title: Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules.
abstract: Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5'-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate.
authors.affiliation: Department of Macromolecular Structures, Centro Nacional de Biotecnología, CSIC, Darwin 3, 28049 Cantoblanco, Madrid, Spain.
authors.forename: Cesar L
authors.initials: CL
authors.lastname: Pastrana
authors.affiliation: Department of Macromolecular Structures, Centro Nacional de Biotecnología, CSIC, Darwin 3, 28049 Cantoblanco, Madrid, Spain.
authors.forename: Carolina
authors.initials: C
authors.lastname: Carrasco
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA 15219, USA.
authors.forename: Parvez
authors.initials: P
authors.lastname: Akhtar
authors.affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors.forename: Sanford H
authors.initials: SH
authors.lastname: Leuba
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA 15219, USA.
authors.forename: Saleem A
authors.initials: SA
authors.lastname: Khan
authors.affiliation: Department of Macromolecular Structures, Centro Nacional de Biotecnología, CSIC, Darwin 3, 28049 Cantoblanco, Madrid, Spain Fernando.Moreno@cnb.csic.es.
authors.forename: Fernando
authors.initials: F
authors.lastname: Moreno-Herrero
mesh_terms: D053904:DNA Breaks, Single-Stranded; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D008954:Models, Biological; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D011994:Recombinant Proteins; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases