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Nucleic Acids Research Advance Access originally published online on October 16, 2007
Nucleic Acids Research 2007 35(20):6973-6983; doi:10.1093/nar/gkm695
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Nucleic Acids Research, 2007, Vol. 35, No. 20 6973-6983
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase

Jeremiah W. Hanes and Kenneth A. Johnson*

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

*To whom correspondence should be addressed. Tel: +1 512 471 0434; Fax: +1 512 471 0435; Email: kajohnson{at}mail.utexas.edu

Received June 17, 2007. Revised August 19, 2007. Accepted August 22, 2007.

Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3'-azido-2',3'-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5'-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s–1) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 µM) and chemistry forward and reverse reactions, 0.38 and 0.22 s–1, respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/Km (7 x 10–6 µ M–1 s–1). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5'-triphosphate (8-oxo-dGTP) show this same phenomena.

Present address: Jeremiah W. Hanes, Department of Chemistry and Chemical Biology, Cornell University, 120 Baker Laboratory, Ithaca, NY 14853, USA.


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