Herbert A G, Spitzner J R, Lowenhaupt K, Rich A
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3339-42. doi: 10.1073/pnas.90.8.3339.
A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.
在鸡血细胞核提取物中已鉴定出一种似乎对左手螺旋Z-DNA构象具有高度特异性的蛋白质（Zα）。通过使用由（dC-dG）35寡聚物组成的放射性探针进行凝胶迁移试验来测量Zα活性，该寡聚物中有50%的脱氧胞嘧啶被5-溴脱氧胞嘧啶取代。在10 mM Mg2+存在下，探针转变为Z-DNA构象并被Zα结合。通过与经化学溴化稳定为Z-DNA形式的线性聚（dC-dG）竞争，可特异性阻断Zα与放射性探针的结合，但B型聚（dC-dG）或煮沸的鲑鱼精DNA则不能。此外，含有Z-DNA插入片段的超螺旋质粒可竞争性阻断Zα的结合活性，但线性化质粒或不含形成Z-DNA插入片段的等量亲本超螺旋质粒则不能。Zα可通过紫外线与32P标记的溴化探针交联，由此我们估计Zα 的最小分子量为39 kDa。
