6116–6125 Nucleic Acids Research, 2006, Vol. 34, No. 21 doi:10.1093/nar/gkl862 Published online 3 November 2006 Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay Tracy L. Diamond and Frederic D. Bushman* Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk Philadelphia, PA 19104-6076, USA Received July 27, 2006; Revised September 29, 2006; Accepted October 3, 2006 ABSTRACT 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 transesterifi- cation in which catalysis by human immunodefi- ciency 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 cys- teines. Mn21 binds tightly to the sulfur atoms of the cysteine residues, but Mg21 does not. We found that Mn21, but not Mg21, 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 50 hydroxyl groups as well as 30 hydroxyls as nucleophiles. Lastly, we show that Mn21 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. INTRODUCTION Divalent metal ions are required for catalysis by many enzymes that carry out nucleic acid phosphoryl-transfer reac- tions. It has been proposed for many such enzymes that rate enhancement is mostly due to metal–nucleic acid interactions in the transition state, and the role of the protein scaffold is mainly to bind and position the metal ions and substrates (1,2). This may explain the ability of dissimilar metal binding protein and RNA enzymes to carry out related chemical reac- tions (3–5). The role of bound metals has been studied extensively for members of the ribonuclease H (RNase H)-like enzyme superfamily, which includes RuvC resolvase, bacterial and retroviral RNase Hs, bacterial transposases and retroviral integrases, all of which carry out polynucleotide phosphoryl- transferase reactions. These enzymes have similar secondary structures within their catalytic domains. The protein folds bring together three or four conserved acidic residues that are thought to be involved in metal coordination (4–7). These enzymes can utilize Mn2+ and/or Mg2+ for catalysis, though other metal ions can bind and for some enzymes support partial function (8–12). Three hypotheses for metal binding by RNase H-like enzymes have been proposed. Several RNase H enzyme structures determined by X-ray crystallography have been shown to contain two metal atoms bound at the active site (12–14), but the majority of structures have only one bound metal (9,15–19). The latter observation supports the second hypothesis that only one metal is involved catalytically (16,20,21). However, observation of a single bound metal in the absence of substrate could reﬂect the need for nucleic acid substrate binding to stabilize metal binding at the second site. As a third possibility, it has also been proposed that two metals bind, with the ﬁrst activating catalysis and the second inhibiting (22). The human immunodeﬁciency virus (HIV) integrase (IN) is a member of the RNase H-like superfamily and has been extensively studied for metal binding at the D,DX35E motif (composed of D64, D116 and E152). Several structural stud- ies of retroviral INs in the absence of DNA substrate have reported binding of a single Mg2+ ion between the two aspar- tate residues (8,17,18), but none have shown Mg2+ binding at the second site between residues D64 and E152. Crystal structures of the avian sarcoma virus (ASV) IN in the pres- ence of Cd2+ or Zn2+ ions revealed two bound metals at the catalytic triad (8). However, these complexes did not retain normal catalytic activity, complicating interpretation. *To whom correspondence should be addressed. Tel: +1 215 573 8732; Fax: +1 215 573 4856; Email: bushman@mail.med.upenn.edu 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.