Weissker S N, Müller B F, Homfeld A, Deppert W
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Germany.
Oncogene. 1992 Oct;7(10):1921-32.
Biologically active mutant p53 from Balb/c mouse tumor cells (Meth A) was analysed for its specific interaction with DNA. Restricted phage lambda DNA, representing DNA of high complexity with regard to sequence and secondary structure, was used to probe for such an activity in a target-bound DNA-binding assay, using doubly immunopurified p53. A single lambda DNA fragment was specifically retained with very high affinity (KD = 10(-10) M). Specific DNA binding was shown to be an intrinsic property of p53, as it could be blocked with p53-specific monoclonal antibodies PAb122 and PAb421. The characteristics of the DNA binding of p53 to this lambda DNA fragment, as well as the structural properties of this fragment, suggested the possibility that p53 might be able to interact with nuclear matrix attachment region (MAR) DNA. Indeed, established genomic MAR elements were specifically bound by Meth A p53, whereas no binding was observed to an AT-rich control DNA. The interaction of p53 with MAR elements in vitro is compatible with the idea that p53 in vivo is involved in the regulation of replication and/or expression of cellular DNA. Complex DNA interactions were not restricted to mutant p53 from Meth A cells. Mutant p53 of a different conformational phenotype (PAb246+ 'wild-type' as opposed to PAb246- 'mutant' for p53 from Meth A cells) from minimally transformed T3T3 cells, as well as genotypic wild-type p53 expressed by a recombinant baculovirus in insect cells, exhibited similar DNA-binding properties.
对来自Balb/c小鼠肿瘤细胞（Meth A）的具有生物活性的突变型p53与DNA的特异性相互作用进行了分析。限制性噬菌体λDNA代表了在序列和二级结构方面具有高度复杂性的DNA，在靶标结合DNA结合试验中用于探测这种活性，试验使用了双免疫纯化的p53。一个单一的λDNA片段以非常高的亲和力（KD = 10⁻¹⁰ M）被特异性保留。特异性DNA结合被证明是p53的固有特性，因为它可以被p53特异性单克隆抗体PAb122和PAb421阻断。p53与该λDNA片段的DNA结合特性以及该片段的结构特性表明，p53可能能够与核基质附着区（MAR）DNA相互作用。实际上，已确定的基因组MAR元件被Meth A p53特异性结合，而对富含AT的对照DNA未观察到结合。p53与MAR元件在体外的相互作用与p53在体内参与细胞DNA复制和/或表达调控的观点相符。复杂的DNA相互作用并不局限于来自Meth A细胞的突变型p53。来自极低转化的T3T3细胞的具有不同构象表型的突变型p53（与来自Meth A细胞的p53的PAb246⁻“突变型”相对，为PAb246⁺“野生型”），以及由重组杆状病毒在昆虫细胞中表达的基因型野生型p53，都表现出类似的DNA结合特性。
