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的形式。它们是由环境致癌物暴露（例如紫外线）或化疗过程中使用顺铂 (cDDP) 等 DNA 修饰剂或苯丁酸氮芥 (CLB) 等烷化剂引起的。虽然敏感性、药物稳态、解毒、DNA 修复和细胞凋亡的机制已得到充分研究，但中心分子事件，即加合物的形成，在体内尚不清楚。有证据表明，表观遗传景观和染色质结构影响加合物的形成并介导药物敏感性。因此，需要更好地识别DNA加合物并了解细胞中表观遗传标记之间的关联。目前还没有方法可以确定 DNA 加合物在体内的确切位置，也没有在整个基因组中以高分辨率确定的方法。为了解决这个问题，我们建议开发一种方法 TdT-Seq，该方法将以单碱基对分辨率在全基因组范围内识别这些加合物。研究人员的专业知识包括癌症生物学和铂类药物药理学知识（Howell 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）；成功开发该检测所需的专业知识。 TdT-Seq 测定依赖于体外加合物介导的 DNA 聚合酶抑制。产生的单链 DNA 将通过特定的 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) 不同 cDDP 浓度和读取深度的灵敏度，2) 通过开发位点特异性方法（链特异性加合物检测）和 50 个加合物位点的独立分析来确定特异性，以及 3) 使用增加的 cDDP 浓度和已知的加标对照来确定测定的技术有效性。我们还将进行具体的实验来确定 TdT-Seq 在临床癌症研究中的用途。特别是，我们将优化用于鉴定 UV 或苯丁酸氮芥 (CLB) 诱导的加合物的方案，以扩大其适用性。我们还将开发来自小鼠组织或异质组织样本的少量 DNA 的方案。最后，我们将分析 TdT-Seq 使用转基因细胞系测量 DNA 修复动力学的能力。因此，TdT-Seq 的开发将带来一种稳健且创新的检测方法，并在癌症研究中具有经证实的性能和实用性。 TdT-Seq将生成一种全新类型的数据，可与ENCODE或TCGA联盟的其他全基因组数据集结合使用，以更精确、更全面地描述各种细胞类型和癌症体内DNA损伤和修复的机制。此类研究的长期好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化组合以获得最佳药物功效。