title: Role of nucleotidyl transferase motif V in strand joining by chlorella virus DNA ligase.
abstract: ATP-dependent DNA ligases, NAD(+)-dependent DNA ligases, and GTP-dependent RNA capping enzymes are members of a covalent nucleotidyl transferase superfamily defined by a common fold and a set of conserved peptide motifs. Here we examined the role of nucleotidyl transferase motif V ((184)LLKMKQFKDAEAT(196)) in the nick joining reaction of Chlorella virus DNA ligase, an exemplary ATP-dependent enzyme. We found that alanine substitutions at Lys(186), Lys(188), Asp(192), and Glu(194) reduced ligase specific activity by at least an order of magnitude, whereas substitutions at Lys(191) and Thr(196) were benign. The K186A, D192A, and E194A changes had no effect on the rate of single-turnover nick joining by preformed ligase-adenylate but affected subsequent rounds of nick joining at the ligase adenylation step. Conservative substitutions K186R, D192E, and E194D partially restored activity, whereas K186Q, D192N, and E194Q substitutions did not. Alanine mutation of Lys(188) elicited distinctive catalytic defects, whereby single-turnover nick joining by K188A-adenylate was slowed by an order of magnitude, and high levels of the DNA-adenylate intermediate accumulated. The rate of phosphodiester bond formation at a pre-adenylated nick (step 3 of the ligation pathway) was slowed by the K188A change. Replacement of Lys(188) by arginine reversed the step 3 arrest, whereas glutamine substitution was ineffective. Gel-shift analysis showed that the Lys(188) mutants bound stably to DNA-adenylate. We infer that Lys(188) is involved in the chemical step of phosphodiester bond formation.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: Verl
authors.initials: V
authors.lastname: Sriskanda
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D004254:DNA Nucleotidyltransferases / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D020125:Mutation, Missense; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014764:Viral Proteins
chemical_list: D014764:Viral Proteins; D005973:Glutamine; D013912:Threonine; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D004247:DNA; D004254:DNA Nucleotidyltransferases; C110456:Chlorella virus DNA ligase; D011088:DNA Ligases; D008239:Lysine; D000409:Alanine