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• W2077364814 abstract "Mitochondrial toxicity can result from antiviral nucleotide analog therapy used to control human immunodeficiency virus type 1 infection. We evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase (pol γ) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. Apparent steady-stateKm and kcat values for insertion of 2′,3′-dideoxy-TTP (ddTTP), 3′-azido-TTP (AZT-TP), 2′,3′-dideoxy-CTP (ddCTP), 2′,3′-didehydro-TTP (D4T-TP), (-)-2′,3′-dideoxy-3′-thiacytidine (3TC-TP), and carbocyclic 2′,3′-didehydro-ddGTP (CBV-TP) indicated incorporation of all six analogs, albeit with varying efficiencies. Dideoxynucleotides and D4T-TP were utilized by pol γ in vitro as efficiently as natural deoxynucleotides, whereas AZT-TP, 3TC-TP, and CBV-TP were only moderate inhibitors of DNA chain elongation. Inefficient excision of dideoxynucleotides, D4T, AZT, and CBV from DNA predicts persistencein vivo following successful incorporation. In contrast, removal of 3′-terminal 3TC residues was 50% as efficient as natural 3′ termini. Finally, we observed inhibition of exonuclease activity by concentrations of AZT-monophosphate known to occur in cells. Thus, although their greatest inhibitory effects are through incorporation and chain termination, persistence of these analogs in DNA and inhibition of exonucleolytic proofreading may also contribute to mitochondrial toxicity. Mitochondrial toxicity can result from antiviral nucleotide analog therapy used to control human immunodeficiency virus type 1 infection. We evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase (pol γ) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. Apparent steady-stateKm and kcat values for insertion of 2′,3′-dideoxy-TTP (ddTTP), 3′-azido-TTP (AZT-TP), 2′,3′-dideoxy-CTP (ddCTP), 2′,3′-didehydro-TTP (D4T-TP), (-)-2′,3′-dideoxy-3′-thiacytidine (3TC-TP), and carbocyclic 2′,3′-didehydro-ddGTP (CBV-TP) indicated incorporation of all six analogs, albeit with varying efficiencies. Dideoxynucleotides and D4T-TP were utilized by pol γ in vitro as efficiently as natural deoxynucleotides, whereas AZT-TP, 3TC-TP, and CBV-TP were only moderate inhibitors of DNA chain elongation. Inefficient excision of dideoxynucleotides, D4T, AZT, and CBV from DNA predicts persistencein vivo following successful incorporation. In contrast, removal of 3′-terminal 3TC residues was 50% as efficient as natural 3′ termini. Finally, we observed inhibition of exonuclease activity by concentrations of AZT-monophosphate known to occur in cells. Thus, although their greatest inhibitory effects are through incorporation and chain termination, persistence of these analogs in DNA and inhibition of exonucleolytic proofreading may also contribute to mitochondrial toxicity. human immunodeficiency virus mitochondrial DNA DNA polymerase γ 3′-azido-3′-deoxythymidine 2′,3′-dideoxycytidine 2′,3′-dideoxyinosine 2′,3′-didehydro-3′-deoxythymidine (-)-2′,3′-dideoxy-3′-thiacytidine carbovir or carbocyclic 2′,3′-didehydro-2′,3′-dideoxyguanosine terminal deoxynucleotidyltransferase reverse transcriptase More than 36 million people are infected by the human immunodeficiency virus worldwide, where 5.3 million new infections occurred during 2000 (1UNAIDS, AIDS Epidemic Update, 2000, 1, 28, World Health Organization, Geneva, Switzerland.Google Scholar). Although antiviral therapy effectively extends the life of individuals, the death toll continues to rise; 3 million people, the highest number since the epidemic began, died from AIDS in 2000 (1UNAIDS, AIDS Epidemic Update, 2000, 1, 28, World Health Organization, Geneva, Switzerland.Google Scholar). Nucleoside analogs utilized in antiviral therapy are readily incorporated