Recombination of hybrid target sites by binary combinations of Flp variants: mutations that foster interprotomer collaboration and enlarge substrate tolerance.
Strategies of directed evolution and combinatorial mutagenesis applied to the Flp site-specific recombinase have yielded recombination systems that utilize bi-specific hybrid target sites. A hybrid site is assembled from two half-sites, each harboring a distinct binding specificity. Satisfying the two specificities by a binary combination of Flp variants, while necessary, may not be sufficient to elicit recombination. We have identified amino acid substitutions that foster interprotomer collaboration between partner Flp variants to potentiate strand exchange in hybrid sites. One such substitution, A35T, acts specifically in cis with one of the two partners of a variant pair, Flp(K82M) and Flp(A35T, R281V). The same A35T mutation is also present within a group of mutations that rescue a Flp variant, Flp(Y60S), that is defective in establishing monomer-monomer interactions on the native Flp target site. Strikingly, these mutations are localized to peptide regions involved in interdomain and interprotomer interactions within the recombination complex. The same group of mutations, when transferred to the context of wild-type Flp, can relax its specificity to include non-native target sites. The hybrid Flp systems described here mimic the naturally occurring XerC/XerD recombination system that utilizes two recombinases with distinct DNA binding specificities. The ability to overcome the constraints of binding site symmetry in Flp recombination has important implications in the targeted manipulations of genomes.
Konieczka
Jay H
JH
Molecular Genetics and Microbiology, University of Texas, Austin, 1 University Station A5000, Austin, TX 78712-0162, USA.
Paek
Andrew
A
Jayaram
Makkuni
M
Voziyanov
Yuri
Y
D001483:Base Sequence; D004247:DNA; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D013379:Substrate Specificity
D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase