title: Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase.
abstract: During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN).
authors: Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia, 28049 Madrid, Spain.
authors: José A
authors: JA
authors: Morin
authors: Departamento Física Atómica, Molecular y Nuclear, Universidad Complutense, 28040 Madrid, Spain.
authors: Francisco J
authors: FJ
authors: Cao
authors: Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
authors: José M
authors: JM
authors: Lázaro
authors: Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia) & CNB-CSIC-IMDEA Nanociencia Associated Unit 'Unidad de Nanobiotecnología', 28049 Madrid, Spain.
authors: J Ricardo
authors: JR
authors: Arias-Gonzalez
authors: Centro Nacional de Biotecnología (CNB-CSIC), 28049 Madrid, Spain.
authors: José M
authors: JM
authors: Valpuesta
authors: Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia) & CNB-CSIC-IMDEA Nanociencia Associated Unit 'Unidad de Nanobiotecnología', 28049 Madrid, Spain Centro Nacional de Biotecnología (CNB-CSIC), 28049 Madrid, Spain.
authors: José L
authors: JL
authors: Carrascosa
authors: Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
authors: Margarita
authors: M
authors: Salas
authors: Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia) & CNB-CSIC-IMDEA Nanociencia Associated Unit 'Unidad de Nanobiotecnología', 28049 Madrid, Spain borja.ibarra@imdea.org.
authors: Borja
authors: B
authors: Ibarra
mesh_terms: D001692:Biological Transport; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D007700:Kinetics
chemical_list: D004259:DNA-Directed DNA Polymerase