Nucleic Acids Research, 2006, Vol. 34, No. 21 6123 Figure 5. DNA transesterification reactions in the presence of Mn2 alone. DNA transesterification reactions were carried out on the standard substrate in the absence of IN. (A) Reactions with 50 mM MnCl2 or 100 mM EDTA were not treated (‘no dehyd’) or dehydrated in a SpeedVac (‘dehyd’) followed by heating for the indicated amount of time at 95 C and 1/100 dilutions were analyzed by qPCR. The total product copies generated during the transesterification reaction are graphed. Error bars indicate one standard deviation around the mean of two independent transesterification reactions each analyzed in triplicate by qPCR. The difference between the reactions with Mn2+ versus EDTA was compared for each time point and found to be significant with a P-value of 0.0011, based on a one- tailed Mann–Whitney U-test, for all time points after 1 h. (B) Verification of the sizes of reaction products by PCR amplification with end-labeled primer and 8% denaturing gel electrophoresis. Products for PCRs containing 10-fold dilutions of reactions from the 22 h time point were compared to amplifications of the 97mer oligonucleotide representing reaction product and used in the standard curve. The number of copies of 97mer standard added is indicated above the autoradiogram. steps in the reaction pathway. Thus strictly speaking, compli- cated models are not ruled out in which the Cys–metal com- plexes in the rescue experiments are acting differently than the carboxyl–metal complexes in wild-type IN. However, together with the results on the Tn7 and Tn10 transposases, these results are most simply interpreted as evidence that IN/transposase enzymes of the RNase H-like superfamily probably all use two divalent metal ions for catalysis. We also used the qPCR-based assay to examine catalysis on a reverse polarity substrate, which has a free 50 hydroxyl at the junction of target DNA and the LTR end instead of a 30 hydroxyl. The wild-type HIV-1 IN was capable of catalyz- ing a DNA transesteriﬁcation reaction on this substrate in the presence of Mn2+, but not in the presence of Mg2+. In attempted metal rescue experiments, no activity was detected for any of the cysteine-substituted INs with either Mn2+or Mg2+. Possibly incorrect positioning of the substrate within the enzyme active site made it difﬁcult for the enzyme to act in the presence of further impaired enzyme/metal combi- nations. Whether this reaction has any biological role is unknown. The ﬁnding that a 50 hydroxyl can be recruited by IN to participate in transesteriﬁcation adds to the surpris- ingly wide range of hydroxylated compounds reported to function as nucleophiles in the presence of Mn2+, including 30 hydroxyls of various nucleotides and a variety of vicinal alcohols (42–46). The increased sensitivity of the qPCR-based assay also pro- vides a convenient and quantitative method for investigating novel catalysts of DNA transesteriﬁcation reactions. Here, we investigated metal-mediated catalysis in the absence of a protein enzyme. Previously, it was suggested that, for enzymes binding two metals, the metal atoms may be responsible for most of the catalytic contacts in the transition state, while the protein serves to bind and orient the metals and substrates (2). According to this idea, it might be possible to detect trans- esteriﬁcation in the presence of high concentrations of metals with a sufﬁciently sensitive assay. We subjected the standard substrate presented here (Figure 1B, upper) to high metal con- centrations, high temperature and dehydration, which allowed detection of ampliﬁable product. The reaction required Mn2+, since product accumulation over time was only detected in reactions containing Mn2+, and not Mg2+ or EDTA. The detection of low level transesteriﬁcation in the pres- ence of Mn2+ only allowed us to make a ﬁrst estimation of the rate enhancement due to binding metal and reactant DNAs to the IN-protein scaffold. In previous studies of sta- phylococcal nuclease, the spontaneous hydrolysis rate of phosphodiesters was measured with chemical models, allow- ing the rate enhancement by the enzyme to be estimated as a factor of 1017 (47). Here we estimate that the rate enhance- ment of IN plus Mn2+ over Mn2+ alone is a factor of at least 60 million. This estimate is a lower bound, because the reactions were carried out under quite different condi- tions. For example, it is possible that the polypropylene sur- face of the microcentrifuge tube on which the Mn2+-only reaction took place played a role, as has been proposed for mineral surfaces in pre-biotic chemistry (48,49). If so, the spontaneous rate in the absence of IN would be even lower. Further optimization of conditions for both the IN-catalyzed and Mn2+-alone reactions could also inﬂuence the measured difference in rate. Despite these uncertainties, this estimation of the rate enhancement provides new information potentially useful for quantitative modeling of active site function. Lastly, we note that the qPCR-based disintegration assay is potentially useful for a wide range of applications that require sensitive detection of transesteriﬁcation products. The RAG-1 recombinase and several bacterial transposases are known to carry out disintegration reactions, so the qPCR-based method can be used to study mutants or reaction conditions supporting only low level activity of these enzymes. The new assay also provides a method for screening candidate small molecule inhibitors of IN or other enzymes