Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

Abstract

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. 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. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

Acyclic analogs of nucleosides based on tris(hydroxymethyl)phosphine oxide: synthesis and incorporation into short DNA oligomers

“…2′-O-alkyl [11], LNA [12], 2′-F [13][14][15]). They are widely applied in cell and molecular biology as gene expression inhibitors in antisense or RNAi strategies [16][17][18][19][20], and are useful tools in enzymatic studies of nucleases, topoisomerases or transferases [2,21,22]. Beneficially, certain analogs of oligonucleotides intended to be used in antisense or RNAi strategies exert enhanced affinity towards target messenger RNA, or increased nucleolytic stability.…”