IDENTIFICATION OF ESSENTIAL RESIDUES IN THERMUS-THERMOPHILUS DNA-LIGASE

DNA ligases play a pivotal role in DNA replication, repair and recombi nation. Reactions catalyzed by DNA ligases consist of three steps: ade nylation of the ligase in the presence of ATP or NAD(+), transferring the adenylate moiety to the 5'-phosphate of the nicked DNA substrate ( deadenylation) and sealing the nick through the formation of a phospho diester bond, Thermus thermophilus HB8 DNA ligase (Tth DNA ligase) dif fers from mesophilic ATP-dependent DNA ligases in three ways: (i) it i s NAD(+) dependent; (ii) its optimal temperature is 65 instead of 37 d egrees C; (iii) it has higher fidelity than T4 DNA ligase. In order to understand the structural basis underlying the reaction mechanism of Tth DNA ligase, we performed site-directed mutagenesis studies on nine selected amino acid residues that are highly conserved in bacterial D NA ligases. Examination of these site-specific mutants revealed that: residue K118 plays an essential role in the adenylation step; residue D120 may facilitate the deadenylation step; residues G339 and C433 may be involved in formation of the phosphodiester bond. This evidence in dicates that a previously identified KXDG motif for adenylation of euk aryotic DNA ligases [Tomkinson, A.E., Totty, N.F., Ginsburg, M. and Li ndahl, T. (1991) Proc. Natl. Acad. Sci. USA, 88, 400-404] is also the adenylation site for NAD(+)-dependent bacterial DNA ligases, In a comp anion paper, we demonstrate that mutations at a different Lys residue, K294, may modulate the fidelity of Tth DNA ligase.