LIGATION OF DOUBLE-STRANDED AND SINGLE-STRANDED [OLIGO(DT)] DNA BY VACCINIA VIRUS-DNA LIGASE

Vaccinia virus DNA ligase has been expressed in Escherichia coil, puri fied, and biochemically characterized. The enzyme ligates double-stran ded (ds) DNA substrates with either cohesive or blunt-end termini and the latter reaction is stimulated by PEG, Vaccinia virus DNA ligase ca n also ligate oligo(dT) when annealed to either a poly(dA) or a poly(r A) backbone and, remarkably, free oligo(dT). This ligation of a single -stranded (ss) substrate is unique among eukaryotic DNA ligases. The e nzyme requires high ATP concentrations with a K-m for the overall liga tion of a ssDNA substrate of 0.8 mM, The salt, divalent cation, temper ature, and pH requirements of the enzyme for the optimal ligation of s s and ds substrate are described. (C) 1996 Academic Press, Inc.