Insights into the Molecular Mechanism of Mitochondrial Toxicity by AIDS Drugs

Abstract

Several of the nucleoside analogs used in the treatment of AIDS exhibit a delayed clinical toxicity limiting their usefulness. The toxicity of nucleoside analogs may be related to their effects on the human mitochondrial DNA polymerase (Pol γ), the polymerase responsible for mitochondrial DNA replication. Among the AIDS drugs approved by the FDA for clinical use, two are modified cytosine analogs, Zalcitabine (2′,3′-dideoxycytidine (ddC)) and Lamivudine (β-d-(+)-2′,3′-dideoxy-3′-thiacytidine ((−)3TC])). (−)3TC is the only analog containing an unnaturall(−) nucleoside configuration and is well tolerated by patients even after long term administration. In cell culture (−)3TC is less toxic than its d(+) isomer, (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than ddC. We have investigated the mechanistic basis for the differential toxicity of these three cytosine analogs by comparing the effects of dideoxy-CTP), (+)3TC-triphosphate (TP), and (−)3TC-TP on the polymerase and exonuclease activities of recombinant human Pol γ. This analysis reveals that Pol γ incorporates (−)3TC-triphosphate 16-fold less efficiently than the corresponding (+)isomer and 1140-fold less efficiently than dideoxy-CTP, showing a good correlation between incorporation rate and toxicity. The rates of excision of the incorporated analogs from the chain-terminated 3′-end of the DNA primer by the 3′-5′-exonuclease activity of Pol γ were similar (0.01 s− 1) for both 3TC analogs. In marked contrast, the rate of exonuclease removal of a ddC chain-terminated DNA occurs at least 2 orders of magnitude slower, suggesting that the failure of the exonuclease to remove ddC may play a major role in its greater toxicity. This study demonstrates that direct analysis of the mitochondrial DNA polymerase structure/function relationships may provide valuable insights leading to the design of less toxic inhibitors. Several of the nucleoside analogs used in the treatment of AIDS exhibit a delayed clinical toxicity limiting their usefulness. The toxicity of nucleoside analogs may be related to their effects on the human mitochondrial DNA polymerase (Pol γ), the polymerase responsible for mitochondrial DNA replication. Among the AIDS drugs approved by the FDA for clinical use, two are modified cytosine analogs, Zalcitabine (2′,3′-dideoxycytidine (ddC)) and Lamivudine (β-d-(+)-2′,3′-dideoxy-3′-thiacytidine ((−)3TC])). (−)3TC is the only analog containing an unnaturall(−) nucleoside configuration and is well tolerated by patients even after long term administration. In cell culture (−)3TC is less toxic than its d(+) isomer, (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than ddC. We have investigated the mechanistic basis for the differential toxicity of these three cytosine analogs by comparing the effects of dideoxy-CTP), (+)3TC-triphosphate (TP), and (−)3TC-TP on the polymerase and exonuclease activities of recombinant human Pol γ. This analysis reveals that Pol γ incorporates (−)3TC-triphosphate 16-fold less efficiently than the corresponding (+)isomer and 1140-fold less efficiently than dideoxy-CTP, showing a good correlation between incorporation rate and toxicity. The rates of excision of the incorporated analogs from the chain-terminated 3′-end of the DNA primer by the 3′-5′-exonuclease activity of Pol γ were similar (0.01 s− 1) for both 3TC analogs. In marked contrast, the rate of exonuclease removal of a ddC chain-terminated DNA occurs at least 2 orders of magnitude slower, suggesting that the failure of the exonuclease to remove ddC may play a major role in its greater toxicity. This study demonstrates that direct analysis of the mitochondrial DNA polymerase structure/function relationships may provide valuable insights leading to the design of less toxic inhibitors. human immunodeficiency virus acquired immunodeficiency syndrome 3′-azido-3′-deoxythymidine 2′,3′-dideoxycytidine 2′,3′-dideoxyinosine deoxy dideoxy DNA polymerase γ monophosphate triphosphate reverse transcriptase β-d-(+)-2′,3′-dideoxy-3′-thiacytidine-5′-TP β-l-(−)-3TC-TP 2′,3′-dideoxy-3′-thiacytidine base pair(s) The in vitro and in vivo mechanisms of cytotoxicity of the nucleoside analogs used as anti-HIV1 drugs are not well understood. Various forms of toxicity, including peripheral neuropathy, myopathy, and pancreatitis, are observed after long term use of nucleoside analogs 3′-azido-3′-deoxythymidine (AZT) (1Wilde M.I. Langtry H.D. Drugs. 