Single-strand DNA cleavages by eukaryotic topoisomerase II.
Single-strand DNA cleavages by eukaryotic topoisomerase II.
复制标题
DOI：
10.1021/bi00422a012
复制
发表时间：
1988-11
期刊：
影响因子：
2.9
通讯作者：
M. Muller;J. Spitzner;J. DiDonato;V. Mehta;K. Tsutsui
中科院分区：
文献类型：
--
作者：
M. Muller;J. Spitzner;J. DiDonato;V. Mehta;K. Tsutsui
描述了真核 II 型 DNA 拓扑异构酶 (EC 5.99.1.3) 的新纯化方法，并对禽类酶进行了纯化和表征。切割反应的分析表明，拓扑异构酶 II 可以作为含有双链和单链断裂 DNA 的 DNA-酶共价复合物被捕获。数据表明，拓扑异构酶 II 进行的 DNA 切割通过相对链（相隔 4 bp）上的两个不对称单链切割和重新密封步骤进行，在添加蛋白质变性剂后具有独立的被捕获概率。单链裂解在强拓扑异构酶 II 和弱拓扑异构酶 II 位点均得到直接证实。因此，与脊椎动物拓扑异构酶 II 共有序列（序列；见正文）匹配（N 是任何碱基，切割发生在 -1 和 +1 之间）[Spitzner, J.R., & Muller, M.T. (1988) 核酸研究。 16, 5533-5556)] 不能预测切割位点是单链还是双链；然而，由拓扑异构酶 II 切割的包含两个保守共有碱基（+2 处的 G 残基和 +4 处的 T）的位点通常会产生双链切割，而缺乏这两个共有元件的识别位点会产生单链切割。最后，拓扑异构酶 II 的单链裂解似乎不是由受损酶分子引起的人为因素，因为新鲜制备的粗提物中的拓扑异构酶 II 也显示出单链裂解的特性。
A new purification method for eukaryotic type II DNA topoisomerase (EC 5.99.1.3) is described, and the avian enzyme has been purified and characterized. An analysis of the cleavage reaction has revealed that topoisomerase II can be trapped as a DNA-enzyme covalent complex containing DNA with double-stranded and single-stranded breaks. The data indicate that DNA cleavage by topoisomerase II proceeds by two asymmetric single-stranded cleavage and resealing steps on opposite strands (separated by 4 bp) with independent probabilities of being trapped upon addition of a protein denaturant. Single-strand cleavages were directly demonstrated at both strong and weak topoisomerase II sites. Thus, a match to the vertebrate topoisomerase II consensus sequence (sequence; see text) (N is any base, and cleavage occurs between -1 and +1) [Spitzner, J.R., & Muller, M.T. (1988) Nucleic Acids Res. 16, 5533-5556)] does not predict whether a cleavage site will be single stranded or double stranded; however, sites cleaved by topoisomerase II that contain two conserved consensus bases (G residue at +2 and T at +4) generally yield double-strand cleavage whereas recognition sites lacking these two consensus elements yield single-strand cleavages. Finally, single-strand cleavages with topoisomerase II do not appear to be an artifact caused by damaged enzyme molecules since topoisomerase II in freshly prepared, crude extracts also shows the property of single-strand cleavages.