Genome-wide DNA Secondary Structure Analysis to Investigate DNA Fragility

项目摘要/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，促成人类基因组不稳定的结果。DNA脆弱性 由另一种DNA二级结构产生的DNA是许多人类疾病的已知原因，也会发生 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 双链在DNA复制和转录等过程中被解离，因此可能会受到 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 在细胞过程中展开时有可能形成稳定的二级结构，可以 作为拓扑异构酶II(TOP2)识别和切割的信号，并导致多余的 超级卷曲。我们已经对整个可用的人类基因组序列进行了计算评估 为了优化将单链序列折叠成多个发夹结构的能力，并识别高度 基因组中稳定的DNA二级结构。我们将在以下位置测量TOP2介导的DNA断裂 这些位点根据细胞活动的DNA超螺旋变化，然后分析TOP2裂解位点来鉴定 结构特征，并检查DNA二级结构是否影响TOP2裂解的移除 复合体。TOP2介导的断裂也常常与TOP2的使用引起的病理损伤有关 作为抗癌药物的抑制剂。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，包括急性髓系白血病(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 致癌重排的潜在发展。我们将首先测试DNA在基因区域的脆弱性 急性髓系白血病重排对各种化疗药物敏感，如果这种敏感性导致 人类细胞中AML重排的形成。然后，测试这种敏感性是否可以预测重排 为了在患者中形成DNA，我们将用 急性髓系白血病重排作为评估个体易感性的一种手段 致AML。这些实验将为利用DNA脆性作为生物标志物的临床应用提供便利。使用 考虑到个性化医学，我们将评估自然发生的序列变异对 易破基因区域和AML易位参与基因区域的脆性，以进一步鉴定 导致DNA脆弱性的结构特征，并揭示了 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 通过改变区域频率的大小或类型，形成二级结构的能力。这项建议 将阐明DNA结构驱动的脆性的机制和功能特征，并提供基础 为未来脆性部位断裂在疾病诊断中的临床应用奠定基础。

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.