into DNA by the HIV-11 reverse transcriptase. Although viral replication is effectively inhibited by DNA chain terminators, cellular side effects also result. The continuous antiviral therapy required to keep the HIV infection under control has increased the chance for severe antiviral analog induced toxicity. Current antiviral nucleoside analog therapy against HIV clearly results in compromised mitochondrial function due to inhibition of the mitochondrial DNA polymerase (2Kakuda T.N. Clin. Ther. 2000; 22: 685-708Abstract Full Text PDF PubMed Scopus (512) Google Scholar, 3Panel on Clinical Practices for the Treatment of HIV Infection (2001)Guidelines for the Use of Antiretroviral Agents in HIV-infected Adults and Adolescents, http://www.hivatis.org/trtgdlns.html#AdultAdolescent.Google Scholar). AZT was the first analog to be approved for anti-HIV therapy in 1985. In 1990, Dalakas et al. (4Dalakas M.C. Illa I. Pezeshkpour G.H. Laukaitis J.P. Cohen B. Griffin J.L. N. Engl. J. Med. 1990; 322: 1098-1105Crossref PubMed Scopus (745) Google Scholar) were the first to report mitochondrial myopathies in HIV-infected individuals undergoing AZT treatment. Control studies thereafter demonstrated that these induced myopathies, most notably visualized histologically as ragged red fibers, were indeed caused by AZT treatment and were not a consequence of the HIV infection (5Arnaudo E. Dalakas M. Shanske S. Moraes C.T. DiMauro S. Schon E.A. Lancet. 1991; 337: 508-510Abstract PubMed Scopus (466) Google Scholar). This study revealed reduced amounts of mitochondrial DNA in AZT-treated skeletal muscle (5Arnaudo E. Dalakas M. Shanske S. Moraes C.T. DiMauro S. Schon E.A. Lancet. 1991; 337: 508-510Abstract PubMed Scopus (466) Google Scholar). Further clinical evidence has demonstrated that mitochondrial myopathy slowly and cumulatively develops during AZT treatment (6Peters B.S. Winer J. Landon D.N. Stotter A. Pinching A.J. Q. J. Med. 1993; 86: 5-15PubMed Google Scholar). The second class of antiviral nucleoside analogs approved for HIV therapy are the dideoxynucleoside analogs ddI and ddC. These chain terminators also cause toxic side effects by inhibiting mitochondrial function. The use of 2′-3′-dideoxycytidine (ddC) causes a reversible peripheral neuropathy in many patients (7Yarchoan R. Perno C.F. Thomas R.V. Klecker R.W. Allain J.P. Wills R.J. McAtee N. Fischl M.A. Dubinsky R. McNeely M.C. Pluda M.C. Leuther M. Collins J.M. Broder S. Lancet. 1988; 1: 76-81Abstract PubMed Scopus (398) Google Scholar). Treatment of human Molt-4 cells with ddC results in delayed cytotoxicity with a concomitant loss of mitochondrial DNA (8Chen C.H. Cheng Y.C. J. Biol. Chem. 1989; 264: 11934-11937Abstract Full Text PDF PubMed Google Scholar), indicating the cellular target is likely mitochondrial DNA replication. Treatment of human CEM cells with ddC, D4T, and ddI results in a significant decrease of mtDNA and ultrastructural changes of the mitochondria (9Medina D.J. Tsai C.H. Hsiung G.D. Cheng Y.C. Antimicrob. Agents Chemother. 1994; 38: 1824-1828Crossref PubMed Scopus (131) Google Scholar). Both AZT and ddC treatment result in depletion of mitochondrial DNA, and both drugs have been shown to cause an increase in mtDNA deletions (10Wang H. Lemire B.D. Cass C.E. Weiner J.H. Michalak M. Penn A.M. Fliegel L. Biochim. Biophys. Acta. 1996; 1316: 51-59Crossref PubMed Scopus (31) Google Scholar). Mitochondrial DNA is replicated by an assembly of proteins and enzymes including DNA polymerase γ, single-stranded DNA-binding protein, DNA helicase, multiple transcription factors, and a number of accessory proteins (11Shadel G.S. Clayton D.A. Annu. Rev. Biochem. 