Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201-1180, USA.
Inorg Chem. 2011 Jun 20;50(12):5442-50. doi: 10.1021/ic102252a. Epub 2011 May 16.
ZIF268, a member of the classical zinc finger protein family, contains three Cys(2)His(2) zinc binding domains that together recognize the DNA sequence 5'-AGCGTGGGCGT-3'. These domains can be fused to an endonuclease to make a chimeric protein to target and cleave specific DNA sequences. A peptide corresponding to these domains, named ZIF268-3D, has been prepared to determine if the zinc finger domain itself can promote DNA cleavage when a redox active metal ion, Fe(II), is coordinated. The UV-vis absorption spectrum of Fe(II)-ZIF268-3D is indicative of Fe(II) coordination. Using fluorescence anisotropy, we demonstrate that Fe(II)-ZIF268-3D binds selectively to its target DNA in the same manner as Zn(II)-ZIF268-3D. In the presence of added oxidant, H(2)O(2) or O(2), DNA cleavage is not observed by Fe(II)-ZIF268-3D. Instead, the peptide itself is rapidly oxidized. Similarly, Zn(II)-ZIF268-3D and apo-ZIF268-3D are rapidly oxidized by H(2)O(2) or O(2), and we propose that ZIF268-3D is highly susceptible to oxidation.
ZIF268 是经典锌指蛋白家族的一员，包含三个 Cys(2)His(2)锌结合结构域，这些结构域共同识别 DNA 序列 5'-AGCGTGGGCGT-3'。这些结构域可以与内切酶融合，形成嵌合蛋白，以靶向和切割特定的 DNA 序列。已经制备了与这些结构域相对应的肽，命名为 ZIF268-3D，以确定当配位有氧化还原活性金属离子 Fe(II)时，锌指结构域本身是否可以促进 DNA 切割。Fe(II)-ZIF268-3D 的紫外可见吸收光谱表明存在 Fe(II)配位。通过荧光各向异性，我们证明 Fe(II)-ZIF268-3D 以与 Zn(II)-ZIF268-3D 相同的方式选择性地结合其靶 DNA。在添加氧化剂 H(2)O(2)或 O(2)的情况下，Fe(II)-ZIF268-3D 不会观察到 DNA 切割。相反，肽本身被迅速氧化。类似地，Zn(II)-ZIF268-3D 和 apo-ZIF268-3D 被 H(2)O(2)或 O(2)迅速氧化，我们提出 ZIF268-3D 极易被氧化。
