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)

DNA amplification systems are powerful technologies with the potential to impact a wide range of diagnostic applications. In this study we explored the feasibility and limitations of a modified ligase chain reaction (Gap-LCR) in detection and discrimination of DNAs that differ by a single base. LCR is a DNA amplification technology based on the ligation of two pairs of synthetic oligonucleotides which hybridize at adjacent positions to complementary strands of a target DNA. Multiple rounds of denaturation, annealing and ligation with a thermostable ligase result in the exponential amplification of the target DNA. A modification of LCR, Gap-LCR was developed to reduce the background generated by target-independent, blunt-end ligation. In Gap-LCR, DNA polymerase fills in a gap between annealed probes which are subsequently joined by DNA ligase. We have designed synthetic DNA targets with single base pair differences and analyzed them in a system where three common probes plus an allele-specific probe were used. A single base mismatch either at the ultimate 3' end or penultimate 3' end of the allele specific probe was sufficient for discrimination, though better discrimination was obtained with a mismatch at the penultimate 3' position. Comparison of Gap-LCR to allele-specific PCR (ASPCR) suggested that Gap-LCR has the advantage of having the additive effect of polymerase and ligase on specificity. As a model system, Gap-LCR was tested on a mutation in the reverse transcriptase gene of HIV, specifically, one of the mutations that confers AZT resistance. Mutant DNA could be detected and discriminated in the presence of up to 10,000-fold excess of wild-type DNA.
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PMID:Detection of point mutations with a modified ligase chain reaction (Gap-LCR). 753 8

A 3'-->5' exonuclease has been highly purified from the cytosol of human acute lymphoblastic leukemia H9 cells. The apparent molecular weight of this enzyme was approximately 50,000, as indicated by its sedimentation in glycerol gradients. The exonuclease did not copurify with DNA polymerase activity, required MgCl2 for its exonucleolytic activity, and was inhibited by KCl above 60 mM. The enzyme was active on single-stranded DNA, DNA duplexes and DNA/RNA duplexes, and it was efficient at removing 3'-terminal mispairs from DNA. The products of the exonucleolytic reaction were deoxynucleoside 5'-monophosphates. The behavior of the exonuclease was examined on DNA terminated at the 3' end with a variety of dideoxynucleosides that are potent against human immunodeficiency virus type 1. The exonuclease has a broad substrate specificity; however, the rate of the enzymatic reaction varied among the D dideoxynucleosides tested (ddAMP = ddCMP > d4TMP > AZTMP). Similarly, the enzyme was examined for its reactivity with DNA terminated by either the D or L enantiomers of ddC, SddC or FddC. The removal of analogs with the native D configuration was at least 6-fold more rapid than that of the L-compounds, and the type of structural modification had an impact on the rate at which the D enantiomers were removed (SddCMP > ddCMP > FddCMP). The monophosphate forms of AZT, D4T, L-FddC and L-ddC were potent inhibitors of the exonuclease at micromolar concentrations, while D-ddCMP partially inhibited the enzyme at millimolar concentrations. Based on its physical and enzymatic properties, this exonuclease represents a novel enzyme that may have an important role in determining the relative potencies of dideoxynucleosides against human immunodeficiency virus type 1.
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PMID:Removal of anti-human immunodeficiency virus 2',3'-dideoxynucleoside monophosphates from DNA by a novel human cytosolic 3'-->5' exonuclease. 757 43

