Ligation of double-stranded and single-stranded [oligo(dT)] DNA by vaccinia virus DNA ligase.
Vaccinia virus DNA ligase has been expressed in Escherichia coli, purified, and biochemically characterized. The enzyme ligates double-stranded (ds) DNA substrates with either cohesive or blunt-end termini and the latter reaction is stimulated by PEG. Vaccinia virus DNA ligase can also ligate oligo(dT) when annealed to either a poly(dA) or a poly(rA) backbone and, remarkably, free oligo(dT). This ligation of a single-stranded (ss) substrate is unique among eukaryotic DNA ligases. The enzyme requires high ATP concentrations with a Km for the overall ligation of a ssDNA substrate of 0.8 mM. The salt, divalent cation, temperature, and pH requirements of the enzyme for the optimal ligation of ss and ds substrate are described.
Odell
M
M
Sir William Dunn School of Pathology, University of Oxford, United Kingdom.
Kerr
S M
SM
Smith
G L
GL
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology*
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C027903:oligo (dT); D000255:Adenosine Triphosphate; D004247:DNA; D011088:DNA Ligases