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J. Biol. Chem., Vol. 281, Issue 27, 18610-18617, July 7, 2006

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The AddAB Helicase/Nuclease Forms a Stable Complex with Its Cognate Sequence During Translocation^*

From the Sections of Microbiology and of Molecular and Cellular Biology, Center for Genetics and Development, University of California, Davis, California 95616

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 (). Recognition of triggers attenuation of the 3'- to 5'-nuclease, which permits the generation of recombinogenic 3'-overhanging, single-stranded DNA (ssDNA), terminating at . Although the RecBCD enzyme briefly pauses at, no specific binding of RecBCD to during translocation has been documented. Here, we show that the AddAB enzyme transiently binds to its cognate sequence (_Bs: 5'-AGCGG-3') during translocation. The binding of AddAB enzyme to the 3'-end of the _Bs-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, _Bs-specific ssDNA that had been stripped of all protein does not restore nuclease protection, indicating that AddAB enzyme binds to_Bs with high affinity only during translocation. Finally, protection of _Bs-specific ssDNA is still observed when translocation occurs in the presence of competitor _Bs-carrying ssDNA, showing that binding occurs in cis. We suggest that this transient binding of AddAB to_Bs is an integral part of the AddAB-_Bs interaction and propose that this molecular event underlies a general mechanism for regulating the biochemical activities and biological functions of RecBCD-like enzymes.

Received for publication, January 30, 2006 , and in revised form, April 14, 2006.

^* This work was supported by a Wellcome Trust Traveling Research Fellowship (to M. S. D.), a Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowship for Research Abroad (to N. H.), and a Grant GM-41347 from the National Institutes of Health (to S. C. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Present address: University of Tokyo, Dept. of Medical Genome Sciences, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

³ Present address: University of Bristol, Dept. of Biochemistry, Bristol BS8 1TD, UK.

⁴ To whom correspondence should be addressed. E-mail: sckowalczykowski{at}ucdavis.edu.

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