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Rolling-circle plasmid pKYM re-initiates DNA replication

H Yasukawa1, Y Masamune

  • 1Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Japan.

DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes
|June 30, 1997
PubMed
Summary

Rolling-circle plasmid pKYM demonstrates re-initiation of DNA replication, challenging previous beliefs. This unique plasmid utilizes the initiator protein RepK for both initiation and re-initiation, similar to bacteriophage phi-X174.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • Rolling-circle replication is a mechanism used by plasmids and viruses for DNA synthesis.
  • Previously, rolling-circle plasmids were thought to be incapable of re-initiating replication to maintain copy number, unlike phages such as phi-X174.

Purpose of the Study:

  • To investigate whether the rolling-circle plasmid pKYM can re-initiate DNA replication.
  • To understand the role of the initiator protein RepK in pKYM replication.

Main Methods:

  • Constructed a plasmid containing both the pKYM origin (fragment 13) and a truncated origin (fragment 32) in the same orientation.
  • Assessed the binding of RepK to different origin fragments.
  • Analyzed plasmid products generated in the presence of RepK.

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Main Results:

  • The constructed plasmid yielded two smaller plasmids in the presence of RepK.
  • RepK bound to fragment 13 but not to fragment 32.
  • Replication initiated at fragment 13 and terminated at fragment 32, with subsequent re-initiation from the fragment 32 region by RepK.

Conclusions:

  • The rolling-circle plasmid pKYM is capable of re-initiating DNA replication.
  • RepK plays a crucial role in both initiation and re-initiation of pKYM replication.
  • pKYM exhibits a unique replication strategy, similar to bacteriophage phi-X174, by re-initiating DNA replication.