期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 106, 期 50, 页码 21109-21114出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0908640106
关键词
Klenow Fragment; polymerase and exonuclease site; single molecule fluorescence; single nucleotide resolution; structural dynamics
资金
- National Institutes of Health [CA40605, GM085116]
- National Science Foundation CAREER Award [MCB0747285]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [0747285] Funding Source: National Science Foundation
The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3'-hydroxyl of the growing DNA chain. Here we report a single-molecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule Forster resonance energy transfer intensity drops in discrete steps to values consistent with single-nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer/template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.
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