Generality of the shared active site among yeast family site-specific recombinases. The R site-specific recombinase follows the Flp paradigm [corrected].
Mutations of the invariant Int family tetrad residues, the RHR triad, and the active site tyrosine, within the Zygosaccharomyces rouxii site-specific recombinase R cause the same "step-arrest" phenotypes as they do in the Flp recombinase of Saccharomyces cerevisiae. In "half-site" recombinations, the R recombinase exhibits catalytic complementation between an RHR triad mutant and an active site tyrosine mutant. Strand cutting by R follows the "trans" DNA cleavage rule. These results are best explained by the assembly of a functional active site from partial active sites harbored by the ARg monomers. Complementation tests using single and double step-arrest ARg mutants verify critical predictions of the "shared active site" model. A wild type monomer paired with an RHR triad-Tyr358 double mutant is a catalytically inactive combination. Pairwise combinations of a single or a double RHR mutant with R(Y358F) yield comparable levels of catalytic complementation. These results strongly imply conservation of the mechanism of active site assembly and the mode of substrate cleavage within the yeast family site-specific recombinases, and perhaps within the larger Int family recombinases [corrected].
Yang
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SH
Department of Microbiology, University of Texas at Austin 78712.
Jayaram
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D001120:Arginine / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism; D005816:Genetic Complementation Test; D006639:Histidine / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D004718:Saccharomycetales / Q000201:enzymology*; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism
D004271:DNA, Fungal; D009838:Oligodeoxyribonucleotides; D014443:Tyrosine; D006639:Histidine; D001120:Arginine; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; C074994:R recombinase