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Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21109-14. doi: 10.1073/pnas.0908640106. Epub 2009 Dec 2.

Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution.

Author information

1
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.

Abstract

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 Förster 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.

PMID:
19955412
PMCID:
PMC2795520
DOI:
10.1073/pnas.0908640106
[Indexed for MEDLINE]
Free PMC Article

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