Mutations That Improve the Binding of Yeast Flp Recombinase to Its Substrate

AUTOR(ES)

Lebreton, B.

RESUMO

When yeast FLP recombinase is expressed from the phage λ P(R) promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.

Documentos Relacionados

• Domain of a yeast site-specific recombinase (Flp) that recognizes its target site.
• The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site.
• Activity of yeast FLP recombinase in maize and rice protoplasts.
• Mechanism of cleavage and ligation by FLP recombinase: classification of mutations in FLP protein by in vitro complementation analysis.
• A tetramer of the Flp recombinase silences the trimers within it during resolution of a Holliday junction substrate