Nick recognition by DNA ligases.
Phage T7 DNA ligase seals nicked DNA substrates and is a representative member of the ATP-dependent class of DNA ligases. Although the catalytic mechanism of DNA ligases has been delineated, little is known about the nature of nick recognition by these enzymes. Here, we show that T7 ligase discriminates, at the nick-binding step, between nicks containing either a 5'-phosphate 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 protein interface covering between 12 and 14 bp of DNA. Based on molecular modelling studies we propose a structural model of the ligase-DNA complex consistent with these and other data. Using photo-crosslinking and site-directed mutagenesis we have identified two residues, K238 and K240, critical for the transadenylation and nick-sealing reactions. Sequence conservation and structural analysis supports the premise that these two lysine residues are critical for both nucleotide binding and DNA nick recognition. The implications of these results on the ligation mechanism are discussed.
Doherty
A J
AJ
Structural Medicine Unit Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease Cambridge Institute for Medical Research, and Department of Haematology, University of Cambridge, Hills Rd, Cambridge, CB2 2XY, UK. aidan@mrc-lmb.cam.ac.uk
Dafforn
T R
TR
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000528:radiation effects; D002384:Catalysis; D017124:Conserved Sequence / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics*; D018983:DNA Footprinting; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding / Q000528:radiation effects; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D009838:Oligodeoxyribonucleotides; D010710:Phosphates; D004247:DNA; D011088:DNA Ligases; D008239:Lysine