The addAB helicase/nuclease forms a stable complex with its cognate χ sequence during translocation

Frederic Chedin, Naofumi Handa, Mark S. Dillingham, Stephen C. Kowalczykowski

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39 Scopus citations


The Bacillus subtilis AddAB enzyme possesses ATP-dependent helicase and nuclease activities, which result in the unwinding and degradation of double-stranded DNA (dsDNA) upon translocation. Similar to its functional counterpart, the Escherichia coli RecBCD enzyme, it also recognizes and responds to a specific DNA sequence, referred to as Chi (χ). Recognition of χ triggers attenuation of the 3′- to 5′-nuclease, which permits the generation of recombinogenic 3′-overhanging, single-stranded DNA (ssDNA), terminating at χ. Although the RecBCD enzyme briefly pauses at χ, no specific binding of RecBCD to χ during translocation has been documented. Here, we show that the AddAB enzyme transiently binds to its cognate χ sequence (χBs: 5′-AGCGG-3′) during translocation. The binding of AddAB enzyme to the 3′-end of the χBs-specific ssDNA results in protection from degradation by exonuclease I. This protection is gradually reduced with time and lost upon phenol extraction, showing that the binding is non-covalent. Addition of AddAB enzyme to processed, χBs-specific ssDNA that had been stripped of all protein does not restore nuclease protection, indicating that AddAB enzyme binds to χBs with high affinity only during translocation. Finally, protection of χBs-specific ssDNA is still observed when translocation occurs in the presence of competitor χBs-carrying ssDNA, showing that binding occurs in cis. We suggest that this transient binding of AddAB to χBs is an integral part of the AddAB-χBs interaction and propose that this molecular event underlies a general mechanism for regulating the biochemical activities and biological functions of RecBCD-like enzymes.

Original languageEnglish (US)
Pages (from-to)18610-18617
Number of pages8
JournalJournal of Biological Chemistry
Issue number27
StatePublished - Jul 7 2006

ASJC Scopus subject areas

  • Biochemistry


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