The inhibitory effects of the diphosphate of (S)-1-(3-hydroxy-2-phosphonylmethoxy-propyl)cytosine (HPMPCpp) toward human DNA polymerases beta and gamma were studied. The Ki values of HPMPCpp were compared with the Ki values of the triphosphates of 3'-azidothymidine (AZTTP), 2',3'-dideoxycytidine (ddCTP) and 5-iodo-2'-fluoroarabinosyluridine (FIAUTP). The Ki values toward DNA polymerase beta in increasing order were 1.32, 1.43, 140, and 520 microM for ddCTP, FIAUTP, AZTTP and HPMPCpp, respectively. The Ki values toward DNA polymerase gamma in increasing order were 0.034, 0.031, 18.3 and 299 microM for ddCTP, FIAUTP, AZTTP and HPMPCpp, respectively. Therefore, HPMPC would be expected to have less inhibitory effects on DNA repair (DNA polymerase beta) and mitochondrial DNA synthesis (DNA polymerase gamma) than ddC, FIAU or AZT.
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PMID:Kinetic analysis of the interaction between the diphosphate of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, ddCTP, AZTTP, and FIAUTP with human DNA polymerases beta and gamma. 798 13

The human immunodeficiency virus (HIV), the human T cell lymphotropic virus (HTLV-1), the human foamy retrovirus and the simian immunodeficiency viruses have been associated with the development of an inflammatory myopathy in humans and primates. The myopathy caused by HIV and HTLV-1 is not due to direct infection of the muscle by these viruses, but rather due to an immunopathologic process triggered by the viruses, mediated by autoaggressive CD8+ cells in the context of MHC-class I antigen expression. This has been based on a series of studies utilizing immunocytochemistry, in situ hybridization, polymerase chain reaction, and co-cultivation of human myotubes with the viruses or with HIV-1 and HTLV-1-infected homologous lymphoid cells. Because the clinical, histological and immunological picture of patients with retroviral-associated inflammatory myopathies is identical to that of patients with retroviral-negative inflammatory myopathy, there is a reasonable possibility that retroviruses may be candidate viruses in triggering inflammatory myopathies. In recent years, the antiretroviral drug AZT (Zidovudine), commonly used for the treatment of AIDS, has been shown to cause a distinct mitochondrial myopathy characterized by depletion of the muscle mitochondrial DNA due to AZT's ability to inhibit the gamma-DNA polymerase of the mitochondrial matrix. Distinction of the AZT-myopathy is clinically important because it responds to discontinuation of AZT and to administration of another antiretroviral agent such as ddI or ddC.
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PMID:Retroviruses and inflammatory myopathies in humans and primates. 815 47

Zidovudine (azidothymidine [AZT]) inhibits human immunodeficiency virus replication and reduces the severity of acquired immunodeficiency syndrome. A limiting side effect of AZT is a mitochondrial cardiac and skeletal myopathy in which the pharmacologically active derivative of AZT (AZT triphosphate) plays a critical role. The present study determined biochemical mechanisms of AZT-induced mitochondrial toxicity and identified AZT triphosphate as an inhibitor of DNA polymerase-gamma in vitro. Inhibition kinetics were defined using purified bovine cardiac mitochondrial DNA polymerase-gamma and AZT triphosphate in vitro. The Km for deoxythymidine triphosphate was 0.8 +/- 0.3 mumol/L. AZT triphosphate incubation with DNA polymerase-gamma in vitro resulted in mixed kinetics with a competitive Ki of 1.8 +/- 0.2 mumol/L and a noncompetitive Ki' of 6.8 +/- 1.7 mumol/L. These Ki and Ki' values were strikingly higher than values for retroviral reverse transcriptase but lower than values for other cellular DNA polymerases. These data support previous molecular and morphological findings in clinical AZT mitochondrial myopathy and in models of AZT myopathy in vivo. Biochemical findings suggest that inhibition of mitochondrial DNA polymerase-gamma may be integral to the pathogenesis of AZT-induced myopathy.
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PMID:Cardiac mitochondrial DNA polymerase-gamma is inhibited competitively and noncompetitively by phosphorylated zidovudine. 829 72

