6124 Nucleic Acids Research, 2006, Vol. 34, No. 21 using very low amounts of reactants, made possible by the sensitive detection method. Finally, the qPCR-based assay also provides a sensitive means for scanning candidate cata- lysts to identify possible reactions important in pre-biotic nucleic acid chemistry. ACKNOWLEDGEMENTS The authors thank members of the Bushman laboratory for helpful discussions. This work was supported by NIH grant GM068408, the James B. Pendleton Charitable Trust and Robin and Frederic Withington. T.L.D. is a fellow of The Leukemia and Lymphoma society (5217-06). Funding to pay the Open Access publication charges for this article was provided by NIH grant GM068408. Conflict of interest statement. None declared REFERENCES 1. Beese,L.S. and Steitz,T.A. (1991) Structural basis for the 30-50 exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J., 10, 25–33. 2. Steitz,T.A. and Steitz,J.A. (1993) A general two-metal-ion mechanism for catalytic RNA. Proc. Natl Acad. Sci. USA, 90, 6498–6502. 3. Pan,T., Long,D.M. and Uhlenbeck,O.C. (1993) Divalent metal ions in RNA folding and catalysis. In Gesteland,R.F. and Atkins,J.F. (eds), The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 271–302. 4. Rice,P., Craigie,R. and Davies,D.R. (1996) Retroviral integrases and their cousins. Curr. Opin. Struct. Biol., 6, 76–83. 5. Yang,W. and Steitz,T.A. (1995) Recombining the structures of HIV integrase, RuvC, and RNase H. Structure, 3, 131–134. 6. Yang,Z.N., Mueser,T.C., Bushman,F.D. and Hyde,C.C. (2000) Crystal structure of an active two-domain derivative of rous sarcoma virus integrase. J. Mol. Biol., 296, 535–548. 7. Dyda,F., Hickman,A.B., Jenkins,T.M., Engelman,A., Craigie,R. and Davies,D.R. (1994) Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science, 266, 1981–1986. 8. Bujacz,G., Alexandratos,J., Wlodawer,A., Merkel,G., Andrake,M., Katz,R.A. and Skalka,A.M. (1997) Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity. J. Biol. Chem., 272, 18161–18168. 9. Bujacz,G., Jaskolski,M., Alexandratos,J., Wlodawer,A., Merkel,G., Katz,R.A. and Skalka,A.M. (1996) The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations. Structure, 4, 89–96. 10. Bushman,F.D. (2001) Lateral DNA Transfer: Mechanisms and Consequences. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 11. Craig,N.L., Craigie,R., Gellert,M. and Lambowitz,A.M. (2002) Mobile DNA II. ASM Press. 12. Nowotny,M., Gaidamakov,S.A., Crouch,R.J. and Yang,W. (2005) Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis. Cell, 121, 1005–1016. 13. Davies,J.F., Hostomska,Z., Hostomsky,Z., Jordan,S.R. and Matthews,D.A. (1991) Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. Science, 252, 88–95. 14. Nowotny,M. and Yang,W. (2006) Stepwise analyses of metal ions in RNase H catalysis from substrate destabilization to product release. EMBO J., 25, 1924–1933. 15. Goldgur,Y., Dyda,F., Hickman,A.B., Jenkins,T.M., Craigie,R. and Davies,D.R. (1998) Three new structures of the core domain of HIV-1 integrase: an active site that binds magnesium. Proc. Natl Acad. Sci. USA, 95, 9150–9154. 16. Katayanagi,K., Okumura,M. and Morikawa,K. (1993) Crystal structure of Escherichia coli RNase HI in complex with Mg2+ at 2.8 A resolution: proof for a single Mg(2+)-binding site. Proteins, 17, 337–346. 17. Lubkowski,J., Yang,F., Alexandratos,J., Wlodawer,A., Zhao,H., Burke,T.R.J., Neamati,N., Pommier,Y., Merkel,G. and Skalka,A.M. (1998) Structure of the catalytic domain of avian sarcoma virus integrase with a bound HIV-1 integrase-targeted inhibitor. Proc. Natl Acad. Sci. USA, 95, 4831–4836. 