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: 8505609
• 负责人: YUH-HWA WANG
• 金额: $ 4.45万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 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 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8505609
• 关键词: Address; Affect; Aphidicolin; Base Sequence; Binding; Cancer Etiology; Carcinogen Screening; Carcinogens; Cells; Characteristics; Chemical Exposure; Chromosome Fragile Sites; Chromosomes; Chromosomes, Human, Pair 10; Clinical; Coupled; Cytogenetic Analysis; DNA; DNA Sequence; DNA Sequence Rearrangement; DNA Structure; DNA biosynthesis; DNA-Directed DNA Polymerase; Databases; Development; Diagnostic; Diagnostic tests; Disease; Dose; Environmental Exposure; Environmental Risk Factor; Evaluation; Exhibits; Exposure to; FHIT gene; Fluorescent in Situ Hybridization; Future; Gene Rearrangement; Genes; Genome; Genomics; Genotype; Goals; Health; Hereditary Disease; Human; Human Genome; In Vitro; Individual; Investigation; Ions; Knowledge; Lead; Ligation; Malignant Neoplasms; Maps; Measures; Mediating; Metabolic; Metabolism; Metaphase; Methods; Milieu Therapy; Molecular; Mutagens; Mutation; Nature; Nucleotides; Oncogenic; PTCH gene; Papillary thyroid carcinoma; Pathway interactions; Pharmaceutical Preparations; Pilot Projects; Predisposition; Prevention; Process; Residual state; Rest; Role; Second Primary Cancers; Site; Stress; Structure; Structure-Activity Relationship; Technology; Testing; Therapeutic Agents; Work; base; carcinogenesis; clinical application; database structure; environmental agent; genome sequencing; genome-wide; genome-wide analysis; human disease; insight; next generation sequencing; programs; research study; response; stem; tool

DESCRIPTION (provided by applicant): Alternative DNA secondary structures are mutagenic and prone to breakage, which can lead to 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. 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, especially multiple stem-loop structures. To our knowledge, there is no such structural database available to the public. Such a 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 for the potential to form multiple stem-loop structures, using the MFOLD program 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 displayed a higher propensity to fold into stable secondar structures compared to the rest of the chromosome. This work will also refine the current cytogenetically-defined large fragile sites, and define additional fragile sites in non-fragile regions. The goal is to compile a list of gene regions possessing high potential to fold into stabl secondary structures. These regions will be validated for 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 work towards clinical application of DNA fragility to a DNA diagnostic test, we will develop a high-throughput DNA breaksite mapping strategy to identify and quantitate breaksites within secondary structure-rich and translocation-participating gene regions. We have coupled ligation-mediated PCR breaksite mapping with massively parallel DNA sequencing using an Ion Torrent Personal Genome Machine. Finally, we will examine whether environmental and therapeutic agents generate DNA breaks at these secondary structure-rich and cancer-specific translocation-participating gene regions. These experiments 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.

描述（由申请人提供）： 替代DNA二级结构具有致突变性，易于断裂，可导致遗传疾病和癌症。当DNA双链体在代谢DNA过程如DNA复制过程中解绕时，这些结构的形成可能发生，并导致这些过程中的异常。随着人们越来越认识到DNA二级结构在促进基因重排中的重要性，对整个人类基因组序列形成二级结构，特别是多个茎环结构的能力进行无偏倚评估是及时和关键的。据我们所知，没有这样的结构性数据库可供公众使用。这样的数据库可以作为未来研究的基础，例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脆性的影响。 在这个提议中，我们将首先分析在全基因组分析中形成DNA二级结构的倾向，并使用它来识别脆弱位点的结构特征。将使用MFOLD程序创建结构数据库，评估整个可用的人类基因组序列形成多个茎环结构的潜力。这些信息将用于直接检查二级结构形成能力是否与DNA脆性相关。我们对10号染色体的分析揭示了令人兴奋的发现，其中与染色体的其余部分相比，由aphidicolin诱导的所有脆性位点都显示出更高的折叠成稳定的次级结构的倾向。这项工作也将完善目前的细胞遗传学定义的大的脆性位点，并确定在非脆性区域的其他脆性位点。目标是编制一份具有高潜力折叠成稳定二级结构的基因区域清单。将验证这些区域的体外二级结构形成和细胞中的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脆性的潜在因素。然后，对DNA脆性的DNA诊断测试的临床应用的工作，我们将开发一个高通量的DNA断裂位点映射策略，以确定和定量断裂位点内的二级结构丰富和易位参与基因区域。我们使用离子激流个人基因组机器将连接介导的PCR断裂位点作图与大规模并行DNA测序相结合。最后，我们将研究环境和治疗剂是否会在这些富含二级结构和癌症特异性易位参与基因区域产生DNA断裂。这些实验将为脆性部位断裂在诊断学中的临床应用铺平道路。该提案将为结构研究提供有用的工具，解决DNA脆弱性的性质，并进一步推进我们对环境暴露对人类疾病发展影响的认识。