1993; 46: 515-578Crossref PubMed Scopus (99) Google Scholar), 2′,3′-dideoxyinosine (ddI) (2Faulds D. Brogden R.N. Drugs. 1992; 44: 94-116Crossref PubMed Scopus (116) Google Scholar), 2′,3′-dideoxycytidine (ddC) (3Whittington R. Brogden R.N. Drugs. 1992; 44: 656-683Crossref PubMed Scopus (57) Google Scholar, 4Adkins J.C. Peters D.H. Faulds D. Drugs. 1997; 53: 1054-1080Crossref PubMed Scopus (36) Google Scholar), and 2′,3′-didehydro-3′-deoxythymidine (5Hitchcock M.J.M. Antiviral Chem. Chemother. 1991; 2: 125-132Crossref Google Scholar). The profile for toxicity varies with each analog. For instance, whereas all of the three, AZT, ddI, and ddC, exert cytotoxic effects on human muscle cells and induce functional alternation of mitochondria, only AZT, but not ddI and ddC, can induce a myopathy in HIV-1-infected patients (6Benbrik E. Chariot P. Bonavaud S. Ammi-Said M. Frisdal E. Rey C. Gherardi R. Barlovatz-Meimon G. J. Neurol. Sci. 1997; 149: 19-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The fact that toxicity of different drugs is cell-, tissue-, and organ-specific (6Benbrik E. Chariot P. Bonavaud S. Ammi-Said M. Frisdal E. Rey C. Gherardi R. Barlovatz-Meimon G. J. Neurol. Sci. 1997; 149: 19-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) indicates that the differences in subcellular bioavailability and pharmacokinetic profile involving the activation and deactivation of the drugs can all contribute to the clinical toxicity. Many mechanisms have been proposed for the origins of nucleoside analog toxicity, but the most obvious sites of action are the enzymes responsible for host cell DNA replication and repair: DNA polymerases α, δ, and ε, which are responsible for chromosomal DNA replication; DNA polymerase β, which is involved in DNA repair; and DNA polymerase γ (Pol γ), which carries out mitochondrial DNA synthesis (7Frick 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 (112) Google Scholar, 8Hall E.T. Yan J.P. Melancon P. Kuchta R.D. J. Biol. Chem. 1994; 269: 14355-14358Abstract Full Text PDF PubMed Google Scholar, 9Kewn S. Veal G.J. Hoggard P.G. Barry M.G. Back D.J. Biochem. Pharmacol. 1997; 54: 589-595Crossref PubMed Scopus (69) Google Scholar). The toxicity toward proliferating tissues, such as the well documented bone marrow toxicity of AZT, is believed to be due to the inhibition of one or more of the chromosomal DNA polymerases (10Parker W.B. Cheng Y.-C. J. Natl. Inst. Health Res. 1994; 6: 57-61Google Scholar). The toxicity manifested in many nonproliferating tissues is thought to result more specifically from the effects of inhibition of DNA Pol γ by nucleoside analogs (10Parker W.B. Cheng Y.-C. J. Natl. Inst. Health Res. 1994; 6: 57-61Google Scholar, 11Horn D.M. Neeb L.A. Colacino J.M. Richardson F.C. Antiviral Res. 1997; 34: 71-74Crossref PubMed Scopus (28) Google Scholar, 12Colacino J.M. Antiviral Res. 1996; 29: 125-139Crossref PubMed Scopus (57) Google Scholar, 13Honkoop P. Scholte H.R. de Man R.A. Schalm S.W. Drug Safety. 1997; 17: 1-7Crossref PubMed Scopus (67) Google Scholar, 14Barile M. Valenti D. Quagliariello E. Passarella S. Gen. Pharmacol. 1998; 31: 531-538Crossref PubMed Scopus (57) Google Scholar). 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Sci. 1987; 77: 285-304Abstract Full Text PDF PubMed Scopus (57) Google Scholar); moreover, prolonged exposure of the cell to nucleoside analogs results in decreased mitochondrial DNA synthesis, decreased cell viability, and changes in the mitochondrial ultrastructure (19Medina D.J. Tsai C.H. Hsiung G.D. Cheng Y.C. Antimicrob. Agents Chemother. 