Genome-wide DNA Secondary Structure Analysis to Investigate DNA Fragility

项目概要/摘要： DNA代谢过程中发生的生理性DNA断裂和病理性DNA断裂 应对广泛的压力，有助于人类基因组不稳定的结果。DNA脆性 由替代DNA二级结构产生的蛋白质是许多人类疾病的已知原因， 在正常的DNA过程中。这些结构的形成可以由单链DNA产生，当DNA 双链体在DNA过程如复制和转录过程中解旋，因此可以受到 细胞活动、核苷酸序列和化学暴露。在这里，我们将检查DNA区域是否 当在细胞过程中解绕时具有形成稳定的二级结构的潜力， 作为拓扑异构酶II（TOP 2）识别和切割的信号，并导致去除过量的 超螺旋我们已经对整个可用的人类基因组序列进行了计算评估 为了获得将单链序列折叠成多发夹结构的最佳能力， 整个基因组中稳定的DNA二级结构。我们将测量TOP2介导的DNA断裂， 这些位点的DNA超螺旋的变化，然后分析TOP 2切割位点，以确定 结构特征，并检查DNA二级结构是否影响TOP2切割的去除 配合物TOP2介导的断裂也经常与由于使用TOP2而引起的病理损伤相关 抑制剂作为抗癌药物。许多DNA二级结构丰富和脆弱的区域位于癌症内， 特定的易位参与基因区域，包括急性髓性白血病（AML）重排区域。 我们将确定这些区域的DNA脆弱性是否可以作为评估DNA脆弱性的生物标志物。 潜在的致癌基因重排。我们将首先测试基因区域的DNA脆弱性 AML重排对各种化疗药物敏感，如果这种敏感性导致AML重排， 在人类细胞中形成AML重排。然后，为了测试这种灵敏度是否可以预测重排， 我们将在AML患者的正常细胞中检测这些区域的DNA断裂， AML重排，与正常个体相比，作为评估个体易感性的一种手段 在AML。这些实验将促进DNA脆性作为生物标志物的临床应用。与 考虑到个性化医疗，我们将评估自然发生的序列变异对 脆性的断裂倾向和AML易位参与基因区域，以进一步确定 结构特征有助于DNA的脆弱性，并揭示了一个未开发的后果， 非编码变体。我们的初步结果表明，序列变异可以影响DNA断裂 通过改变二级结构形成能力的程度或类型来改变该区域的频率。这项建议 将阐明DNA结构驱动的脆弱性的机制和功能特征，并提供一个基础 用于将来疾病诊断中脆性部位断裂的临床应用。

Project Summary/Abstract: Both physiological DNA breaks occurring during DNA metabolic processes and pathological DNA breaks responding to a wide range of stresses, contribute to the outcome of human genome instability. DNA fragility generated by alternative DNA secondary structures is a known cause of many human diseases, and also occurs in normal DNA processes. Formation of these structures can arise from single-stranded DNA when the DNA duplex is unwound during DNA processes such as replication and transcription, and thus can be affected by cellular activities, nucleotide sequences, and chemical exposures. Here we will examine if DNA regions having potential to form stable secondary structures when unwound during cell processes, can serve as signals for topoisomerase II (TOP2) to recognize and cleave, and lead to the removal of the excessive supercoiling. We have carried out a computational evaluation of the entire available human genome sequence for optimal ability to fold single-stranded sequences into multiple-hairpin structures, and identified sites of highly stable DNA secondary structures throughout the genome. We will measure TOP2-mediated DNA breaks at these sites upon changes in DNA supercoiling from cell activities, then analyze TOP2-cleaved sites to identify structural features, and examine if DNA secondary structures influence the removal of TOP2 cleavage complexes. TOP2-mediated breaks are also often associated with pathological damage due to the use of TOP2 inhibitors as anticancer drugs. Many DNA secondary structure-rich and fragile regions are located within cancer- specific translocation-participating gene regions, including acute myeloid leukemia (AML)-rearranged regions. We will determine whether DNA fragility at these regions can serve as a biomarker for assessing the potential development of cancer-causing rearrangements. We will first test if DNA fragility at gene regions of AML rearrangements is sensitive to various chemotherapeutic agents, and if this sensitivity leads to the formation of AML rearrangements in human cells. Then, to test if this sensitivity can predict the rearrangement formation in patients, we will examine DNA breakage at these regions in normal cells of AML patients with the AML rearrangements, compared to that of normal individuals, as a means to evaluate individual susceptibility to AML. These experiments will facilitate the clinical application of using DNA fragility as a biomarker. With personalized medicine in mind, we will evaluate the effect of naturally occurring sequence variants on the fragility of the break-prone and AML translocation-participating gene regions, to further identify structure characteristics contributing to DNA fragility, and to reveal an unexploited consequence of non-coding variants. Our preliminary results suggest that sequence variants can influence DNA break frequency of the region by changing the extent or the type of secondary structure forming ability. This proposal will elucidate the mechanistic and functional features of DNA structure-driven fragility and provide a foundation for future clinical use of fragile site breakage in disease diagnostics.