In the present study, templates containing a specific segment of the G gamma-globin gene were constructed and incorporation of 3'-azido-3'-deoxythymidine 5'-triphosphate (AZT-TP) or 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) into these templates was compared to that observed in M13 bacteriophage DNA. Investigations on the intrinsic fidelity of T7 DNA polymerase in reactions with AZT-TP and without deoxythymidine 5'-triphosphate, resulted in DNA synthesis beyond the first T site, suggesting that other normal deoxynucleotides misincorporated at these T sites. Modified T7 DNA polymerase incorporated AZT-TP into T sites of elongating DNA strands. Chain termination at noncomplementary sites was also observed with AZT-TP when a genomic DNA template was used and interestingly, this phenomenon was not detected in the presence of a M13 DNA template. These DNA template-dependent effects were not detected with either ddTTP, 2',3'-dideoxycytidine 5'-triphosphate, or 2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate (D4T-TP). A variation in the extent of chain termination at T sites was observed with D4T-TP suggesting that each 2',3'-dideoxynucleoside may exhibit unique chain termination patterns along with the template sequence.
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PMID:Influence of template primary structure on 3'-azido-3'-deoxythymidine triphosphate incorporation into DNA. 833 52

When crystals of human DNA polymerase beta (pol beta) complexed with DNA [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742-12761] are soaked in the presence of dATP and Mn2+, X-ray structural analysis shows that nucleotidyl transfer to the primer 3'-OH takes place directly in the crystals, even though the DNA is blunt-ended at the active site. Under similar crystal-soaking conditions, there is no evidence for a reaction when Mn2+ is replaced by Mg2+, which is thought to be the divalent metal ion utilized by most polymerases in vivo. These results suggest that one way Mn2+ may manifest its mutagenic effect on polymerases is by promoting greater reactivity than Mg2+ at the catalytic site, thereby allowing the nucleotidyl transfer reaction to take place with little or no regard to instructions from a template. Non-template-directed nucleotidyl transfer is also observed when pol beta-DNA cocrystals are soaked in the presence of dATP and Zn2+, but the reaction products differ in that the sugar moiety of the incorporated nucleotide appears distorted or otherwise cleaved, in agreement with reports that Zn2+ may act as a polymerase inhibitor rather than as a mutagen [Sirover, M. A., & Loeb, L. A. (1976) Science 194, 1434-1436]. Although no reaction is observed when crystals are soaked in the presence of dATP and other metal ions such as Ca2+, Co2+, Cr3+, or Ni2+, X-ray structural analyses show that these metal ions coordinate the triphosphate moiety of the nucleotide in a manner that differs from that observed with Mg2+. In addition, all metal ions tested, with the exception of Mg2+, promote a change in the side-chain position of aspartic acid 192, which is one of three highly conserved active-site carboxylate residues. Soaking experiments with nucleotides other than dATP (namely, dCTP, dGTP, dTTP, ATP, ddATP, ddCTP, AZT-TP, and dATP alpha S) reveal a non-base-specific binding site on pol beta for the triphosphate and sugar moieties of a nucleotide, suggesting a possible mechanism for nucleotide selectivity whereby triphosphate-sugar binding precedes a check for correct base pairing with the template.
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PMID:A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta. 884 Nov 19

