Abstract
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).
Highlights
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
In the strand joining reaction, the 5Ј-hydroxyl group can be replaced by polyhydric alcohols, by water, and by the adjacent 2Ј-hydroxyl group from a ribonucleotide [10, 11]
The 5Ј-hydroxyl attack can be promoted by a Flp monomer lacking Tyr-343, but properly positioned with respect to a 3Ј-phosphate covalently linked to tyrosine or a short tyrosyl peptide; the same substrate-protein configuration fails to support glycerolysis
Summary
Purification of Flp—Wild type Flp and the Flp variants were purified as described previously [17]. Of Activated Substrates—The activated substrates were obtained following the same general hybridization strategy described for the nonactivated substrates, except that the 3Ј-phosphate end of the labeled strand was linked to a short Flp peptide via Tyr-343. For this purpose, the labeled strand was prepared as follows. Assays for the Formation of the Cleaved Protein-DNA Complex from Half-site or Full Site Substrates—The activated half- or full sites, appropriately labeled at one 5Ј-end, were treated with wild type Flp or Flp(H305L) under the conditions described for the strand transfer assays (see above). The samples were subjected to electrophoresis in 6% nondenaturing polyacrylamide gels (3% bisacrylamide), and the DNA-protein complexes were visualized by autoradiography (or phosphor imaging)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
