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Characterization of a T7 DNA Polymerase Mutant with a Completely Rate‐limiting Conformational Change Step during Nucleotide Incorporation
Author(s) -
Jin Zhinan,
Johnson Kenneth A.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a657-a
Subject(s) - nucleotide , chemistry , conformational change , polymerase , mutant , dna , dna polymerase , biophysics , dna replication , biochemistry , stereochemistry , biology , gene
Previous studies of the T7 DNA polymerase using a conformational sensitive fluorophore (CSF) showed that a conformational change step provided selectivity by committing a correct nucleotide to incorporation while favoring dissociation of a mismatched nucleotide. In this study, we performed kinetic analysis of mutants in two flexible loops (Gly‐Ala‐Gly) connecting helices within the recognition domain. Mutation of all four glycines to alanines eliminated the DNA replication activity. Mutation of two glycines to alanines (G542A/G544A) in one loop reduced the rate of correct nucleotide incorporation to 0.5 s −1 . The absence of a thio elemental effect suggested that the conformational change is completely rate‐limiting. In support of this conclusion, stopped‐flow experiments using the CSF labeled enzyme showed that the forward rate of the conformational change was 0.5 s −1 . Analysis of processive polymerization showed that the first nucleotide incorporation (0.5 s −1 ) was slower than the ensuing steps (15 s −1 ), suggesting that the resting state of this mutant may differ from the nucleotide‐free state formed during processive polymerization. Our results imply that the conformation change reported by the CSF probe is dependent upon the flexible segments within the recognition domain and the transition from open to closed does not occur through a rigid body movement.