title: dNTP-dependent conformational transitions in the fingers subdomain of Klentaq1 DNA polymerase: insights into the role of the "nucleotide-binding" state.
abstract: BACKGROUND
  Conformational selection plays a key role in the polymerase cycle.
  
  
  RESULTS
  Klentaq1 exists in conformational equilibrium between three states (open, closed, and “nucleotide-binding”) whose level of occupancy is determined by the bound substrate.
  
  
  CONCLUSIONS
  The “nucleotide-binding” state plays a pivotal role in the reaction pathway.
  
  
  CONCLUSIONS
  Direct evidence is provided for the role of a conformationally distinct “nucleotide-binding” state during dNTP incorporation. DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the "nucleotide-binding" state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state.
authors.affiliation: Chair for Molecular Physical Chemistry, Heinrich-Heine University, Universitätsstraβe 1, 40225 Düsseldorf, Germany. p.rothwell@mail.cryst.bbk.ac.uk
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Rothwell
authors.forename: William J
authors.initials: WJ
authors.lastname: Allen
authors.forename: Evangelos
authors.initials: E
authors.lastname: Sisamakis
authors.forename: Stanislav
authors.initials: S
authors.lastname: Kalinin
authors.forename: Suren
authors.initials: S
authors.lastname: Felekyan
authors.forename: Jerker
authors.initials: J
authors.lastname: Widengren
authors.forename: Gabriel
authors.initials: G
authors.lastname: Waksman
authors.forename: Claus A M
authors.initials: CA
authors.lastname: Seidel
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D004256:DNA Polymerase I / Q000737:chemistry*; D003838:Deoxyadenine Nucleotides / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006834:Hydrazines / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D013194:Staining and Labeling; D013379:Substrate Specificity; D013824:Thermus / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D003838:Deoxyadenine Nucleotides; D005456:Fluorescent Dyes; D006834:Hydrazines; D013942:Thymine Nucleotides; D004256:DNA Polymerase I; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate
keywords: Conformational Selection; DNA Enzymes; DNA Polymerase; Enzyme Kinetics; Fluorescence Resonance Energy Transfer (FRET); Single Molecule Biophysics