FLP RIBONUCLEASE-ACTIVITIES - MECHANISTIC SIMILARITIES AND CONTRASTS TO SITE-SPECIFIC DNA RECOMBINATION

The ribonuclease active site harbored by the Flp site-specific recombi nase can act on two neighboring phosphodiester bonds to yield mechanis tically 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 ac tivity). The second activity (Flp RNase II) targets the phosphodiester immediately to the 3' side but appears not to involve an enzyme-linke d intermediate. Flp RNase I is absolutely dependent upon Tyr-343 of Fl p and is competitive with respect to the normal strand joining reactio n. It can utilize the 2'-hydroxyl group from any one of the four ribon ucleotides with comparable efficiencies in the cleavage reaction. On t he other hand, the RNase II reaction mediated by Flp(Y343F) is specifi c 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 s ensitive to the 3'-neighboring base. Although dT is functional, the ac tivity is stimulated by U or U-2'-OMe, The Flp RNase II reaction effec tively competes with the normal strand cleavage reaction mediated by T yr-343, even though their phosphodiester targets are not the same.