1997; 66: 409-435Crossref PubMed Scopus (804) Google Scholar). In vitro analysis from several laboratories has demonstrated that among the cellular replicative DNA polymerases, the mitochondrial DNA polymerase γ is the enzyme most sensitive to the antiviral nucleotide analogs currently approved to control HIV-1 infection (12Kaguni L.S. Wernette C.M. Conway M.C. Yang-Cashman P. Eukaryotic DNA Replication. 6. Cold Spring Harbor Press, Cold Spring Harbor, NY1988: 425-432Google Scholar, 13Martin J.L. Brown C.E. Matthews-Davis N. Reardon J.E. Antimicrob. Agents Chemother. 1994; 38: 2743-2749Crossref PubMed Scopus (307) Google Scholar, 14Hart G.J. Orr D.C. Penn C.R. Figueiredo H.T. Gray N.M. Boehme R.E. Cameron J.M. Antimicrob. Agents Chemother. 1992; 36: 1688-1694Crossref PubMed Scopus (145) Google Scholar, 15Parker W.B. White E.L. Shaddix S.C. Ross L.J. Buckheit Jr., R.W. Germany J.M. Secrist IIII, J.A. Vince R. Shannon W.M. J. Biol. Chem. 1991; 266: 1754-1762Abstract Full Text PDF PubMed Google Scholar, 16Lewis W. Simpson J.F. Meyer R.R. Circ. Res. 1994; 74: 344-348Crossref PubMed Scopus (142) Google Scholar, 17Copeland W.C. Chen M.S. Wang T.S. J. Biol. Chem. 1992; 267: 21459-21464Abstract Full Text PDF PubMed Google Scholar, 18Eriksson S. Xu B. Clayton D.A. J. Biol. Chem. 1995; 270: 18929-18934Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 19Nickel W. Austermann S. Bialek G. Grosse F. J. Biol. Chem. 1992; 267: 848-854Abstract Full Text PDF PubMed Google Scholar, 20Huang P. Farquhar D. Plunkett W. J. Biol. Chem. 1990; 265: 11914-11918Abstract Full Text PDF PubMed Google Scholar, 21Huang P. Farquhar D. Plunkett W. J. Biol. Chem. 1992; 267: 2817-2822Abstract Full Text PDF PubMed Google Scholar). As a thymidylate analog, AZT-TP is a competitive inhibitor for dTTP with pol γ (16Lewis W. Simpson J.F. Meyer R.R. Circ. Res. 1994; 74: 344-348Crossref PubMed Scopus (142) Google Scholar). Partially purified human DNA pol γ is strongly inhibited by dideoxynucleotide triphosphates and D4T-TP, whereas AZT-TP, 3TC-TP, and CBV-TP inhibit pol γ to a lesser but significant degree (13Martin J.L. Brown C.E. Matthews-Davis N. Reardon J.E. Antimicrob. Agents Chemother. 1994; 38: 2743-2749Crossref PubMed Scopus (307) Google Scholar). Purified recombinant yeast pol γ can readily incorporate dideoxynucleotides and didehydroCTP, but this enzyme is less efficient in the incorporation of AZT (18Eriksson S. Xu B. Clayton D.A. J. Biol. Chem. 1995; 270: 18929-18934Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). 3TC-TP has also been shown to be a substrate for human DNA pol γ as well as for HIV-RT (22Gray N.M. Marr C.L. Penn C.R. Cameron J.M. Bethell R.C. Biochem. Pharmacol. 1995; 50: 1043-1051Crossref PubMed Scopus (69) Google Scholar). These results clearly show that pol γ is a primary cellular target for analog-induced mitochondrial toxicity. This acquired mitochondrial toxicity may be caused by 1) direct inhibition of DNA pol γ without incorporation, 2) chain termination by incorporation of these analogs into mitochondrial DNA by DNA pol γ, 3) alteration of the fidelity of DNA synthesis by pol γ, 4) the persistence of these analogs in mtDNA due to inefficient excision, or 5) any combination thereof. To understand the mechanism of this acquired mitochondrial toxicity, a thorough understanding of the interaction of nucleotides and analogs with pol γ is needed. Such information may help in the design of nucleotide analogs that selectively inhibit the HIV reverse transcriptase without inducing mitochondrial dysfunction. We and others have cloned and overproduced the catalytic subunit of human DNA polymerase γ in insect cells via a recombinant baculovirus (23Ropp P.A. Copeland W.C. Genomics. 1996; 36: 449-458Crossref PubMed Scopus (241) Google Scholar, 24Longley M.J. Ropp P.A. Lim S.E. Copeland W.C. Biochemistry. 