Nucleic Acids Research Advance Access originally published online on November 3, 2006
Nucleic Acids Research 2006 34(21):6116-6125; doi:10.1093/nar/gkl862
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Nucleic Acids Research, 2006, Vol. 34, No. 21 6116-6125
© 2006 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 |
Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay
Department of Microbiology, University of Pennsylvania School of Medicine 3610 Hamilton Walk Philadelphia, PA 19104-6076, USA
*To whom correspondence should be addressed. Tel: +1 215 573 8732; Fax: +1 215 573 4856; Email: bushman{at}mail.med.upenn.edu
Received July 27, 2006. Revised September 29, 2006. Accepted October 3, 2006.
Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn2+ binds tightly to the sulfur atoms of the cysteine residues, but Mg2+ does not. We found that Mn2+, but not Mg2+, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5' hydroxyl groups as well as 3' hydroxyls as nucleophiles. Lastly, we show that Mn2+ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold.
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