Abstract
In this study, we have used multiple strategies to characterize the mechanisms of the type I and type II RNA cleavage activities harbored by the Flp (pronounced here as "flip") site-specific DNA recombinase (Flp-RNase I and II, respectively). Reactions using half-sites pre-bound by step-arrest mutants of Flp agree with a "shared active site" being responsible for the type I reaction (as is the case with normal DNA recombination). In a "pre-cleaved" type I substrate containing a 3'-phosphotyrosyl bond, the Flp-RNase I activity can be elicited by either wild type Flp or by Flp(Y343F). Kinetic analyses of the type I reaction are consistent with the above observations and support the notion that the DNA recombinase and type I RNase active sites are identical. The type II RNase activity is expressed by Flp(Y343F) in a half-site substrate and is unaffected by the catalytic constitution of a Flp monomer present on a partner half-site. Reaction conditions that proscribe the assembly of a DNA bound Flp dimer have no effect on Flp-RNase II. These biochemical results, together with kinetic data, are consistent with the reaction being performed from a "non-shared active site" contained within a single Flp monomer. The Flp-related recombinase Cre, which utilizes a non-shared recombination active site, exhibits the type I RNA cleavage reaction. So far, we have failed to detect the type II RNase activity in Cre. Despite their differences in active site assembly, Cre functionally mimics Flp in being able to provide two functional active sites from a trimer of Cre bound to a three-armed (Y-shaped) substrate.
Highlights
The yeast site-specific recombinase Flp is encoded by the 2-m circle, a highly persistent, multicopy extrachromosomal DNA element found in most strains of Saccharomyces cerevisiae
We have used multiple strategies to characterize the mechanisms of the type I and type II RNA cleavage activities harbored by the Flp site-specific DNA recombinase (Flp-RNase I and II, respectively)
Conventions Used in Substrate Representations—For clarity and uniformity, the following conventions are followed in schematically depicting the various half-site substrates used for type I or type II Flp-RNase assays
Summary
The yeast site-specific recombinase Flp is encoded by the 2-m circle, a highly persistent, multicopy extrachromosomal DNA element found in most strains of Saccharomyces cerevisiae (reviewed in Ref. 1). Based on the chemistry of recombination and the active site residues involved in the reaction [5, 6], Flp has been assigned to the integrase/tyrosine family of recombinases. The active site nucleophiles in Flp and Cre (Tyr-343 and Tyr-324, respectively) form 3Ј-phosphotyrosyl bonds during the DNA cleavage reaction. The 5Ј-hydroxyl groups generated by cleavage provide the active nucleophiles for the strand exchange step They attack the phosphotyrosyl bonds across DNA partners to bring about strand joining in the recombinant mode. In Cre, all active site residues are provided by a single monomer so that the catalytic tyrosine attacks the scissile phosphodiester bond immediately adjacent to the DNA-bound Cre monomer. The final product of the Flp-RNase II reaction contains a 3Ј-phosphate
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