Fig. 2. JGT is specific for hmU containing DNA but does not possess sequence specificity.

(A) Radiolabeled in vitro glucosyltransferase reaction. Recombinant JGT and UDP-[³H] glucose was incubated with 36nt long dsDNA substrates listed in supplementary material and methods containing one hmU modification (hmU) or unmodified dsDNA (T). CPM (counts per minute), indicative of the transfer of glucose to DNA, were read for each sample. Experiment was performed in triplicate and error bars are representative of standard error. (B) UDP-Glo in vitro glucosyltransferase reaction. Recombinant JGT was incubated with 36nt long dsDNA substrates listed in Table 1 containing one hmU modification (hmU) or unmodified dsDNA (T). The amount of UDP Cleaved, indicative of the transfer of glucose to DNA, was estimated from a standard curve of UDP for each sample. Experiment was performed in triplicate and error bars are representative of standard error. (C) Representative substrate–velocity curve of recombinant JGT. Recombinant JGT activity with the 15nt long hmU-containing ds DNA substrate (WT substrate) listed in Table I. Glucosylation reactions were conducted with hmU DNA substrate concentrations of 0, 0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, and 25 μM and fixed enzyme and UDP-glucose concentrations of 18 μM and 1 mM, respectively. Kinetic experiments were performed in triplicate and error bars are representative of standard error. UDP cleaved is plotted vs substrate concentration, and nonlinear regression was performed to determine kinetic parameters.