Electrostatic suppression allows tyrosine site-specific recombination in the absence of a conserved catalytic arginine.

The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3'-phosphotyrosyl intermediate. The 5'-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3'-5' phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5'-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.