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J. Biol. Chem., Vol. 277, Issue 17, 14530-14538, April 26, 2002

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Site-specific Photo-cross-linking between  Integrase and Its DNA Recombination Target^*

Margaret J. Kovach, Radhakrishna Tirumalai^§, and Arthur Landy^¶

From the ^¶ Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912,  Southern Illinois University School of Medicine, Springfield, Illinois 62794-9626, and ^§ Celera Genomics, Proteomics Division, Rockville, Maryland 20850

The site-specific recombinase (Int) of bacteriophage  is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to "arm-type" sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the "core-type" sequences where DNA cleavage and ligation are executed. We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position. When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically. The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of ~20%. The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly. Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the  Int family.

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