Li R, Hannon G J, Beach D, Stillman B
Cold Spring Harbor Laboratory, New York 11724, USA.
Curr Biol. 1996 Feb 1;6(2):189-99. doi: 10.1016/s0960-9822(02)00452-9.
The p21 protein binds to both cyclin-dependent kinases (Cdks) and the proliferating cell nuclear antigen (PCNA). In mammalian cells, DNA damage results in an increase in the level of p53 protein, which stimulates expression of the gene encoding p21, which in turn leads to an inhibition of Cdk activity. Biochemical studies have shown that the direct interaction between p21 and PCNA blocks the latter's function in DNA replication but not in DNA repair. In addition to the p53-dependent damage response, the stimulation of quiescent cells with serum can also cause a p53-independent elevation in p21 gene expression. It is not clear, however, whether the induction of p21 protein under these two circumstances serves the same purpose. In this study, we have investigated the kinetics of p21 induction by DNA damage and serum stimulation and the consequent effects on cell-cycle progression. Using both normal and repair-deficient human cells, we have also analyzed the nuclear distribution of p21 in relation to that of PCNA.
In vivo immunofluorescence staining experiments indicate that, following UV damage, DNA repair is not inhibited by the presence of a large amount of p21 protein in the nucleus; in contrast, cells undergoing DNA replication during S phase contain very low amounts of p21. The addition of serum induced a transitory elevation of p21 levels, whereas UV damage to cells resulted in a sustained, high level of p21 that was more tightly associated with the nuclear structure. Interestingly, cells deficient in global nucleotide excision-repair displayed a distinct pattern of detergent-insoluble p21 that co-localized with PCNA.
The in vivo studies presented here, which are consistent with our previous findings in vitro, indicate that p21 has a differential effect on DNA replication and DNA repair, and that the induction of p21 by serum and DNA damage may have different consequences. Furthermore, the co-localization of p21 and PCNA in the nucleus of normal and repair-deficient human cells indicates that p21 and PCNA interact during post-damage events.
p21蛋白可与细胞周期蛋白依赖性激酶（Cdks）和增殖细胞核抗原（PCNA）结合。在哺乳动物细胞中，DNA损伤会导致p53蛋白水平升高，p53蛋白会刺激编码p21的基因表达，进而导致Cdk活性受到抑制。生化研究表明，p21与PCNA之间的直接相互作用会阻断PCNA在DNA复制中的功能，但不会影响其在DNA修复中的功能。除了p53依赖的损伤反应外，用血清刺激静止细胞也会导致p21基因表达出现不依赖p53的升高。然而，尚不清楚在这两种情况下p21蛋白的诱导是否具有相同的目的。在本研究中，我们研究了DNA损伤和血清刺激诱导p21的动力学以及对细胞周期进程的后续影响。我们还使用正常和修复缺陷的人类细胞分析了p21与PCNA在细胞核中的分布关系。
体内免疫荧光染色实验表明，紫外线损伤后，细胞核中大量p21蛋白的存在不会抑制DNA修复；相反，在S期进行DNA复制的细胞中p21含量非常低。添加血清会导致p21水平短暂升高，而细胞受到紫外线损伤会导致p21持续高水平表达，且与核结构的关联更为紧密。有趣的是，全局核苷酸切除修复缺陷的细胞呈现出一种与PCNA共定位的、不溶于去污剂的p21独特模式。
此处呈现的体内研究结果与我们之前的体外研究结果一致，表明p21对DNA复制和DNA修复具有不同的影响，血清和DNA损伤诱导p21可能会产生不同的后果。此外，p21与PCNA在正常和修复缺陷人类细胞的细胞核中共定位，表明p21与PCNA在损伤后事件中相互作用。
