In a previous study, binding of divalent metal ions by the HIV-1 IN catalytic triad was investigated using metal rescue of cysteine substitutions (23). The metal rescue technique has been used to map metal binding sites for ribozymes, such as the Tetrahymena group I ribozyme or the human spliceosome (24–27) and protein enzymes, such as NAD- dependent isocitrate dehydrogenase, Escherichia coli DNA polymerase I and RNase H-like enzyme superfamily mem- bers (28–33). Metal rescue takes advantage of the unique property of Mn2+ to bind tightly to sulfur atoms, while Mg2+ ions bind much more weakly. For analysis of ribozymes, sulfur substitutions have been carried out with oxygens in the substrate or nucleophile RNA, while for pro- tein enzymes, suspected metal binding residues can be tested by replacement with cysteines. If the mutated motif has a necessary interaction with metal ions, then the enzyme will not be functional in reactions with Mg2+, but may be functional in the presence of Mn2+, indicating a requirement for metal binding. These assays have been used to analyze metal binding by HIV-1 IN, and indicated functional metal binding by residues D64 and D116, but did not show activity with either metal for E152C (23). It was unclear whether the absence of activity for the E152C IN was due to a lack of metal binding by that residue or a lack in assay sensitivity. Here, we report an ultra-sensitive assay for measuring nucleic acid transesteriﬁcation reactions in vitro and its use to investigate metal-mediated catalysis by HIV-1 IN. The assay is based on the HIV-1 IN disintegration reaction (34), which is a DNA transesteriﬁcation reaction that reverses the normal integration reaction catalyzed by HIV-1 IN (Figure 1A, see legend for details). In disintegration, the 30 hydroxyl of the target DNA at the insertion site attacks the DNA backbone between the viral and target DNA, thereby joining the target DNA segments and releasing the viral LTR end. Similar activities have been reported for the RAG-1 recombinase involved in V(D)J recombination (35), and the Mu transposase (36). To boost the sensitivity, we developed lengthened disintegration substrates, which allowed PCR ampliﬁcation and TaqMan detection (Figure 1B), which greatly increases sensitivity over previous assays. We used this assay to probe three aspects of metal- mediated catalysis by HIV-1 IN. First, we demonstrate metal rescue for HIV-1 IN derivatives containing cysteine substitutions at all three residues of the catalytic triad, including E152. These studies established that two metal ions are likely involved in catalysis and support the idea that this is generally true for the retroviral INs. Second, we used this assay to measure DNA transesteriﬁcation reactions on a related substrate with a 50 hydroxyl func- tioning as the nucleophile instead of a 30 hydroxyl. We found that HIV-1 IN was able to catalyze transesteriﬁcation at a low level on this substrate as well, but was more selective for metal ion usage than seen for the standard substrate. In the third application, we took advantage of the exceptional sensitivity of the assay to detect transesteri- ﬁcation in the presence of Mn2+ alone in the absence of a protein enzyme scaffold. This allowed us to estimate the rate enhancement contributed by binding of the metal and DNA substrate to the IN-protein scaffold as at least a factor of 60 million. Nucleic Acids Research, 2006, Vol. 34, No. 21 6117 Figure 1. Disintegration and the PCR-based disintegration assay. (A) Diagram of integration and disintegration. During normal integration, IN first cleaves 2 nt off the 30 end of each LTR DNA. IN then catalyzes a coupled DNA breaking and joining reaction, which covalently joins the viral DNA 30 end to host cell DNA. Disintegration is the reversal of the integration reaction. In disintegration, the 30 end in the target DNA at the junction with the viral DNA attacks the nearby phosphodiester, rejoining the target DNA segment and releasing the viral DNA. For standard disintegration assays in vitro, a 32P-radiolabel is attached at the indicated 50 end (*), allowing detection of the larger product formed by disintegration (34). (B) The qPCR- based disintegration assay. The assay substrate contains four annealed oligonucleotides resembling the integration product (upper). DNA transester- ification can occur on this substrate, yielding the 97mer disintegration product (middle). The product can be quantitated by TaqMan qPCR (lower). Nucleic acid 50 ends are represented by black dots. LTR ends are represented by thick lines, while target is represented by thin lines. MATERIALS AND METHODS Enzymes Protein expression vectors used encode IN derived from the NL4-3 strain of HIV-1 fused at the N-terminus to a hexahis- tidine tag and thrombin cleavage site. All vectors were gen- erated using site-directed mutagenesis and were described