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，并有助于转移和对癌症的耐药性 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 已知会造成DNA损伤。然而，转录和DNA拓扑异构酶的产生机制 人类细胞中的加合物相关突变，由这些过程引起的突变的类型，以及它们 对癌症突变的贡献还知之甚少。利用人类细胞中的一种新的突变报告，我们 观察到突变谱以2-5个碱基对(BP)缺失和明显的较大缺失为主。这 突变谱反映了DNA拓扑异构酶1依赖的、转录相关的突变 表明转录诱导的DNA-拓扑异构酶加合物在 人类细胞。我们还发现，2到5个碱基的缺失在高表达的基因中丰富。 原发乳腺癌，提示DNA-拓扑异构酶加合物是癌症病因的重要因素 由于≥、2BP的缺失，肿瘤抑制基因的所有失活突变占3%到12%。 这项建议的目标是表征转录相关的基本决定因素，DNA- 拓扑异构酶加合物诱变人细胞及其对细胞诱变作用的评价 癌症。AIM1将确定转录相关的DNA拓扑异构酶加合物的机制基础- 在人类细胞中诱导突变，并定义由这一过程产生的突变的谱。这将是 通过使用突变报告器测量突变率和谱来实现，我们将 通过CRISPRi和CRISPRA调节转录和/或增加DNA拓扑异构酶加合物 拓扑异构酶变异体和抑制剂。此外，我们还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。AIM 2将确定这两种DNA的全基因组分布- Top1利用一种新的基因组学方法在单核苷酸分辨率下加成，DNA-TOP1加成- 通过人类细胞全基因组测序的依赖突变。这些不偏不倚、相辅相成 加合物和突变的分布将与转录本等基因组特征的位置进行比较 和R-环，以评估它们对DNA-TOP1加合物的发生和光谱的影响 突变。这些加合物和突变的分布将进一步与分布的DNA- Top1在测序的人类肿瘤中加成签名突变以评估DNA-TOP1的作用 加合物在癌症中的诱变作用。完成这些目标将提供对DNA- 拓扑异构酶加合物在肿瘤病因学中的作用，确定产生转录的主要机制(S) 相关突变，并创建实验系统，以促进未来对DNA影响的研究 结构、遗传决定因素和环境暴露对这一未被充分研究的诱变途径有影响。

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.