A Novel Carcinogen-Induced Cell Cycle Checkpoint

描述（由申请人提供）：本提案的长期目标是阐明苯并甲芘（B[a]P）诱导的s期检查点的分子基础。B[a]P是一种含量丰富且普遍存在的环境致癌物，可在细胞内代谢生成苯并[a]芘二氢二醇环氧化物（BPDE）。bpde诱导的DMA损伤触发协调基因组复制和修复的s期检查点信号通路。然而，检查点信号调节DNA复制的分子机制尚不完全清楚。受损DNA的不准确修复和复制会导致基因组不稳定，这是癌细胞的一个特征。bpde诱导的s期检查点可能有助于维持B[a]P暴露后的基因组稳定性和预防癌症。该应用程序将研究DNA损伤激活s期检查点的机制，以及能够从检查点恢复的跨病变合成（TLS）介导的机制。我们的研究表明，Cdc45 （DNA复制因子）和DNA聚合酶Pol kappa （Pol?）分别参与了s期检查点的激活和恢复。我们也证明了Pol？由E3泛素连接酶Rad18调控。本研究的具体目的是：(1)验证Cdc45是bpde诱导的S期检查点靶点的假设。(2)阐明Rad18在bpde诱导的s期检查点恢复中的作用。(3)阐明Pol？在检查点恢复中相互作用的DNA聚合酶Rev1。Aims 1和2将识别和突变Cdc45泛素化（Aim 1）和Rad18磷酸化（Aim 2）的位点。然后，我们将研究通过检查点信号对泛素化和磷酸化抗性突变体的调节。这些实验将测试Cdc45泛素化和Rad18磷酸化与s期检查点的关系。在目标3中，我们将确定消融Rev1（它在物理和遗传上与Pol?相互作用）对Pol?的影响。监管。这些研究将确定Pol？和Rev1调控s期检查点。总之，这些实验将为DNA复制机制与DNA修复蛋白协调响应B[a]P（和）的机制提供一个新的范例。可能是其他基因毒素)来维持基因组的稳定性。我们的研究结果可能有助于识别环境B[a] p诱发疾病的高危人群。此外，我们的研究可以帮助确定癌症治疗的新药物靶点：与B[a]P相似，许多化疗具有基因毒性并激活检查点通路。我们已经证明了Pol？或Rad18-缺乏使细胞对B[a] p诱导的死亡敏感。潜在的，针对Rad18的小分子，Pol？或其他TLS酶可以使癌细胞对化疗药物的杀伤变得敏感。

DESCRIPTION (provided by applicant): The broad long-term objective of this proposal is to elucidate the molecular basis of the Benzofalpyrene (B[a]P)-induced S-phase checkpoint. B[a]P is an abundant and ubiquitous environmental carcinogen that is metabolized intracellularly to generate Benzo[a]pyrene Di-hydrodiol-Epoxide (BPDE). BPDE-induced DMA damage triggers S-phase checkpoint signaling pathways that coordinate replication and repair of the genome. However, the molecular mechanism(s) by which checkpoint signaling regulates DNA replication are incompletely understood. Inaccurate repair and replication of damaged DNA can result in genomic instability, a hallmark of cancer cells. The BPDE-induced S-phase checkpoint is likely to help maintain genomic stability and prevent cancer after B[a]P exposure. This application will investigate the mechanisms by which S-phase checkpoints are activated by DNA damage, and the Trans-Lesion Synthesis (TLS)-mediated mechanisms that enable recovery from the checkpoint. Our studies indicate that Cdc45 (a DNA replication factor) and DNA polymerase Pol kappa (Pol?) are involved in S-phase checkpoint activation and recovery respectively. We have also shown that Pol? is regulated by the E3 ubiquitin ligase Rad18. The Specific Aims of this proposal are: (1) To test the hypothesis that Cdc45 is a target of the BPDE-induced S- phase checkpoint. (2) To elucidate the role of Rad18 in recovery from the BPDE-induced S-phase checkpoint. (3) To elucidate the role of the Pol?-interacting DNA polymerase Rev1 in checkpoint recovery. Aims 1 and 2 will identify and mutate sites of Cdc45 ubiquitination (Aim 1) and Rad18 phosphorylation (Aim 2). Then we will study the regulation of ubiquitination and phosphorylation-resistant mutants by checkpoint signaling. These experiments will test the significance of Cdc45 ubiquitination and Rad18 phosphorylation in relation to the S-phase checkpoint. In Aim 3 we will determine the effect of ablating Rev1 (which interacts physically and genetically with Pol?) on Pol? regulation. These studies will determine how interactions between Pol? and Rev1 regulate the S-phase checkpoint. Together, these experiments will provide a novel paradigm for the mechanisms by which the DNA replication machinery is coordinated with DNA repair proteins in response to B[a]P (and .possibly other genotoxins) to maintain genomic stability. Results of our studies might help identify individuals that are at high-risk for environmental B[a]P-induced disease. Moreover, our studies could help identify novel drug targets for cancer therapy: Similar to B[a]P, many chemotherapies are genotoxic and activate checkpoint pathways. We have shown that Pol? or Rad18- deficiency sensitizes cells to B[a]P-induced death. Potentially, small molecules that target Rad18, Pol?, or other TLS enzymes could sensitize cancer cells to killing by chemotherapeutic agents.