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J. Biol. Chem., Vol. 277, Issue 12, 9661-9667, March 22, 2002
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From the Molecular Biology Program, Sloan-Kettering Institute, New
York, New York 10021
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 (184LLKMKQFKDAEAT196) in
the nick joining reaction of Chlorella virus DNA ligase, an
exemplary ATP-dependent enzyme. We found that alanine
substitutions at Lys186, Lys188,
Asp192, and Glu194 reduced ligase specific
activity by at least an order of magnitude, whereas substitutions at
Lys191 and Thr196 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 Lys188 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 Lys188 by arginine reversed
the step 3 arrest, whereas glutamine substitution was ineffective.
Gel-shift analysis showed that the Lys188 mutants bound
stably to DNA-adenylate. We infer that Lys188 is involved
in the chemical step of phosphodiester bond formation.
Role of Nucleotidyl Transferase Motif V in Strand Joining by
Chlorella Virus DNA Ligase*
*
This work was supported by National Institutes of Health
Grant GM63611.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. E-mail:
s-shuman@ski.mskcc.org.
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