title: Kinetic mechanism of initiation by RepD as a part of asymmetric, rolling circle plasmid unwinding.
abstract: Some bacterial plasmids carry antibiotic resistance genes and replicate by an asymmetric, rolling circle mechanism, in which replication of the two strands is not concurrent. Initiation of this replication occurs via an initiator protein that nicks one DNA strand at the double-stranded origin of replication. In this work, RepD protein from the staphylococcal plasmid pC221 carries this function and allows PcrA helicase to bind and begin unwinding the plasmid DNA. This work uses whole plasmid constructs as well as oligonucleotide-based mimics of parts of the origin to examine the initiation reaction. It investigates the phenomenon that nicking, although required to open a single-stranded region at the origin and so allow PcrA to bind, is not required for another function of RepD, namely to increase the processivity of PcrA, allowing it to unwind plasmid lengths of DNA. A kinetic mechanism of RepD initiation is presented, showing rapid binding of the origin DNA. The rate of nicking varies with the structure of the DNA but can occur with a rate constant of >25 s(-1) at 30 °C. The equilibrium constant of the nicking reaction, which involves a transesterification to form a phosphotyrosine bond within the RepD active site, is close to unity.
authors.affiliation: MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Claudia
authors.initials: C
authors.lastname: Arbore
authors.forename: Lori M
authors.initials: LM
authors.lastname: Lewis
authors.forename: Martin R
authors.initials: MR
authors.lastname: Webb
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*; D004261:DNA Replication / Q000235:genetics; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics / Q000493:pharmacokinetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*; D011499:Protein Processing, Post-Translational / Q000235:genetics; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C058757:RepD protein, Staphylococcus aureus; D015534:Trans-Activators; C084217:pcrA protein, Bacteria; C045264:replication initiator protein; D004265:DNA Helicases