18. Maignan,S., Guilloteau,J.P., Zhou-Liu,Q., Clement-Mella,C. and Mikol,V. (1998) Crystal structures of the datalytic domain of HIV-1 integrase free and complexed with its metal cofactor: High level of similarity of the active site with other viral integrases. J. Mol. Biol., 282, 359–368. 19. Tsunaka,Y., Takano,K., Matsumura,H., Yamagata,Y. and Kanaya,S. (2005) Identification of single Mn(2+) binding sites required for activation of the mutant proteins of E.coli RNase HI at Glu48 and/or Asp134 by X-ray crystallography. J. Mol. Biol., 345, 1171–1183. 20. Black,C.B. and Cowan,J.A. (1994) Magnesium activation of ribonuclease H. Evidence for one catalytic metal ion. Inorg. Chem., 33, 5805–5808. 21. Lai,B., Li,Y., Cao,A. and Lai,L. (2003) Metal ion binding and enzymatic mechanism of Methanococcus jannaschii RNase HII. Biochemistry, 42, 785–791. 22. Keck,J.L., Goedken,E.R. and Marqusee,S. (1998) Activation/attenuation model for RNase H. A one-metal mechanism with second-metal inhibition. J. Biol. Chem., 273, 34128–34133. 23. Gao,K., Wong,S. and Bushman,F. (2004) Metal binding by the D,DX35E motif of human immunodeficiency virus type 1 integrase: selective rescue of Cys substitutions by Mn2+ in vitro. J. Virol., 78, 6715–6722. 24. Gordon,P.M., Sontheimer,E.J. and Piccirilli,J.A. (2000) Metal ion catalysis during the exon-ligation step of nuclear pre-mRNA splicing: extending the parallels between the spliceosome and group II introns. RNA, 6, 199–205. 25. Hougland,J.L., Kravchuk,A.V., Herschlag,D. and Piccirilli,J.A. (2005) Functional identification of catalytic metal ion binding sites within RNA. PLoS Biol., 3, e277. 26. Sontheimer,E.J., Sun,S. and Piccirilli,J.A. (1997) Metal ion catalysis during splicing of premessenger RNA. Nature, 388, 801–805. 27. Yoshida,A., Sun,S. and Piccirilli,J.A. (1999) A new metal ion interaction in the Tetrahymena ribozyme reaction revealed by double sulfur substitution. Nature Struct. Biol., 6, 318–321. 28. Allingham,J.S., Pribil,P.A. and Haniford,D.B. (1999) All three residues of the Tn 10 transposase DDE catalytic triad function in divalent metal ion binding. J. Mol. Biol., 289, 1195–1206. 29. Kim,D.R., Dai,Y., Mundy,C.L., Yang,W. and Oettinger,M.A. (1999) Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase. Genes Dev., 13, 3070–3080. 30. Landree,M., Wibbenmeyer,J. and Roth,D. (1999) Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination. Genes Dev., 13, 3059–3069. 31. Sarnovsky,R.J., May,E.W. and Craig,N.L. (1996) The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products. EMBO J., 15, 6348–6361. 32. Soundar,S., O’Hagan,M., Fomulu,K.S. and Colman,R.F. (2006) Identification of Mn2+-binding aspartates from alpha, beta and gamma subunits of human NAD-dependent isocitrate dehydrogenase. J. Biol. Chem., 281, 21073–21081. 33. Curley,J.F., Joyce,C.M. and Piccirilli,J.A. (1997) Functional evidence that the 30-50 exonuclease domain of Escherichia coli DNA polymerase I employs a divalent metal ion in leaving group stabilization. J. Am. Chem. Soc., 119, 12691–12692. 34. Chow,S.A., Vincent,K.A., Ellison,V. and Brown,P.O. (1992) Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus. Science, 255, 723–726. 35. Melek,M. and Gellert,M. (2000) RAG1/2-mediated resolution of transposition intermediates: two pathways and possible consequences. Cell, 101, 625–633. 36. Au,T.K., Pathania,S. and Harshey,R.M. (2004) True reversal of Mu integration. EMBO J., 23, 3408–3420. 37. Gao,K., Butler,S.L. and Bushman,F.D. (2001) Human immunodeficiency virus type 1 integrase: arrangement of protein domains in active cDNA complexes. EMBO J., 20, 3565–3576.