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Journal of Bacteriology, September 2008, p. 5781-5796, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00170-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of Bacteriophage P22 Xis Protein{triangledown}

Aras N. Mattis,1 Richard I. Gumport,2 and Jeffrey F. Gardner3*

Department of Pathology, University of California, San Francisco, San Francisco, California,1 Department of Biochemistry and College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois,2 Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois3

Received 3 February 2008/ Accepted 15 May 2008

The temperate bacteriophages {lambda} and P22 share similarities in their site-specific recombination reactions. Both require phage-encoded integrase (Int) proteins for integrative recombination and excisionase (Xis) proteins for excision. These proteins bind to core-type, arm-type, and Xis binding sites to facilitate the reaction. {lambda} and P22 Xis proteins are both small proteins ({lambda} Xis, 72 amino acids; P22 Xis, 116 amino acids) and have basic isoelectric points (for P22 Xis, 9.42; for {lambda} Xis, 11.16). However, the P22 Xis and {lambda} Xis primary sequences lack significant similarity at the amino acid level, and the linear organizations of the P22 phage attachment site DNA-binding sites have differences that could be important in quaternary intasome structure. We purified P22 Xis and studied the protein in vitro by means of electrophoretic mobility shift assays and footprinting, cross-linking, gel filtration stoichiometry, and DNA bending assays. We identified one protected site that is bent approximately 137 degrees when bound by P22 Xis. The protein binds cooperatively and at high protein concentrations protects secondary sites that may be important for function. Finally, we aligned the attP arms containing the major Xis binding sites from bacteriophages {lambda}, P22, L5, HP1, and P2 and the conjugative transposon Tn916. The similarity in alignments among the sites suggests that Xis-containing bacteriophage arms may form similar structures.

* Corresponding author. Mailing address: Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801. Phone: (217) 333-7289. Fax: (217) 244-6697. E-mail: jeffgard{at}uiuc.edu

{triangledown} Published ahead of print on 23 May 2008.

Journal of Bacteriology, September 2008, p. 5781-5796, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00170-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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