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Cited by 121DNA and Nucleic Acid ChemistryProtein Structure and DynamicsDNA Repair Mechanisms

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

Purohit, Vandana, Grindley, Nigel D. F., Joyce, Catherine M.·2003·Biochemistry
DOI 10.1021/bi0341206
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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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  • A recent study using stopped-flow fluorescence has suggested that the conformations adopted during correct and incorrect nucleotide incorporation are distinct and that specificity for the correct substrate could be generated if these conformational differences persist into the transition states, consistent with an induced fit mechanism.

    “Indeed, a recent study (41) using stopped-flow fluorescence has suggested that the conformations adopted during correct and incorrect nucleotide incorporation are distinct and that specificity for the correct substrate could be generated if these conformational differences persist into the transition states, consistent with an induced fit mechanism (for further discussions, see Refs.41 and 42).”

    From DNA Replication Fidelity (2004) · cites this paper as [41]

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Authors
Purohit, V., Grindley, N. D. F., & Joyce, C. M.
Affiliations
Yale University (US)
Corresponding author
Joyce, Catherine M.
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
2003-08-09
Volume
42
Issue
34
Pages
10200-10211
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi0341206
OpenAlex
W2029605473
Cited by
121
References
16
Citations by year
2026: 1 · 2025: 2 · 2024: 1 · 2023: 1 · 2022: 5 · 2021: 4 · 2020: 3 · 2019: 1 · 2018: 2 · 2017: 1 · 2016: 4 · 2015: 5 · 2014: 5 · 2013: 8 · 2012: 5
FWCI
2.30
Type
journal article
Language
en
Open access
closed
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
Funders
National Institute of General Medical Sciences (GM-28550, R01GM028550)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Keywords
DNA polymerase I Klenow fragment, nucleotide incorporation, conformational change, 2-aminopurine fluorescence, ternary complex formation, dNTP binding, misincorporation, stopped-flow fluorescence, polymerase mechanism
MeSH terms
Base Sequence, Binding Sites, Deoxyribonucleotides, DNA Polymerase I, Fluorescent Dyes, Kinetics, Magnesium, Manganese, Models, Molecular, Protein Binding, Protein Conformation, Spectrometry, Fluorescence, Substrate Specificity, Templates, Genetic, Thymine Nucleotides, 2-Aminopurine, DNA Primers

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