Identification of essential residues in Thermus thermophilus DNA ligase.
DNA ligases play a pivotal role in DNA replication, repair and recombination. Reactions catalyzed by DNA ligases consist of three steps: adenylation 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 phosphodiester bond. Thermus thermophilus HB8 DNA ligase (Tth DNA ligase) differs from mesophilic ATP-dependent DNA ligases in three ways: (i) it is NAD+ dependent; (ii) its optimal temperature is 65 instead of 37 degrees 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 DNA 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 indicates that a previously identified KXDG motif for adenylation of eukaryotic DNA ligases [Tomkinson, A.E., Totty, N.F., Ginsburg, M. and Lindahl, T. (1991) Proc. Natl. Acad. Sci. USA, 88, 400-404] is also the adenylation site for NAD+-dependent bacterial DNA ligases. In a companion paper, we demonstrate that mutations at a different Lys residue, K294, may modulate the fidelity of Tth DNA ligase.
Luo
J
J
Department of Microbiology, Strang Cancer Prevention Center, Cornell University Medical College, NY 10021, USA.
Barany
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004261:DNA Replication; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010755:Organophosphates / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D010755:Organophosphates; D011994:Recombinant Proteins; D011088:DNA Ligases; D008239:Lysine