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An orthogonal DNA replication system in yeast


An extranuclear replication system, consisting of an orthogonal DNA plasmid–DNA polymerase pair, was developed in Saccharomyces cerevisiae. Engineered error-prone DNA polymerases showed complete mutational targeting in vivo: per-base mutation rates on the plasmid were increased substantially and remained stable with no increase in genomic rates. Orthogonal replication serves as a platform for in vivo continuous evolution and as a system whose replicative properties can be manipulated independently of the host's.

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Figure 1: An orthogonal replication system based on the p1/2 replication system.


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This research was funded by generous startup funds from the University of California–Irvine to C.C.L. and a US National Science Foundation Graduate Research Fellowship to A.R. We are greatly indebted to A.P. Arkin and members of the Miller Institute of the University of California–Berkeley for early encouragement of this idea. We thank R. Rezvani, K.F. Kearns and K.H. Spencer for experimental assistance. We thank J.E. Dueber (University of California–Berkeley) for helpful discussions and the gift of certain yeast expression plasmids. We thank P.G. Schultz, J.A. Wells and G.A. Weiss for valuable comments on our work.

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C.C.L. conceived this project; A.R. and C.C.L. designed all of the experiments; A.R., A.A. and C.C.L. performed all of the experiments; and A.R. and C.C.L. analyzed all of the data and wrote the paper.

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Correspondence to Chang C Liu.

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A provisional patent on this work has been filed.

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Supplementary Results, Supplementary Figures 1–7 and Supplementary Tables 1–6. (PDF 2105 kb)

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Ravikumar, A., Arrieta, A. & Liu, C. An orthogonal DNA replication system in yeast. Nat Chem Biol 10, 175–177 (2014).

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