Skip Navigation



Nucleic Acids Research Advance Access published online on October 16, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm695
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (314K) Freely available
Right arrow Screen PDF (334K) Freely available
Right arrowOA All Versions of this Article:
35/20/6973    most recent
gkm695v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Hanes, J. W.
Right arrow Articles by Johnson, K. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hanes, J. W.
Right arrow Articles by Johnson, K. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 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.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.