1994; 38: 1824-1828Crossref PubMed Scopus (131) Google Scholar). Some in vitro studies have shown that for many of the nucleoside analogs tested, Pol γ is more sensitive than any of the other DNA polymerases (20Kukhanova M. Liu S.-H. Mozzherin D. Lin T.-S. Chu C.K. Cheng Y.-C. J. Biol. Chem. 1995; 270: 23055-23059Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), but there are conflicting reports (19Medina D.J. Tsai C.H. Hsiung G.D. Cheng Y.C. Antimicrob. Agents Chemother. 1994; 38: 1824-1828Crossref PubMed Scopus (131) Google Scholar, 21Martin J.L. Brown C.E. Matthews-Davis N. Reardon J.E. Antimicrob. Agents Chemother. 1994; 38: 2743-2749Crossref PubMed Scopus (314) Google Scholar). Earlier studies to assess the interaction of nucleoside analogs have used partially purified preparations of native enzyme. However, recently, highly purified homogenous preparations of recombinant human Pol γ have become available through cloning and overexpression in a baculovirus expression system (22Gray H. Wong T.W. J. Biol. Chem. 1992; 267: 5835-5841Abstract Full Text PDF PubMed Google Scholar, 23Graves S.W. Johnson A.A. Johnson K.A. Biochemistry. 1998; 37: 6050-6058Crossref PubMed Scopus (77) Google Scholar, 24Longley M.J. Ropp P.A. Lim S.E. Copeland W.C. Biochemistry. 1998; 37: 10529-10539Crossref PubMed Scopus (146) Google Scholar, 25Lim S.E. Longley M.J. Copeland W.C. J. Biol. Chem. 1999; 274: 32803-38197Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 26Johnson A.A. Tsai Y. Graves S.W. Johnson K.A. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (124) Google Scholar). Human DNA Pol γ is composed of a catalytic subunit of 140 kDa and an accessory subunit of ∼54 kDa (22Gray H. Wong T.W. J. Biol. Chem. 1992; 267: 5835-5841Abstract Full Text PDF PubMed Google Scholar). The large catalytic subunit provides both polymerase and 3′-5′-exonuclease activities (23Graves S.W. Johnson A.A. Johnson K.A. Biochemistry. 1998; 37: 6050-6058Crossref PubMed Scopus (77) Google Scholar, 24Longley M.J. Ropp P.A. Lim S.E. Copeland W.C. Biochemistry. 1998; 37: 10529-10539Crossref PubMed Scopus (146) Google Scholar), whereas the smaller accessory subunit (25Lim S.E. Longley M.J. Copeland W.C. J. Biol. Chem. 1999; 274: 32803-38197Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) facilitates substrate binding and dNTP incorporation, thereby improving A.A. Tsai Y. Graves S.W. Johnson K.A. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (124) Google Scholar). The and of the human Pol γ a highly purified a more analysis of the incorporation and removal by the exonuclease Liu Cheng Y.C. Biochem. Pharmacol. 1995; 50: PubMed Scopus (28) Google Scholar). Among the nucleoside analogs used the is the only one with the and most been shown to be more and less toxic than the isomer, of (−)3TC are well tolerated 1998; Full Text PDF PubMed Scopus Google Scholar, R. J.M. Drug Safety. 1996; PubMed Scopus Google Scholar, P. de Man R.A. Scholte H.R. Schalm S.W. 1997; PubMed Scopus (47) Google Scholar), and there is or of mitochondrial P. Scholte H.R. de Man R.A. Schalm S.W. Drug Safety. 1997; 17: 1-7Crossref PubMed Scopus (67) Google Scholar, P. de Man R.A. Scholte H.R. Schalm S.W. 1997; PubMed Scopus (47) Google Scholar). In contrast, ddC is one of the more toxic of the nucleoside analogs approved for AIDS by the Drug The mechanistic basis for observed differential toxicity for the two 3TC and toxicity of ddC are not and a of may The differences in toxicity may be a of including Cheng Y.C. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, G. Cheng Y.C. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar), (10Parker W.B. Cheng Y.-C. J. Natl. Inst. Health Res. 1994; 6: 57-61Google Scholar), activation Cheng Y.C. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, G. Cheng Y.C. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, J.M. Biochem. Pharmacol. 1995; 50: PubMed Scopus Google Scholar), incorporation Cheng Y.C. