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 compose d of an N-terminal domain (domain 1, containing nucleotidyl transferas e motifs I, III, IIIa and IV) and a C-terminal domain (domain 2, conta ining motif VI) with an intervening cleft, Motif V links the two struc tural domains. Deletion analysis of the 298 amino acid Chlorella virus DNA ligase indicates that motif VI plays a critical role in the react ion of ligase with ATP to form ligase-adenylate, but is dispensable fo r the two subsequent steps in the ligation pathway; DNA-adenylate form ation and strand closure. We find that formation of a phosphodiester a t a pre-adenylated nick is subject to a rate limiting step that does n ot apply during the sealing of nicked DNA by ligase-adenylate. This st ep, presumably conformational, is accelerated or circumvented by delet ing five amino acids of motif VI, The motif I lysine nucleophile (Lys2 7) is not required for strand closure by wild-type ligase, but this re sidue enhances the closure rate by a factor of 16 when motif VI is tru ncated. We find that a more extensively truncated ligase consisting of only N-terminal domain 1 and motif V is inert in ligase-adenylate for mation, but competent to catalyze strand closure at a pre-adenylated n ick. These results suggest that different enzymic catalysts facilitate the three steps of the DNA ligase reaction.