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Papers In Press, published online ahead of print December 20, 2001
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021
Corresponding Author: s-shuman{at}ski.mskcc.org
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 pre-formed 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.
J. Biol. Chem, 10.1074/jbc.M110613200
Submitted on November 5, 2001
Revised on December 20, 2001
Accepted on December 17, 2001
Role of nucleotidyl transferase motif V in strand joining by Chlorella virus DNA ligase
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