Zidovudine (AZT), didanosine (ddI) and zalcitabine (ddC) are the reference antiretroviral therapy in patients with AIDS. A toxic mitochondrial myopathy can be observed in patients treated with AZT, but not with ddI and ddC. All 3 compounds can inhibit mitochondrial (mt)DNA polymerase and cause termination of synthesis of growing mtDNA strands and mtDNA depletion. The propensity to injure particular target tissues is unexplained. In our work, cultured muscle cells prepared from human muscle biopsies, were exposed to various concentrations of AZT (4-5000 micromol/l), ddI (5-1000 micromol/l) and ddC (1-1000 micromol/l) for 10 days. We evaluated cell proliferation and differentiation and measured lipid droplet accumulation, lactate production and respiratory chain enzyme activities. All 3 compounds induced a dose-related decrease of cell proliferation and differentiation. AZT seemed to be the most potent inhibitor of cell proliferation. AZT, ddI and ddC induced cytoplasmic lipid droplet accumulations, increased lactate production and decreased activities of COX (complex IV) and SDH (part of complex II). NADHR (complex I) and citrate sinthase activities were unchanged. Zalcitabine (ddC) and, to a lesser extent, ddI, were the most potent inhibitors of mitochondrial function. In conclusion, AZT, ddI and ddC all exert cytotoxic effects on human muscle cells and induce functional alterations of mitochondria possibly due to mechanisms other than the sole mtDNA depletion. Our results provide only a partial explanation of the fact that AZT, but not ddI and ddC, can induce a myopathy in HIV-infected patients. AZT myopathy might not simply result from a direct mitochondrial toxic effect of crude AZT.
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PMID:Cellular and mitochondrial toxicity of zidovudine (AZT), didanosine (ddI) and zalcitabine (ddC) on cultured human muscle cells. 916 61

1. The subject of this review is the interaction between AZT (zidovudine) and mitochondria as described in papers dealing with AZT therapy both in AIDS patients and in model systems--that is, in cultured cells and in isolated mitochondria. 2. The structure and function of mitochondria are briefly described with discussion of the theoretical frame for a detailed bioenergetic investigation. 3. Experimental work is reported showing that mitochondria are cell AZT targets: changes in the structure and function induced by long-term AZT therapy as investigated both in AIDS patients and in model systems. 4. The AZT inhibition of energy-supplying reactions is considered in detail in studies dealing with long-term treatment and studies in which AZT was added to isolated mitochondria. In particular, adenylate kinase, ADP/ATP translocase and DNA polymerase gamma are reported as molecular targets of AZT. 5. Some perspectives of AZT therapy from the study of the effect of AZT on mitochondrion biochemistry are briefly reported.
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PMID:Mitochondria as cell targets of AZT (zidovudine). 979 11

The multiple mutations associated with high-level AZT resistance (D67N, K70R, T215F, K219Q) arise in two separate subdomains of the viral reverse transcriptase (RT), suggesting that these mutations may contribute differently to overall resistance. We compared wild-type RT with the D67N/K70R/T215F/K219Q, D67N/K70R, and T215F/K219Q mutant enzymes. The D67N/K70R/T215F/K219Q mutant showed increased DNA polymerase processivity; this resulted from decreased template/primer dissociation from RT, and was due to the T215F/K219Q mutations. The D67N/K70R/T215F/K219Q mutant was less sensitive to AZTTP (IC50 approximately 300 nM) than wt RT (IC50 approximately 100 nM) in the presence of 0.5 mM pyrophosphate. This change in pyrophosphate-mediated sensitivity of the mutant enzyme was selective for AZTTP, since similar Km values for TTP and inhibition by ddCTP and ddGTP were noted with wt and mutant RT in the absence or in the presence of pyrophosphate. The D67N/K70R/T215F/K219Q mutant showed an increased rate of pyrophosphorolysis (the reverse reaction of DNA synthesis) of chain-terminated DNA; this enhanced pyrophosphorolysis was due to the D67N/K70R mutations. However, the processivity of pyrophosphorolysis was similar for the wild-type and mutant enzymes. We propose that HIV-1 resistance to AZT results from the selectively decreased binding of AZTTP and the increased pyrophosphorolytic cleavage of chain-terminated viral DNA by the mutant RT at physiological pyrophosphate levels, resulting in a net decrease in chain termination. The increased processivity of viral DNA synthesis may be important to enable facile HIV replication in the presence of AZT, by compensating for the increased reverse reaction rate.
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PMID:Phenotypic mechanism of HIV-1 resistance to 3'-azido-3'-deoxythymidine (AZT): increased polymerization processivity and enhanced sensitivity to pyrophosphate of the mutant viral reverse transcriptase. 984 96


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