Figure 3
ATP independence of the K97A MthRnl mutant. (A) The reactions of 5’ pre-adenylated 3’-amino-blocked DNA donor (AppDNA17-NH2) with ssDNA acceptor (DNA25) and K97A MthRnl mutant without ATP or with increasing (5–500 μM) concentration of ATP. (B) The reaction of 5’-phosphorylated 3’-amino-blocked DNA donor (pDNA17-NH2) with ssDNA acceptor (DNA25) and K97A MthRnl mutant without ATP or with increasing (5–500 μM) concentration of ATP. (C) The reactions of 5’ pre-adenylated 3’-amino blocked DNA donor (AppDNA17-NH2) with ssDNA acceptor (DNA25) and wild type MthRnl without ATP or with increasing (5–500 μM) concentration of ATP. The ligation reactions were carried out as described in the Methods. The products were separated on 15% urea-PAGE and visualized with SYBR Gold stain. The concentration of ATP used in reactions is indicated at the bottom. Positions of substrates and ligated products are indicated on the right. Mr are molecular weight RNA markers. The ‘input’ indicates reactions without enzyme.