1998; 37: 10529-10539Crossref PubMed Scopus (144) Google Scholar, 25Graves S.W. Johnson A.A. Johnson K.A. Biochemistry. 1998; 37: 6050-6058Crossref PubMed Scopus (77) Google Scholar). In this report, we have determined the insertion efficiency of the currently approved anti-HIV analogs into DNA by purified recombinant human DNA polymerase γ, and we have investigated the efficiency of removing these analogs from DNA by the intrinsic 3′-5′ exonuclease activity of pol γ. Poly(rA)·oligo(dT)12–18, dideoxynucleoside triphosphates, dNTPs, and radioisotopes ([α-32P]dTTP, [α-32P]dGTP, [α-32P]dCTP, and [γ-32P]ATP) were fromAmersham Pharmacia Biotech. Oligonucleotides were purchased from Oligos Etc. or Life Technologies, Inc. dTMP and dTDP were purchased from United States Biochemical Corp. AZT-MP, AZT-DP, and AZT-TP were purchased from Moravek. CBV-TP and the minus enantiomer of 3TC-TP were generous gifts from GlaxoWellcome. D4T-TP was a gift from Triangle Pharmaceuticals, Inc. The recombinant wild type histidine-tagged human DNA polymerase γ (wild type pol γ) was purified from baculoviral-infected insect cells as described (24Longley M.J. Ropp P.A. Lim S.E. Copeland W.C. Biochemistry. 1998; 37: 10529-10539Crossref PubMed Scopus (144) Google Scholar). To make the histidine-tagged exonuclease-deficient DNA pol γ (Exo−pol γ), the wild typeBamH-NotI fragment of pHuγpQE9 (24Longley M.J. Ropp P.A. Lim S.E. Copeland W.C. Biochemistry. 1998; 37: 10529-10539Crossref PubMed Scopus (144) Google Scholar) was replaced with BamHI-NotI fragment of Exo−100/103huγpVL. The EcoRI-NotI fragment of the resulting plasmid was then inserted into the baculovirus transfer vector pVL1393 and resulting recombinant baculovirus, Exo−pQVSL11.4, selected. The Exo−pol γ was purified from Exo−pQVSL11.4 baculoviral infected cells like the wild type pol γ. Reverse transcriptase activity of pol γ was determined using poly(rA)·oligo(dT)12–18 in reactions (50 μl) containing 25 mm Hepes-OH, pH 8.0, 1 mm 2-mercaptoethanol, 50 μg/ml acetylated bovine serum albumin, 0.5 mm MnCl2, 25 μm[α-32P]TTP (2000 cpm/pmol), 75 mm NaCl, 50 μg/ml poly(rA)·oligo(dT)12–18, and 2.5 ng of pol γ as described previously (24Longley M.J. Ropp P.A. Lim S.E. Copeland W.C. Biochemistry. 1998; 37: 10529-10539Crossref PubMed Scopus (144) Google Scholar). One unit is the amount of enzyme required to catalyze the incorporation of 1 pmol of dTMP into trichloroacetic acid-precipitable DNA in 1 h at 37 °C using poly(rA)/oligo(dT). Inhibition of reverse transcriptase activity of pol γ by antiviral nucleotides was determined in this standard assay in the presence of 0.2–1000 nm ddTTP or D4T-TP, or 0.2–438 μmAZT-TP. Exonuclease activity was determined by incubating 0.5 pmol of 5′-32P-end-labeled 18-mer with 0, 10, 40, or 120 ng of wild type pol γ or Exo−pol γ in 25 mm Hepes-OH, pH 7.5, 5 mm 2-mercaptoethanol, 1 μg of acetylated bovine serum albumin, and 5 mm MgCl2 at 37 °C for 30 min. Reaction was terminated at 90 °C for 3 min in 10 μl of formamide loading dye (95% deionized formamide, 0.01 mEDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol). The reaction products were separated on a 20% polyacrylamide-urea gel, and products were visualized and quantified with a Molecular Dynamics PhosphorImager Storm860 and NIH Image 1.61 software. The kinetics of antiviral nucleotide analog insertion into DNA by Exo−pol γ was measured using the gel-based oligonucleotide extension assay (26Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 14689-14696Abstract Full Text PDF PubMed Google Scholar, 27Mendelman L.V. Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1989; 264: 14415-14423Abstract Full Text PDF PubMed Google Scholar) as modified for incorporation of antiviral nucleoside analogs (17Copeland W.C. Chen M.S. Wang T.S. J. Biol. Chem. 1992; 267: 21459-21464Abstract Full Text PDF PubMed Google Scholar). The primer-template sets used for each type of analog follow. TTP analog primer­template set:18­mer:5′­TGA CCA TGT AAC AGA GAG­3′36­mer:3′­ACT GGT ACA TTG TCT CTC,ATT CTC TCT CTC TTC TCT­5′ dCTP analog primer­template set:18­mer:5′­TGA CCA TGT AAC AGA GAG­3′36­mer:3′­ACT GGT ACA TTG TCT CTC,GTT CTC TCT CTC TTC TCT­5′ dGTP analog primer­template set:18­mer:5′­TGA CCA TGT AAC AGA GAG­3′36­mer:3′­ACT GGT ACA TTG TCT CTC,CAG TAG GTT ATG TGT AGA­5′ SEQUENCES1–3 The positions for analog insertion are in bold type. The gel-purified 18-mer was labeled at the 5′ end with [γ-32P]ATP and annealed in a 1:1.4 ratio to the respective templates in 10 mm Tris-HCl, pH 7.5, by heating to 90 °C for 5 min followed by slow cooling to room temperature. One pmol of primer:template (32P-end-labeled primer) was incubated with 1.4 ng of Exo−pol γ (10 fmol of enzyme) in a 10-μl reaction containing 25 mm Hepes-OH, pH 7.5, 2 mm 2-mercaptoethanol, 0.1 mm EDTA, 5 mm MgCl2, 50 μg/ml acetylated bovine serum albumin, and variable dNTP concentrations (3 nm to 1.0 mm). Reactions were incubated at 37 °C for 10 min and stopped on ice by the addition of 10 μl of formamide loading dye. The products were separated on a 15% polyacrylamide-urea gel and visualized as described above. The inhibition of a single nucleotide incorporation was performed with the primer-template sets described above in the same buffer conditions but with 100 nm [α-32P]dCTP, [α-32P]dTTP, or [α-32P]dGTP. Antiviral nucleotide analog triphosphate was added to these reactions as indicated. The products were separated on a 15% polyacrylamide-urea gel and visualized as described above. Ten fmol of32P-end-labeled 38-mer was added to all reactions and used to normalize the products from gel loading error. To produce single-stranded substrates for exonuclease assay with various antiviral analogs at the 3′ terminus, the 18-mer primer was 32P-end-labeled on the 5′ termini with T4 polynucleotide kinase and then extended at the 3′ terminus with the different analogs using terminal deoxynucleotidyltransferase (TdT) in one-Phor-all buffer (Amersham Pharmacia Biotech). The products of the reaction were desalted and purified on a Sephadex G-25 column followed by centrifugation in a Microcon-3 microconcentrator. Purified HIV-RT was used to label the 3′ termini of the 18-mer in the 18/36-mer duplex with analog by incubating with 1 mm of the indicated analog triphosphate for 1 h at 37 °C. The reactions were heat-inactivated and duplex DNA purified on a Sephadex G-25 spin column and washed in a Microcon-3 microconcentrator with six volumes of distilled H2O. To examine pol γ exonuclease activity with these analog-containing primer-templates, 0.2 pmol of32P-end-labeled oligonucleotide containing the designated analog at the 3′ end was incubated with 70–840 fmol of wild type pol γ, as indicated. The exonuclease activity was carried out in 33 mm Hepes-OH, pH 7.5, 13 mm KCl, 1.3 mm DTT, and 3.3 mm MgCl2 at 37 °C for 30 min. The products were separated by denaturing PAGE and visualized as described above. To determine the inhibition of pol γ exonuclease activity by AZT mono- and di- phosphate, 21 fmol of wild type pol γ was incubated with 0.5 pmol of 32P-labeled 18-mer primer in a reaction containing 25 mm Hepes-OH, pH 7.5, 50 μg/ml acetylated bovine serum albumin, 2 mm 2-mercaptoethanol, 0.01 mm EDTA, and 10 mm MgCl2 at 37 °C for 15 min in the presence of nucleotide mono- or diphosphate, as indicated. The reaction was stopped by heat denaturation and separated on a gel as described above. We sought to identify