6120 Nucleic Acids Research, 2006, Vol. 34, No. 21 product were detected in the qPCR-based disintegration assay in reactions containing the RNA Tbottom-32 strand (data not shown). Subsequent experiments showed that it was not necessary to add RNase, possibly because the RNA strand was destroyed during thermocycling. Comparison of disinte- gration reactions using 32P-labeled substrates with either RNA or DNA Tbottom-32 strands showed indistinguishable amounts of product formation, indicating that the RNA strand was inﬂuencing the PCR detection step and not the disinte- gration reaction itself (data not shown). The Tbottom-32 RNA strand was thus used in all subsequent assays. In order to assess the sensitivity of the qPCR-based disin- tegration assay, we carried out disintegration reactions in the presence of decreasing concentrations of HIV-1 IN and quan- tiﬁed them using the qPCR and the 32P methods. For the qPCR-based detection, reaction products were diluted up to 250 000-fold in order to obtain measurements in the linear range of the standard curve (Figure 2B). In our test reactions, the lowest concentration of IN used, 0.0016 mM, generated product that was well within the linear range, even at a 50 000-fold dilution. Reactions performed in the absence of IN did not yield product after 40 cycles of ampliﬁcation. Figure 2C displays products of transesteriﬁcation reactions carried out using 32P-labeled substrate and analyzed by 8% denaturing-PAGE. Product formation was detectable for the three highest concentrations of IN, but was less evident with 0.08 mM or 0.0016 mM IN. The product size matched the labeled 97mer oligonucleotide marker as expected. The percent of substrate converted to product was measured using ImageQuant software and converted to the total number of product copies (Figure 2D). The dashed line in the graph represents background in the no IN control. The 0.008 mM IN reaction product is slightly above background, while the 0.0016 mM IN sample is below background. We have not made a speciﬁc effort to determine the lowest amount of product detectable using the qPCR-based assay. However, comparison of the product conversion measured under condi- tions of very low activity (see below) indicate that the qPCR- based disintegration assay is at least 1000-fold more sensitive than the 32P-based assay. Metal rescue analysis of cysteine substitutions in the HIV-1 IN active site We examined the disintegration capabilities of HIV-1 IN variants containing alanine and cysteine substitutions within the conserved D,DX35E catalytic triad in order to assess the role of metal binding in the active site (Figure 3). It has been hypothesized that the D64, D116 and E152 residues of HIV-1 IN coordinate two divalent metal ions (Figure 3A). The wild-type glutamate and aspartate amino acids are capa- ble of binding both Mn2+ and Mg2+. In contrast, cysteines can Figure 3. Functional metal binding by all three residues of the conserved D,DX35E motif of HIV-1 IN. (A) Two divalent metal ions proposed to bind to the active site are shown interacting with the conserved acidic residues of HIV-1 IN. This model was adapted from the structure of avian sarcoma leukosis virus (1VSJ.pdb) in complex with cadmium ions (8). (B) Assays of WT IN and substitutions at D64 and D116. (C) Assays of substitutions at E152. PCR-based disintegration assays were carried out with the indicated IN derivatives in the presence of 10 mM Mn2+ or 20 mM Mg2+. Disintegration reactions were incubated for 4 h at 37 C. The amount of product copies was determined using 250 000-fold dilutions of WT IN, 50 000-fold dilutions of D64 and D116 substituted INs and 10 000- fold dilutions or E152 substituted INs in the qPCR. The total product copies generated in the disintegration reaction is graphed. A one-tailed Mann–Whitney U-test was used to compare three independent disintegration reactions with qPCR in at least triplicate for each of the E152A and E152C Mn2+ reactions or the E152C Mn2+ and Mg2+ reactions to show that the difference between the sets was statistically significant with P < 0.0001. (D) A time course for disintegration activity with E152C IN. All error bars indicate one standard deviation around the mean of qPCR performed in triplicate. (E) Verification of the sizes of disintegration reaction products by PCR amplification and end labeling.