HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Knudsen, B. R. Articles by Jayaram, M. Search for Related Content PubMed PubMed Citation Articles by Knudsen, B. R. Articles by Jayaram, M. Social Bookmarking                      What's this?

J Biol Chem, Vol. 273, Issue 34, 22028-22036, August 21, 1998

Alcoholysis and Strand Joining by the Flp Site-specific Recombinase
MECHANISTICALLY EQUIVALENT REACTIONS MEDIATED BY DISTINCT CATALYTIC CONFIGURATIONS

Birgitta Ruth Knudsen, Jehee Lee^§, Michael Lisby, Ole Westergaard, and Makkuni Jayaram^¶

From the  Department of Molecular and Structural Biology, University of Aarhus, C. F. Møllers Allé Building 130, Aarhus C, DK-8000, Denmark, ^§ Faculty of Applied Marine Sciences, Cheju University, Cheju City 690756, South Korea, and the ^¶ Department of Microbiology and Institute of Cell and Molecular Biology, University of Texas, Austin, Texas 78712

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).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. K. Sau, G. D. Tribble, I. Grainge, R. F. Frohlich, B. R. Knudsen, and M. Jayaram Biochemical and Kinetic Analysis of the RNase Active Sites of the Integrase/Tyrosine Family Site-specific DNA Recombinases J. Biol. Chem., November 30, 2001; 276(49): 46612 - 46623. [Abstract] [Full Text] [PDF]