the mechanisms by which AZT-TP, ddCTP, 3TC-TP, D4T-TP, and carbovir-TP inhibit the human DNA polymerase γ. All of these analogs lack the 3′ hydroxyl group and consequently act as chain terminators once incorporated into DNA. For comparison and reference the structures of these analogs are shown in Fig.1. We used the purified recombinant human DNA polymerase γ overproduced in baculovirus-infected insect cells. This recombinant DNA polymerase γ and an exonuclease-deficient catalytic subunit has been characterized previously in our laboratory and shown to possess polymerase properties identical to the native catalytic subunit of DNA polymerase γ (24Longley M.J. Ropp P.A. Lim S.E. Copeland W.C. Biochemistry. 1998; 37: 10529-10539Crossref PubMed Scopus (144) Google Scholar). To simplify the analysis of analog incorporation into DNA without the complication of proofreading, we generated a histidine-tagged exonuclease-deficient DNA polymerase. The specific polymerase activity of both the wild type and exonuclease-deficient histidine-tagged pol γ was 32 units/ng in the poly(rA)/oligo(dT) assay (data not shown). We first addressed the inhibition and incorporation of these antiviral nucleotide analogs into DNA, and then we tested the efficiency of excising analogs from DNA by the 3′-5′ exonuclease activity. As a first approximation, the IC50 values for inhibiting DNA synthesis were determined with two different assays. We designed these assays to specifically measure the ability of analogs to inhibit the incorporation of the cognitive nucleotide. First, the incorporation of a single normal α-32P-labeled dNMP into an 18/36-mer primer-template was assayed in the presence of increasing concentrations of competing antiviral nucleotide analog. Inhibition was monitored by gel electrophoresis and quantified to determined the IC50 concentrations. Graphical results for all five analogs are shown in Fig. 2 A. These results demonstrated that both dideoxycytidine and D4T-TP had strong inhibition profiles, whereas AZT-TP, 3TC-TP, and CBV-TP showed modest inhibition. The IC50 for ddNTP and D4T-TP was 8 and 20 μm, respectively, while 3TC-TP and CBV-TP had an IC50 of 80 μm. The analog AZT-TP had an IC50 of 130 μm. The severity of inhibition is better demonstrated in the second assay, which measures multiple incorporation events. Inhibition by the thymidine analogs was determined here in our standard poly(rA)·oligo(dT) assay using 25 μm dTTP. On this substrate pol γ has a Km for ddTTP of 4.5 μm (28Longley M.J. Prasad R. Srivastava D.K. Wilson S.H. Copeland W.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12244-12248Crossref PubMed Scopus (175) Google Scholar). Results are shown in Fig. 2 B and demonstrate that ddTTP and D4T-TP are potent inhibitors in vitro while AZT-TP required higher levels to inhibit pol γ. Addition of AZT-TP resulted in an IC50 of ∼25 μm, which was also the concentration of normal dTTP in this assay. Dideoxythymidine triphosphate and D4T-TP showed IC50 at ∼15 and 150 nm, respectively, more than 2 orders of magnitude lower than AZT-TP. Given this relative ranking as inhibitors, we wanted to determine the mode of inhibition for each analog. We specifically wanted to determine whether chain termination was the primary mechanism of inhibition or whether inhibition of polymerase activity could occur prior to incorporation of the analog into DNA. Additionally, once incorporated into DNA how efficiently could the analogs be removed by the 3′-5′ exonuclease activity of pol γ? To determine relative efficiencies with which these analogs could be incorporated into DNA, we performed primer extension reactions and analyzed the products by gel electrophoresis. We used the exonuclease-deficient