Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D

NAD(+)-dependent DNA ligases from thermophilic bacteria Thermus species are highly homologous with amino acid sequence identities ranging from 85 to 9 8%. Thermus species AK16D ligase, the most divergent of the seven Thermus i solates collected worldwide, was cloned, expressed in Escherichia coli and purified to homogeneity. This Thermus ligase is similar to Thermus thermoph ilus HB8 ligase with respect to pH, salt, NAD(+), divalent cation profiles and steady-state kinetics. However, the former is more discriminative towar d T/G mismatches at the 3'-side of the ligation junction, as judged by the ratios of initial ligation rates of matched and mismatched substrates. The two wild-type Thermus ligases and a Tth ligase mutant (K294R) demonstrate 1 -2 orders of magnitude higher fidelity than viral T4 DNA ligase. Both Therm us ligases are active with either the metal cofactor Mg2+, Mn2+ or Ca2+ but not with Co2+, Ni2+, Cu2+ or Zn2+. While the nick closure step with Ca2+ b ecomes rate-limiting which results in the accumulation of DNA-adenylate int ermediate, Ni2+ only supports intermediate formation to a limited extent. B oth Thermus ligases exhibit enhanced mismatch ligation when Mn2+ is substit uted for Mg2+, but the Tsp. AK16D ligase remains more specific toward perfe ctly matched substrate.