Nick recognition by DNA ligases

Phage T7 DNA ligase seals nicked DNA substrates and is a representative mem ber of the ATP-dependent class of DNA ligases. Although the catalytic mecha nism of DNA ligases has been delineated, Little is known about the nature o f nick recognition by these enzymes. Here, we show that T7 ligase discrimin ates, at the nick-binding step, between nicks containing either a 5'-phosph ate or a 5'-OH. T7 ligase binds preferentially to phosphorylated nicks and catalyses the sealing reaction. We also show using DNA footprinting studies , that T7 Ligase binds asymmetrically to nicks as a monomer, with the prote in interface covering between 12 and 14 bp of DNA. Based on molecular model ling studies we propose a structural model of the ligase-DNA complex consis tent with these and other data. Using photo-crosslinking and site-directed mutagenesis we have identified two residues, K238 and K240, critical for th e transadenylation and nick-sealing reactions. Sequence conservation and st ructural analysis supports the premise that these two lysine residues are c ritical for both nucleotide binding and DNA nick recognition. The implicati ons of these results on the ligation mechanism are discussed. (C) 2000 Acad emic Press.