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Related Experiment Videos

Monomeric RecBCD enzyme binds and unwinds DNA

A F Taylor1, G R Smith

  • 1Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, USA.

The Journal of Biological Chemistry
|October 13, 1995
PubMed
Summary

The RecBCD enzyme, crucial for DNA repair in E. coli, functions as a monomer. This monomeric form binds and unwinds double-stranded DNA ends, challenging previous models of its action.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • RecBCD enzyme is vital for homologous recombination and DNA repair in Escherichia coli.
  • Its precise functional oligomeric state has been a long-standing question due to variable reported molecular weights.
  • The enzyme possesses potent ATP-dependent helicase activity for unwinding DNA.

Purpose of the Study:

  • To elucidate the functional oligomeric state of the RecBCD enzyme.
  • To investigate the enzyme's DNA binding and unwinding capabilities in its different forms.

Main Methods:

  • Isolation and characterization of distinct RecBCD enzyme oligomeric forms (monomeric and dimeric).
  • Determination of DNA binding affinities (K_D) using ds DNA ends and Mg2+ ions.
  • Assessment of the DNA unwinding competence of the isolated enzyme forms.

Main Results:

  • Two oligomeric forms of RecBCD enzyme were isolated: monomeric (one copy each of RecB, RecC, RecD) and dimeric (two copies each).
  • The monomeric form (M(r) ~330,000) efficiently forms stable initiation complexes on ds DNA ends with high affinity (K_D ~0.1-0.7 nM).
  • The monomeric RecBCD-DNA complex is competent for DNA unwinding, and these findings challenge models requiring a dimeric enzyme form.

Conclusions:

  • The monomeric form of RecBCD enzyme is the functional unit capable of initiating DNA binding and unwinding.
  • This discovery refutes general helicase models that necessitate a dimeric enzyme complex for DNA processing.
  • The study clarifies the molecular mechanism of RecBCD enzyme in DNA recombination and repair pathways.

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