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.

p21蛋白（Cip1, Waf1, Sdi1）是细胞周期蛋白依赖性激酶（CDKs）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）的相互作用来阻断DNA复制，PCNA是聚合酶δ的辅助因子；除了在复制过程中发挥作用外，PCNA显然还参与了核苷酸切除修复（NER）的修复再合成步骤。由于PCNA参与DNA修复和复制，推测p21也可能通过与PCNA的相互作用调控NER。以往关于p21在NER中的作用的研究得出了相互矛盾的结果，使得p21在NER中的确切作用难以达成普遍共识。我们利用含有cdk结合域或pna结合域的p21纯化片段，在体外和体内研究了p21对NER的影响。结果表明，p21蛋白的c端结构域与PCNA结合，在体外和体内均抑制NER。当p21 c端与PCNA单体的比例为50:1时，对体外NER的抑制率为50%。这是p21的c端特有的功能，因为该蛋白的n端对核苷酸切除修复没有影响。我们的结果表明，抑制发生在修复过程的再合成步骤。我们进一步证明DNA修复的抑制是通过p21与PCNA的结合介导的，因为添加纯化的PCNA蛋白可以缓解这种抑制。我们得出结论，p21对DNA修复的影响发生在远高于其对DNA复制的影响的浓度。P21可能是这两个过程的细胞内调节因子，我们目前正在探索P21是否也参与另一个主要的DNA修复途径——碱基切除修复（BER）。PCNA参与长贴片BER修复，我们预计p21也会影响这一途径。此外，正在研究p21是否也影响线粒体中的DNA修复，其中碱基切除也起作用。