Restoration of catalytic functions in Cre recombinase mutants by electrostatic compensation between active site and DNA substrate.
Two conserved catalytic arginines, Arg-173 and Arg-292, of the tyrosine site-specific recombinase Cre are essential for the transesterification steps of strand cleavage and joining in native DNA substrates containing scissile phosphate groups. The active site tyrosine (Tyr-324) provides the nucleophile for the cleavage reaction, and forms a covalent 3'-phosphotyrosyl intermediate. The 5'-hydroxyl group formed during cleavage provides the nucleophile for the joining reaction between DNA partners, yielding strand exchange. Previous work showed that substitution of the scissile phosphate (P) by methylphosphonate (MeP) permits strand cleavage by a Cre variant lacking Arg-292. We now demonstrate that MeP activation and cleavage are not blocked by substitution of Arg-173 or even simultaneous substitutions of Arg-173 and Arg-292 by alanine. Furthermore, Cre(R173A) and Cre(R292A) are competent in strand joining, Cre(R173A) being less efficient. No joining activity is detected with Cre(R173A, R292A). Consistent with their ability to cleave and join strands, Cre(R173A) and Cre(R292A) can promote recombination between two MeP-full-site DNA partners. These findings shed light on the overall contribution of active site electrostatics, and tease apart distinctive contributions of the individual arginines, to the chemical steps of recombination. They have general implications in active site mechanisms that promote important phosphoryl transfer reactions in nucleic acids.
Kachroo
Aashiq H
AH
Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712, USA.
Ma
Chien-Hui
CH
Rowley
Paul A
PA
Maciaszek
Anna D
AD
Guga
Piotr
P
Jayaram
Makkuni
M
D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D055162:Biocatalysis; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D053837:DNA Cleavage; D004706:Endodeoxyribonucleases / Q000378:metabolism; D006868:Hydrolysis; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009943:Organophosphorus Compounds / Q000737:chemistry; D011995:Recombination, Genetic; D055672:Static Electricity; D014443:Tyrosine / Q000737:chemistry
D009943:Organophosphorus Compounds; C032627:methylphosphonic acid; D014443:Tyrosine; D004247:DNA; D001120:Arginine; C045073:Cre recombinase; D019426:Integrases; D004706:Endodeoxyribonucleases