Methylphosphonate modiﬁcation, combined with ap- propriate active site mutations, provides a useful and convenient way of probing electrostatic attributes of phos- phoryl transfer mechanisms. An added bonus is that the analysis may also speak to the potential signiﬁcance of substrate and active site electrostatics as a water repelling strategy. ACKNOWLEDGEMENTS The authors thank K. A. Johnson for his generous help with the use of the KinTek Global Kinetic Explorer software FUNDING National Institutes of Health award (GM035654 to M.J.); Robert F Welch Foundation grant (F-1274); UT Austin Faculty research Award. Funding for open access charge: National Institutes of Health award (GM035654 to M.J.). Conﬂict of interest statement. None declared. REFERENCES 1. Chen,Y., Narendra,U., Iype,L.E., Cox,M.M. and Rice,P.A. (2000) Crystal structure of a Flp recombinase-Holliday junction complex: assembly of an active oligomer by helix swapping. Mol. Cell, 6, 885–897. 2. Guo,F., Gopaul,D.N. and Van Duyne,G.D. (1997) Structure of Cre recombinase complexed with DNA in a site-speciﬁc recombination synapse. Nature, 389, 40–46. 3. Van Duyne,G.D. (2002) A structural view of tyrosine recombinase site-speciﬁc recombination. In Craig,N.L., Craigie,R., Gellert,M. and Lambowitz,A.M. (eds), Mobile DNA II. ASM Press, Washington DC, pp. 93–117. 4. Krogh,B.O. and Shuman,S. (2000) Catalytic mechanism of DNA topoisomerase IB. Mol. Cell, 5, 1035–1041. 5. Redinbo,M.R., Stewart,L., Kuhn,P., Champoux,J.J. and Hol,W.G. (1998) Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science, 279, 1504–1513. 6. Chen,Y. and Rice,P.A. (2003) New insight into site-speciﬁc recombination from Flp recombinase-DNA structures. Annu. Rev. Biophys. Biomol. Struct., 32, 135–159. 7. Ma,C.H., Kachroo,A.H., Maciaszek,A., Chen,T.Y., Guga,P. and Jayaram,M. (2009) Reactions of Cre with methylpzhosphonate DNA: similarities and contrasts with Flp and vaccinia topoisomerase. PLoS ONE, 4, e7248. Nucleic Acids Research, 2010, Vol. 38, No. 19 6601 8. Ma,C.H., Rowley,P.A., Maciaszek,A., Guga,P. and Jayaram,M. (2009) Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination. EMBO J., 28, 1745–1756. 9. Tian,L., Claeboe,C.D., Hecht,S.M. and Shuman,S. (2003) Guarding the genome: electrostatic repulsion of water by DNA suppresses a potent nuclease activity of topoisomerase IB. Mol. Cell, 12, 199–208. 10. Tian,L., Claeboe,C.D., Hecht,S.M. and Shuman,S. (2005) Mechanistic plasticity of DNA topoisomerase IB: phosphate electrostatics dictate the need for a catalytic arginine. Structure, 13, 513–520. 11. Rowley,P.A., Kachroo,A.H., Ma,C.H., Maciaszek,A.D., Guga,P. and Jayaram,M. Electrostatic suppression allows tyrosine site-speciﬁc recombination in the absence of a conserved catalytic arginine. J. Biol. Chem., doi:10.1074/jbc.M110.112292. 12. Cheng,C., Kussie,P., Pavletich,N. and Shuman,S. (1998) Conservation of structure and mechanism between eukaryotic topoisomerase I and site-speciﬁc recombinases. Cell, 92, 841–850. 13. Perry,K., Hwang,Y., Bushman,F.D. and Van Duyne,G.D. (2006) Structural basis for speciﬁcity in the poxvirus topoisomerase. Mol. Cell, 23, 343–354. 14. Chen,J.W., Lee,J. and Jayaram,M. (1992) DNA cleavage in trans by the active site tyrosine during Flp recombination: switching protein partners before exchanging strands. Cell, 69, 647–658. 15. Lee,J., Jayaram,M. and Grainge,I. (1999) Wild-type Flp recombinase cleaves DNA in trans. EMBO J., 18, 784–791. 16. Lee,J., Whang,I. and Jayaram,M. (1996) Assembly and orientation of Flp recombinase active sites on two-, three- and four-armed DNA substrates: implications for a recombination mechanism. J. Mol. Biol., 257, 532–549. 17. Shaikh,A.C. and Sadowski,P.D. (1997) The Cre recombinase cleaves the lox site in trans. J. Biol. Chem., 272, 5695–5702. 18. Johnson,K.A., Simpson,Z.B. and Blom,T. (2009) Global kinetic explorer: a new computer program for dynamic simulation and ﬁtting of kinetic data. Anal. Biochem., 387, 20–29. 19. Johnson,K.A., Simpson,Z.B. and Blom,T. (2009) FitSpace explorer: an algorithm to evaluate multidimensional parameter space in ﬁtting kinetic data. Anal. Biochem., 387, 30–41. 20. Ghosh,K., Lau,C.K., Gupta,K. and Van Duyne,G.D. (2005) Preferential synapsis of loxP sites drives ordered strand exchange in Cre-loxP site-speciﬁc recombination. Nat. Chem. Biol., 1, 275–282. 21. Woods,K.C., Martin,S.S., Chu,V.C. and Baldwin,E.P. (2001) Quasi-equivalence in site-speciﬁc recombinase structure and function: crystal structure and activity of trimeric Cre recombinase bound to a three-way Lox DNA junction. J. Mol. Biol., 313, 49–69. 22. Hoess,R.H., Wierzbicki,A. and Abremski,K. (1986) The role of the loxP spacer region in P1 site-speciﬁc recombination. Nucleic Acids Res., 14, 2287–2300.