Biochemical characterization of an ATP-dependent DNA ligase from the hyperthermophilic crenarchaeon Sulfolobus shibatae

A gene encoding a putative ATP-dependent DNA ligase was identified in the genome of the hyperthermophilic archaeon Sulfolobus shibatae and expressed in Escherichia coli. The 601 amino acid recombinant polypeptide was a monomeric protein capable of strand joining on a singly nicked DNA substrate in the presence of ATP (K-m = 34 muM) and a divalent cation (Mn2+, Mg2+, or Ca2+). dATP was partially active in supporting ligation catalyzed by the protein, but GTP, CTP, UTP, dGTP, dCTP, dTTP, and NAD+ were inactive. The cloned Ssh ligase showed an unusual metal cofactor requirement; it was significantly more active in the presence of Mn2+ than in the presence of Mg2+ or Ca2+. Unexpectedly, the native Ssh ligase preferred Mg2+ and Ca2+ rather than Mn2+. Both native and recombinant enzymes displayed optimal nick-joining activity at 60-80degreesC. Ssh ligase discriminated against substrates containing mismatches on the 3'-side of nick junction and was more tolerant of mismatches at the 5'-end than of those at the penultimate 5'-end. The enzyme showed little activity on a 1-nucleotide gapped substrate. This is the first biochemical study of a DNA ligase from the crenarchaeotal branch of the archaea domain.