Single-Molecule Measurements of Synthesis by DNA Polymerase with Base-Pair Resolution

Abstract

The catalytic mechanism of DNA polymerases has been shown to involve 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 (smFRET) intensity drops in discrete steps to values consistent with single nucleotide additions to the primer terminus.