MULTIPLE COMPONENTS ARE INVOLVED IN THE EFFICIENT JOINING OF DOUBLE-STRANDED DNA BREAKS IN HUMAN CELL-EXTRACTS
MULTIPLE COMPONENTS ARE INVOLVED IN THE EFFICIENT JOINING OF DOUBLE-STRANDED DNA BREAKS IN HUMAN CELL-EXTRACTS
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DOI：
10.1093/nar/20.16.4145
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发表时间：
1992-08-25
影响因子：
14.9
通讯作者：
THACKER, J
中科院分区：
文献类型：
--
作者：
FAIRMAN, MP;JOHNSON, AP;THACKER, J
我们描述了一种快速有效的体外系统，用于基于人类 293 细胞提取物重新连接 DNA 中的双链断裂。使用该系统作为分析，我们将核提取物分离成参与断裂重新连接的几种成分。未分级系统可以转换大约。 100% 输入的 DNA，用限制性内切酶线性化，在低温（17 摄氏度）和 37 摄氏度的生理温度下转化为高分子量材料，我们已经证明提取物中的竞争活性也可以作用于 DNA 模板。我们提出了提取物的分级分离和新因子的部分纯化，该因子将刺激粗重连接活性，此外还增加了纯化的 DNA 连接酶 I 的活性。我们还部分纯化了断裂连接活性，并表明色谱特性与先前描述的三种 DNA 连接酶并不直接对应，表明观察到的活性可能不是由于单一酶种类所致。通过研究双链 DNA 断裂的重新连接作为一个生化过程，我们已经证明，这种断裂的有效连接还需要 DNA 连接酶之外的因素。
We describe a rapid and efficient in vitro system for the rejoining of double stranded breaks in DNA based on extracts of human 293 cells. Using this system as an assay, we have separated the nuclear extract into several components involved in break rejoining. The unfractionated system can convert approx. 100% of the input DNA, linearized with a restriction enzyme, to high molecular weight material at low temperature (17-degrees-C), and at the physiological temperature of 37-degrees-C we have shown that competing activities in the extract can also act on the DNA template. We present the fractionation of the extract and the partial purification of a novel factor which will stimulate a crude rejoin activity and in addition increases the activity of purified DNA ligase I. We have also partially purified the break joining activity and show that the chromatographic properties do not directly correspond with the three DNA ligases previously described, indicating that the activity observed may not be due to a single enzyme species. By studying the rejoining of double stranded DNA breaks as a biochemical process, we have demonstrated that the efficient joining of such breaks requires factors in addition to DNA ligases.