Propagation of genetic information is a fundamental property of living organisms. Escherichia coli has a 4.6 Mb circular chromosome with a replication origin, oriC. While the oriC replication has been reconstituted in vitro more than 30 years ago, continuous repetition of the replication cycle has not yet been achieved. Here, we reconstituted the entire replication cycle with 14 purified enzymes (25 polypeptides) that catalyze initiation at oriC, bidirectional fork progression, Okazaki-fragment maturation and decatenation of the replicated circular products. Because decatenation provides covalently closed supercoiled monomers that are competent for the next round of replication initiation, the replication cycle repeats autonomously and continuously in an isothermal condition. This replication-cycle reaction (RCR) propagates ∼10 kb circular DNA exponentially as intact covalently closed molecules, even from a single DNA molecule, with a doubling time of ∼8 min and extremely high fidelity. Very large DNA up to 0.2 Mb is successfully propagated within 3 h. We further demonstrate a cell-free cloning in which RCR selectively propagates circular molecules constructed by a multi-fragment assembly reaction. Our results define the minimum element necessary for the repetition of the chromosome-replication cycle, and also provide a powerful in vitro tool to generate large circular DNA molecules without relying on conventional biological cloning.
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