Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments
Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments
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DOI：
10.1038/sj.onc.1202710
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发表时间：
1999-06-17
期刊：
影响因子：
8
通讯作者：
Vojtesek, B
中科院分区：
文献类型：
--
作者：
Palecek, E;Brázdová, M;Vojtesek, B
最近，我们已经证明，在体外存在和不存在p53共识序列（p53CON）的情况下，温和型人p53蛋白都优先与超螺旋（sc） DNA结合。这种结合在琼脂糖凝胶上产生了一个阶梯状的延迟带。用抗体DO-1进行免疫印迹，我们发现得到的条带对应于乙二胺染色的DNA，这表明梯子的每个条带都含有DNA-p53复合物。这些条带的强度和数量随锌离子的生理浓度而降低。在较高的锌浓度下，p53与scDNA的结合被完全抑制。附加锌离子与p53的结合似乎比核心结构域DNA结合位点的内在锌离子的结合要弱得多。先前发表的数据表明，100 μ M锌离子不会影响DNA寡核苷酸中p53与p53CON的结合，与此相反，我们发现5-20 μ M锌能有效抑制DNA片段中p53与p53CON的结合。我们还表明，相对较低浓度的二硫苏糖醇而不是2-巯基乙醇会降低游离锌离子的浓度，从而阻止它们对p53与DNA结合的抑制作用。镍和钴离子抑制p53与scDNA及其在线性DNA片段中的一致序列的结合的效率低于锌；钴离子效率最低，需要100 μ M Co2+才能完全抑制p53的结合。生理锌浓度对p53与DNA结合的调节可能是体内调节p53活性的新途径。
Recently we have shown that mild-type human p53 protein binds preferentially to supercoiled (sc) DNA in vitro in both the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on an agarose gel. Using immunoblotting with the antibody DO-1, me show that the bands obtained correspond to ethidium-stained DNA, suggesting that each band of the ladder contains a DNA-p53 complex. The intensity and the number of these bands are decreased by physiological concentrations of zinc ions. At higher zinc concentrations, binding of p53 to scDNA is completely inhibited. The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data suggesting that 100 mu M zinc ions do not influence p53 binding to p53CON in a DNA oligonucleotide, we show that 5-20 mu M zinc efficiently inhibits binding of p53 to p53CON in DNA fragments. We also show that relatively low concentrations of dithiothreitol but not of 2-mercaptoethanol decrease the concentration of free zinc ions, thereby preventing their inhibitory effect on binding of p53 to DNA. Nickel and cobalt ions inhibit binding of p53 to scDNA and to its consensus sequence in linear DNA fragments less efficiently than zinc; cobalt ions are least efficient, requiring > 100 mu M Co2+ for full inhibition of p53 binding. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a novel pathway that regulates p53 activity in vivo.