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Related Experiment Videos

Viewing single lambda site-specific recombination events from start to finish.

Jeffrey P Mumm1, Arthur Landy, Jeff Gelles

  • 1Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, J Walter Wilson Laboratories, Providence, RI 02912, USA.

The EMBO Journal
|September 16, 2006
PubMed
Summary
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Bacteriophage lambda DNA excision from E. coli involves multi-protein complexes. Single-molecule microscopy revealed stable complexes form rapidly, with excision efficiency near 100% once synapsis occurs.

Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • Bacteriophage lambda DNA integration and excision are crucial for its life cycle within Escherichia coli.
  • Understanding the precise molecular mechanisms and kinetics of these site-specific recombination events is complex.
  • Previous studies faced challenges in dissecting individual steps within the entire pathway.

Purpose of the Study:

  • To investigate the step-by-step process of bacteriophage lambda DNA excision from the E. coli chromosome.
  • To analyze the kinetics and efficiency of individual reaction steps using advanced microscopy.
  • To elucidate the factors contributing to the directionality of this recombination pathway.

Main Methods:

  • Utilized single-molecule light microscopy to observe the entire excision pathway in real-time.

Related Experiment Videos

  • Characterized the formation and stability of DNA-protein complexes involving integrase, IHF, and Xis.
  • Performed kinetic analysis to identify rate-limiting steps and determine reaction directionality.

    • Main Results:

    • Stable bent-DNA complexes formed rapidly on recombination sites (attL and attR) with integrase, IHF, and Xis.
    • Synapsis of partner complexes occurred rapidly, followed by integrase-mediated DNA cleavage for stabilization.
    • Approximately 50% of synapsed complexes yielded recombinant product with nearly 100% efficiency.
    • The rate-limiting step was identified post-synapsis, preceding or coinciding with Holliday junction formation.

    Conclusions:

    • Single-molecule microscopy provides unprecedented insight into the bacteriophage lambda DNA excision pathway.
    • The pathway involves rapid complex formation, efficient synapsis, and highly efficient recombination upon stabilization.
    • Irreversibility of multiple reaction steps confers directionality to the site-specific recombination process.