Recognition of DNA modified by antitumor cisplatin by "latent" and "active" protein p53
Recognition of DNA modified by antitumor cisplatin by "latent" and "active" protein p53
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DOI：
10.1016/s0006-2952(03)00078-9
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发表时间：
2003-04-15
影响因子：
5.8
通讯作者：
Brabec, V
中科院分区：
文献类型：
--
作者：
Fojta, M;Pivonkova, H;Brabec, V
肿瘤抑制蛋白 p53 拥有两个 DNA 结合位点。位于其核心结构域内的一个负责蛋白质的序列特异性 DNA 结合、与单链或双链 DNA 的内部片段以及某些类型的非 B DNA 结构的非特异性结合。蛋白质 C 末端包含的另一种物质与受损的 DNA 结合。使用琼脂糖凝胶电泳迁移率变动分析和免疫印迹分析研究了活性、潜伏和体外激活的 p53 蛋白与抗肿瘤顺铂修饰的 DNA 片段的结合。我们发现，潜在的和活性的 p53 形式与顺铂全局修饰的 DNA 随机序列结合，比未修饰的 DNA 具有更高的亲和力。有趣的是，潜在形式对铂化 DNA 表现出比活性 p53 更明显的选择性。与这一观察结果一致，潜在形式对铂化 DNA 的偏好降低是由于潜在 p53 通过其 C 末端内的蛋白激酶 C 位点磷酸化或通过单克隆抗体 Bp53-10.1 的结合而激活的结果。涉及 p53 20 bp 共有序列的竞争实验表明，当活性 p53 与缺乏共有序列但经过顺铂修饰的 DNA 片段结合时，p53 核心结构域是活性 p53 的主要结合位点。此外，发现潜在蛋白可能通过其 C 末端选择性地与顺铂修饰的 DNA 相互作用。 (C) 2003 Elsevier Science Inc. 保留所有权利。
Tumor suppressor protein p53 possesses two DNA-binding sites. One that is located within its core domain is responsible for sequence-specific DNA binding of the protein, non-specific binding to internal segments of single- or double-stranded DNA, and to certain kinds of non-B DNA structures. The other that is contained in the C-terminus of the protein binds to damaged DNA. Binding of active, latent, and in vitro-activated p53 protein to DNA fragments modified by antitumor cisplatin was studied using electrophoretic mobility shift assay in agarose gels and immunoblotting analysis. We found that both latent and active p53 forms bound to random sequences of DNA globally modified by cisplatin with a higher affinity than to unmodified DNA. Interestingly, the latent form exhibited a more pronounced selectivity for platinated DNA than the active p53. Consistently with this observation, the preference of the latent form for platinated DNA decreased as a consequence of the activation of latent p53 by phosphorylation at the protein kinase C site within its C-terminus or by binding of the monoclonal antibody Bp53-10.1. Competition experiments involving a 20-bp consensus sequence of p53 suggested that the p53 core domain was a primary binding site of the active p53 when it bound to DNA fragments lacking consensus sequence, but modified by cisplatin. In addition, the latent protein was found to selectively interact with DNA modified by cisplatin probably via its C-terminus. (C) 2003 Elsevier Science Inc. All rights reserved.