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Nucleic Acids Res. 2015 Apr 20;43(7):3643-52. doi: 10.1093/nar/gkv204. Epub 2015 Mar 23.

Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase.

Author information

1
Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia, 28049 Madrid, Spain.
2
Departamento Física Atómica, Molecular y Nuclear, Universidad Complutense, 28040 Madrid, Spain.
3
Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
4
Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia) & CNB-CSIC-IMDEA Nanociencia Associated Unit 'Unidad de Nanobiotecnología', 28049 Madrid, Spain.
5
Centro Nacional de Biotecnología (CNB-CSIC), 28049 Madrid, Spain.
6
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.
7
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.

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).

PMID:
25800740
PMCID:
PMC4402526
DOI:
10.1093/nar/gkv204
[Indexed for MEDLINE]
Free PMC Article

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