The Role of DNA Bending in Flp-mediated Site-specific Recombination

Abstract

The Flp recombinase of the 2 μm plasmid of Saccharomyces cerevisiaebinds to a recognition target site, induces DNA bending and catalyses DNA cleavage and strand exchange to bring about recombination. The minimal Flp recognition target site contains two Flp binding sequences flanking an 8 bp core region; binding of Flp results in the formation of two Flp:DNA complexes (complexes I and II). Binding of a Flp monomer to a single symmetry element generates a DNA bend of about 60 ° (a type I bend), whereas binding of two Flp monomers to the FRT site generates a DNA bend of>144 ° (a type II bend). We have used circular permutation analysis to locate the centre of the type I and type II DNA bends induced by Flp, and the Flp peptides P27 (27 kDa; amino acid residues 124 to 346) and P32 (32 kDa; amino acid residues 124 to 423). The location of the centre of the type I bend depends upon whether the substrate contains one or two Flp binding elements. When the substrate contains one symmetry element, the centre of the type I bend induced by Flp is located at the core-distal end of the belement. However, it is located at the core-proximal end of the belement when the substrate contains two Flp-binding elements. The P27 and P32 peptides, which lack the NH 2-terminal 13 kDa region of Flp, do not show this behavior. We deduce that the 13 kDa region of Flp is critical for the positioning of the type I bend centre on a minimal Flp recognition site. We propose a model in which a single molecule of Flp interactions with two symmetry elements to account for these results. The centre of the type II bend induced by Flp is in the middle of the core region. We used ligation-defective Flp proteins to determine the location of the type II bend centres in complexes where either the top or bottom strand was cleaved. The bend centres of such complexes depend upon which strand is cleaved. We propose a model which associates the position of Flp-induced type II bends with a defined order of strand exchanges in the recombination reaction.