Nucleic Acids Research, Vol 26, Issue 20 4618-4625, Copyright © 1998 by Oxford University Press
V Sriskanda and S Shuman
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.
ARTICLES
Mutational analysis of Chlorella virus DNA ligase: catalytic roles of domain I and motif VI
Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA.
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