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Single‐molecule experiments provide evidence for a new step in the catalytic mechanism of DNA polymerase

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 dynamics of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base‐pair resolution. Following nucleotide incorporation, the polymerase is observed translocating past the next pre‐insertion site, a distance equivalent to approximately two bases, and then backs up one base. Rate differences are observed between incorporation of pyrimidines and purines. When a mismatched primer/template junction is used, we observe the primer moving into the polymerase exonuclease site at a position clearly distinct from the translocation site. A revised model for DNA polymerase mechanism is proposed that includes a post‐insertion fidelity checking site that monitors the formation of a complementary nascent base‐pair as part of the proofreading mechanism. This study was supported by grants from the National Institutes of Health (CA40605,GM085116) and the National Science Foundation (MCB0747285).