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

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。它们是由于暴露于环境致癌物（如紫外线）或化疗期间使用DNA修饰剂如顺铂（cDDP）或烷基化剂如氯苯（CLB）造成的。虽然敏感性、药物稳态、解毒、DNA修复和细胞凋亡的机制已经得到了很好的研究，但中心分子事件，即加合物的形成，在体内还没有得到很好的理解。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药物敏感性。因此，有必要更好地识别DNA加合物，并了解细胞中表观遗传标记之间的关系。目前还没有办法确定DNA加合物在体内的确切位置，也没有办法在整个基因组中进行高分辨率的确定。为了解决这个问题，我们建议开发一种方法，TdT-Seq，将识别这些加合物在单碱基对分辨率全基因组。研究人员的专业知识包括癌症生物学和铂类药物药理学。Howell和Abada)以及在高通量基因组分析和计算分析方面的经验（Harismendy博士）；成功开发检测所需的专业知识。TdT-Seq检测依赖于体外加合物介导的DNA聚合酶抑制。产生的单链DNA将被特定的TdT介导的连接捕获，富集，然后高通量测序。我们建议通过以下方法来确定该方法的技术有效性：1)在不同cDDP浓度和读取深度下的敏感性；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；3)使用增加的cDDP浓度和已知的峰值控制来确定定量。我们还将进行具体的实验，以建立TdT-Seq在临床癌症研究中的应用。特别是，我们将优化UVor氯苯（CLB）诱导加合物的鉴定方案，以扩大其适用性。我们还将为来自小鼠组织或异质组织标本的低量DNA制定方案。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的开发将带来一种强大而创新的检测方法，具有已证明的性能和用于癌症研究的实用性。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，从而更精确和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。此类研究的长期益处包括预测药物敏感性或研究表观遗传修饰化合物以使组合合理化以获得最佳药物疗效。

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.