Abstract

The Escherichia coli RecBCD holoenzyme and the individual constituent subunits have been purified from overproducing strains. The purified RecBCD holoenzyme has a native molecular mass of approximately 330 kDa, indicative of a heterotrimer subunit assembly. The RecB, RecC, and RecD subunits can associate in vitro to give nuclease, helicase, ATPase, and Chi-specific endonuclease activities which are indistinguishable from those of the RecBCD holoenzyme. At concentrations at which the reconstituted RecB + C + D enzyme is very active, none of the individual RecB, RecC, or RecD subunits have readily detectable activities of the holoenzyme, except RecB protein which had previously been shown to exhibit DNA-dependent ATPase activity (Hickson, I. D., Robson, C. N., Atkinson, K. E., Hutton, L., and Emmerson, P. T. (1985) J. Biol. Chem. 260, 1224-1229). At higher concentrations and with shorter DNA substrates reconstituted RecBC protein exhibits low levels of helicase and exonuclease activity.

Highlights

  • The Escherichia coli RecBCD holoenzyme and the Amundsen et at., 1990)

  • The purified RecBCD holoenzyme catalyzes a wide range of activities: 1)it acts as an ATP-dependentexonuclease on single- and double-strandedDNA (Wright et al, 1971; Goldmark and Linn, 1972; Eichler and Lehman, 1977); 2) it is a n ATP-stimulated endonuclease acting on singlestranded circular DNA (Goldmark and Linn, 1972); 3) it is an ATP-dependenthelicase using theenergy of ATP hydrolysis to unwind duplex DNA

  • Ase, with the ATP being hydrolyzed to ADP and Pi without further breakdown of the ADP (Goldmark and Linn, 1972; The RecBCD protein (EC 3.1.11.5) of Escherichia coli K12, known as Exonuclease V, is a multifunctional, multisubunit enzyme whichperforms critical functions ihnomologous genetic recombination, DNA repair,maintenance of cell viability, and degradation of foreign or damaged DNA

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Summary

Reconstitution of RecEBnCfzrDyomme

Its Subunits stranded (5s) DNA-agarose was obtained fromBethesdaResearch Laboratories. Antibiotics, heparin-agarose, DNase I, and lysozyme were obtained from Sigma. SSB protein was prepared from an overproducing strain Hfr H (pRLM55). This strain and thepurification protocol were kindly provided by Dr R.

Purification of the RecBCD Holoenzyme
Purification of the RecB Protein
Purification of the RecC Protein
Purification of the RecD Protein
DNA Substrates
Enzyme Assays
Determination of Protein
Purification of the RecBCD Protein
Native Molecular Mass of RecBCD
Reconstitution of RecBCD Enzyme fromIts Subunits
Reconstitution of RecEBnCzDyme from Its Subunits
DISCUSSION
The RecBCD protein canbe purified fromstrains harboring
Findings
Reconstitution of RecBCD Enzyme froSmubIutsnits

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