The C-terminal domain of p21 inhibits nucleotide excision repair in vitro and in vivo
The C-terminal domain of p21 inhibits nucleotide excision repair in vitro and in vivo
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DOI：
10.1091/mbc.10.7.2119
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发表时间：
1999-07-01
影响因子：
3.3
通讯作者：
Bohr, VA
中科院分区：
文献类型：
--
作者：
Cooper, MP;Balajee, AS;Bohr, VA
蛋白质p21（Cip 1，）（Waf 1，）（Sdi 1）是细胞周期蛋白依赖性激酶（CDKs）的有效抑制剂。p21还可以通过其与增殖细胞核抗原（PCNA）的相互作用来阻断DNA复制，增殖细胞核抗原是聚合酶δ的辅助因子。PCNA还涉及核苷酸切除修复（NER）的修复再合成步骤。以前的研究已经产生了矛盾的结果，是否p21调节NER通过其与PCNA的相互作用。解决这一争议是有趣的，因为它将有助于了解DNA修复和复制是如何调节的。因此，我们已经研究了NER的p21在体外和体内使用纯化的片段的p21含有CDK结合结构域（N端）或PCNA结合结构域（C端）的蛋白质的影响。在体外研究中，使用紫外线照射损伤的质粒在正常人成纤维细胞提取物中测定DNA修复合成。在体内研究中，我们使用完整的和透化的细胞。结果表明，p21蛋白的C端在体外和体内均抑制NER。这些是第一个在体内研究中，这个问题已经被检查，我们证明，抑制NER的p21不仅仅是一个人工的体外效应。当p21 C-末端片段与PCNA单体的摩尔比为50：1时，对体外NER的抑制率为50%。p21差异调节DNA修复和复制，修复比复制对抑制的敏感性低得多。我们的体内研究结果表明，抑制发生在修复过程的再合成步骤。它也出现在修复位点的PCNA的预组装减轻p21的抑制作用。我们进一步证明，抑制DNA修复介导的p21与PCNA的结合。p21的N端对DNA修复无影响，而p21的C端对DNA修复的抑制作用可被纯化的PCNA蛋白所解除。
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.