Activity of the MthRnl and the K246A, K246Q and K97A mutants in different steps of the ligation reaction. (A) pH-dependence of self-adenylation activity of enzymes (step 1) in reactions with radioactive ATP. The reactions were carried out as described in the Methods and the products were resolved by an SDS-PAGE. Autoradiographs of the gels are shown. (B) pH-dependence of adenylation of 5’-phosphorylated, 3’-amino-blocked ssDNA (pDNA17-NH2) (step 2) with wild type and the mutants of MthRnl under reaction conditions described in the Methods. The reaction products were separated by 15% urea-PAGE and visualized with SYBR Gold stain. (C) pH-dependence of deadenylation/hydrolysis of 5’ pre-adenylated DNA (AppDNA17-NH2) with wild type and the mutants of MthRnl under reaction conditions described in the Methods. The products were analyzed on 15% urea-PAGE and stained with SYBR Gold. Enzymes used in each step of ligation reactions are indicated on the left. Position of DNA donor substrates and products are indicated on the right. The pH of the buffer is indicated at the bottom. The control (c) indicates the reaction without enzyme.