Mutational analysis of bacterial NAD+-dependent DNA ligase: role of motif IV in ligation catalysis.
The bacterial DNA ligase as a multiple domain protein is involved in DNA replication, repair and recombination. Its catalysis of ligation can be divided into three steps. To delineate the roles of amino acid residues in motif IV in ligation catalysis, site-directed mutants were constructed in a bacterial NAD+-dependent DNA ligase from Thermus sp. TAK16D. It was shown that four conserved residues (D286, G287, V289 and K291) in motif IV had significant roles on the overall ligation. Under single turnover conditions, the observed apparent rates of D286E, G287A, V289I and K291R mutants were clearly reduced compared with that of WT ligase on both match and mismatch nicked substrates. The effects of D286E mutation on overall ligation may not only be ascribed to the third step. The G287A mutation has a major effect on the second step. The effects of V289I and K291R mutation on overall ligation are not on the third step, perhaps other aspects, such as conformation change of ligase protein in ligation catalysis, are involved. Moreover, the amino acid substitutions of above four residues were more sensitive on mismatch nicked substrate, indicating an enhanced ligation fidelity.
Key Laboratory of Bio-resource and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China. hfengcd@yahoo.com.cn
Hong
H
Feng
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020137:Base Pair Mismatch / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013824:Thermus / Q000201:enzymology / Q000235:genetics; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D011088:DNA Ligases; C120640:DNA ligase (NAD)