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Related Experiment Videos

Substrate binding and turnover by the highly specific I-PpoI endonuclease

P K Wittmayer1, R T Raines

  • 1Department of Biochemistry, University of Wisconsin-Madison 53706-1569, USA.

Biochemistry
|January 23, 1996
PubMed
Summary

Intron-encoded endonucleases like I-PpoI bind and bend DNA substrates. This study reveals I-PpoI

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Biochemistry

Background:

  • Intron-encoded endonucleases cleave double-stranded DNA with high specificity.
  • I-PpoI endonuclease from Physarum polycephalum is a small enzyme acting on a 15-bp asymmetric DNA sequence.

Purpose of the Study:

  • To examine the interactions of I-PpoI with its DNA substrate during binding and catalysis.
  • To elucidate the chemical mechanisms of substrate binding and turnover for intron-encoded endonucleases.

Main Methods:

  • Circular permutation assays to determine DNA bending.
  • Gel mobility shift and fluorescence polarization assays for binding kinetics.
  • Steady-state kinetics to analyze catalytic efficiency and metal ion dependence.

Main Results:

  • I-PpoI bends its substrate by 38 degrees upon binding.
  • Tight substrate binding with Kd values from 3.3 to 112 nM, sensitive to NaCl concentration.
  • Catalytic efficiency (kcat/Km) approaches 10(8) M-1 s-1, with Mg2+ being the most efficient metal cofactor.

Conclusions:

  • I-PpoI exhibits significant substrate bending and tight binding.
  • Catalysis is dependent on metal ion cofactors, with Mg2+ showing highest efficiency.
  • Provides the first chemical insights into the binding and turnover mechanisms of intron-encoded endonucleases.

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