Published online 28 May 2010 Nucleic Acids Research, 2010, Vol. 38, No. 19 6589–6601 doi:10.1093/nar/gkq466 Restoration of catalytic functions in Cre recombinase mutants by electrostatic compensation between active site and DNA substrate Aashiq H. Kachroo1, Chien-Hui Ma 1 , Paul A. Rowley1, Anna D. Maciaszek2, Piotr Guga2 and Makkuni Jayaram1,* 1Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712, USA and 2Department of Bio-organic Chemistry, Center for Molecular and Macromolecular studies, Polish Academy of Sciences, 90-363 Lodz, Poland Received March 6, 2010; Revised May 10, 2010; Accepted May 11, 2010 ABSTRACT 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 sub- strates containing scissile phosphate groups. The active site tyrosine (Tyr-324) provides the nucleo- phile for the cleavage reaction, and forms a covalent 30-phosphotyrosyl intermediate. The 50-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 phos- phate (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 ar- ginines, to the chemical steps of recombination. They have general implications in active site mech- anisms that promote important phosphoryl transfer reactions in nucleic acids. INTRODUCTION Tyrosine family site-speciﬁc recombinases and type IB topoisomerases are characterized by a catalytic pentad, Arg-Lys-(His/Lys)-Arg-(His/Trp), that assists the strand cleavage and strand joining steps of DNA recombination and relaxation, respectively (1–6). In Cre recombinase, the pentad residues are Arg-173, Lys-201, His-289, Arg-292 and Trp-315. The nucleophile for strand cleavage, Tyr-324, attacks the scissile phosphodiester bond to form a 30 covalent adduct with the cleaved strand. The 50-hydroxyl group formed at the cleavage step carries out nucleophilic attack on the tyrosyl intermediate to promote strand joining. Recombination results when the joining reaction is performed between, rather than, within DNA partners. A recombination event is completed in two steps of concerted single strand exchanges. The ﬁrst gives rise to a Holliday junction intermediate; the second resolves it into reciprocal recombinant products (3). Arg-173 and Arg-292 are critical for both strand cleavage and strand joining reactions, presumably to balance the negative charge on the non-bridging phos- phate oxygen atoms in the transition state. Consistent with this function, the crystal structures of Cre reveal hydrogen bonding interactions between these two argin- ines and the scissile phosphate in the ‘disengaged’ (sans- cleavage) and ‘engaged’ (post-cleavage) active sites (2) (Figure 1). A modiﬁed DNA substrate in which the negative charge on the scissile phosphate (P) is neutralized by methylphosphonate (MeP) substitution is a poor sub- strate for strand cleavage by Cre. However, when Arg-292 is replaced by alanine, Cre(R292A) cleaves the MeP–DNA to form the tyrosyl intermediate, which can undergo sub- sequent hydrolysis (7). This hydrolytic activity promoted by Cre(R292A) has been termed the type I endonuclease activity to distinguish it from a potential type II *To whom correspondence should be addressed. Tel: +1 512 471 0966; Fax: +1 512 471 5546; Email: Jayaram@icmb.utexas.edu The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.