Kinetic mechanism of initiation by RepD as a part of asymmetric, rolling circle plasmid unwinding

Biochemistry. 2012 May 1;51(17):3684-93. doi: 10.1021/bi300172p. Epub 2012 Apr 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / pharmacokinetics*
  • DNA Helicases / chemistry*
  • DNA Helicases / genetics
  • DNA Helicases / pharmacokinetics*
  • DNA Replication / genetics
  • DNA, Bacterial / biosynthesis
  • DNA, Bacterial / genetics
  • DNA, Bacterial / pharmacokinetics
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / pharmacokinetics*
  • Plasmids / chemistry*
  • Plasmids / genetics
  • Plasmids / pharmacokinetics*
  • Protein Processing, Post-Translational / genetics
  • Staphylococcus aureus / enzymology
  • Staphylococcus aureus / genetics
  • Trans-Activators / chemistry*
  • Trans-Activators / genetics
  • Trans-Activators / pharmacokinetics*

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • DNA-Binding Proteins
  • RepD protein, Staphylococcus aureus
  • Trans-Activators
  • pcrA protein, Bacteria
  • replication initiator protein
  • DNA Helicases