GENOMIC INSTABILITY

工作概述：p21蛋白(Cip1、Waf1、Sdi1)是细胞周期蛋白依赖性蛋白激酶(CDKs)和细胞周期蛋白的有效抑制因子。P21还可通过与作为聚合酶增量辅助因子的增殖细胞核抗原相互作用来阻断DNA复制；除了在复制中的作用外，增殖细胞核抗原明显参与核苷酸切除修复(NER)的修复再合成步骤。由于增殖细胞核抗原既参与DNA修复，又参与DNA复制，推测p21也可能通过与增殖细胞核抗原的相互作用来调节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，这使得人们很难就p21在NER中的确切作用达成普遍共识。我们利用纯化的含有p21蛋白CDK结合域和增殖细胞核抗原结合域的p21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末端结构域与增殖细胞核抗原结合，在体外和体内都能抑制NER。当p21C末端与增殖细胞核抗原单体的比例为50：1时，NER的体外抑制率为50%。这是p21的C末端的一种特殊功能，因为蛋白质的N末端对核苷酸切除修复没有影响。我们的结果表明，这种抑制发生在修复过程的重新合成步骤。我们进一步证明，DNA修复的抑制是通过p21与增殖细胞核抗原的结合来实现的，因为加入纯化的增殖细胞核抗原蛋白可以缓解这种抑制作用。我们得出结论，p21对DNA修复的影响在比它对DNA复制的影响更高的浓度下发生。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是否也参与了另一个主要的DNA修复途径，即碱基切除修复(BER)。增殖细胞核抗原参与了长斑块的误码率修复，我们预计p21也会影响这一通路。此外，正在研究p21是否也影响线粒体的DNA修复，在线粒体中，碱基切除也起作用。

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.