Cox L S, Hupp T, Midgley C A, Lane D P
Department of Biochemistry, University of Dundee, UK.
EMBO J. 1995 May 1;14(9):2099-105. doi: 10.1002/j.1460-2075.1995.tb07201.x.
p53 is a transcriptional activator and repressor, but recent evidence suggests that some of its many biological functions may not be dependent on transcription. To determine whether p53 exerts a direct influence on nuclear DNA replication, purified human p53 was added to a transcription-free DNA replication extract from Xenopus eggs. Full-length human p53 that inhibits SV40 DNA replication in vitro had no effect on nuclear DNA synthesis in the Xenopus system. In contrast, a C-terminal truncated form of p53 (p53 delta 30), which is constitutively active for DNA binding and similar to an alternately spliced form found in vivo, showed a concentration-dependent inhibition of DNA replication in both the soluble SV40 system and eukaryotic nuclei. This inhibition occurred primarily at initiation of DNA synthesis. Oxidation of p53 delta 30, which eliminates DNA binding activity, also abrogated the protein's ability to inhibit nuclear DNA synthesis. The p53 binding DNA consensus sequence enhanced rather than competed away inhibitory activity of p53 delta 30. Therefore, p53 that is constitutively active for DNA binding can inhibit nuclear DNA replication in the absence of transcription. This inhibition may require binding of p53 to DNA, in addition to interactions between p53 and proteins of the replication complex.
p53是一种转录激活因子和阻遏因子，但最近的证据表明，它的许多生物学功能可能不依赖于转录。为了确定p53是否对核DNA复制有直接影响，将纯化的人p53添加到非洲爪蟾卵的无转录DNA复制提取物中。在体外抑制SV40 DNA复制的全长人p53对非洲爪蟾系统中的核DNA合成没有影响。相比之下，p53的C末端截短形式（p53δ30），其对DNA结合具有组成型活性且类似于体内发现的一种可变剪接形式，在可溶性SV40系统和真核细胞核中均表现出浓度依赖性的DNA复制抑制作用。这种抑制主要发生在DNA合成的起始阶段。消除DNA结合活性的p53δ30的氧化也消除了该蛋白抑制核DNA合成的能力。p53结合DNA共有序列增强而非竞争消除p53δ30的抑制活性。因此，对DNA结合具有组成型活性的p53在没有转录的情况下可以抑制核DNA复制。这种抑制可能除了需要p53与复制复合物的蛋白质之间的相互作用外，还需要p53与DNA的结合。
