title: Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion.
abstract: The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.
authors.affiliation: Biological Physics Research Group, Department of Physics, Clarendon Laboratory, University of Oxford, Parks Road, Oxford OX1 3PU, UK.
authors.forename: Johannes
authors.initials: J
authors.lastname: Hohlbein
authors.forename: Louise
authors.initials: L
authors.lastname: Aigrain
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Craggs
authors.forename: Oya
authors.initials: O
authors.lastname: Bermek
authors.forename: Olga
authors.initials: O
authors.lastname: Potapova
authors.forename: Pouya
authors.initials: P
authors.lastname: Shoolizadeh
authors.forename: Nigel D F
authors.initials: ND
authors.lastname: Grindley
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Joyce
authors.forename: Achillefs N
authors.initials: AN
authors.lastname: Kapanidis
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D001411:Geobacillus stearothermophilus / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D009711:Nucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009711:Nucleotides; D004256:DNA Polymerase I