Chi-activated RecBCD enzyme possesses 5′→3′ nucleolytic activity, but RecBC enzyme does not: Evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit

Daniel G. Anderson, Jason J. Churchill, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

46 Scopus citations

Abstract

Background: Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme, and is stimulated by DNA elements known as Chi (x) sites. The RecBCD enzyme is both a helicase and a nuclease. Recognition of x causes both attenuation of the 3′→5′ exonuclease activity of the RecBCD enzyme, and activation of an exonuclease activity with 5′→3′ polarity, while leaving the helicase activity unaffected. A variety of evidence suggests that x-recognition by RecBCD enzyme is accompanied by ejection of the RecD subunit. Results: Through examination of RecBCD exonuclease activity under a variety of conditions, we have shown that recognition of x by the RecBCD enzyme results in a net reduction of nuclease activity. In addition, the exact location of the first cleavage event elicited by x-activation of the 5′→3′ nuclease is dependent upon the concentration of free magnesium ions. Finally, we have demonstrated that purified RecBC enzyme (i.e. without the RecD subunit) possesses no significant exonuclease activity under conditions where the X-modified RecBCD enzyme is an active 5′→3′ exonuclease. Conclusions: We have shown that, despite the activation of a 5′→3′ exonuclease, recognition of x by the RecBCD enzyme results in a net preservation of DNA. This new x-activated nucleolytic action shows surprising variability in the exact location of its initial cleavage. We have demonstrated that purified RecBC enzyme is not an exact analogue of the x-activated RecBCD enzyme, suggesting that the biochemical basis of x-activation is not simply ejection of the RecD subunit.

Original languageEnglish (US)
Pages (from-to)117-128
Number of pages12
JournalGenes to Cells
Volume2
Issue number2
StatePublished - 1997

ASJC Scopus subject areas

  • Cell Biology
  • Genetics

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