title: Electrostatic suppression allows tyrosine site-specific recombination in the absence of a conserved catalytic arginine.
abstract: 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.
authors.affiliation: Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Rowley
authors.forename: Aashiq H
authors.initials: AH
authors.lastname: Kachroo
authors.forename: Chien-Hui
authors.initials: CH
authors.lastname: Ma
authors.forename: Anna D
authors.initials: AD
authors.lastname: Maciaszek
authors.forename: Piotr
authors.initials: P
authors.lastname: Guga
authors.forename: Makkuni
authors.initials: M
authors.lastname: Jayaram
mesh_terms: D001120:Arginine; D001483:Base Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation; D009943:Organophosphorus Compounds / Q000378:metabolism; D045522:Recombinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D055672:Static Electricity; D013379:Substrate Specificity; D014443:Tyrosine
chemical_list: D009943:Organophosphorus Compounds; D045522:Recombinases; C032627:methylphosphonic acid; D014443:Tyrosine; D004247:DNA; D001120:Arginine; D004720:Endonucleases
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