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Mutational analysis of the bacteriophage phi X174 replication origin.

P D Baas1

  • 1Institute of Molecular Biology and Medical Biotechnology, State University of Utrecht, The Netherlands.

Journal of Molecular Biology
|November 5, 1987
PubMed
Summary

Mutations in the bacteriophage phi X174 replication origin (ori) were created. While most mutants grew well, secondary mutations improved growth by enhancing gene A protein binding, though wild-type ori remains superior for reproduction.

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Area of Science:

  • Molecular Biology
  • Virology
  • Genetics

Background:

  • Bacteriophage phi X174 relies on a specific replication origin (ori) for its life cycle.
  • The gene A protein is crucial for initiating replication at the ori.

Purpose of the Study:

  • To investigate the functional significance of the bacteriophage phi X174 replication origin.
  • To identify key sequences within the ori essential for phage replication and gene A protein interaction.

Main Methods:

  • Oligodeoxynucleotide-directed mutagenesis was used to create base substitution mutants within the phi X174 ori.
  • Insertions and deletions were introduced into the ori cloned in a plasmid.
  • In vitro assays assessed gene A protein cleavage, and in vivo experiments evaluated viral packaging and growth.

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Main Results:

  • 18 viable base substitution mutants were generated, most exhibiting wild-type plaque morphology and burst size.
  • Mutants with reduced growth acquired secondary mutations, enhancing gene A protein binding to the mutated ori.
  • Small insertions/deletions in the ori impaired single-stranded DNA packaging, despite allowing in vitro gene A protein cleavage.

Conclusions:

  • The wild-type phi X174 ori sequence provides a superior reproductive advantage over mutated sequences.
  • Secondary mutations can compensate for ori defects by improving gene A protein recognition, highlighting the adaptability of viral replication machinery.
  • Specific structural elements within the ori are critical for efficient DNA packaging, not just initiation of replication.