Flp ribonuclease activities. Mechanistic similarities and contrasts to site-specific DNA recombination.

The ribonuclease active site harbored by the Flp site-specific recombinase can act on two neighboring phosphodiester bonds to yield mechanistically distinct chain breakage reactions. One of the RNase reactions apparently proceeds via a covalent enzyme intermediate and targets the phosphodiester position involved in DNA recombination (Flp RNase I activity). The second activity (Flp RNase II) targets the phosphodiester immediately to the 3' side but appears not to involve an enzyme-linked intermediate. Flp RNase I is absolutely dependent upon Tyr-343 of Flp and is competitive with respect to the normal strand joining reaction. It can utilize the 2'-hydroxyl group from any one of the four ribonucleotides with comparable efficiencies in the cleavage reaction. On the other hand, the RNase II reaction mediated by Flp(Y343F) is specific for U and cannot utilize the 2'-hydroxyl group from ribo-A, -G, or -C under standard reaction conditions. The RNase II activity is also sensitive to the 3'-neighboring base. Although dT is functional, the activity is stimulated by U or U-2'-OMe. The Flp RNase II reaction effectively competes with the normal strand cleavage reaction mediated by Tyr-343, even though their phosphodiester targets are not the same.