Mutational /Functional Analysis of p53 Tumor Suppressor

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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.