Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle.
Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle.
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DOI：
10.1093/nar/gkx822
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发表时间：
2017-11-16
影响因子：
14.9
通讯作者：
Tsujimoto H
中科院分区：
文献类型：
--
作者：
Su'etsugu M;Takada H;Katayama T;Tsujimoto H
遗传信息的传播是生物体的一项基本属性。大肠杆菌有一条4.6Mb的环形染色体，其复制起点为ORIC。虽然ORIC复制在30多年前就已经在体外重组，但复制周期的连续重复尚未实现。在这里，我们用14种纯化的酶(25个多肽)重构了整个复制周期，这些酶催化ORIC的启动、双向分叉进展、Okazaki-片段成熟和复制环状产物的降解。因为十链提供了共价闭合的超螺旋单体，能够胜任下一轮的复制启动，所以复制周期在等温条件下自主地连续重复。这种复制循环反应以完整的共价闭合分子的形式指数地传播∼10kb环状DNA，甚至从单个DNA分子传播，倍增时间为∼8分钟，并且具有极高的保真度。在3小时内成功地繁殖了高达0.2Mb的超大DNA。我们进一步展示了一种无细胞克隆，在这种克隆中，RCR选择性地传播由多片段组装反应构建的环状分子。我们的结果定义了重复染色体复制周期所需的最小元素，并提供了一个强大的体外工具来产生大的环状DNA分子，而不依赖于传统的生物克隆。
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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DOI：
10.1073/pnas.86.23.9104
发表时间：
1989-12-01
影响因子：
11.1
作者：
LEE, EH;KORNBERG, A;HORIUCHI, T
通讯作者：
HORIUCHI, T
影响因子：
7.8
作者：
Kaguni, Jon M.
通讯作者：
Kaguni, Jon M.
影响因子：
5.6
作者：
Fujii, S;Akiyama, M;Maki, H
通讯作者：
Maki, H
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
48
作者：
Gibson, Daniel G.
通讯作者：
Gibson, Daniel G.