Tyr60 variants of Flp recombinase generate conformationally altered protein-DNA complexes. Differential activity in full-site and half-site recombinations.
The tyrosine at position 60 of the Flp recombinase of the Saccharomyces cerevisiae plasmid, 2 mu circle, is invariant among site-specific recombinases of the "yeast plasmid family". Alterations of this residue give rise to Flp variants that show no recombination activity when assayed in vivo in Escherichia coli. Upon purification, they bind substrate, execute DNA cleavage and catalyze recombination. The efficiency of strand cleavage follows the order: Flp(Y60F) greater than Flp greater than Flp(Y60S) greater than Flp(Y60D); efficiency of recombination between Flp sites on a linear substrate and a circular one follows the order: Flp greater than Flp(Y60F) greater than Flp(Y60S) greater than Flp(Y60D). Methylation footprints of the DNA-protein complexes formed by two of the Flp variants, Flp(Y60S) and Flp(Y60D), do not show hypermethylation of the G residues within the substrate core that is characteristic of complexes formed by wild-type Flp. The third variant, Flp(Y60F), causes significant distortion (although less than wild-type Flp) of the substrate core, as indicated by enhanced G-methylation. Binding profiles with circularly permuted substrates indicate that Flp(Y60S) and Flp(Y60D), but not Flp(Y60F), are defective in bending substrate DNA. In recombination between two Flp half-sites, the variant proteins are significantly more active than in normal full-site recombination.
Chen
J W
JW
Department of Microbiology, University of Texas at Austin, Austin 78712.
Evans
B R
BR
Zheng
L
L
Jayaram
M
M
D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D014644:Genetic Variation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D011485:Protein Binding; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014443:Tyrosine
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D014443:Tyrosine; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase