Stepwise manipulation of DNA specificity in Flp recombinase: progressively adapting Flp to individual and combinatorial mutations in its target site.
The Flp protein from Saccharomyces cerevisiae is one of the site-specific tyrosine family recombinases that are used widely in genomic engineering. As a first step towards mediating directed DNA rearrangements at non-native Flp recombination targets (mFRTs), we have evolved three separate groups of Flp variants that preferentially act on mFRTs containing substitutions at the first, seventh or both positions of the Flp-binding elements. The variants that recombine the double-mutant mFRT contain a subset of the mutations present in those that are active on the single-mutant mFRTs, plus additional mutations. Specificity for and discrimination between target sites, effected primarily by amino acid residues that contact DNA, can be modulated by those that do not interact with DNA or with a DNA-contacting residue. The degree of modulation can range from relaxed DNA specificity to almost completely altered specificity. Our results suggest that combined DNA shuffling and mutagenesis of libraries of Flp variants active on distinct mFRTs can yield variants that can recombine mFRTs containing combinations of the individual mutations.
Voziyanov
Yuri
Y
Section of Molecular Genetics and Microbiology, University of Texas, Austin, TX 78712-1095, USA. voziyanov@mail.utexas.edu
Konieczka
Jay H
JH
Stewart
A Francis
AF
Jayaram
Makkuni
M
D020029:Base Pairing; D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D017930:Genes, Reporter / Q000235:genetics; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation; D015202:Protein Engineering; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D008239:Lysine