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：
10887019
负责人：
STEVEN A ROBERTS
金额：
$ 13.12万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
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/10887019
关键词：
Base Pairing Bioinformatics CRISPR interference CRISPR-mediated transcriptional activation Camptothecin Cancer Etiology Cell Line Cells Clustered Regularly Interspaced Short Palindromic Repeats DNA DNA Adduction DNA Adducts DNA Damage DNA Double Strand Break DNA Repair DNA Structure DNA Topoisomerases DNA biosynthesis DNA mapping DNA-PKcs Data Defect Deletion Mutation 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 Insertion Mutation Ligation Malignant Neoplasms 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 cancer genome cancer initiation carcinogenesis de novo mutation density driver mutation experimental study genome sequencing genome-wide genomic locus 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，并导致转移和对癌症的抗性治疗。在人类细胞中，扰动转录和诱导的DNA--------- 已知会导致DNA损伤。但是，产生转录和DNA-昆虫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对癌症突变的贡献知之甚少。在人类细胞中使用新型突变报告基因，我们观察到的突变频谱由2至5碱基对（BP）缺失和明显的较大缺失支配。这突变光谱反映了DNA拓扑异构酶1依赖性，转录相关诱变的谱。 S. cerevisiae，表明转录诱导的DNA昆虫酶加合物产生了这些突变人类细胞。我们还发现，在高度表达的基因中，2至5 bp的缺失富集原发性乳腺癌，表明DNA--昆虫酶加合物是癌症病因的重要因素由于≥2bp的缺失占肿瘤抑制基因中所有灭活突变的3％至12％。该建议的目的是表征转录相关，DNA-的基本确定拓扑异构酶加合物诱导的人类细胞中诱导的诱变，并评估其对诱变的贡献癌症。 AIM1将确定与转录相关的，DNA-昆虫酶加合物的机械基础 - 诱导人类细胞中的诱变并定义了该过程产生的突变谱。这会可以通过使用突变记者来测量突变率和光谱来实现，我们将通过CRISPRI和CRISPRA调节转录和/或增加DNA - topoisomerase加合物。拓扑异构酶变体和抑制剂。另外，我们将确定连接DNA的各种端的贡献双链破裂修复途径和R环分辨率，以促进或限制特定类型的DNA- 拓扑酶加合物诱导的突变。 AIM 2将确定两个DNA-的全基因组分布采用新型基因组方法和DNA-TOP1加合物 - 通过人类细胞的整个基因组测序的依赖突变。这些公正的完善将加合物和突变的分布与基因组特征（例如转录本）的位置进行比较和R环评估其对DNA-TOP1加合物诱导的发生的影响和光谱突变。这些加合物和突变的分布将与分布DNA-相比在测序的人类肿瘤中的TOP1加合物特征突变，以估计DNA-TOP1的贡献癌症诱变的加合物。这些目标的完成将提供有关角色DNA-的见解癌症病因中的拓扑异构酶加合物确定产生转录的主要机制相关的突变，并创建实验系统，以促进对DNA影响的未来研究结构，遗传决定和环境暴露在这一理解的诱变途径上。