6122 Nucleic Acids Research, 2006, Vol. 34, No. 21 Figure 4. DNA transesterification activity of HIV-1 on a reverse polarity substrate. (A) Schematic of the reverse polarity substrate, which contains a free 50 hydroxyl at the DNA three-way junction in place of the usual 30 hydroxyl. The same primers and probe are used to measure product copies for the two substrates. The 50 ends are noted by black dots. (B) A time course for DNA transesterification activity of WT IN on the reversed polarity substrate in the presence of 10 mM MnCl2 or 20 mM MgCl2. Error bars indicate one standard deviation around the mean of qPCR performed in triplicate. To show that the difference seen between reactions with Mn2+ and Mg2+ were significantly different, three independent reactions from the 2 h time point (the time point for which we had the most replicates) with triplicate qPCR for each were compared using a one-tailed Mann–Whitney U-test (P < 0.0001). Figure 5A shows an example of a time course carried out at 95 C in the presence of either 50 mM MnCl2 or 100 mM EDTA. Product for the MnCl2 reactions was distinguishable from the EDTA control reactions after 1 h of incubation, and continued product formation was seen up to 22 h in the presence of MnCl2. We consistently saw a reduced rate of product generation at longer times, perhaps due to competing DNA degradation under the extreme conditions used. We also carried out assays under the same conditions in the presence of 50 mM MgCl2, but no product was detected (data not shown). The product structure was characterized by PCR ampliﬁcation with labeled primers, followed by electrophor- esis on DNA-sequencing-type gels (Figure 5B). The product of the reaction in the presence of Mn2+ showed the expected 97mer length. Small amounts of product were detected in the EDTA control as well, but these were lower in abundance than the 50-copy standard and are of uncertain signiﬁcance. These measurements allow us to make an initial estimate of the rate enhancement in the disintegration reaction resulting from binding the metal atoms and DNA to the IN-protein scaf- fold. The reaction conditions in the presence and absence of IN are quite different, so the value represents an initial estima- tion of a lower limit. We compared the initial rate of product formation per minute between reactions at 37 C with 1 mM WT IN (ﬁrst 60 min) and 10 mM MnCl2 to reactions at 95 C with no IN and 50 mM MnCl2 (ﬁrst 180 min). For WT IN the rate of product copies generated per minute was 1.23 ± 0.18 (·109) and for the reactions without IN the rate of product copies generated per minute was 1.09 ± 0.10 (·103). In order to account for the difference in temperature, we corrected by the temperature coefﬁcient (Q10), estimating a 2-fold increase in rate for every 10 C increase in temperature (41). Thus if the enzymatic reactions were carried out at 95 C (and the enzyme was still stable) the rate would be increased theoretically by a factor of 56, to 6.9 · 1010 product copies generated per minute. That indicates that the protein scaffold of the HIV-1 IN increased the rate of DNA transesteriﬁcation by a factor of at least 60 million. DISCUSSION In this study, we present a sensitive and quantitative assay for measuring DNA transesteriﬁcation reactions based on the dis- integration reaction of retroviral INs. This was made possible by adding a TaqMan real-time PCR detection step, which was found to increase sensitivity at least 1000-fold over previous assays using radiolabeled substrates. We used the new method to investigate three issues surrounding the role of metal atoms in catalysis by HIV IN. These are discussed sequentially below. Previously, our laboratory investigated the role of the con- served acidic residues of HIV-1 IN by monitoring metal res- cue of cysteine substitutions (23). No activity was detected for E152C, leaving open the role of this residue. Similar stud- ies have been carried out on other RNase H-like superfamily members, such as Tn7 TnsA (31), Tn10 transposase (28) and RAG1 (29,30). For Tn10, all three residues of the catalytic triad were found to coordinate metal ions, while the other studies only showed functional metal binding by metal rescue for one of the three residues. For the enzyme family more broadly, there has been debate over whether the enzymes uti- lize one or two metal mechanisms for catalysis (16,20,21). Thanks to the increased sensitivity provided by the real- time PCR step, we were able to observe metal rescue for the E152C IN (Figure 3C), as well as conﬁrm rescue of the D64C and D116C INs (Figure 3B). As expected, product formation was not observed for the E152A IN with either metal ion and no product was formed with E152C IN in the presence of the Mg2+. The activity with E152C in Mn2+ was only 1% that of WT IN, but the speciﬁcity for the cysteine versus the alanine residue and Mn2+ versus Mg2+metal supports the idea that E152 is normally involved in metal binding. The activity of E152C IN may have been reduced compared to wild-type IN because the E152 side chain makes additional contacts besides binding to a single metal atom and/or because the cysteine side chain is shorter than that of glutamate, potentially resulting in suboptimal posi- tioning of the metal atom in the active site. These results sup- port the model shown in Figure 3A, where all three catalytic residues coordinate the binding of two metal ions that carry out catalysis, and argue against the idea that the second metal might be inhibiting catalysis. We disfavor the view that all three acidic residues bind a single metal ion, since that would be inconsistent with the geometry in Figure 3A. Because IN-protein is does not turnover efﬁciently in vitro and is not amenable to detailed kinetic analysis, it has not been possible to assign the effects of metal atoms to speciﬁc