Fig. 7.

Comparison of deaminase activity of WT and FW mutant A3G. a Labeled ssDNA construct 118 nts in length with potential deamination sites 17 nts from the 5′-end and 36 nts from the 3′-end. The 63-nt segment between the deamination sites contains a fluorescently labeled thymine. After deamination, the label will be on a 100-, 81- or 63-nt-long strand if the 5′-site, 3′-site or both sites are cleaved, respectively. b Gel electrophoresis used to determine deaminase activity of WT (left) and FW mutant (right) A3G. A3G (25 nM) is preincubated with a 69-nt unlabeled ssDNA construct without deamination sites for 3 min before the labeled ssDNA is added at 100 nM and allowed to deaminate for 10 min. In lanes 2 through 5, a concentration of 0, 0.5, 1 and 5 mM unlabeled DNA is used. Lane 1 is a control with the labeled ssDNA construct only. Labeled ssDNA with the 5′-site, 3′-site or both sites deaminated appear in distinct bands. c Percentage of potential sites deaminated as a function of unlabeled DNA concentration for both WT and FW mutant A3G. Error bars show SD of at least three independent experiments. Preincubating WT A3G with unlabeled DNA before the introduction of the labeled DNA significantly reduces deaminase activity. In contrast, FW mutant A3G continues to deaminate the labeled DNA even when preincubated with large amounts of unlabeled DNA. Results indicate that the deaminase activity of A3G is strongly repressed by A3G oligomerization. d Representation of effect of A3G oligomerization on catalytic activity. During pre-incubation, WT A3G forms dimers, which inhibits the deamination of the subsequently added labeled DNA. In contrast, the FW mutant A3G remains in the monomeric state during pre-incubation and retains its deaminase activity