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Related Experiment Videos

FokI dimerization is required for DNA cleavage

J Bitinaite1, D A Wah, A K Aggarwal

  • 1New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA.

Proceedings of the National Academy of Sciences of the United States of America
|September 2, 1998
PubMed
Summary

FokI endonuclease requires catalytic domain dimerization for DNA cleavage. Studies show variants support this model, impacting DNA binding and cleavage rates.

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Structural Biology

Background:

  • FokI is a type IIs restriction endonuclease with distinct DNA recognition and catalytic domains.
  • Structural data suggests potential dimerization of FokI catalytic domains, similar to BamHI.
  • Previous research indicated a dimerization interface within FokI catalytic domains.

Purpose of the Study:

  • To provide evidence that FokI catalytic domain dimerization is essential for DNA cleavage.
  • To investigate the mechanism of DNA binding, dimerization, and cleavage by FokI.
  • To characterize FokI variants affecting dimerization and DNA cleavage activity.

Main Methods:

  • Enzyme kinetics assays to analyze DNA cleavage rates at varying protein concentrations.
  • Construction and characterization of FokI variants (FokN13Y, FokD483A, R487A) with altered DNA binding or dimerization properties.

Related Experiment Videos

  • Analysis of DNA cleavage activity in the presence of wild-type FokI and variant proteins.

    • Main Results:

    • DNA cleavage rates were not directly proportional to FokI concentration, indicating cooperativity.
    • A FokI variant (FokN13Y) unable to bind DNA enhanced wild-type FokI cleavage rates when mixed.
    • A FokI variant (FokD483A, R487A) with mutations at the putative dimerization interface showed significantly reduced DNA cleavage activity.

    Conclusions:

    • FokI catalytic domain dimerization is a prerequisite for DNA cleavage.
    • A model for FokI function involving sequential DNA binding, catalytic domain dimerization, and DNA cleavage is supported.
    • Understanding FokI dimerization provides insights into type IIs restriction endonuclease mechanisms.