Mutational analysis of Chlorella virus DNA ligase: catalytic roles of domain I and motif VI.
A conserved catalytic core of the ATP-dependent DNA ligases is composed of an N-terminal domain (domain 1, containing nucleotidyl transferase motifs I, III, IIIa and IV) and a C-terminal domain (domain 2, containing motif VI) with an intervening cleft. Motif V links the two structural domains. Deletion analysis of the 298 amino acid Chlorella virus DNA ligase indicates that motif VI plays a critical role in the reaction of ligase with ATP to form ligase-adenylate, but is dispensable for the two subsequent steps in the ligation pathway; DNA-adenylate formation and strand closure. We find that formation of a phosphodiester at a pre-adenylated nick is subject to a rate limiting step that does not apply during the sealing of nicked DNA by ligase-adenylate. This step, presumably conformational, is accelerated or circumvented by deleting five amino acids of motif VI. The motif I lysine nucleophile (Lys27) is not required for strand closure by wild-type ligase, but this residue enhances the closure rate by a factor of 16 when motif VI is truncated. We find that a more extensively truncated ligase consisting of only N-terminal domain 1 and motif V is inert in ligase--adenylate formation, but competent to catalyze strand closure at a pre-adenylated nick. These results suggest that different enzymic catalysts facilitate the three steps of the DNA ligase reaction.
Sriskanda
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Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA.
Shuman
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics*; D002708:Chlorella / Q000821:virology; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D014764:Viral Proteins
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004247:DNA; C110456:Chlorella virus DNA ligase; D011088:DNA Ligases; D000409:Alanine