DNA polymerase γ in this assay to avoid degradation of the primer by the proofreading function and to simplify interpretation of results. This strategy became imperative due to the relatively high amount of enzyme and the longer incubation times required to detect incorporation with some of these analogs. Fig.3 depicts the incorporation of dTMP, ddTMP, AZT-MP, and D4T-MP into DNA. Rate was determined as the fraction of primer extended by one nucleotide per unit time, and Michaelis-Menten kinetic constants were determined by plotting the rate as a function of nucleotide analog concentration (26Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 14689-14696Abstract Full Text PDF PubMed Google Scholar). Human pol γ displayed high affinity (low apparent Km) for normal nucleotides (TablesTable I, Table II, Table III), which is in agreement with other kinetic studies of pol γ (13Martin J.L. Brown C.E. Matthews-Davis N. Reardon J.E. Antimicrob. Agents Chemother. 1994; 38: 2743-2749Crossref PubMed Scopus (307) Google Scholar, 14Hart G.J. Orr D.C. Penn C.R. Figueiredo H.T. Gray N.M. Boehme R.E. Cameron J.M. Antimicrob. Agents Chemother. 1992; 36: 1688-1694Crossref PubMed Scopus (145) Google Scholar, 15Parker W.B. White E.L. Shaddix S.C. Ross L.J. Buckheit Jr., R.W. Germany J.M. Secrist IIII, J.A. Vince R. Shannon W.M. J. Biol. Chem. 1991; 266: 1754-1762Abstract Full Text PDF PubMed Google Scholar,18Eriksson S. Xu B. Clayton D.A. J. Biol. Chem. 1995; 270: 18929-18934Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). All the analogs could be incorporated into our DNA substrate, but different concentrations of the analog were required. The dideoxynucleotide analogs ddCTP and ddTTP were the easiest to incorporate and had kcat values similar to their normal nucleotide counterpart (Tables Table I, Table II). The apparentKm was 2–5-fold higher than the normal nucleotide. The effect of these analogs on competitive incorporation can be assessed by taking the ratio of the kinetic constants, (fin). This value is equivalent theoretically to measurements made using competing substrates. Thus, ddTTP would get incorporated one in four incorporation events if the concentration of TTP and ddTTP were equal (Table I). The apparent Kmfor D4T-TP incorporation was similar to dideoxynucleotides but had a slightly decreased kcat (Table I). These results predict D4T to be as inhibitory as the dideoxynucleotides. Thefin values for these analogs determined with pol γ followed the general trend of inhibition observed in Fig. 2. Pol γ exerted most of its discrimination through Kmeffects, whereas the kcat was only modestly reduced for most of these analogs. Incorporation of AZT-TP, 3TC-TP, and CBV-TP required much higher concentrations than ddNTP or D4T-TP. However, apparent Km values were still in the micromolar range (Tables Table I, Table II, Table III), indicating AZT, 3TC, and CBV are only moderately incorporated as compared with dideoxynucleotides and D4T. The dCTP analog, 3TC-TP, had the lowest kcat as well as a high apparent Km (Table II).Table IApparent kinetic parameters of recombinant DNA polymerase γ with thymidine analogs in 18-mer/36-mer single nucleotide extension assayNucleoside triphosphateKmkcatkcat/Kmfin1-afin = (kcat/Km(TTP))/(kcat/Km(analog)).μmmin−1min−1μm−1TTP0.0382.976ddTTP0.0601.1184AZT-TP940.560.00612,700D4T-TP0.050.651361-a fin = (kcat/Km(TTP))/(kcat/Km(analog)). Open table in a new tab Table IIApparent kinetic parameters of recombinant DNA polymerase γ with deoxycytidine analogs in 18-mer/36-mer single nucleotide extension assayNucleoside triphosphateKmkcatkcat/Kmfin2-afin = (kcat/Km(dCTP))/(kcat/Km(analog)).