GENOMIC INSTABILITY

基因组不稳定

• 批准号: 6097869
• 负责人: VILHELM A. BOHR
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6097869
• 关键词: DNA damage; DNA repair; DNA replication; aging; gene expression; gene interaction; genome; guanine nucleotide binding protein; human tissue; neoplasm /cancer genetics; oncoprotein p21; telomere; tissue /cell culture; tumor suppressor genes

Summary of work The protein p21 (Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin dependent kinases (CDKs) and cyclins. P21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA) which is an auxiliary factor for polymerase delta;. In addition to its role on replication, PCNA is clearly implicated in the repair resynthesis step of nucleotide excision repair (NER). Since PCNA particpates in both DNA repair and replication, it has been speculated that p21 might also regulate NER through its interaction with PCNA. Previous studies on the role of p21 on NER have yielded contradictory results that make it difficult to reach a general consensus with regard to the precise role of p21 in NER. In this study, 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 or the PCNA-binding domain of the protein. In the in vitro studies DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by ultraviolet (UV) irradiation. A permeabilized cell system and electroporation of intact cells were utilized for in vivo studies. The results show that the C-terminal domain of the p21 protein, which binds to PCNA, inhibits NER both in vitro and in vivo. A 50% percent inhibition of in vitro NER occurred at a ratio of 50:1 p21 C-terminus to PCNA monomer. Our results suggest that the inhibition occurs at the resynthesis step of the repair process. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA since it is relieved by addition of purified PCNA protein.

蛋白质p21（Cip 1，Waf 1， Sdi 1）是细胞周期蛋白依赖性激酶（CDK）的有效抑制剂， 细胞周期蛋白P21还可以通过与DNA的相互作用来阻断DNA的复制。 增殖细胞核抗原（PCNA）是一种 聚合酶δ辅助因子。除了其作用外， 在复制过程中，PCNA明显参与修复再合成步骤 核苷酸切除修复（NER）由于PCNA参与了 DNA修复和复制，推测p21 也可能通过与PCNA的相互作用调节NER。 先前关于p21在NER中的作用的研究已经产生了 矛盾的结果，使其难以达到普遍的 关于p21在NER中的确切作用的共识。在这 在这项研究中，我们研究了p21对NER的影响， 以及在体内使用纯化的p21片段， 蛋白质的CDK结合结构域或PCNA结合结构域。 在体外研究中，DNA修复合成被测量， 从正常人成纤维细胞中提取， 紫外线（UV）照射。透化细胞系统， 完整细胞的电穿孔用于体内研究。的 结果表明，p21蛋白的C-末端结构域， 结合PCNA，在体外和体内抑制NER。A 50% 体外NER的抑制百分比为50：1 p21 C-末端至PCNA单体。我们的研究结果表明， 抑制发生在修复过程的再合成步骤。我们 进一步证明DNA修复的抑制是介导的， 通过p21与PCNA的结合，因为它通过添加 纯化的PCNA蛋白。