Janus F, Albrechtsen N, Knippschild U, Wiesmüller L, Grosse F, Deppert W
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, D-20251 Hamburg, Germany.
Mol Cell Biol. 1999 Mar;19(3):2155-68. doi: 10.1128/MCB.19.3.2155.
In this study we further characterized the 3'-5' exonuclease activity intrinsic to wild-type p53. We showed that this activity, like sequence-specific DNA binding, is mediated by the p53 core domain. Truncation of the C-terminal 30 amino acids of the p53 molecule enhanced the p53 exonuclease activity by at least 10-fold, indicating that this activity, like sequence-specific DNA binding, is negatively regulated by the C-terminal basic regulatory domain of p53. However, treatments which activated sequence-specific DNA binding of p53, like binding of the monoclonal antibody PAb421, which recognizes a C-terminal epitope on p53, or a higher phosphorylation status, strongly inhibited the p53 exonuclease activity. This suggests that at least on full-length p53, sequence-specific DNA binding and exonuclease activities are subject to different and seemingly opposing regulatory mechanisms. Following up the recent discovery in our laboratory that p53 recognizes and binds with high affinity to three-stranded DNA substrates mimicking early recombination intermediates (C. Dudenhoeffer, G. Rohaly, K. Will, W. Deppert, and L. Wiesmueller, Mol. Cell. Biol. 18:5332-5342), we asked whether such substrates might be degraded by the p53 exonuclease. Addition of Mg2+ ions to the binding assay indeed started the p53 exonuclease and promoted rapid degradation of the bound, but not of the unbound, substrate, indicating that specifically recognized targets can be subjected to exonucleolytic degradation by p53 under defined conditions.
在本研究中，我们进一步对野生型p53固有的3'-5'核酸外切酶活性进行了表征。我们发现，这种活性与序列特异性DNA结合一样，是由p53核心结构域介导的。p53分子C末端30个氨基酸的截短使p53核酸外切酶活性增强了至少10倍，这表明这种活性与序列特异性DNA结合一样，受到p53 C末端碱性调节结构域的负调控。然而，激活p53序列特异性DNA结合的处理，如识别p53 C末端表位的单克隆抗体PAb421的结合，或更高的磷酸化状态，会强烈抑制p53核酸外切酶活性。这表明，至少对于全长p53而言，序列特异性DNA结合和核酸外切酶活性受到不同且看似相反的调节机制的影响。继我们实验室最近发现p53能以高亲和力识别并结合模拟早期重组中间体的三链DNA底物（C. Dudenhoeffer、G. Rohaly、K. Will、W. Deppert和L. Wiesmueller，《分子细胞生物学》18:5332 - 5342）之后，我们询问这种底物是否会被p53核酸外切酶降解。在结合测定中添加Mg2+离子确实启动了p53核酸外切酶，并促进了结合底物而非未结合底物的快速降解，这表明在特定条件下，p53能够对特异性识别的靶标进行核酸外切酶降解。
