoxynucleotides should prove useful for studies of chemical mutagenesis.
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PMID:Oligodeoxynucleotides containing synthetic abasic sites. Model substrates for DNA polymerases and apurinic/apyrimidinic endonucleases. 244 Aug 61

3-Methylthymine was synthesized into DNA copolymers and deoxynucleoside triphosphate to study its effect on DNA synthesis by the Klenow fragment of Escherichia coli polymerase I and avian myeloblastosis virus reverse transcriptase. Both polymerases were greatly inhibited by template 3-methylthymine. In response to 3-methylthymine, misincorporation of dTTP increased slightly, but occurred only at low levels consistent with spontaneous misincorporation in vitro. Surprisingly, template 3-methylthymine resulted in a striking decrease in background misincorporation, relative to normal incorporation by the Klenow fragment, of dGTP and, to a lesser extent, of dATP and dCTP. The incorporation of 3-methyl-dTTP into DNA was studied using DNA sequencing technology. The Klenow fragment failed to incorporate 3-methyl-dTTP even at 1 mM. Reverse transcriptase incorporated 3-methyl-dTTP opposite adenine, cytosine, and thymine, but at only about 1/40,000th the efficiency of complementary deoxynucleoside triphosphate incorporation. Furthermore, synthesis generally stalled at sites of 3-methyl-thymine incorporation. From these results, we conclude that damage at the central hydrogen-bonding position of thymine abolishes its base-pairing capabilities during DNA synthesis.
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PMID:DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis. 244 69

An aqueous extract from the marine red alga, Schizymenia pacifica has been tested in a cell free system for its effect on reverse transcriptase from avian retrovirus (avian myeloblastosis virus), and mammalian retrovirus (Rauscher murine leukemia virus). The extract inhibited reverse transcriptase from both these retroviruses but showed almost no effect, if any, on the activity of cellular DNA polymerase alpha and RNA polymerase II in vitro. Consequently it is unlikely to have an adverse effect on the growth of cultured cell. The inhibitory activity of the extract was stable over a relatively wide pH range (pH 1-11) and was not lost after pronase digestion. Inhibitory activity of the extract was lost after boiling at 100 degrees C in 0.67 N HCl, and after treatment with 100 mM NaIO4. The active principle in the extract has an apparent molecular weight in excess of 100,000 daltons. This new reverse transcriptase inhibitor is probably a polysaccharide.
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PMID:Antiretroviral activity in a marine red alga: reverse transcriptase inhibition by an aqueous extract of Schizymenia pacifica. 244 71


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