Site-specific photo-cross-linking between lambda integrase and its DNA recombination target.
The site-specific recombinase (Int) of bacteriophage lambda 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 approximately 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 lambda Int family.
Kovach
Margaret J
MJ
Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
Tirumalai
Radhakrishna
R
Landy
Arthur
A
D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D017931:DNA Primers; D006868:Hydrolysis; D019426:Integrases / Q000378:metabolism*; D010777:Photochemistry; D045522:Recombinases; D011995:Recombination, Genetic
D017931:DNA Primers; D045522:Recombinases; D004254:DNA Nucleotidyltransferases; D019426:Integrases