A Novel Fret-Based Structure Of Dna Polymerase Complexed With Kinked Gapped-Dna

DNA polymerase I Klenow Fragment (Pol) adopts multiple conformations during high-fidelity DNA replication and repair, many of which have not been resolved by conventional techniques because of their transient and dynamic nature. Early in the DNA repair process, Pol must recognise and bind a one-nucleotide-gapped DNA substrate. Both the structure of this complex and the recognition mechanism by which it is formed are currently unknown. After demonstrating the sub-angstrom accuracy of our distance measurements using duplex DNA standards, we present a single-molecule FRET-restrained structural model of the DNA-Pol complex and show that binding of the gapped-DNA is consistent with a conformational-selection mechanism.Experiments on donor-acceptor-labelled gapped-DNA alone showed it adopted a low-FRET (unkinked) and a higher-FRET (kinked) conformation. Increasing concentrations of Pol stabilised the higher-FRET conformer, whilst also leading to the appearance of a third state, attributed to binding of a second Pol. We selected the DNA-Pol binary species from this dynamic equilibrium for structural characterization, obtaining >30 DNA-DNA and DNA-Pol distances, sufficient to restrain a unique molecular model of the complex (a structure which has not been resolved by NMR or crystallography). In this structure, the DNA exhibits a ∼90 degree bend which is likely important for both the search for the site of polymerization and for subsequent catalysis.To further investigate the Pol-DNA binding mechanism we developed a protein induced fluorescence enhancement (PIFE)-FRET coupled assay, which reported simultaneously on the DNA conformation and the presence of the Pol. This assay showed that all bound DNAs were kinked. Taken together, these data are consistent with a conformational-selection mechanism, in which the gapped-DNA substrate is recognised in its kinked state. This mechanism contrasts with experiments on the nucleotide binding, which we show to proceed via an induced-fit mechanism.