Reversible inactivation of the Escherichia coli RecBCD enzyme by the recombination hotspot χ in vitro: Evidence for functional inactivation or loss of the RecD subunit

Dan A. Dixon, Jason J. Churchill, Stephen C. Kowalczykowski

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Abstract

Genetic recombination in Escherichia coli is stimulated by a RecBCD enzyme-mediated event at DNA sequences known as Chi (χ) sites (5'-GCTGGTGG- 3'). Previously, it was shown that χ acts to regulate the nuclease activity of RecBCD; here, we demonstrate that, under appropriate conditions, interaction with χ sites can also result in an inactivation of helicase activity of RecBCD. The unwinding of double-stranded DNA-containing χ sites, under conditions of limiting Mg2+ ion, results in the reversible inactivation of RecBCD; addition of excess Mg2+ to the reaction reactivates all activities of RecBCD. Inactivation is the consequence of a χ-dependent modification of RecBCD that appears to result from an inability of the χ- modified RecBCD to reinitiate unwinding of intact DNA molecules. This characteristic behavior of RecBCD and χ is displayed by the reconstituted RecBC (i.e., without the RecD subunit), except that it is not dependent on χ interaction. This biochemical similarity between the χ-modified RecBCD and RecBC enzymes implies that recognition of χ results in a dissociation or functional inactivation of RecD subunit and lends support to the hypothesis that interaction with χ results in ejection of the RecD subunit.

Original languageEnglish (US)
Pages (from-to)2980-2984
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number8
StatePublished - Apr 12 1994

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ASJC Scopus subject areas

  • Genetics
  • General

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