DNA Repair and Antitopoisomerase Drug Effects

描述(由申请人提供)：以拓扑异构酶II为靶点的药物对多种人类肿瘤有效。稳定共价复合体，将拓扑异构酶II转化为DNA损伤，是这些药物杀死细胞的一个重要方面。我们利用最新开发的酵母基因组工具来鉴定编码DNA修复或DNA损伤耐受功能的基因，这些功能在针对拓扑异构酶的药物敏感性中发挥关键作用。这些工具的成功利用，特别是一组删除所有非必要开放阅读框架的菌株的成功应用，要求我们开发新的方法来有效地提高酵母细胞对拓扑异构酶II靶向药物的敏感性。我们开发了几种策略来显著提高酵母细胞对拓扑异构酶靶向药物的敏感性，包括开发抑制药物外排基因表达的嵌合转录调节因子。最近利用酵母缺失集的发现表明，几个修复复合体的核酸酶活性在TOP2靶向剂作用下的细胞存活后是重要的。我们还表明，在DNA损伤后调节蛋白质稳定性的蛋白质在修复TOP2介导的DNA损伤中也发挥着关键作用。在下一个资助期间，我们将使用这些工具来识别在接触TOP2靶向药物后影响细胞存活的酵母基因，但不会影响对其他类型DNA损伤的敏感性。实验将阐明处理拓扑异构酶产生的DNA损伤的蛋白质的生化作用。这些蛋白质包括调节RNA聚合酶对DNA损伤反应的稳定性的因子，以及针对DNA结构改变的核酸酶。这些关于DNA修复功能对拓扑异构酶靶向药物敏感性影响的研究，其长期目标是了解影响临床重要药物如依托泊苷和阿霉素疗效的因素。这项建议中的实验结果应该会加深我们对这些药物针对DNA拓扑异构酶的作用机制的理解。这项工作将解决的一个关键问题是，为什么针对不同拓扑异构酶的药物对细胞存活有不同的影响，从而产生不同的临床疗效。回答这些问题也可能为规避这些临床上重要的抗癌药物的耐药性提供策略。

DESCRIPTION (provided by applicant): Agents targeting topoisomerase II are active against a wide range of human tumors. Stabilization of covalent complexes, converting topoisomerase II into DNA damage, is an essential aspect of cell killing by these drugs. We have taken advantage of newly developed yeast genomic tools to identify genes encoding DNA repair or DNA damage tolerance functions that play key roles in sensitivity to drugs targeting topoisomerases. Successful utilization of these tools, notably a set of strains deleting all non-essential open reading frames has required us to develop new approaches to efficiently enhance the sensitivity of yeast cells to drugs targeting topoisomerase II. We developed several strategies for dominantly increasing the sensitivity of yeast cells to topoisomerase targeting drugs, including the development of chimeric transcriptional regulators that repress the expression of drug efflux genes. Recent findings that have taken advantage of the yeast deletion set have demonstrated that the nuclease activities of several repair complexes are important following cell survival following exposure to Top2 targeting agents. We have also shown that proteins that regulate protein stability following exposure to DNA damage also play key roles in repairing Top2 mediated DNA damage. During the next grant period we will use these tools to identify yeast genes that affect cell survival following exposure to Top2 targeting drugs, but do not affect sensitivity to other types of DNA damage. Experiments will elucidate the biochemical roles of proteins that process the DNA damage generated by topoisomerases. These proteins include factors that regulate the stability of RNA polymerase in response to DNA damage and nucleases that are specific for altered DNA structures. These studies of the role of DNA repair functions on sensitivity to topoisomerase targeting agents have the long-term goal of understanding factors that contribute to the efficacy of clinically important agents such as etoposide and doxorubicin. The results from experiments in this proposal should enhance our understanding of the mechanisms of action of these drugs targeting DNA topoisomerases. A key question that this work will address is why drugs targeting different topoisomerases have different effects on cell survival, and therefore different clinical efficacies. Answering these questions may also suggest strategies for circumventing resistance to these clinically important anticancer drugs.