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Related Experiment Videos

Hin recombinase mutants functionally disrupted in interactions with Fis.

O Z Nanassy1, K T Hughes

  • 1Department of Microbiology, University of Washington, Seattle, Washington 98195, USA.

Journal of Bacteriology
|December 15, 2000
PubMed
Summary

This study investigates Hin recombinase mutants affecting DNA recombination. Increasing Fis protein concentration partially rescued one mutant, suggesting Fis plays a postsynaptic role in the inversion reaction.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • A prior genetic screen classified Hin recombinase mutants based on recombination block stages.
  • Certain mutants, proficient in DNA binding but deficient in recombination, were predicted to be defective before invertasome formation and interact with Fis protein.

Purpose of the Study:

  • To biochemically characterize specific Hin recombinase mutants (R123Q, T124I, A126T) and elucidate their roles in DNA recombination.
  • To investigate the relationship between genetic classification and biochemical function, particularly concerning Fis protein interactions.

Main Methods:

  • Purification of Hin recombinase mutants R123Q, T124I, and A126T.
  • Characterization of DNA cleavage and recombination activities in vitro.
  • In vivo and in vitro rescue experiments using varying Fis protein concentrations.

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Main Results:

  • The R123Q mutant was inactive, while T124I and A126T showed partial activity.
  • The A126T mutant was less defective than T124I and could be partially rescued by increased Fis protein concentration.
  • Successful rescue of A126T required DNA binding-proficient Fis protein.

Conclusions:

  • The results suggest a postsynaptic role for Fis protein in the Hin-mediated DNA inversion reaction.
  • Biochemical characterization refines the understanding of Hin recombinase function and its interaction with Fis.