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

表征转录相关 DNA 拓扑异构酶加合物对癌症诱变的贡献

基本信息

批准号：
10444838
负责人：
STEVEN A ROBERTS
金额：
$ 20.22万
依托单位：
WASHINGTON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10444838
关键词：
Base Pairing Bioinformatics CRISPR interference CRISPR-mediated transcriptional activation Camptothecin Cancer Etiology Cell Line Cells Clustered Regularly Interspaced Short Palindromic Repeats Conflict (Psychology)DNA DNA Adducts DNA Damage DNA Double Strand Break DNA Repair DNA Structure DNA Topoisomerases DNA biosynthesis DNA-PKcs Data Defect Development Direct Repeats Double Strand Break Repair Environmental Exposure Etiology Etoposide Foundations Frequencies Future Gene Deletion Generations Genes Genetic Genetic Transcription Genomic DNA Genomic approach Genomics Goals Human Induced Mutation Ligation Location Malignant Neoplasms Maps Measurement Measures Mediating Methods Mutagenesis Mutation Mutation Spectra Neoplasm Metastasis Nonhomologous DNA End Joining Nucleotides Outcome Pathologic Mutagenesis Pathway interactions Process Proteins Proto-Oncogenes Reporter Resistance Resolution Role Saccharomyces cerevisiae Source System TOP1 gene TOP2A gene Therapeutic Topoisomerase Topoisomerase Inhibitors Transcript Tumor Suppressor Genes Tumor Suppressor Proteins Variant Work adduct base cancer genome carcinogenesis cell type de novo mutation density driver mutation experimental study genome sequencing genome-wide human genome sequencing inhibitor insertion/deletion mutation insight malignant breast neoplasm novel overexpression repaired therapy resistant tumor tumorigenesis whole genome

项目摘要

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.

抽象的突变是癌症的根本原因，有助于转移和抵抗癌症在人类细胞中，干扰转录和诱导 DNA-拓扑异构酶加合物都是重要的。已知会导致 DNA 损伤，但是产生转录和 DNA 拓扑异构酶的机制。人类细胞中与加合物相关的突变、这些过程引起的突变类型及其我们对人类细胞中突变的贡献知之甚少。观察到以 2 至 5 个碱基对 (bp) 缺失和明显较大缺失为主的突变谱。突变谱反映了 DNA 拓扑异构酶 1 依赖性转录相关突变酿酒酵母，表明转录诱导的 DNA 拓扑异构酶加合物在我们还发现，人类细胞中高表达基因中富含 2 至 5 bp 的缺失。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因为 ≥ 2 bp 的缺失占抑癌基因所有失活突变的 3% 至 12%。该提案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类细胞诱变并评估其对诱变的贡献 Aim1 将确定转录相关的 DNA 拓扑异构酶加合物的机制基础。诱导人类细胞突变并定义该过程产生的突变谱。通过利用突变产生来测量突变率和谱来完成，我们将为此通过 CRISPRi 和 CRISPRa 调节转录和/或通过利用增加 DNA-拓扑异构酶加合物此外，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- 目标 2 将确定拓扑异构酶加合物诱导的突变。使用新型基因组学方法和 DNA-TOP1 加合物以单核苷酸分辨率获得 TOP1 加合物 - 通过对人类细胞进行全基因组测序来确定依赖突变。加合物和突变的分布将与基因组特征（例如转录本）的位置进行比较和 R 环来评估它们对 DNA-TOP1 加合物诱导发生的影响和谱这些加合物和突变的分布将进一步与DNA-分布进行比较。已测序的人类肿瘤中的 TOP1 加合物特征突变可估计 DNA-TOP1 的贡献完成这些目标将有助于深入了解 DNA 的作用。癌症病因学中的拓扑异构酶加合物，确定产生转录的主要机制相关突变，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尚未充分研究的诱变途径有影响。