| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2014: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000)
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| Outline of Final Research Achievements |
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 purified enzymes 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 large circular DNA up to 0.2 Mb as intact covalently closed molecules. RCR provides a powerful in vitro tool to propagate large circular DNA molecules
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