title: Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms.
abstract: DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intra-protein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s(-1), much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.
authors.affiliation: Department of Physics and Biological Physics Research Group, Clarendon Laboratory, University of Oxford, Parks Road, Oxford OX1 3PU, United Kingdom.
authors.forename: Geraint W
authors.initials: GW
authors.lastname: Evans
authors.affiliation: Department of Physics and Biological Physics Research Group, Clarendon Laboratory, University of Oxford, Parks Road, Oxford OX1 3PU, United Kingdom.
authors.forename: Johannes
authors.initials: J
authors.lastname: Hohlbein
authors.affiliation: Department of Physics and Biological Physics Research Group, Clarendon Laboratory, University of Oxford, Parks Road, Oxford OX1 3PU, United Kingdom.
authors.forename: Timothy
authors.initials: T
authors.lastname: Craggs
authors.affiliation: Department of Physics and Biological Physics Research Group, Clarendon Laboratory, University of Oxford, Parks Road, Oxford OX1 3PU, United Kingdom.
authors.forename: Louise
authors.initials: L
authors.lastname: Aigrain
authors.affiliation: Department of Physics and Biological Physics Research Group, Clarendon Laboratory, University of Oxford, Parks Road, Oxford OX1 3PU, United Kingdom a.kapanidis1@physics.ox.ac.uk.
authors.forename: Achillefs N
authors.initials: AN
authors.lastname: Kapanidis
mesh_terms: D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D009038:Motion; D011487:Protein Conformation
chemical_list: D003854:Deoxyribonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase