Mutational /Functional Analysis of p53 Tumor Suppressor

真核生物基因组不断面临外源和内源诱变剂的破坏威胁。因此，哺乳动物细胞进化出一个复杂的防御网络来维持基因组的稳定，例如细胞周期检查点、DNA修复和细胞凋亡。这些细胞过程中的缺陷可导致与肿瘤发生相关的突变子表型，例如许多家族性癌症易患疾病，包括色素性干皮病、Bloom综合征、Rothmund-Thomson综合征、共济失调毛细血管扩张症、Werner综合征和Li-Fraumeni综合征。P53位于这些途径的十字路口，为P53成为人类癌症体细胞突变的主要靶点提供了生物学基础。我们正在研究与这些途径相关的分子机制。考虑到P53是在1979年被发现的，它是一种与SV-40病毒大T结合的细胞蛋白，是一种3‘到5’的DNA解旋酶，我们假设P53在物理和功能上都与细胞解旋酶相互作用。例如，我们在20世纪90年代中期的S首先发现，P53通过与两个DNA解旋酶XPB和XPD相互作用，与基础转录和核苷酸切除修复复合体TFIIH结合，并且P53失活的细胞的DNA修复活性降低。利用遗传学方法，我们还发现XPB和XPD参与了P53介导的细胞凋亡。这些数据表明，P53可能通过与TFIIH相关的DNA解旋酶结合并调节其活性来调节DNA修复和细胞凋亡。 目前，我们正在研究P53与其他DNA解旋酶(WRN、BLM和RTS)以及参与DNA重组的蛋白质(RAD51、RAD54和MUS81)之间的物理和功能相互作用。WRN或RTS的胚系突变在患有早衰和癌症易感综合征的患者中发现，这些综合征被称为Werner综合征和Rothmund-Thomson综合征。虽然Bloom综合征不被认为是一种过早衰老的情况，但生殖系BLM突变使个人容易患癌症，包括结肠癌。我们与Steve Gruber和Nathan Ellis合作，发现BLM基因的单一缺失也容易患结肠癌。由于BLm和WRN可能通过促进依赖三磷酸腺苷的Holliday连接(HJ)的易位来抑制不适当的同源重组修复，我们首次证明了P53在体外调节BLm和WRN螺旋酶破坏合成X连接的能力，在体内，模型HJ与BLm、RAD51和RAD54在细胞核内与BLM、RAD51和RAD54共定位和共沉淀，位于停滞的DNA复制分叉和链断裂的位置，在S早期，通过姐妹染色单体交换测量，并调节同源重组。这些数据表明，BLM将P53转运到停滞的DNA复制叉的这些位置，这些蛋白质与DNA修复和重组蛋白的复合体合作来解决Holliday连接和修复DNA双链断裂。 我们还在研究细胞蛋白对BLM和WRN解旋酶活性的调节。例如，激活的野生型P53比248WP53和273HP53突变体更有效地抑制BLM和WRN的解旋酶活性。激活的P53对解旋酶的调节主要是通过其去磷酸化的C末端与BLM或WRN结合来实现的。通过Pab421(p53羧基末端特异性抗体)的结合或蛋白激酶C对丝氨酸376和378的磷酸化，P53的C末端修饰可降低P53对BLM或WRN解旋酶活性的抑制。相比之下，错配修复蛋白hMSH2和hMSH6可以增强WRN和BLM解旋酶的活性，并推翻P53对它们的抑制。这些结果和其他结果为P53介导的DNA重组和修复的调节提供了一条分子途径和生理机制。这些和其他以前发表的数据进一步支持了这样的假设，即p53可以通过调节特定的RecQ DNA解旋酶来诱导细胞凋亡，并可能对这些基因组不稳定疾病中观察到的癌症易感性有一定的影响。

The eukaryote genome is constantly facing the threat of damage from exogenous and endogenous mutagens. Mammalian cells, therefore, have evolved an intricate network of defenses to maintain genomic stability, e.g., cell cycle checkpoints, DNA repair, and apoptosis. Defects in these cellular processes can result in a mutator phenotype associated with tumorigenesis, as exemplified by a number of familial cancer-prone disorders, including xeroderma pigmentosum, Bloom syndrome, Rothmund-Thomson Syndrome, ataxia telangiectasia, Werner syndrome, and Li-Fraumeni syndrome. p53 is at the crossroads of these pathways, and provides a biological basis for p53 being a prime target of somatic mutations in human cancers. We are investigating the molecular mechanisms related to these pathways. Remembering that p53 was discovered in 1979 as a cellular protein binding to SV-40 viral large T, a 3' to 5' DNA helicase, we hypothesized that p53 would physically and functionally interact with cellular helicases. For example, we were the first to discover in the mid-1990's that p53 binds to the basal transcription and nucleotide excision repair complex, TFIIH, through interaction with two DNA helicases, XPB and XPD, and cells with p53 inactivation have a reduced DNA repair activity. Using a genetic approach, we also showed that XPB and XPD contribute to p53-mediated apoptosis. These data indicate that p53 may modulate DNA repair and apoptosis by binding to and regulating the activity of the TFIIH-associated DNA helicases. Currently, we are investigating the physical and functional interactions between p53 and other DNA helicases (WRN, BLM, and RTS) and proteins involved in DNA recombination (RAD51, RAD54, and MUS81). Germline mutations in either WRN or RTS are found in patients with the premature aging and cancer susceptibility syndromes, known as the Werner Syndrome and the Rothmund-Thomson Syndrome. Although Bloom Syndrome is not considered a premature aging condition, germline BLM mutations predispose individuals to cancer including colon cancer. We have collaborated with Steve Gruber and Nathan Ellis, and discovered that haplosufficiency of BLM also predisposes individuals to colon cancer. Because BLM and WRN may be involved in suppressing inappropriate homologous recombinational repair by promoting ATP-dependent translocation of Holliday junctions (HJ), we have shown for the first time that p53 modulates, in vitro, the ability of the BLM and WRN helicases to disrupt synthetic X-junctions and that model HJ, colocalizes and co-immunoprecipitates, in vivo, with BLM, RAD51, and RAD54 in nuclear foci at sites of stalled DNA replication forks and strand breaks of cells arrested by hydroxyurea in early S-phase, and regulates homologous recombination, as measured by sister chromatid exchanges. These data indicate that BLM transports p53 to these sites of stalled DNA replication forks and these proteins cooperate with a complex of DNA repair and recombinogenic proteins to resolve Holliday junctions and to repair DNA double-strand breaks. We are also investigating the regulation of BLM and WRN helicase activities by cellular proteins. For example, activated wild-type p53 inhibits the helicase activities of BLM and WRN more efficiently than do the 248Wp53 and 273Hp53 mutants. The helicase modulation by activated p53 is primarily through its dephosphorylated C-terminus binding to either BLM or WRN. p53-mediated inhibition of either BLM or WRN helicase activity is reduced by modifications of the C-terminus of p53, through either the binding of Pab421, a p53 carboxyl terminal domain-specific antibody, or the phosphorylation of serines 376 and 378 by protein kinase C. In contrast, hMSH2 and hMSH6, mismatch repair proteins, enhance WRN and BLM helicase activities and override their suppression by p53. These and other results provide a molecular pathway and a physiological mechanism for p53-mediated regulation of DNA recombination and repair. These and other previously published data further support the hypothesis that p53 can induce apoptosis through the modulation of specific RecQ DNA helicases and may have implications for the cancer predisposition observed in these genomic instability diseases.