title: Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution.
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.
authors: Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.
authors: Thomas D
authors: TD
authors: Christian
authors: Louis J
authors: LJ
authors: Romano
authors: David
authors: D
authors: Rueda
mesh_terms: D020029:Base Pairing; D002384:Catalysis; D004256:DNA Polymerase I / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008722:Methods; D009038:Motion; D009711:Nucleotides / Q000378:metabolism*; D013698:Templates, Genetic
chemical_list: D029968:Escherichia coli Proteins; D009711:Nucleotides; D004256:DNA Polymerase I