DNA Repair and Antitopoisomerase Drug Effects

描述（由申请人提供）：靶向拓扑异构酶II的试剂对多种人类肿瘤具有活性。共价复合物的稳定化，将拓扑异构酶II转化为DNA损伤，是这些药物杀死细胞的重要方面。我们已经利用新开发的酵母基因组工具来识别编码DNA修复或DNA损伤耐受功能的基因，这些基因在靶向拓扑异构酶的药物敏感性中发挥关键作用。成功利用这些工具，特别是一组删除所有非必需开放阅读框架的菌株，需要我们开发新的方法来有效地提高酵母细胞对靶向拓扑异构酶II的药物的敏感性。我们开发了几种策略，占主导地位的增加酵母细胞的拓扑异构酶靶向药物的敏感性，包括开发嵌合转录调控抑制药物外排基因的表达。利用酵母缺失集的最新发现已经证明，在暴露于Top2靶向剂后的细胞存活后，几种修复复合物的核酸酶活性是重要的。我们还表明，在暴露于DNA损伤后调节蛋白质稳定性的蛋白质在修复Top2介导的DNA损伤中也起着关键作用。在下一个资助期间，我们将使用这些工具来鉴定酵母基因，这些基因在暴露于Top2靶向药物后影响细胞存活，但不影响对其他类型DNA损伤的敏感性。实验将阐明蛋白质的生化作用，处理由拓扑异构酶产生的DNA损伤。这些蛋白质包括调节RNA聚合酶响应DNA损伤的稳定性的因子和对改变的DNA结构特异的核酸酶。DNA修复功能对拓扑异构酶靶向药物敏感性的作用的这些研究的长期目标是了解有助于临床重要药物（如依托泊苷和多柔比星）疗效的因素。实验结果表明，这些药物对DNA拓扑异构酶的作用机制有一定的认识。这项工作将解决的一个关键问题是为什么靶向不同拓扑异构酶的药物对细胞存活有不同的影响，因此具有不同的临床疗效。探讨这些问题也可能提出规避这些临床上重要的抗癌药物耐药性的策略。

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.