Genome-wide DNA Secondary Structure Analysis to Investigate DNA Fragility

用于研究 DNA 脆弱性的全基因组 DNA 二级结构分析

• 批准号: 8775363
• 负责人: YUH-HWA WANG
• 金额: $ 28.12万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8775363
• 关键词: Genome; wide; DNA; Secondary; Structure

Alternative DNA secondary structures are mutagenic and prone to breakage, which can lead to the etiology of human genetic diseases and cancers. Formation of these structures can occur when the DNA duplex is unwound during metabolic DNA processes such as DNA replication, and cause abnormalities in these processes. Both nucleotide sequences and cellular activities can determine the formation of a highly stable secondary structure at a given DNA region, and the influence of environmental exposures is also reported. With increasing recognition of the importance of DNA secondary structures in promoting gene rearrangements, it is timely and critical to carry out a bias-free assessment of the ability of the entire human genome sequence to form secondary structures. This structure database can serve as a basis for future studies, such as exploration of structure-function relationships of chromosome components, investigation of the influence of DNA structure on DNA metabolic processes, and the impact of environmental exposures on DNA fragility. In this proposal, we will first analyze the propensity to form DNA secondary structures in a genome- wide analysis, and use it to identify structural characteristics of fragile sites. The entire available human genome sequence will be evaluated using the MFOLD program for the potential to form DNA secondary structures, to create a structure database. This information will be used to directly examine whether the secondary structure-forming ability correlates with DNA fragility. Our analysis of chromosome 10 revealed exciting findings in which all fragile sites induced by aphidicolin display a higher propensity to fold into stable secondary structures compared to the rest of the chromosome. Also, the current cytogenetically-defined large fragile sites can be refined, while additional fragile sites in non-fragile regions are identified. The goal is to compile a list of gene regions possessing high potential to fold into stable secondary structures. These regions will be validated for the secondary structure formation in vitro and for DNA breakage in cells, to directly test whether the propensity to form highly stable secondary structure is an underlying factor for DNA fragility. Then, to examine the effect of these regions on DNA replication and its instability, we will use the SV40 replication system to evaluate cis-elements such as replication direction, and trans-factors such as components of the cell cycle checkpoints in causing DNA breakage and replication delay. Finally, we will examine whether environmental and therapeutic agents generate DNA breaks at these secondary structure-rich and cancer-specific translocation-participating gene regions. This experiment will pave the way for the clinical application of using fragile site breakage in diagnostics. This proposal will generate useful tools for structural studies, address the nature of DNA fragility, and further advance our knowledge about the impact of environmental exposures in human disease development.

替代的DNA二级结构是致突变的，并且易于断裂，这可以导致DNA的断裂。 人类遗传疾病和癌症的病因学。这些结构的形成可以发生在DNA 双链体在代谢DNA过程如DNA复制过程中解旋，并导致DNA复制异常。 这些过程。核苷酸序列和细胞活性都可以决定一种高度依赖性的蛋白质的形成。 稳定的二级结构在一个给定的DNA区域，和环境暴露的影响也是 报道随着人们对DNA二级结构在基因启动子中重要性的认识， 重新安排，这是及时和关键的进行无偏见的评估，整个人类的能力， 基因组序列形成二级结构。该结构数据库可以作为未来的基础 研究，如探索染色体组分的结构-功能关系， DNA结构对DNA代谢过程的影响，以及环境暴露对 DNA脆弱性 在这个提议中，我们将首先分析在基因组中形成DNA二级结构的倾向- 广泛的分析，并使用它来识别脆弱网站的结构特征。整个可用的人类 将使用MFOLD程序评估基因组序列，以确定形成DNA二级结构的可能性。 创建一个结构数据库。此信息将用于直接检查 二级结构形成能力与DNA脆性相关。我们对10号染色体的分析显示 令人兴奋的发现，其中所有由aphidicolin诱导的脆性位点都显示出更高的倾向， 与染色体的其余部分相比，二级结构。此外，目前的细胞遗传学定义的大 可以对脆弱场址进行改进，同时在非脆弱区域确定更多的脆弱场址。目标是 编辑一份具有高度折叠成稳定二级结构潜力的基因区域清单。这些 将验证区域的体外二级结构形成和细胞中的DNA断裂， 直接测试形成高度稳定的二级结构的倾向是否是DNA的潜在因素 脆弱然后，为了检查这些区域对DNA复制及其不稳定性的影响，我们将使用 SV 40复制系统评估顺式元件如复制方向和反式因子如 细胞周期检查点的组成部分导致DNA断裂和复制延迟。最后我们将 检查环境和治疗剂是否会在这些次级DNA断裂， 结构丰富和癌症特异性易位参与基因区。这个实验将为 脆性部位断裂在诊断学中的临床应用途径。 这一提议将为结构研究提供有用的工具，解决DNA脆弱性的本质， 进一步加深我们对环境暴露对人类疾病发展的影响的认识。