GENOMIC INSTABILITY

基因组不稳定

• 批准号: 6431454
• 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/6431454
• 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. 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. 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. This is a specific function of the C-terminal end of p21 since the N-terminal of the protein had no effect on nucleotide excision repair. 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. We conclude that the effect of p21 on DNA repair occurs at much higher concentrations than its effect on DNA replication. P21 might serve as an intracellular regulator of these two processesWe are currently exploring if P21 also participates in another major DNA repair pathway, that of Base excision repair (BER). PCNA is involved in the long patch BER repair and we expect that p21 will influence that pathway as well. Further, it is being examined whether p21 also affects the DNA repair in mitochondria, where base excision also operates.

工作摘要蛋白p21（CIP1，WAF1，SDI1）是细胞周期蛋白依赖激酶（CDK）和细胞周期蛋白的有效抑制剂。 p21还可以通过与增殖细胞核抗原（PCNA）相互作用来阻止DNA复制，这是聚合酶三角洲的辅助因子； 除了其在复制方面的作用外，PCNA还与核苷酸切除修复（NER）的修复重合步骤明显有关。 由于DNA修复和复制中的PCNA颗粒物，因此据推测P21也可能通过与PCNA的相互作用来调节NER。先前关于p21对NER的作用的研究产生了矛盾的结果，使得在NER中P21的确切作用方面很难达成共识。我们使用含有p21的纯化片段，其中含有CDK结合结构域或蛋白质的PCNA结合结构域，研究了p21对体外和体内NER的影响。 结果表明，与PCNA结合的p21蛋白的C末端结构域抑制了体外和体内的NER。 50％的体外抑制作用以50：1 p21 c-末端与PCNA单体的比率发生。这是p21的C末端的特定功能，因为该蛋白的N末端对核苷酸切除修复没有影响。 我们的结果表明，抑制作用发生在修复过程的重新合成步骤中。 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，因为通过添加纯化的PCNA蛋白可以缓解它。我们得出的结论是，p21对DNA修复的影响的浓度远高于其对DNA复制的影响。 P21可能是这两个过程的细胞内调节剂，我们目前正在探索P21是否还参与了另一个主要的DNA修复途径，即基础切除修复（BER）。 PCNA参与了长片ber修复，我们希望p21也会影响该途径。 此外，正在研究p21是否还影响线粒体的DNA修复，在该线粒体中也可以进行碱基切除。