In vivo detection and genome-wide location analysis of DNA-adducts

DNA 加合物的体内检测和全基因组定位分析

• 批准号: 8547505
• 负责人: STEPHEN B HOWELL
• 金额: $ 23.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-03 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8547505
• 关键词: Address; Alkylating Agents; Antineoplastic Agents; Apoptosis; Base Pairing; Biological; Biological Assay; Cancer Biology; Carcinogen exposure; Cell Death; Cell Line; Cell physiology; Cells; Chlorambucil; Chromatin Structure; Cisplatin; Clinical; Computer Analysis; DNA; DNA Adduction; DNA Adducts; DNA Damage; DNA Methylation; DNA Repair; DNA Sequence; DNA lesion; DNA-Directed DNA Polymerase; Data; Data Set; Detection; Development; Doctor of Philosophy; Drug Metabolic Detoxication; Environmental Carcinogens; Environmental Risk Factor; Epigenetic Process; Event; Exposure to; Genes; Genome; Genomics; Goals; Homeostasis; Implant; In Vitro; Individual; Kinetics; Knowledge; Lead; Libraries; Ligation; Location; Malignant Neoplasms; Maps; Measurement; Measures; Mediating; Methods; Molecular; Mus; Mutation; Normal tissue morphology; Nucleotides; Pathway interactions; Pattern; Performance; Pharmaceutical Preparations; Pharmacology; Physiological; Platinum; Predisposition; Process; Proteins; Protocols documentation; Reading; Reproducibility; Research; Research Personnel; Resolution; Sampling; Scientist; Single-Stranded DNA; Specificity; Specimen; Tail; Testicular Germ Cell Tumor; The Cancer Genome Atlas; Therapeutic; Time; Tissue Sample; Tissues; Tumor Tissue; UV induced DNA damage; Ultraviolet Rays; Validation; adduct; anticancer research; base; cancer cell; carcinogenesis; cell type; chemotherapeutic agent; chemotherapy; drug carcinogenesis; drug efficacy; drug sensitivity; epigenome; experience; genetically modified cells; genome-wide; improved; in vivo; innovation; novel; preference; public health relevance; repaired; research study; response; tumor; tumor xenograft; ultraviolet damage

DESCRIPTION (provided by applicant): DNA adducts are the hallmark and most common form of DNA damage in the cell. They result from environmental carcinogen exposure (such as UV) or during chemotherapy using DNA modifying agents like cisplatin (cDDP) or alkylators such as chlorambucil (CLB). While mechanisms underlying sensitivity, agent homeostasis, detoxification, DNA repair and apoptosis, have been well investigated, the central molecular event, the formation of adducts, is not well understood in vivo. Evidence suggests that the epigenetic landscape and the structure of the chromatin influences the formation of adducts and mediates drug sensitivity. Therefore, there is a need to better identify DNA adducts and understand the association between the epigenetic marks in the cell. Currently there is no method to determine the exact location of DNA adducts in vivo nor at a high-resolution across the genome. In order to address this, we propose to develop a method, TdT-Seq, that will identify these adducts genome-wide at the single base pair resolution. The expertise of the investigators include knowledge in cancer biology and platinum drug pharmacology (Drs. Howell and Abada) as well as experience in high-throughput genomic assays and computational analysis (Dr. Harismendy); expertise that will be needed to successfully develop the assay. The TdT-Seq assay relies on adduct-mediated inhibition of the DNA polymerase in vitro. The resulting single strand DNA will be captured by a specific TdT mediated ligation, enriched, then sequenced in high throughput. We propose to establish the technical validity of the assay by determining 1) sensitivity at various cDDP concentrations and read depth, 2) specificity by the development of a locus specific method (Strand Specific Adduct Detection) and independent analysis of 50 adduct loci, and 3) quantativity using increasing cDDP concentrations and known spike-in controls. We will also perform specific experiments to establish TdT-Seq's use for clinical cancer research. In particular, we will optimize the protocol for the identification of UVor chlorambucil (CLB) induced adducts to broaden its applicability. We will also develop the protocol for low amounts of DNA originating from mouse tissues or heterogeneous tissue specimens. Finally, we will analyze the ability of TdT-Seq to measure the kinetics of DNA repair using genetically modified cell lines. TdT-Seq's development will therefore lead to a robust and innovative assay, with demonstrated performance and utility for cancer research. TdT-Seq will generate an entirely new type of data, which can be used in combination of other whole genome datasets from the ENCODE or TCGA consortium to provide a more precise and comprehensive description of the mechanism of DNA damage and repair in vivo in various cell types and cancers. The long-term benefits of such research include the prediction of drug sensitivity or the study of epigenetic modifying compounds to rationalize combinations for optimal drug efficacy.

描述（由申请人提供）：DNA加合物是细胞中DNA损伤的标志和最常见形式。它们是由环境致癌物暴露（如紫外线）或在使用DNA修饰剂如顺铂（cDDP）或烷化剂如苯丁酸氮芥（CLB）的化疗期间引起的。虽然敏感性、药物稳态、解毒、DNA修复和细胞凋亡的潜在机制已经得到了很好的研究，但在体内对加合物的形成这一中心分子事件还没有很好的理解。有证据表明，表观遗传景观和染色质的结构影响加合物的形成和介导的药物敏感性。因此，需要更好地鉴定DNA加合物并了解细胞中表观遗传标记之间的关联。目前还没有方法来确定DNA加合物在体内的确切位置，也没有在整个基因组中的高分辨率。为了解决这个问题，我们建议开发一种方法，TdT-Seq，将确定这些加合物的基因组范围内的单碱基对分辨率。研究人员的专业知识包括癌症生物学和铂类药物药理学方面的知识（豪厄尔博士和Abada博士）以及高通量基因组测定和计算分析方面的经验（Harismendy博士）;成功开发该测定所需的专业知识。TdT-Seq测定依赖于加合物介导的体外DNA聚合酶抑制。所得单链DNA将通过特异性TdT介导的连接捕获，富集，然后以高通量测序。我们建议通过确定1）在不同cDDP浓度和读取深度下的灵敏度，2）通过开发基因座特异性方法（链特异性加合物检测）和独立分析50个加合物基因座的特异性，以及3）使用增加的cDDP浓度和已知加标对照品的定量性，来确定测定的技术有效性。我们还将进行特定的实验，以确定TdT-Seq在临床癌症研究中的用途。特别是，我们将优化的协议，用于鉴定紫外线或苯丁酸氮芥（CLB）诱导的加合物，以扩大其适用性。我们还将开发用于源自小鼠组织或异质组织标本的少量DNA的方案。最后，我们将分析TdT-Seq使用遗传修饰细胞系测量DNA修复动力学的能力。因此，TdT-Seq的开发将带来一种强大而创新的检测方法，并在癌症研究中表现出良好的性能和实用性。TdT-Seq将产生一种全新类型的数据，可以与ENCODE或TCGA联盟的其他全基因组数据集结合使用，以提供对各种细胞类型和癌症体内DNA损伤和修复机制的更精确和全面的描述。这种研究的长期益处包括预测药物敏感性或研究表观遗传修饰化合物，以合理组合以获得最佳药物疗效。