Genome-wide DNA Secondary Structure Analysis to Investigate DNA Fragility

替代的DNA二级结构是突变的，容易断裂，这可能导致 人类遗传性疾病和癌症的病因学。这些结构的形成可能发生在DNA 双链在新陈代谢DNA过程中解旋，如DNA复制，并导致 这些过程。核苷酸序列和细胞活性都可以决定高度 在给定DNA区域稳定的二级结构，以及环境暴露的影响也是 据报道。随着人们对DNA二级结构在促进基因表达中的重要性的认识日益加深 重新安排，及时和关键的是对整个人类的能力进行无偏见的评估 基因组序列以形成二级结构。该结构化数据库可作为今后数据库开发的基础 研究，如探索染色体组分的结构-功能关系，调查 DNA结构对DNA代谢过程的影响以及环境暴露对DNA代谢过程的影响 DNA的脆弱性。 在这个提案中，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，并用它来识别脆弱部位的结构特征。所有可用的人类 基因组序列将使用MFOLD程序进行评估，以确定形成DNA二级结构的可能性 结构，以创建结构数据库。这些信息将被用来直接检查 二级结构形成能力与DNA脆性有关。我们对10号染色体的分析揭示了 令人兴奋的发现，在这些发现中，由蜂毒灵诱导的所有脆性部位都显示出更高的折叠成稳定的倾向 次生结构与染色体的其余部分相比。此外，目前细胞遗传学定义的大型 可以细化脆弱部位，同时确定非脆弱区域中的更多脆弱部位。我们的目标是 编制一张具有高折叠成稳定二级结构潜力的基因区域列表。这些 这些区域将在体外验证二级结构的形成和细胞中的DNA断裂，以 直接测试形成高度稳定二级结构的倾向是否是DNA的潜在因素 脆弱。然后，为了研究这些区域对DNA复制及其不稳定性的影响，我们将使用 SV40复制系统用于评估诸如复制方向之类的顺式元件，以及诸如 导致DNA断裂和复制延迟的细胞周期检查点的组成部分。最后，我们会 检查环境和治疗药物是否在这些继发性疾病中产生DNA断裂 富含结构和癌症特异性易位参与基因区域。这项实验将为 脆性部位断裂在诊断中的临床应用途径。 这一提议将产生用于结构研究的有用工具，解决DNA脆弱性的性质，以及 进一步增进我们对环境暴露在人类疾病发展中的影响的知识。

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.