Binding and Catalytic Contributions to Site Recognition by Flp Recombinase
نویسندگان
چکیده
منابع مشابه
DNA recognition by the FLP recombinase of the yeast 2 mu plasmid. A mutational analysis of the FLP binding site.
The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of the FLP protein and two inverted recombination sites on the plasmid. The minimal fully functional substrate for in-vitro recombination in this system consists of two FLP protein binding sites separated by an eight base-pair spacer sequence. We have used site-directed mutagenesis to ...
متن کاملAlcoholysis and strand joining by the Flp site-specific recombinase. Mechanistically equivalent reactions mediated by distinct catalytic configurations.
The strand joining step of recombination mediated by the Flp site-specific recombinase involves the attack of a 3'-phosphotyrosyl bond by a 5'-hydroxyl group from DNA. The nucleophile in this reaction, the 5'-OH, can be substituted by glycerol or other polyhydric alcohols. The strand joining and glycerolysis reactions are mechanistically equivalent and are competitive to each other. The target ...
متن کاملFLP Recombinase-Mediated Site-Specific Recombination in Silkworm, Bombyx mori
A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In th...
متن کاملStepwise 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 b...
متن کاملDomain of a yeast site-specific recombinase (Flp) that recognizes its target site.
Binding of a partial proteolytic digest by V8 enzyme of the yeast site-specific recombinase Flp to its target site gives rise to DNA-protein complexes that are smaller than those produced by the full-sized protein. The smallest of these complexes (occupancy of one peptide monomer per site) contains either one of two polypeptides (32 and 28 kDa) of the V8 digestion mixture. The amino termini of ...
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ژورنال
عنوان ژورنال: Journal of Biological Chemistry
سال: 2008
ISSN: 0021-9258
DOI: 10.1074/jbc.m800106200