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Related Experiment Videos

Cre mutants with altered DNA binding properties

M Hartung1, B Kisters-Woike

  • 1Institute for Genetics, University of Cologne, Weyertal 121, D-50931 Cologne, Germany.

The Journal of Biological Chemistry
|August 29, 1998
PubMed
Summary
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Researchers studied Cre recombinase mutants to understand DNA binding. A specific mutant, N317A, showed altered target recognition, binding a variant DNA sequence and offering insights into recombinase mechanisms.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Cre recombinase from bacteriophage P1 is a site-specific recombinase.
  • It catalyzes DNA rearrangements, crucial for genetic engineering.
  • Understanding its DNA recognition mechanism is key to controlling these processes.

Purpose of the Study:

  • To investigate the DNA binding specificity of Cre recombinase.
  • To identify amino acid residues critical for target DNA recognition.
  • To explore how mutations affect Cre recombinase activity and specificity.

Main Methods:

  • Construction and characterization of Cre recombinase mutants with targeted amino acid substitutions.
  • In vitro DNA binding assays to assess target sequence recognition.

Related Experiment Videos

  • In vivo recombination assays to evaluate mutant protein activity.

    • Main Results:

    • Most mutations in the putative DNA-binding region resulted in loss of binding or altered specificity.
    • The N317A mutant showed reduced efficiency with the wild-type loxP site.
    • Notably, the N317A mutant recombined a symmetric variant of the loxP site, unlike wild-type Cre.

    Conclusions:

    • Amino acid substitutions significantly impact Cre recombinase DNA binding and specificity.
    • The N317A mutation provides a tool to study DNA recognition flexibility.
    • A common multihelical DNA binding motif is proposed for integrases and recombinases, suggesting conserved structural rearrangements upon DNA binding.