Journal of Biological Chemistry
Volume 286, Issue 16, 22 April 2011, Pages 14480-14492
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Enzymology
Distinct Complexes of DNA Polymerase I (Klenow Fragment) for Base and Sugar Discrimination during Nucleotide Substrate Selection*

https://doi.org/10.1074/jbc.M111.218750Get rights and content
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During each catalytic cycle, DNA polymerases select deoxyribonucleoside triphosphate (dNTP) substrates complementary to a templating base with high fidelity from a pool that includes noncomplementary dNTPs and both complementary and noncomplementary ribonucleoside triphosphates (rNTPs). The Klenow fragment of Escherichia coli DNA polymerase I (KF) achieves this through a series of conformational transitions that precede the chemical step of phosphodiester bond formation. Kinetic evidence from fluorescence and FRET experiments indicates that discrimination of the base and sugar moieties of the incoming nucleotide occurs in distinct, sequential steps during the selection pathway. Here we show that KF-DNA complexes formed with complementary rNTPs or with noncomplementary nucleotides can be distinguished on the basis of their properties when captured in an electric field atop the α-hemolysin nanopore. The average nanopore dwell time of KF-DNA complexes increased as a function of complementary rNTP concentration. The increase was less than that promoted by complementary dNTP, indicating that the rNTP complexes are more stable than KF-DNA binary complexes but less stable than KF-DNA-dNTP ternary complexes. KF-DNA-rNTP complexes could also be distinguished from KF-DNA-dNTP complexes on the basis of ionic current amplitude. In contrast to complementary rNTPs, noncomplementary dNTPs and rNTPs diminished the average nanopore dwell time of KF-DNA complexes in a concentration-dependent manner, suggesting that binding of a noncomplementary nucleotide keeps the KF-DNA complex in a less stable state. These results imply that nucleotide selection proceeds through a series of complexes of increasing stability in which substrates with the correct moiety promote the forward transitions.

DNA Enzymes
DNA Polymerase
DNA-Protein Interaction
DNA Replication
Single Molecule Biophysics

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*

This work was supported, in whole or in part, by National Institutes of Health Grants 1RC2HG005553 from the NHGRI (to M. A.) and 1R01GM087484-01A2 from the NIGMS (to K. R. L. and M. A.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1, Figs. S1–S4, and additional mathematical methods.

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Supported in part by National Science Foundation Grant DMS-0719361.