THE YEAST SITE-SPECIFIC RECOMBINASE FLP MEDIATES ALCOHOLYSIS AND HYDROLYSIS OF THE STRAND CLEAVAGE PRODUCT - MIMICKING THE STRAND-JOINING REACTION WITH NON-DNA NUCLEOPHILES

The yeast site-specific recombinase Flp is covalently linked to DNA vi a a 3'-phosphotyrosyl bond during the strand-breakage step of recombin ation. We show that this phosphotyrosyl diester bond formed between Fl p and DNA can serve as the target for alcoholysis or hydrolysis in an Flp-assisted reaction. Flp does not mediate alcoholysis of the labile phosphodiester bond within the DNA chain under our assay conditions. T he body of available evidence supports the notion that the alcoholysis /hydrolysis reaction is mechanistically analogous to the strand-joinin g step of the recombination pathway. The only difference is that the D NA 5'-hydroxyl group that acts as the nucleophile during recombination is substituted by a non-DNA nucleophile. We find that the alcoholysis reaction occurs only within the normal cleavage complex produced by t he ''shared active site'' assembled at the interface of two Flp monome rs. Unlike the strand-joining reaction, alcoholysis does not occur on an activated DNA substrate Linked at its 3'-phosphate end to a short t yrosyl peptide (not to the full-length Flp), and bound non-covalently by a Flp monomer. However, even in this substrate that mimics the stra nd-cleaved state, the joining reaction is competitively inhibited by a polyhydric alcohol such as glycerol. (C) 1997 Academic Press Limited.