Kinetics of single-turnover nick sealing by Rnl2 at correctly paired 3′-OH ends. (A) The singly nicked duplex substrate for sealing by Rnl2–AMP is shown with the 5′ 32P-label at the nick denoted by •. The 3′-OH strand is all RNA. The 3′ N:X base pair at the nick (highlighted in the shaded box) is variable. A product analysis by denaturing PAGE of a sealing reaction of Rnl2 at a 3′ C:I nick (where I is inosine) is shown. The times at which the reactions were quenched are specified above the lanes. The positions of the radiolabeled 18-mer 5′-PO4 strand (pNick), the 5′-adenylylated intermediate (AppNick), and the sealed product are indicated on the left. (B) The distributions of radiolabeled AppNick intermediate and sealed product during the reaction of Rnl2 at a 3′ C:I nick are plotted as a function of time. Each datum in the graph is the average of three separate experiments. The curve fits to the kinetic scheme are shown. (C) The step 2 and step 3 rate constants for sealing nicks with the indicated 3′ N:X pairs.
