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 via a 3'-phosphotyrosyl bond during the strand-breakage step of recombination. We show that this phosphotyrosyl diester bond formed between Flp 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. The body of available evidence supports the notion that the alcoholysis/hydrolysis reaction is mechanistically analogous to the strand-joining step of the recombination pathway. The only difference is that the DNA 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 the "shared active site" assembled at the interface of two Flp monomers. Unlike the strand-joining reaction, alcoholysis does not occur on an activated DNA substrate linked at its 3'-phosphate end to a short tyrosyl peptide (not to the full-length Flp), and bound non-covalently by a Flp monomer. However, even in this substrate that mimics the strand-cleaved state, the joining reaction is competitively inhibited by a polyhydric alcohol such as glycerol.
Knudsen
B R
BR
Department of Molecular and Structural Biology, University of Aarhus, Denmark.
Dahlstrøm
K
K
Westergaard
O
O
Jayaram
M
M
D000438:Alcohols / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D005816:Genetic Complementation Test; D005990:Glycerol / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D000438:Alcohols; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D005990:Glycerol