Mapping the functional domains of bacteriophage lambda integrase protein.
Bacteriophage lambda encodes a site-specific recombination system that promotes the movement of the phage genome into and out of the host bacterial chromosome. The phage-encoded integrase (Int) is composed of 356 amino acid residues and carries out the required strand exchanges by means of a type I topoisomerase activity. Int also contains two distinct DNA-binding domains that interact with two different, specific sequences (arm-type and core-type sites) on DNA. In order to help understand the mechanism of site-specific recombination, we have used a genetic approach to isolate mutants defective in different steps in the recombination reaction. We developed a genetic screen for Int mutants that are defective in catalyzing excisive recombination in vivo. These mutants were screened for proficiency in binding to the P'123 arm-type sites using the bacteriophage P22 challenge-phage assays. In all, 78 such mutants were isolated and the mutational changes mapped and sequenced. These mutants have been further characterized (1) for their ability to bind the P'1 and P'123 arm-type sites and for their ability to form the attL complex in vivo, (2) for negative dominance in vitro, (3) for the presence of type I topoisomerase activity, and (4) for the ability to resolve artificially constructed recombination intermediates. We found that (1) residues in a stretch of 88 amino acids in the middle of the protein may be involved in Int-Int interactions, (2) a region around Arg212 is involved in the catalytic site, (3) residues near the carboxyl terminus play a role in enhancing Int binding to its arm-type sites, possibly by interacting with the small amino-terminal region that has been shown to be responsible for specific recognition of the arm-type sites, and (4) residues at the very carboxyl end of the protein may be involved in modulating the cleavage or religation activities of the Int protein.
Han
Y W
YW
Department of Microbiology, University of Illinois at Urbana-Champaign 61801.
Gumport
R I
RI
Gardner
J F
JF
D010582:Bacteriophage lambda / Q000235:genetics*; D004254:DNA Nucleotidyltransferases / Q000737:chemistry*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D019426:Integrases; D008242:Lysogeny; D016296:Mutagenesis; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry*
D014764:Viral Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D004264:DNA Topoisomerases, Type I