RepC protein of the octopine-type Ti plasmid binds to the probable origin of replication within repC and functions only in cis

Mol Microbiol. 2011 Sep;81(6):1593-606. doi: 10.1111/j.1365-2958.2011.07789.x. Epub 2011 Aug 23.

Abstract

Vegetative replication and partitioning of many plasmids and some chromosomes of alphaproteobacteria are directed by their repABC operons. RepA and RepB proteins direct the partitioning of replicons to daughter cells, while RepC proteins are replication initiators, although they do not resemble any characterized replication initiation protein. Here we show that the replication origin of an Agrobacterium tumefaciens Ti plasmid resides fully within its repC gene. Purified RepC bound to a site within repC with moderate affinity, high specificity and with twofold cooperativity. The binding site was localized to an AT-rich region that contains a large number of GANTC sites, which have been implicated in replication regulation in related organisms. A fragment of RepC containing residues 26-158 was sufficient to bind DNA, although with limited sequence specificity. This portion of RepC is predicted to have structural homology to members of the MarR family of transcription factors. Overexpression of RepC in A. tumefaciens caused large increases in copy number in cis but did not change the copy number of plasmids containing the same oriV sequence in trans, confirming other observations that RepC functions only in cis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium tumefaciens / genetics*
  • Agrobacterium tumefaciens / metabolism
  • Binding Sites
  • DNA Helicases / isolation & purification
  • DNA Helicases / metabolism*
  • DNA Mutational Analysis
  • DNA Replication
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Models, Molecular
  • Plant Tumor-Inducing Plasmids / metabolism*
  • Protein Binding
  • Protein Structure, Tertiary
  • Replication Origin*
  • Sequence Deletion
  • Trans-Activators / isolation & purification
  • Trans-Activators / metabolism*

Substances

  • DNA-Binding Proteins
  • Trans-Activators
  • replication initiator protein
  • DNA Helicases