DNA replication is one of the central functions of the cell. The complexity of modern DNA replication systems raises a question: is it possible to achieve a simpler continuous isothermal DNA replication using fewer proteins? Here, we searched such replication using an evolutionary approach. Through a long-term serial dilution experiment with phi29 DNA polymerase, we found that large repetitive DNAs spontaneously appear and continuously replicate. The repetitive sequence is critical for replication. Arbitrary sequences can replicate if they contain many repeats. We also demonstrated continuous DNA replication using expressed polymerase from the DNA for 10 rounds. This study revealed that continuous isothermal DNA replication can be achieved in a scheme simpler than that employed by modern organisms, providing an alternative strategy for simpler artificial cell synthesis and a clue to possible primitive forms of DNA replication.
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