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

This Article Full Text Full Text (PDF) All Versions of this Article: 277/17/14530    most recent M108197200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kovach, M. J. Articles by Landy, A. Search for Related Content PubMed PubMed Citation Articles by Kovach, M. J. Articles by Landy, A. Social Bookmarking                      What's this?

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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