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Bacterial plasmid replication initiation is controlled by DNA supercoiling and cruciform formation. The RepD protein nicks DNA only when negatively supercoiled, regulating chloramphenicol resistance plasmid duplication.

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Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • Bacterial antibiotic resistance is frequently mediated by plasmids, which are circular DNA molecules.
  • Plasmids like pC221 from Staphylococcus aureus replicate via asymmetric rolling circle replication.
  • The initiation of this replication process is regulated by the plasmid-encoded RepD protein, which nicks the origin of replication (oriD).

Purpose of the Study:

  • To investigate the regulatory mechanisms controlling the initiation of plasmid replication.
  • To determine the role of DNA supercoiling and secondary structures in RepD-mediated nicking at oriD.
  • To elucidate the importance of the ICRII sequence within oriD for efficient nicking.

Main Methods:

  • Magnetic tweezers were used to precisely control DNA linking number and supercoiling.
  • Nicking assays were performed with wild-type and mutant RepD proteins.
  • Quenched-flow experiments were utilized to study the kinetics of nicking on intact plasmids.

Main Results:

  • RepD-mediated nicking at oriD was observed exclusively under conditions of negative DNA supercoiling.
  • A non-nicking RepD mutant (RepDY188F) stabilized secondary DNA structures at oriD upon binding.
  • The inverted complementary repeat sequence, ICRII, within oriD was identified as crucial for rapid nicking.

Conclusions:

  • Cruciform formation at the oriD is a critical regulatory step for initiating plasmid replication.
  • DNA supercoiling plays a vital role in controlling the activity of the RepD initiator protein.
  • Understanding these mechanisms can inform strategies against antibiotic resistance spread.