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, J.M. Biochem. Pharmacol. 1995; 50: PubMed Scopus Google G.J. J.M. Antimicrob. Agents Chemother. 1992; PubMed Scopus Google Scholar), and removal or from the system (10Parker W.B. Cheng Y.-C. J. Natl. Inst. Health Res. 1994; 6: 57-61Google Scholar, Liu Cheng Y.C. Biochem. Pharmacol. 1995; 50: PubMed Scopus (28) Google Scholar, J.M. Biochem. Pharmacol. 1995; 50: PubMed Scopus Google Scholar, N. P. J.M. Biochem. Pharmacol. 1992; PubMed Scopus Google Scholar, Chu C.K. Tsai C.H. Cheng Y.C. D. R. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). analysis of each is The of inhibition of DNA Pol γ by the nucleoside analogs in vivo to the triphosphate and incorporation the DNA primer in in vitro studies have on the triphosphate of Earlier studies have the of the and (−)3TC-TP incorporation DNA by Pol γ J.L. Brown C.E. Matthews-Davis N. Reardon J.E. Antimicrob. Agents Chemother. 1994; 38: 2743-2749Crossref PubMed Scopus (314) Google Scholar, Cheng Y.C. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) and a in However, these studies were that a of Pol and the to of for of the A.A. Tsai Y. Graves S.W. Johnson K.A. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (124) Google Scholar), the polymerase not have as the the of studies native purified is due to the activity from the of the subunit to many of the DNA Pol γ 3′-5′-exonuclease which is believed to a (22Gray H. Wong T.W. J. Biol. Chem. 1992; 267: 5835-5841Abstract Full Text PDF PubMed Google M.W. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). is that the failure of 3′-5′-exonuclease of Pol γ to efficiently remove may play a role in mitochondrial toxicity of nucleoside analogs. However, in the the of the exonuclease in removal of these analogs and correlation to toxicity. been proposed that removal of by the 3′-5′-exonuclease activity of Pol γ be responsible for the toxicity of (−)3TC J.M. Biochem. Pharmacol. 1995; 50: PubMed Scopus Google Scholar); similar rates of exonuclease removal were for the excision of and from the of a DNA even there are differences in mitochondrial toxicity J.M. Biochem. 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Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (124) Google Scholar). We analysis of incorporation and excision of triphosphate (+)3TC, and with the of these modified cytosine analogs with the Pol γ in to of the of inhibition of the DNA γ polymerase is in associated with cytotoxicity with nucleoside analogs. of the mechanistic and differences may in for the interaction with the DNA polymerase reverse the DNA γ polymerase and thereby in the design of AIDS The large and of Pol γ were purified as (23Graves S.W. Johnson A.A. Johnson K.A. Biochemistry. 1998; 37: 6050-6058Crossref PubMed Scopus (77) Google Scholar, 26Johnson A.A. Tsai Y. Graves S.W. Johnson K.A. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (124) Google Scholar). The at with the and for the large and DNA Biochemistry. 1999; 38: PubMed Scopus Google Scholar), used in which the base for incorporation of the were at the for the and the primer with and The and were from and the and (−)3TC-TP were the of at to were as Biochemistry. 1999; 38: PubMed Scopus Google Scholar). were K.A. PubMed Scopus Google Scholar). all to after on the the were by the DNA with large and in of this with an of a of analogs and in The were by of at were at incorporation by of were on on a and were by the The were to a the of the that with the of in is the observed rate for dNTP incorporation, is the observed rate The for dNTP binding to the of Pol γ and DNA is by the the is the rate of dNTP incorporation, is the corresponding of is the for the interaction of dNTP with the The were by the corresponding analogs the of DNA analogs and in at for The DNA were purified by The exonuclease activity by the rate of of the in the of or analogs. from the were as a of The of the by the in the and a for exonuclease activity The by a of catalytic subunit accessory