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J. Biol. Chem., Vol. 278, Issue 33, 31210-31217, August 15, 2003

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Effect of Mutations in the C-terminal Domain of Mu B on DNA Binding and Interactions with Mu A Transposase^*

Colin J. Coros  , Yukiko Sekino ¶, Tania A. Baker ¶ || ** and George Chaconas   

From the Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada, the Department of Biochemistry and Molecular Biology and the Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada, the ^¶Department of Biology and the ^||Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Bacteriophage Mu transposition requires two phage-encoded proteins, the transposase, Mu A, and an accessory protein, Mu B. Mu B is an ATP-dependent DNA-binding protein that is required for target capture and target immunity and is an allosteric activator of transpososome function. The recent NMR structure of the C-terminal domain of Mu B (Mu B_223–312) revealed that there is a patch of positively charged residues on the solvent-exposed surface. This patch may be responsible for the nonspecific DNA binding activity displayed by the purified Mu B_223–312 peptide. We show that mutations of three lysine residues within this patch completely abolish nonspecific DNA binding of the C-terminal peptide (Mu B_223– ³¹²). To determine how this DNA binding activity affects transposition we mutated these lysine residues in the full-length protein. The full-length protein carrying all three mutations was deficient in both strand transfer and allosteric activation of transpososome function but retained ATPase activity. Peptide binding studies also revealed that this patch of basic residues within the C-terminal domain of Mu B is within a region of the protein that interacts directly with Mu A. Thus, we conclude that this protein segment contributes to both DNA binding and protein-protein contacts with the Mu transposase.

Received for publication, April 9, 2003 , and in revised form, June 4, 2003.

^* This work was supported by the Canadian Institutes of Health Research, the Alberta Heritage Fund for Medical Research and the National Institutes of Health (GM49224). 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.

Present address: Wadsworth Center, P.O. Box 22002, Albany, NY 12201-2002.

^** An employee of the Howard Hughes Medical Institute.

Supported by a Distinguished Scientist Award from the Canadian Institutes of Health Research and by a Scientist Award from the Alberta Heritage Fund for Medical Research. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology and the Department of Microbiology and Infectious Diseases, 3330 Hospital Dr. N.W., University of Calgary, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-210-9692; Fax: 403-283-8731; E-mail: chaconas{at}ucalgary.ca.

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