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: 8881219
• 负责人: YUH-HWA WANG
• 金额: $ 32.72万
• 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/8881219
• 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 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; Genomic Segment; Genotype; Goals; Hereditary Disease; High-Throughput Nucleotide Sequencing; Human; In Vitro; Individual; Investigation; Ions; Knowledge; Lead; Ligation; Malignant Neoplasms; Maps; Massive Parallel Sequencing; 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-wide; genome-wide analysis; human disease; human genome sequencing; insight; next generation sequencing; programs; public health relevance; 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)： 替代的DNA二级结构具有突变性，容易断裂，这可能导致遗传性疾病和癌症。在DNA复制等代谢DNA过程中，当DNA双链解开时，这些结构的形成可能会导致这些过程中的异常。随着人们对DNA二级结构在促进基因重排中的重要性的日益认识，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见的评估是及时和关键的。据我们所知，目前还没有这样的结构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，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。在这个提案中，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，并利用它来识别脆弱部位的结构特征。将使用MFOLD程序创建结构数据库，对整个可用的人类基因组序列进行评估，以确定形成多个茎环结构的可能性。这些信息将被用来直接检查二级结构形成能力是否与DNA脆弱性相关。我们对10号染色体的分析显示了令人兴奋的发现，与染色体的其他部分相比，由aphidicolin诱导的所有脆性部位都显示出更高的折叠成稳定的次级结构的倾向。这项工作还将提炼目前在细胞遗传学上定义的大的脆弱部位，并在非脆弱区域定义更多的脆弱部位。目标是编制一份具有高折叠成稳定二级结构潜力的基因区域的列表。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的二级结构的倾向是否是DNA脆弱性的潜在因素。然后，为了将DNA脆性用于DNA诊断测试，我们将开发一种高通量DNA断裂点定位策略，以识别和定量二级结构丰富和易位参与的基因区域的断裂点。我们使用Ion Torrent Personal Genome Machine将连接介导的PCR断点测序与大规模并行DNA测序相结合。最后，我们将检查环境和治疗药物是否在这些二级结构丰富和癌症特异性易位参与的基因区域产生DNA断裂。这些实验将为脆性部位断裂在诊断中的临床应用铺平道路。这一建议将为结构研究提供有用的工具，解决DNA脆弱性的性质，并进一步促进我们对环境暴露在人类疾病发展中的影响的了解。