Single-Molecule Conformational Dynamics of E. coli DNA Polymerase I

Abstract

The extraordinary accuracy of DNA replication is due in part to the coordinated action of various enzymatic activities exhibited by many DNA polymerases. Structural studies have revealed that many of these functions are localized in spatially separated protein domains, but what is not known is how these domains are coordinated with respect to each other and the DNA substrate in order to ensure efficient and accurate DNA synthesis. One such polymerase is DNA polymerase I (Pol I) from E. coli, which is composed of the Klenow fragment (KF), containing the 5’-3’ polymerase and 3’-5’ exonuclease activities, and a separate 5’ nuclease domain attached via a flexible linker. Here we have used single-molecule FRET (smFRET) microscopy to determine the position of the 5’ nuclease domain of Pol I relative to the downstream strand of various immobilized model DNA substrates. A high-FRET state was observed for a substrate containing a downstream flap - the natural substrate for the 5’ nuclease activity - suggesting that the 5’ nuclease domain is docked with the downstream strand of the duplex, as expected. Interestingly, a similar high-FRET state was also observed for a gapped substrate, suggesting that the 5’ nuclease is also near the downstream strand during polymerase activity. Together, these observations may explain the increased processivity of Pol I versus KF during DNA synthesis and suggest that the 5’ nuclease domain may also serve to mediate the switch between strand-displacement synthesis and RNA primer removal during Okazaki fragment processing. Conversely, no FRET was observed for a substrate containing a double mismatch at the 3’ end of the primer, indicating that the 5’ nuclease domain is remote from the downstream strand of the substrate during proofreading activity.