μmmin−1min−1μm−1dCTP0.0252.9116ddCTP0.1302.11673TC-TP10.80.110.01111,3002-a fin = (kcat/Km(dCTP))/(kcat/Km(analog)). Open table in a new tab Table IIIApparent kinetic parameters of recombinant DNA polymerase γ with carbovir in 18-mer/36-mer single nucleotide extension assayNucleoside triphosphateKmkcatkcat/Kmfin3-afin = (kcat/Km(dGTP))/(kcat/Km(CBV)).μmmin−1min−1μm−1dGTP0.0202.8143CBV-TP4.30.240.05625503-a fin = (kcat/Km(dGTP))/(kcat/Km(CBV)). Open table in a new tab The inhibitory effect of a chain terminator is limited by its ability to persist in DNA once incorporated. The persistence of all of these analogs in DNA has largely been ignored in the literature. This concept is critical to the understanding the toxicity because, even though many of these analogs are only moderate inhibitors in vitro and are not readily incorporated, their resistance to exonucleolytic removal increases their ability to thwart DNA replication and presumably cause cytotoxicity. Since DNA polymerase γ has an intrinsic 3′-5′ exonuclease function, we investigated whether human DNA polymerase γ could remove these antiviral nucleotide analogs from DNA termini. Single-stranded DNA substrates bearing the analog at the 3′ terminus were constructed with TdT. Since TdT did not effectively add AZT-MP to the ends of DNA we used HIV-1 reverse transcriptase to insert AZT-MP onto the 3′ end of the 18/36-mer substrate. The exonuclease activity by pol γ on this dsDNA substrate was compared with the degradation of the normal 18/36-mer substrate, as well as a 19/36-mer dsDNA bearing either D4T-MP or ddTMP. At an equal molar ratio of wild type pol γ and either ssDNA or dsDNA, we observed efficient exonucleolytic removal of normal nucleotides, but very little detectable removal of the analogs with the exception of 3TC. At this stoichiometric level of polymerase and substrate, 10–20% of the 3TC was removed from the 3′ termini as compared with the control. Pol γ did not remove detectable amounts of the other analogs at these enzyme concentrations (data not shown). However, when enzyme concentrations exceeded substrate concentrations, we detected removal of the analogs from the 3′ terminus (Fig.4 A). A 1.3-fold molar excess of enzyme was only able to remove 10% or the terminally incorporated analogs for single or double-stranded substrate. The exception was 3TC, where >50% of the analog was removed in 30 min (Fig. 4 B). The remaining terminally incorporated analogs required a 3–4-fold molar excess of pol γ to remove >50% in 30 min. Through uptake and phosphorylation, AZT-MP is known to accumulate in millimolar concentrations in cells (29Frick L.W. Nelson D.J. St. Clair M.H. Furman P.A. Krenitsky T.A. Biochem. Biophys. Res. Commun. 1988; 154: 124-129Crossref PubMed Scopus (110) Google Scholar, 30Furman P.A. Fyfe J.A. St Clair M.H. Weinhold K. Rideout J.L. Freeman G.A. Lehrman S.N. Bolognesi D.P. Broder S. Mitsuya H. Barry D.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8333-8337Crossref PubMed Scopus (1228) Google Scholar). Since normal deoxynucleoside monophosphates inhibit the 3′-5′exonucleolytic activity by product inhibition, we tested the ability of the mono- and diphosphate forms of AZT to inhibit the exonuclease activity of pol γ. Inhibition of exonucleolytic digestion of the 18-mer by increasing concentrations of TMP, AZT-MP, TDP, or AZT-DP was monitored by gel electrophoresis. The fraction of 18-mer was plotted for the indicated concentration of each analog (Fig. 5). The" @default.
• W2077364814 created "2016-06-24" @default.
• W2077364814 creator A5033411994 @default.
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• W2077364814 title "Differential Incorporation and Removal of Antiviral Deoxynucleotides by Human DNA Polymerase γ" @default.
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