| Summary: | Vaccinia virus DNA ligase has been expressed in <em>Escherichia coli</em>, 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 <i>K<sub>m</sub></i> for the overall ligation of a ssDNA substrate of 0.8 m<i>M</i>. The salt, divalent cation, temperature, and pH requirements of the enzyme for the optimal ligation of ss and ds substrate are described.
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