Ahumada A, Tse-Dinh Y C
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York, 10595, USA.
Biochem Biophys Res Commun. 1998 Oct 20;251(2):509-14. doi: 10.1006/bbrc.1998.9500.
Escherichia coli DNA topoisomerase I binds three Zn(II) with three tetracysteine motifs. Three subclones containing these tetracysteine motifs were expressed and purified. Subclone ZD1 contained the minimal tetracysteine motifs sequence. A larger subclone ZD2 corresponded to a region bordered by two protease sensitive sites. Subclone ZD3 also included the 14-kDa C-terminal domain that has been shown to bind DNA. Subclones ZD1 and ZD2 were found to bind one and two Zn(II), respectively, and neither had detectable DNA binding activity. ZD3 could bind three Zn(II) and had higher DNA binding affinity than the 14-kDa C-terminal domain. The complex formed between ZD3 and a single-stranded 31mer could be detected by the gel shift assay while the complex formed by the 14-kDa C-terminal domain was not stable under gel electrophoresis conditions. The three Zn(II) binding motifs appeared to be part of a high-affinity DNA binding domain.
大肠杆菌DNA拓扑异构酶I通过三个四半胱氨酸基序结合三个锌离子（Zn(II)）。表达并纯化了包含这些四半胱氨酸基序的三个亚克隆。亚克隆ZD1包含最小的四半胱氨酸基序序列。较大的亚克隆ZD2对应于由两个蛋白酶敏感位点界定的区域。亚克隆ZD3还包括已证明能结合DNA的14 kDa C末端结构域。发现亚克隆ZD1和ZD2分别结合一个和两个锌离子（Zn(II)），且两者均无可检测到的DNA结合活性。ZD3能结合三个锌离子（Zn(II)），并且比14 kDa C末端结构域具有更高的DNA结合亲和力。通过凝胶迁移试验可检测到ZD3与单链31聚体形成的复合物，而14 kDa C末端结构域形成的复合物在凝胶电泳条件下不稳定。这三个锌离子（Zn(II)）结合基序似乎是高亲和力DNA结合结构域的一部分。
