Thermophilic DNA ligase. Purification and properties of the enzyme from Thermus thermophilus HB8.

Thermophilic and thermostable DNA ligase was purified to near homogeneity from the extract of Ther- mus thermophilus HBS.The purified enzyme has an isoelectric point at pH 6.6 and consists of a single polypeptide of about 79,000 in molecular weight on the bases of sodium dodecyl sulfate-polyacrylamide gel electrophoresis data and an equilibrium sedimentation method.The enzyme requires divalent cations, Mg2+ or Mn2+, and the optimum concentration of these ions being 5-9 X M and 3-6 X M, respectively.The enzyme also requires NAD as a cofactor.The apparent K,,, for NAD is 1.85 X lo-' M and that of (dT)lo is 1.4 x M. The pH optimum is 7.4-7.6 in Tris-HC1 and 8.0 in collidine/HCl buffer.The joining reaction is activated by K+ and NHZ at a concentration of 2-100 m M and inhibited by Na+ above 25 mM.The optimum temperatures of the joining of thymidylate oligomers in the presence of poly(dA) as a template are 27.5 "C for p(dT)', 34.5 O C for p(dT)lo, and 37 "C for p(dT)12-l~ and that of cohesive-end DNA restriction fragments is 24-37 "C.The nick-closing activity of the enzyme was observed over a wide range of the temperature from 15 to 85 "C and the optimum temperature is 65-72 "C.The temperature dependency of ligation with HB8 DNA ligase for various substrates was found to shift to a region of 7-10 "C higher than that of T4 DNA ligase and the activity of HBS DNA ligase decreased remarkably below 4 O C .The enzyme was stable for 1 week at 37 "C, its activity dropped by 50% within 2 days at 65 "C.DNA ligases catalyze the formation of phosphodiester linkages between DNA chains.They have been isolated from a variety of sources.In particular the enzymes from Escherichia coli and T4 phage have been studied extensively (1-4).Recently, various enzymes from thermophilic bacteria have been isolated such as DNA-dependent RNA polymerase (5) and nuclease TT1 (6) from Thermus therrnophilus HB8 and restriction endonucleases from T. thermophilus 111 (7, 8).These enzymes are thermostable and have high optimum temperatures as expected in consideration of their source.An attempt was thus made to find a new thermostable and thermophilic DNA ligase in thermophilic bacteria.It was worth-while to determine whether the ligation of a thermophilic DNA ligase would proceed at a high temperature, con-