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Related Experiment Videos

Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory

J F Thompson, D Waechter-Brulla, R I Gumport

    Journal of Bacteriology
    |December 1, 1986
    PubMed
    Summary

    Integration host factor (IHF) controls lambda site-specific recombination. Mutations affecting IHF binding at the H1 site alter recombination direction, suggesting IHF regulates the phage lytic cycle.

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    Area of Science:

    • Molecular Biology
    • Genetics
    • Microbiology

    Background:

    • Site-specific recombination is crucial for viral genome integration and excision.
    • Integration host factor (IHF) is a known regulator of bacteriophage lambda DNA manipulation.
    • Understanding IHF's role is key to deciphering viral life cycle control.

    Purpose of the Study:

    • To investigate how Integration Host Factor (IHF) regulates lambda site-specific recombination.
    • To analyze the impact of specific DNA mutations on IHF binding and recombination activity.
    • To elucidate the mechanism by which IHF directs the integration or excision of the lambda phage genome.

    Main Methods:

    • Analysis of wild-type and mutant attP DNA in integrative and excisive recombination assays.

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  • In vitro and in vivo experiments to assess protein-DNA binding affinities.
  • Quantification of IHF requirements for recombination events.

    • Main Results:

    • Mutations in the H1 site significantly reduced IHF binding (over 500-fold) but increased IHF requirement for integration (8-fold).
    • A severe integration defect was observed in vitro for a mutant attP with nine base changes, less so in vivo.
    • Both mutations relieved IHF inhibition of excisive recombination, indicating H1 site occupancy dictates recombination direction.

    Conclusions:

    • H1 site occupancy by IHF is critical for determining the directionality of lambda site-specific recombination.
    • IHF-mediated inhibition of excision acts as a cellular sensor for induction stimulus and nutritional status.
    • This regulatory mechanism allows prophage excision only under conditions favorable for the lytic cycle completion.