Novel Mechanisms by which RAD18 and POLZ affect Response to Anticancer Agents

描述（由申请人提供）：通过直接交联或捕获拓扑异构酶I （TOP1）复合物靶向DNA的药物是一些最有效的癌症治疗方法。更全面地了解影响癌细胞如何应对这些病变的分子途径，可能会为药物开发确定新的靶点，从而通过联合化疗提高治疗效果。E3泛素连接酶Rad18在增殖后修复（PPR）中起着至关重要的作用，通过单泛素化PCNA来响应停止的复制分叉。该事件通过招募一组特殊的DNA聚合酶，通过称为转化DNA合成（TLS）的过程复制含有DNA病变的DNA模板，从而促进病变旁路。TLS被认为对顺铂加合物的复制旁路很重要，可能是肿瘤细胞抵抗治疗的重要机制。为了进一步探讨TLS如何影响化疗，我们使用siRNA来消耗人类细胞中的Rad18、Rev3 （Pol ?的催化亚基）或另外两种TLS聚合酶Rev1和DNA聚合酶Eta，这四种酶都被认为参与了含有顺铂加合物的DNA的复制。我们发现所有四种基因产物都是防止顺铂暴露后复制叉停滞所必需的，这与病变旁路是通过PCNA单泛素化和多个TLS聚合酶的协调来完成的模型一致。出乎意料的是，我们发现Rad18和rev3缺失的细胞对顺铂过敏，这表明它们在顺铂耐受中发挥了额外的作用。我们还观察到，当细胞暴露于电离辐射、喜树碱、丝裂霉素C或顺铂时，Rad18与DNA双链断裂（DSB）共定位，并且Rev3的消耗导致细胞表型与DSB修复缺陷一致。我们假设Rad18和Pol 6除了执行PPR之外还具有其他功能。我们认为这些功能对于分解复制相关的dsb很重要，这是icl修复过程中的一种常见中间体，也是由TOP1抑制剂引起的复制相关dsb。为了解决这一假设，我们提出以下三个具体目标：1。确定是Rad18还是Pol ？当细胞受到诱导复制相关的dsb的攻击时，促进细胞存活并防止染色体畸变的积累。2. 确定是Rad18还是Pol ？Rad18或Pol 6是否促进喜树碱和icl诱导剂触发的HR，可以通过测量细胞内HR活性和在染色体水平上药物诱导的姐妹染色单体交换频率来实现。3. 通过检查定位是否依赖于DNA复制以及PCNA泛素化是否与此事件相关，确定Rad18定位到dsb位点的功能意义。我们还将描述Rad18中定位所必需的功能域，以及这些域是否对细胞抵抗复制相关的dsb和指导DNA修复很重要。公共卫生相关性：靶向拓扑异构酶I并引入链间DNA交联的药物是一些用于治疗癌症的最广泛使用的药物。我们假设Rad18和DNA聚合酶Zeta两种酶对于促进DNA双链断裂的修复是重要的，这与这些药物的治疗效果有关。这里提出的研究将促进我们对DNA修复活动的理解，这些修复活动调节癌细胞对这些抗癌药物的敏感性，并为细胞如何防止基因组不稳定（癌症的常见标志）提供新的见解。

DESCRIPTION (provided by applicant): Drugs which target DNA through direct crosslinking or trapping of topoisomerase I (TOP1) complexes are some of the most effective treatments for cancer. A more complete understanding of the molecular pathways that influence how cancer cells cope with these lesions may identify new targets for drug development with the goal of increasing therapeutic effectiveness through combination chemotherapy. The E3 ubiquitin ligase Rad18 plays a crucial role in postreplication repair (PPR) by monoubiquitinating PCNA in response to stalled replication forks. This event promotes lesion bypass by recruiting a group of specialized DNA polymerases that copy DNA templates containing DNA lesions through a process termed Translesion DNA synthesis (TLS). TLS is believed to be important for replicative bypass of cisplatin adducts and may be an important mechanism by which tumor cells resist therapy. To further probe into how TLS may impact chemotherapy, we used siRNA to deplete human cells of Rad18, Rev3 (the catalytic subunit of Pol ?), or two additional TLS polymerases, Rev1 and DNA Polymerase Eta, all four believed to be involved in replicating DNA containing cisplatin adducts. We found that all four gene products were necessary to prevent replication fork stalling following exposure to cisplatin consistent with a model that lesion bypass is accomplished by PCNA monoubiquitination and coordination of multiple TLS polymerases. Unexpectedly, we found that Rad18 and Rev3-depleted cells are hypersensitive to cisplatin suggesting they play additional roles in tolerance to cisplatin. We also observed that Rad18 colocalizes with DNA double strand breaks (DSBs) when cells are exposed to ionizing radiation, camptothecin, Mitomycin C, or cisplatin and that depletion of Rev3 leads to cellular phenotypes consistent with cells deficient in DSB repair. We hypothesize that Rad18 and Pol 6 possess alternative functions in addition to performing PPR. We believe these functions are important for resolution of replication-associated DSBs, a common intermediate during repair of ICLs and replication associated DSBs caused by TOP1 inhibitors. To address this hypothesis, we propose the following three specific aims: 1. Determine whether Rad18 or Pol ? promotes cell survival and prevents accumulation of chromosomal aberrations when cells are challenged with agents that induce replication-associated DSBs. 2. Determine whether Rad18 or Pol ? is necessary for the efficient repair of replication-associated DSB and whether Rad18 or Pol 6 facilitates HR triggered by camptothecin and ICL-inducing agents by measuring HR activity in cells and frequencies of drug-induced sister chromatid exchanges at the chromosomal level. 3. Determine the functional significance of localization of Rad18 to sites of DSBs by examining whether localization is dependent upon DNA replication and whether PCNA ubiquitination is associated with this event. We will also characterize the functional domains within Rad18 that are necessary for localization and whether those domains are important for cellular resistance to replication-associated DSBs and directing DNA repair. PUBLIC HEALTH RELEVANCE: Agents which target Topoisomerase I and introduce interstrand DNA crosslinks are some of the most widely used drugs used to treat cancer. We hypothesize that two enzymes, Rad18 and DNA Polymerase Zeta, are important for facilitating repair of DNA double stranded breaks associated with the therapeutic effectiveness of these agents. The studies proposed here will advance our understanding of DNA repair activities that modulate the sensitivity of cancer cells to these anticancer agents and provide new insight into how cells prevent genomic instability, a common hallmark of cancer.