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Molecular and General Genetics MGG

, Volume 249, Issue 1, pp 43–50 | Cite as

Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01

  • J. F. M. L. Seegers
  • W. J. J. Meijer
  • G. Venema
  • S. Bron
  • A. C. Zhao
  • S. A. Khan
Original Paper

Abstract

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 by region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR 1) showed some homology with other previously described SSOs of the SSOA type, as well as with the conversion signal of the Escherichia coli phage ΦX174. This IR was shown to have a partial, RNA polymerise-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerise-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).

Key words

Rolling-circle plasmid Lagging strand synthesis Single-strand origin RNA polymerise Lactococcus lactis 

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Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • J. F. M. L. Seegers
    • 1
  • W. J. J. Meijer
    • 1
  • G. Venema
    • 1
  • S. Bron
    • 1
  • A. C. Zhao
    • 2
  • S. A. Khan
    • 2
  1. 1.Department of GeneticsGroningen Biomolecular Sciences and Biotechnology InstituteHarenThe Netherlands
  2. 2.Department of Molecular Genetics and BiochemistryUniversity of Pittsburgh, School of MedicinePittsburghUSA

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