Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system.
Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system.
复制标题
通过纯化的 T4 噬菌体复制系统在体外有效复制双链 DNA 模板。
DOI：
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发表时间：
1980
影响因子：
4.8
通讯作者：
B. Alberts
中科院分区：
文献类型：
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作者：
N. Sinha;C. Morris;B. Alberts
多种双链DNA模板在体外DNA复制系统中广泛复制，所述体外DNA复制系统含有由7个T4噬菌体DNA复制基因指定的纯化蛋白质（32、41、43、44、62、45和61）。在有利的条件下，这种多蛋白系统在30至60分钟的孵育中催化输入DNA模板的几个拷贝的合成。在体外产生的复制叉以高度进行性的方式移动，大约以每秒500个核苷酸的体内速率移动。在体外复制叉的滞后侧合成的DNA是不连续的，就像在体内一样，产生平均长度约为10，000个核苷酸的“冈崎片段”;相反，DNA在体外复制叉的前导侧以连续的方式聚合。虽然7蛋白体外DNA复制系统传播复制叉的机制与体内机制非常相似，但它缺乏去除RNA引物、重新密封冈崎片段和在确定的DNA起点启动复制叉的能力;需要补充额外的T4特异性复制蛋白来促进后三种功能。
A wide variety of double-stranded DNA templates are replicated extensively in an in vitro DNA replication system containing the purified proteins specified by seven T4 bacteriophage DNA replication genes (32, 41, 43, 44, 62, 45, and 61). In favorable conditions, this multiprotein system catalyzes the synthesis of several copies of the input DNA template in a 30- to 60-min incubation. The replication forks produced in vitro move in a highly processive fashion, at approximately the in vivo rate of 500 nucleotides per s. The DNA synthesized on the lagging side of the in vitro replication fork is made discontinuously, as it is in vivo, giving rise to "Okazaki pieces" averaging some 10,000 nucleotides in length; in contrast, DNA is polymerized in a continuous manner on the leading side of the in vitro fork. Although the mechanism by which the seven-protein in vitro DNA replication system propagates replication forks closely resembles the in vivo mechanism, it lacks the capacity to remove RNA primers, to reseal Okazaki pieces, and to initiate replication forks at defined DNA origins; supplementation of the system with additional T4-specific replication proteins will be required to facilitate these latter three functions.