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Antimicrob. Agents Chemother. doi:10.1128/AAC.00778-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Exonuclease Removal of Dideoxycytidine (Zalcitabine) by the Human Mitochondrial DNA Polymerase

From the Department of Chemistry & Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712

^* To whom correspondence should be addressed. Email: kajohnson{at}mail.utexas.edu.

	Abstract

The toxicity of nucleoside analogs used for the treatment of HIV infection is primarily due to the inhibition of replication of the mitochondrial genome by the human mitochondrial DNA polymerase (Pol ). The severity of clinically observed toxicity correlates with the kinetics of incorporation versus excision of each analog as quantified by a toxicity index, spanning over six orders of magnitude. Here we show that the rate of excision of ddC (dideoxycytidine, zalcitabine) was reduced fourfold (to a half-life of 2.4 hours) by the addition of a physiological concentration of dNTPs due to the formation of a tight ternary E-DNA-dNTP complex at the polymerase site. In addition, we provide a more accurate measurement of the rate of excision and show that the slow rate of removal of ddCMP results from both the unfavorable transfer of the primer strand from the polymerase to the exonuclease site and the inefficient binding and/or hydrolysis at the exonuclease site. Compared to normal nucleotides, the analogs ddC (zalcitabine), d4T (stavudine) and ddA (metabolite of didanosine) each bind more tightly at the polymerase site during incorporation and this contributes to slower excision by the proofreading exonuclease, leading to increased toxicity toward mitochondrial DNA replication.