Alcoholysis 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 diester in strand joining can be a 3'-phosphate covalently linked either to a short tyrosyl peptide or to the whole Flp protein via Tyr-343. By contrast, only the latter type of 3'-phosphotyrosyl linkage is a substrate for glycerolysis. As a result, in activated DNA substrates (containing the scissile phosphate linked to a short Flp peptide), Flp(Y343F) can mediate the joining reaction utilizing the 5'-hydroxyl attack but fails to promote glycerolysis. Wild type Flp promotes both reactions in these substrates. The strand joining and glycerolysis reactions are absolutely dependent on the catalytic histidine at position 305 of Flp. Our results fit into a model in which a Flp dimer, with one monomer covalently attached to the 3'-phosphate, is essential for orienting the target diester or the nucleophile (or both) during glycerolysis. The requirement for this dimeric complex is relaxed in the strand joining reaction because of the ability of DNA to orient the nucleophile (5'-OH) by complementary base pairing. The experimental outcomes described here have parallels to the "cleavage-dependent ligation" carried out by a catalytic variant of Flp, Flp(R308K) (Zhu, X.-D., and Sadowski, P. D. (1995) J. Biol. Chem. 270, 23044-23054).
Knudsen
B R
BR
Department of Molecular and Structural Biology, University of Aarhus, C. F. Mollers Allé Building 130, Aarhus C, DK-8000, Denmark.
Lee
J
J
Lisby
M
M
Westergaard
O
O
Jayaram
M
M
D000438:Alcohols / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011995:Recombination, Genetic
D000438:Alcohols; D004271:DNA, Fungal; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase