Characterizing the contribution of transcription-associated DNA-topoisomerase adducts to mutagenesis in cancer

摘要 突变是癌症的根本原因，有助于转移和抵抗癌症 治疗学在人类细胞中，干扰转录和诱导DNA-拓扑异构酶加合物都是 会导致DNA损伤然而，产生转录和DNA拓扑异构酶的机制， 人类细胞中与内收物相关的突变，由这些过程引起的突变类型，以及它们的 对癌症突变的贡献知之甚少。在人类细胞中使用新的突变报告基因， 观察到由2-至5-碱基对（bp）缺失和明显的较大缺失主导的突变谱。这 突变谱反映了DNA拓扑异构酶1依赖的，转录相关的诱变， S.这表明转录诱导的DNA-拓扑异构酶加合物在酿酒酵母中产生这些突变。 人体细胞我们还发现，2- 5-bp的缺失富集在高表达基因中， 原发性乳腺癌，表明DNA拓扑异构酶加合物是癌症病因学的重要贡献者 在所有肿瘤抑制基因失活突变中，≥ 2 bp的缺失占3%-12%。 这项提议的目标是描述转录相关的DNA的基本决定因素， 拓扑异构酶加合物诱导的人细胞诱变，并评估其对 癌Aim 1将确定转录相关的DNA-拓扑异构酶加合物的机制基础， 在人类细胞中诱导诱变，并定义由该过程产生的突变谱。这将 通过利用突变报告子测量突变率和谱来实现， 通过利用CRISPRi和CRISPRa调节转录和/或增加DNA-拓扑异构酶加合物， 拓扑异构酶变体和抑制剂。此外，我们将确定各种末端连接DNA的贡献 双链断裂修复途径和R环解析，以促进或限制特定类型的DNA- 拓扑异构酶加合物诱导的突变。目标2将确定两种DNA的全基因组分布- 使用新的基因组学方法和DNA-TOP 1加合物在单核苷酸分辨率下的TOP 1加合物- 通过人类细胞的全基因组测序的依赖性突变。这些不带偏见的、互补的 加合物和突变的分布将与基因组特征如转录本的位置进行比较 和R环，以评估它们对DNA-TOP 1加合物诱导的 突变。这些加合物和突变的分布将进一步与DNA分布进行比较- 在测序的人类肿瘤中TOP1加合物标记突变以估计DNA-TOP1的贡献 加合物在癌症中致突变。这些目标的完成将提供深入了解DNA的作用， 拓扑异构酶加合物在癌症病因学中的作用，决定了产生转录的主要机制- 相关的突变，并创建实验系统，这将有助于未来的研究，DNA的影响， 结构、遗传决定因素和环境暴露对这一未充分研究的致突变途径的影响。

Abstract Mutations are the underlying cause of cancer and contribute to metastasis and resistance to cancer therapeutics. In human cells, both perturbing transcription and inducing DNA-topoisomerase adducts are known to cause DNA damage. However, the mechanisms generating transcription- and DNA-topoisomerase adduct-associated mutations in human cells, the types of mutations caused by these processes, and their contribution to mutations in cancer are poorly understood. Using a novel mutation reporter in human cells, we observed a mutation spectrum dominated by 2- to 5-base pair (bp) deletions and distinct larger deletions. This mutation spectrum mirrors that of DNA topoisomerase 1-dependent, transcription-associated mutagenesis in S. cerevisiae, which indicates transcription-induced DNA-topoisomerase adducts produce these mutations in human cells. We additionally found that 2- to 5-bp deletions are enriched within highly expressed genes in primary breast cancers, suggesting DNA-topoisomerase adducts are important contributors to cancer etiology as deletions of ≥ 2 bp constitute between 3% and 12% of all inactivating mutations in tumor suppressor genes. The goal of this proposal is to characterize the basic determinants of transcription-associated, DNA- topoisomerase adduct-induced mutagenesis in human cells and assess its contribution to mutagenesis in cancer. Aim1 will determine the mechanistic basis of transcription-associated, DNA-topoisomerase adduct- induced mutagenesis in human cells and define the spectrum of mutations generated by this process. This will be accomplished by measuring mutation rates and spectra utilizing mutation reporters for which we will modulate transcription via CRISPRi and CRISPRa and/or increase DNA-topoisomerase adducts by utilizing topoisomerase variants and inhibitors. Also, we will determine the contribution of various end joining DNA double strand break repair pathways and R-loop resolution to promoting or limiting specific types of DNA- topoisomerase adduct-induced mutations. Aim 2 will determine the genome-wide distribution of both DNA- TOP1 adducts at single nucleotide resolution using a novel genomics approach and DNA-TOP1 adduct- dependent mutations via whole genome sequencing of human cells. These unbiased, complementary distributions of adducts and mutations will be compared to the location of genomic features such as transcripts and R-loops to assess their influence on the occurrence and spectrum of DNA-TOP1 adduct-induced mutations. The distributions of these adducts and mutations will be further compared to the distribution DNA- TOP1 adduct signature mutations in sequenced human tumors to estimate the contribution of DNA-TOP1 adducts to mutagenesis in cancer. Completion of these aims will provide insight into the roles DNA- topoisomerase adducts in cancer etiology, determine the primary mechanism(s) that generate transcription- associated mutations, and create experimental systems that will facilitate future studies on the effects that DNA structures, genetic determinates, and environmental exposures have on this understudied mutagenic pathway.