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Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy
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Published on: June 25, 2013

The lambda Gam protein inhibits RecBCD binding to dsDNA ends.

Kenan C Murphy1

  • 1Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue, North, Worcester, MA 01655, USA. kenan.murphy@umassmed.edu

Journal of Molecular Biology
|June 23, 2007
PubMed
Summary

Lambda Gam protein inhibits Escherichia coli RecBCD enzyme activity, crucial for recombineering. Residual enzyme activity persists, impacting in vivo experiments and requiring revised models for DNA repair and radioresistance.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The lambda Gam protein is essential for recombineering, a gene replacement technology in Escherichia coli.
  • Gam protein is known to inhibit the RecBCD enzyme, but some in vivo effects of recBCD mutants are not fully replicated by Gam expression.
  • Understanding Gam's precise inhibitory mechanism on RecBCD is crucial for optimizing recombineering and interpreting experimental results.

Purpose of the Study:

  • To investigate the mechanism by which lambda Gam protein inhibits the Escherichia coli RecBCD enzyme.
  • To determine if Gam completely inactivates RecBCD or if residual activity remains.
  • To propose a revised model for Gam-induced radioresistance in E. coli.

Main Methods:

  • Biochemical assays to examine Gam's effect on RecBCD binding to DNA ends.
  • Enzyme activity assays measuring RecBCD helicase activity in the presence of Gam and ATP.
  • Comparison of Gam's inhibitory effect on native RecBCD versus Abc2-modified RecBCD.

Main Results:

  • Gam inhibits RecBCD binding to double-stranded DNA ends, even when RecBCD is already bound to DNA.
  • Residual RecBCD helicase activity is detected even with a large excess of Gam, particularly upon ATP addition.
  • Gam's inhibitory effect is diminished on Abc2-modified RecBCD due to tighter DNA binding.

Conclusions:

  • Cells expressing Gam retain a small, uninhibited pool of RecBCD enzyme.
  • The suspected instability of Gam in vivo necessitates caution when interpreting experiments involving Gam-inhibited RecBCD.
  • A revised model for Gam-induced radioresistance in E. coli, considering residual RecBCD activity, is proposed.