A biotin interference assay highlights two different asymmetric interaction profiles for lambda integrase arm-type binding sites in integrative versus excisive recombination.
The site-specific recombinase integrase encoded by bacteriophage lambda promotes integration and excision of the viral chromosome into and out of its Escherichia coli host chromosome through a Holliday junction recombination intermediate. This intermediate contains an integrase tetramer bound via its catalytic carboxyl-terminal domains to the four "core-type" sites of the Holliday junction DNA and via its amino-terminal domains to distal "arm-type" sites. The two classes of integrase binding sites are brought into close proximity by an ensemble of accessory proteins that bind and bend the intervening DNA. We have used a biotin interference assay that probes the requirement for major groove protein binding at specified DNA loci in conjunction with DNA protection, gel mobility shift, and genetic experiments to test several predictions of the models derived from the x-ray crystal structures of minimized and symmetrized surrogates of recombination intermediates lacking the accessory proteins and their cognate DNA targets. Our data do not support the predictions of "non-canonical" DNA targets for the N-domain of integrase, and they indicate that the complexes used for x-ray crystallography are more appropriate for modeling excisive rather than integrative recombination intermediates. We suggest that the difference in the asymmetric interaction profiles of the N-domains and arm-type sites in integrative versus excisive recombinogenic complexes reflects the regulation of recombination, whereas the asymmetry of these patterns within each reaction contributes to directionality.
Hazelbaker
Dane
D
Department of Molecular Biology, Cellular Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
Azaro
Marco A
MA
Landy
Arthur
A
D001287:Attachment Sites, Microbiological; D010582:Bacteriophage lambda / Q000201:enzymology*; D001665:Binding Sites; D001681:Biological Assay / Q000379:methods*; D001710:Biotin / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D045566:DNA, Cruciform / Q000737:chemistry; D019426:Integrases / Q000378:metabolism*; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic / Q000235:genetics*; D017384:Sequence Deletion
D045566:DNA, Cruciform; D001710:Biotin; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C077570:Site-specific recombinase