bind tightly to Mn2+, but not to Mg2+. Cysteine-substituted INs that are functional in the presence of Mn2+, but not Mg2+, reveal amino acids that interact directly with metal ions during enzyme catalysis. Alanine can not bind either divalent metal ion, so alanine substitutions serve as negative controls. In these studies, we analyzed the disintegration activity in the presence of 10 mM MnCl2 or 20 mM MgCl2. These metal concentrations were chosen because they showed the highest amount of product conversion during metal titration experi- ments with wild-type (WT) IN (data not shown). As seen in Figure 3B, WT IN was able to convert 1.44 ± 0.49 (·1011) copies of substrate to product using Mn2+ as the coordinating metal ion during a 4 h disintegration reaction. At optimized metal concentrations, activity in the presence of Mg2+ was 20% lower than in the presence of Mn2+. The initial rate of product formation was estimated at 51.03 ± 7.47 (·10 6) product copies generated per minute per molecule IN for the ﬁrst 60 min in Mn2+ containing reactions, and 15.10 ± 1.23 (·10 6) product copies generated per minute per mole- cule IN in Mg2+ containing reactions. IN is a sluggish enzyme in vitro, perhaps because it needs to carry out each reaction step only once in vivo. Turnover is very slow, but has been detected in speciﬁcally designed experiments (39). The D64A IN did not convert any detectable substrate to product in either Mn2+ or Mg2+. The D64C IN was able to gen- erate disintegration product in the presence of Mn2+, but not Mg2+, indicating functional binding of the Mn2+ ion to the cys- teine residue (Figure 3B). A time course for D64C showed that the initial rate of product conversion for D64C in the presence of Mn2+ was about 2.35 ± 0.21 (·10 6) product copies gener- ated per minute per molecule IN. In the presence of the D116C there was a large increase in activity with Mn2+, while no activity was detected with Mg2+. For the D116A IN there was a low, but measurable, amount of product formed in the presence of Mn2+ (4- to 5-fold lower than the D116C IN at 4 h), but not in the presence of Mg2+. This may indicate that D116 is not as critical for metal bind- ing as the other two members of the catalytic triad. For sev- eral other members of the RNase H family, low levels of activity have also been seen with corresponding amino acid substitutions (12,14,22,40). The rate of product formation during a 60 min time course with Mn2+ was measured and the D116C IN was found to generate about 19.09 ± 1.00 (·10 6) product copies per minute per molecule IN compared to about 1.79 ± 0.14 (·10 6) product copies generated per minute per molecule IN for the D116A IN in the presence Mn2+. We next tested the E152A and E152C INs (Figure 3C), which previously had undetectable activity. The E152A IN showed no detectable activity in a 4 h disintegration reaction. The E152C IN, on the other hand, was able to produce prod- uct in the presence of Mn2+, and not in the presence of Mg2+, revealing functional metal binding by the cysteine residue. As seen in Figure 3D, there was increased product formed over time by the E152C enzyme in the presence of Mn2+. The rate over the ﬁrst 60 min was estimated at 0.564 ± 0.058 (·10 6) product copies generated per minute per molecule IN, which is 1% the rate measured for the WT IN. In order to verify that the product measured in E152C IN reactions was the expected transesteriﬁcation product, we Nucleic Acids Research, 2006, Vol. 34, No. 21 6121 ampliﬁed the reaction product using a radiolabeled Dis F pri- mer instead of carrying out qPCR with the TaqMan probe. The PCR products were compared to a 32P-labeled synthetic 97mer oligonucleotide matching the expected product (Figure 3E). The 97mer product comprised 70–90% of the detectable product in reactions containing WT IN in the pres- ence of either metal cofactor. No product was seen for the E152A IN with either metal ion. For E152C IN, 90% of prod- uct was the expected 97mer and it was only detected in the presence of Mn2+. The doublet seen for WT IN is either a PCR artifact, an artifact resulting from overloading the gel well or the result of a low level of transesteriﬁcation invol- ving another phosphodiester. In the next sections, we present two further uses of the qPCR-based disintegration assay in studies of HIV IN. In the ﬁrst, we show that transesteriﬁcation by IN can involve a DNA 50 end as well as a 30 end. In the second, we detect catalysis in the presence of metal atoms only, allowing an ini- tial estimation of the rate enhancement contributed by the protein scaffold of the IN enzyme. WT IN activity on a substrate with reversed chemical polarity In order to examine alternate transesteriﬁcation reactions, we designed a new substrate (Figure 4A) in which an exposed 50 hydroxyl is present near the joining point of the ‘target’ and ‘LTR’ DNA analogs, rather than the standard 30 hydroxyl. In reactions with this reversed polarity substrate, product will be measurable only if the free 50 hydroxyl is able to act as a nucleophile for attacking the backbone to form the 97mer product. As seen in Figure 4B, the wild-type IN was able to generate product over time in the presence of Mn2+ on the reverse polarity substrate. Product was not observed in the presence of Mg2+. The rate of product formation in the presence of Mn2+ was 1.07 ± 0.14 (·10 6) product copies generated per minute per molecule IN, about 1% of the rate with WT IN. We also attempted to test the alanine and cysteine-substituted enzymes on this substrate. No activity was detected, indicating a lack of metal rescue (data not shown). This may be due to suboptimal positioning of the alternate substrate in the active site of IN confounded by the presence of amino acid substitutions, which alter the conformation of the active site. DNA transesterification reactions in the absence of protein In order to study the rate enhancement contributed by the IN-protein scaffold, we assayed disintegration in reactions containing metal ion cofactors without added protein catalyst. For these reactions we used the standard disintegration sub- strate and buffer conditions as used for IN reactions and com- pared different metals or no added metal (100 mM EDTA) in the absence of IN. We initially scanned a wide range of con- ditions, including comparing incubation at either 37 or 95 C for up to 24 h. In one test, reaction mixtures were dehydrated in a SpeedVac before heating at 95 C. Dehydrated reactions mixtures incubated at 95 C produced measurable amounts of transesteriﬁcation product (Figure 5). In contrast, 37 C incubation (either with or without dehydra- tion) did not yield any detectable product (data not shown).