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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.

摘要