Cooper M P, Balajee A S, Bohr V A
Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224-6823, USA.
Mol Biol Cell. 1999 Jul;10(7):2119-29. doi: 10.1091/mbc.10.7.2119.
The protein p21(Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase delta. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.
蛋白质p21（Cip1、Waf1、Sdi1）是细胞周期蛋白依赖性激酶（CDK）的一种强效抑制剂。p21还可通过与增殖细胞核抗原（PCNA）相互作用来阻断DNA复制，PCNA是聚合酶δ的辅助因子。PCNA也参与核苷酸切除修复（NER）的修复再合成步骤。先前关于p21是否通过与PCNA相互作用来调节NER的研究得出了相互矛盾的结果。解决这一争议很有意义，因为这将有助于理解DNA修复和复制是如何被调控的。因此，我们使用含有该蛋白质CDK结合结构域（N端）或PCNA结合结构域（C端）的p21纯化片段，在体外和体内研究了p21对NER的影响。在体外研究中，使用经紫外线照射损伤的质粒，在正常人成纤维细胞提取物中测量DNA修复合成。在体内研究中，我们使用了完整的和通透的细胞。结果表明，p21蛋白的C端在体外和体内均抑制NER。这些是首次对此问题进行研究的体内研究，我们证明p21对NER的抑制不仅仅是一种人为的体外效应。当p21 C端片段与PCNA单体的摩尔比为50:1时，体外NER受到50%的抑制。p21对DNA修复和复制的调节存在差异，修复对抑制的敏感性远低于复制。我们的体内结果表明，抑制发生在修复过程的再合成步骤。似乎PCNA在修复位点的预组装减轻了p21的抑制作用。我们进一步证明，DNA修复的抑制是通过p21与PCNA的结合介导的。p21的N端对DNA修复没有影响，添加纯化的PCNA蛋白可缓解p21 C端对DNA修复的抑制作用。
