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Use of 2-Aminopurine Fluorescence To Examine Conformational Changes during Nucleotide Incorporation by DNA Polymerase I (Klenow Fragment)

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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520
Cite this: Biochemistry 2003, 42, 34, 10200–10211
Publication Date (Web):August 9, 2003
https://doi.org/10.1021/bi0341206
Copyright © 2003 American Chemical Society

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    Abstract

    We have investigated conformational transitions in the Klenow fragment polymerase reaction by stopped-flow fluorescence using DNA substrates containing the fluorescent reporter 2-aminopurine (2-AP) on the template strand, either at the templating position opposite the incoming nucleotide (designated the 0 position) or 5‘ to the templating base (the +1 position). By using both deoxy- and dideoxy-terminated primers, we were able to distinguish steps that accompany ternary complex formation from those that occur during nucleotide incorporation. The fluorescence changes revealed two extremely rapid steps that occur early in the pathway for correct nucleotide incorporation. The first, detectable with the 2-AP reporter at the 0 position, occurs within the first few milliseconds and is associated with dNTP binding. This is followed by a rapid step involving relative movement of the +1 base, detectable when the 2-AP reporter is at the +1 position. Finally, when the primer had a 3‘-OH, a fluorescence decrease with a rate equal to the rate of nucleotide incorporation was observed with both 0 and +1 position reporters. When the primer was dideoxy-terminated, the only change observed at the rate expected for nucleotide incorporation had a very small amplitude, suggesting that the rate-limiting conformational change does not produce a large fluorescence change, and is therefore unlikely to involve a significant change in the environment of the fluorophore. Fluorescence changes observed during misincorporation were substantially different from those observed during correct nucleotide incorporation, implying that the conformations adopted during correct and incorrect nucleotide incorporation are distinct.

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     This work was supported by Grant GM-28550 from the National Institutes of Health.

    *

     To whom correspondence should be addressed. Phone:  (203) 432-8992. Fax:  (203) 432-3104. E-mail:  [email protected].

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    Examples of curve fitting and residuals for stopped-flow experiments using dP·0T1, dP·+1T1, and ddP·+1T1 substrates (Figures S1−S3). This material is available free of charge via the Internet at http://pubs.acs.org.

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