Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.
Bottom Line: Positional conservation of the arginines does not translate into strict functional conservation.Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions.The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.
Affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA.Show MeSH
Mentions: Reactions of Arg-II mutants of Cre and Flp on pure stereoisomers of MeP-substrates. (A and B) MeP-half-site substrates are schematically diagrammed above the respective reaction panels. The horizontal arrows depict the recombinase binding element, and ‘mp’ denotes the scissile MeP bond (as in Figure 1C). The wavy lines indicate extra nucleotides that are not directly relevant to the reaction. The base pair abutting the scissile MeP, highlighted in bold letters for each site, marks the terminal base pair of the Flp binding element and the immediate neighbor of the Cre binding element. The MeP-half-site for Flp contains a substitution of AT for the native CG at this position. The asterisk stands for the 32P-label at the 5′ end. The 5′-hydroxyl group on the bottom strand is blocked by phosphorylation. This general representation is followed in subsequent figures as well. The reactions were analyzed by phenol-chloroformn extraction, ethanol precipitation of DNA, and electrophoresis in 12% denaturing (urea) polyacrylamide gels. Any covalent recombinase–DNA intermediate would be trapped at the interphase during phenol–chloroform extraction, and would thus be excluded from the analysis. The bands labeled ‘S’ and ‘HP’ represent the substrate and the hydrolysis product, respectively. The stereochemical bias, derived as described under ‘Methods and Materials’, is listed for the indicated reactions. The value for Flp is based on published results (26) and results from similar additional reactions (data not shown). In this figure and subsequent ones, the kinetic plots represent the results, shown as the mean ± SD (standard deviation), from at least three independent assays. The pseudo-first order rate constants for the RP and SP reactions are listed here and in Figures 3, 4 and 6.
Affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA.