TY - JOUR

T1 - Alcoholysis and strand joining by the Flp site-specific recombinase

T2 - Mechanistically equivalent reactions mediated by distinct catalytic configurations

AU - Knudsen, Birgitta Ruth

AU - Lee, Jehee

AU - Lisby, Michael

AU - Westergaard, Ole

AU - Jayaram, Makkuni

PY - 1998/8/21

Y1 - 1998/8/21

N2 - 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. 23044-23054).

AB - 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. 23044-23054).

UR - http://www.scopus.com/inward/record.url?scp=0032555489&partnerID=8YFLogxK

U2 - 10.1074/jbc.273.34.22028

DO - 10.1074/jbc.273.34.22028

M3 - Journal article

C2 - 9705345

AN - SCOPUS:0032555489

VL - 273

SP - 22028

EP - 22036

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 34

ER -