Insertion of an extra stage into the CSR cycle allows the application of CSR to enzymes other than polymerases. For example, two enzymes can cooperate through reciprocal catalysis as shown for nucleoside diphosphate kinase (NDK) and Taq polymerase, whereby NDK produces the substrates required for the replication of its own gene by the polymerase. NDK converts deoxynucleoside diphosphates (dNDPs) to triphosphates (dNTPs), which are substrates for the polymerase. Hence, only ndk genes encoding active NDK are replicated.
Conclusion: CSR applications in molecular evolution and genomics
CSR is a powerful new technology in molecular evolution and can be used to rapidly evolve novel polymerases with useful properties.
It can readily be applied to enzymes (or auxiliary factors) involved in nucleic acid transactions such as polymerases, ligases, helicases etc., but we envisage more diverse uses of the technology. For example, CSR may be used for the functional cloning of enzymes from genomic libraries or directly from diverse cellular populations.
Two (and presumably more) enzymes can cooperate in CSR through reciprocal catalysis. Further stages may be added to a cooperative CSR cycle allowing the evolution of both single enzymes and reaction pathways. We anticipate a generic selection system for catalysis built around the “polymerase engine” of CSR, whereby coupled catalytic reactions either produce replicase substrates or consume inhibitors, thus allowing replication of the genes encoding the enzymes to proceed.
References
[1] Steitz, T.A. (2000) DNA polymerases: structural diversity and common mechanisms. J. Biol. Chem. 274, 17395-17398.
[2] Szostak, J.W., Bartel, D.P. & Luisi, P.L. (2001) Synthesizing life. Nature 409, 387-390.
[3] Ghadessy, F.J., Ong, J.L. & Holliger, P. (2001) Directed evolution of polymerase function by compartmentalized self-replication (CSR). Proc. Natl. Acad. Sci USA, in press.
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