Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding.
Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding.
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DOI：
10.1016/0022-2836(87)90370-6
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发表时间：
1987-03
影响因子：
5.6
通讯作者：
M. Sander;T. Hsieh;A. Udvardy;P. Schedl
中科院分区：
文献类型：
--
作者：
M. Sander;T. Hsieh;A. Udvardy;P. Schedl
研究了果蝇拓扑异构酶II超螺旋松弛和结合活性的序列依赖性。在结合实验中使用的DNA底物是来自果蝇热休克位点87A7的两个片段。这些DNA片段之一包括热休克蛋白hsp70的编码区，另一个包括基因间非编码区，该基因间非编码区在该基因座处分离hsp70基因的两个不同转录拷贝。基因间区域先前被证明具有比hsp70编码区高得多的拓扑异构酶切割位点密度。竞争硝酸纤维素过滤器结合试验表明，优先结合的intergene片段，结合特异性随着离子强度的增加而增加。解离动力学表明拓扑异构酶II复合物与基因间DNA片段的动力学稳定性更高。为了研究拓扑异构酶II松弛活性，我们使用了超螺旋质粒，其中包含作为插入片段克隆的来自基因座87A7的相同片段。两种质粒的相对弛豫速率在离子强度的几种条件下测定，并且当质粒底物被包括在单独的反应中时，或者当它们在单个反应中混合时。这两种质粒的松弛特性可以解释为高亲和力结合位点、强切割位点和拓扑异构酶II催化链通过事件期间使用的位点的重合。因此，拓扑异构酶II催化活性的序列依赖性可能与该酶切割DNA的序列依赖性平行。
The sequence dependence ofDrosophilatopoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments fromDrosophilaheat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of thehsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.