Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic.
Biochem Biophys Res Commun. 2010 Jan 15;391(3):1409-14. doi: 10.1016/j.bbrc.2009.12.076. Epub 2009 Dec 22.
p53 is one of the most important tumor suppressors which responds to DNA damage by binding to DNA and regulating the transcription of genes involved in cell cycle arrest, apoptosis, or senescence. As it was shown previously, p53 binding to DNA is strongly influenced by DNA topology. DNA supercoiling is fundamentally important for a wide range of biological processes including DNA transcription, replication, recombination, control of gene expression and genome organization. In this study, we investigated the cruciform structures formation of various inverted repeats in p53-responsive sequences from p21, RGC, mdm2 and GADD45 promoters under negative superhelical stress, and analyzed the effects of these DNA topology changes on p53-DNA binding. We demonstrated using three different methods (gel retardation analyses, ELISA and magnetic immunoprecipitation assay) that the p53 protein binds preferentially to negatively supercoiled plasmid DNAs with p53-responsive sequence presented as a cruciform structure. Not only the appearance of the cruciform structures within naked supercoiled DNA, but also the potential of the binding sites for adopting the non-B structures can contribute to a more favorable p53-DNA complex.
p53 是最重要的肿瘤抑制因子之一，它通过与 DNA 结合来响应 DNA 损伤，并调节细胞周期停滞、细胞凋亡或衰老相关基因的转录。如前所述，p53 与 DNA 的结合受 DNA 拓扑结构的强烈影响。DNA 超螺旋对于广泛的生物学过程至关重要，包括 DNA 转录、复制、重组、基因表达的控制和基因组组织。在这项研究中，我们在负超螺旋压力下研究了 p53 反应序列中来自 p21、RGC、mdm2 和 GADD45 启动子的各种倒位重复的十字形结构形成，并分析了这些 DNA 拓扑变化对 p53-DNA 结合的影响。我们使用三种不同的方法（凝胶阻滞分析、ELISA 和磁免疫沉淀测定）证明，p53 蛋白优先结合呈现十字形结构的负超螺旋质粒 DNA。不仅在裸露的超螺旋 DNA 中出现十字形结构，而且结合位点采用非 B 结构的潜力也有助于形成更有利于 p53-DNA 复合物。