subunit and DNA in and with For exonuclease activity of the catalytic catalytic subunit and DNA were The mechanisms of toxicity were investigated by the of the two different by human mitochondrial DNA Pol DNA synthesis and 3′-5′-exonuclease This analysis out by the incorporation and two modified analogs, and a for DNA by the recombinant Pol γ The exonuclease investigated by the excision of and from the of the corresponding DNA or primer by the catalytic subunit and the of Pol γ. of the incorporation studies were out at to the Pol γ and on a to a of enzyme. a the rate of the for the binding and the incorporation or for the natural as well as for and (−)3TC-TP in is by the the rate for substrate In the of DNA K.A. Annu. Rev. Biochem. 1993; PubMed Scopus Google Scholar) shown that the binding of nucleoside is in a and the the and the We a to a of and In each a of that at of a after the is and The after incorporation of a is the of the DNA from the A.A. Tsai Y. Graves S.W. Johnson K.A. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (124) Google Scholar). Johnson and In the the of the to a from which the the observed rate of and the observed were of were of and and (−)3TC-TP to the dNTP of the observed rate for each dNTP of and a of observed the rate with the to the rate of incorporation, the were with in of DNA as in A.A. Tsai Y. Graves S.W. Johnson K.A. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (124) Google Scholar) and and Biochemistry. 1999; 38: PubMed Scopus Google Scholar). The interaction of with the by the of the rates to the is the rate of incorporation, is the of the nucleoside and is the for the binding of dNTP with the The of the observed rate for the incorporation of and (−)3TC-TP a by Pol γ is shown in of for each analog is shown in and the for are all in good with studies A.A. Tsai Y. Graves S.W. Johnson K.A. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (124) Google for incorporation of analogs a DNA by human mitochondrial DNA polymerase in a of the analogs reveals that the natural as the substrate for human mitochondrial DNA as by the observed binding and rate of In contrast, is incorporated more than but more the incorporation is only The (−)3TC-TP and analogs are incorporated to more than The and a similar for the or the become for In contrast, the of (−)3TC-TP for the is than that of the analog that of containing the natural nucleoside Pol γ incorporates the at least 16-fold less efficiently than the isomer, as by the in This in incorporation is in good with cell culture studies with the nucleoside of the the been to be and more toxic than the (−)3TC in of inhibition of cell and mitochondrial DNA synthesis, Chu C.K. Tsai C.H. Cheng Y.C. D. R. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). We an analysis of Pol γ 3′-5′-exonuclease removal of ddC and and (−)3TC from DNA with highly purified Pol γ catalytic subunit and analysis out DNA that were with or with the corresponding DNA and containing and We the three DNA that were with dideoxy nucleoside analogs, and the that may be the DNA and containing and were in to containing modified the for the exonuclease is shown in For only have been for or of the rates of exonuclease activity for the catalytic subunit or the of Pol γ different is shown in for the exonuclease activity of human mitochondrial DNA polymerase γ toward different DNA of the used in this study are DNA of the rate of from the that less than of the DNA after of with the or with the catalytic subunit of the rate of from the that less than of the DNA after of with the or with the catalytic subunit of the used in this study are DNA The of the rate of from the that less than of the DNA after of with the or with the catalytic subunit in a The and the excision these rates are and with the of due to the removal of of the at the 3′-end of the K.A. Annu. Rev. Biochem. 