Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits

Christine Masterson, Paul E Boehmer, Fraser McDonald, Subhendu Chaudhuri, Ian D. Hickson, Peter T. Emmerson

Research output: Contribution to journalArticle

36 Citations (Scopus)

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 Chispecific 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.

Original languageEnglish (US)
Pages (from-to)13564-13572
Number of pages9
JournalJournal of Biological Chemistry
Volume267
Issue number19
StatePublished - Jul 5 1992
Externally publishedYes

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Holoenzymes
Escherichia coli
4 alpha-glucanotransferase
Exonucleases
Molecular mass
Adenosine Triphosphatases
Proteins
DNA
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Masterson, C., Boehmer, P. E., McDonald, F., Chaudhuri, S., Hickson, I. D., & Emmerson, P. T. (1992). Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits. Journal of Biological Chemistry, 267(19), 13564-13572.

Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits. / Masterson, Christine; Boehmer, Paul E; McDonald, Fraser; Chaudhuri, Subhendu; Hickson, Ian D.; Emmerson, Peter T.

In: Journal of Biological Chemistry, Vol. 267, No. 19, 05.07.1992, p. 13564-13572.

Research output: Contribution to journalArticle

Masterson, C, Boehmer, PE, McDonald, F, Chaudhuri, S, Hickson, ID & Emmerson, PT 1992, 'Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits', Journal of Biological Chemistry, vol. 267, no. 19, pp. 13564-13572.
Masterson C, Boehmer PE, McDonald F, Chaudhuri S, Hickson ID, Emmerson PT. Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits. Journal of Biological Chemistry. 1992 Jul 5;267(19):13564-13572.
Masterson, Christine ; Boehmer, Paul E ; McDonald, Fraser ; Chaudhuri, Subhendu ; Hickson, Ian D. ; Emmerson, Peter T. / Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 19. pp. 13564-13572.
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AU - Emmerson, Peter T.

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N2 - 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 Chispecific 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.

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