1993; PubMed Scopus Google Scholar). of the and of 3TC than removal of and the of the accessory on this excision The of the accessory subunit of Pol γ decreased the exonuclease activity for the DNA that natural or at their are in to a study that the accessory subunit may the exonuclease activity (25Lim S.E. Longley M.J. Copeland W.C. J. Biol. Chem. 1999; 274: 32803-38197Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). the excision the exonuclease remove the with a of this the rate of mitochondrial DNA the rate of incorporation the toxic In contrast, or DNA observed for after with the catalytic subunit or with the The of the to base excision for a 3′-5′-exonuclease with DNA polymerase γ M.J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). results have been in studies on mitochondrial DNA the incorporated and were not by the 3′-5′-exonuclease activity of Pol γ S. B. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). This may inhibition of mitochondrial DNA synthesis by ddC is studies have shown that (−)3TC-TP is less efficiently incorporated than DNA by Pol and both are incorporated less efficiently than the of incorporation by Pol γ is with toxicity of each nucleoside analog. to incorporation by Pol γ mitochondrial DNA as a major of toxicity of nucleoside analogs. The of preparations of Pol γ a to drugs for toxicity and the toward structure/function studies to in the design of less toxic analogs. 3TC is well tolerated in the 1998; Full Text PDF PubMed Scopus Google Scholar, R. J.M. Drug Safety. 1996; PubMed Scopus Google Scholar), and of mitochondrial toxicity from (−)3TC for have been observed P. de Man R.A. Scholte H.R. Schalm S.W. 1997; PubMed Scopus (47) Google Scholar). to (−)3TC shown of mitochondrial P. Scholte H.R. de Man R.A. Schalm S.W. Drug Safety. 1997; 17: 1-7Crossref PubMed Scopus (67) Google Scholar). The configuration of (−)3TC to play an role in the activity the on the host Pol γ. This other nucleoside analogs may similar with toxicity. In contrast, the effects of inhibition on mitochondrial DNA vivo and in vitro by ddC are C.H. M. Cheng Y.C. Pharmacol. 1991; 39: Google R. Dalakas M. S. PubMed Scopus Google Scholar). The for ddC to the and of 3TC that ddC is and more toxic than the and isomer, Chu C.K. Tsai C.H. Cheng Y.C. D. R. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). of may play including and removal from the but studies have shown a of removal of a that may contribute to toxicity. analogs that the exonuclease removal by Pol γ may exhibit even toxicity, and this may provide a for of less toxic The accessory subunit to by the exonuclease rates of excision by the were observed for DNA and the is by a natural with a the accessory subunit may the excision of this by the of the 3′-end at the polymerase studies are to the of nucleoside analogs that may be to for DNA Pol γ. In studies have that the is a more for human mitochondrial DNA Pol γ than the through binding and incorporation rate a DNA leading to greater toxicity. highly purified Pol there is in the excision of the incorporated and by the 3′-5′-exonuclease activity of Pol γ from the of a DNA this not contribute to the toxicity of studies have shown that a incorporated is not efficiently by the 3′-5′-exonuclease activity of Pol suggesting this may play a role in the of inhibition of ddC on mitochondrial DNA synthesis observed in vitro and in The toxicity of a nucleoside analog toward replication be a of the of incorporation of the and the of excision by the exonuclease to the rate of DNA replication to We can these and to assess the that for Pol γ in each basis for differential toxicity of modified cytosine for each from from the observed exonuclease rate from in which the nucleoside of the analogs is for each from from the observed exonuclease rate from Chu C.K. Tsai C.H. Cheng Y.C. D. R. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, in which the nucleoside of the analogs is in a of the of inhibition of DNA polymerase γ is in the cytotoxicity of drugs toward mitochondrial replication. of the mechanistic and and differences between Pol γ and may for incorporation by the but not by Pol γ the design of less AIDS