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

基本信息

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
项目类别：
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

项目摘要

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二级结构是诱变的，容易破裂，这可能导致遗传疾病和癌症。当DNA双链体在代谢DNA过程（例如DNA复制）中解开并在这些过程中引起异常时，可能会发生这些结构的形成。随着对DNA二级结构在促进基因重排的重要性的越来越多，对整个人类基因组序列形成二级结构的能力进行无偏见的评估是及时且至关重要的，尤其是多个茎环结构。据我们所知，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，例如探索染色体成分的结构 - 功能关系，研究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。在此提案中，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，并使用它来识别脆弱位点的结构特征。将对整个可用的人类基因组序列进行评估，以使用MFOLD程序创建结构数据库，以形成多个茎环结构。该信息将用于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号染色体的分析揭示了令人兴奋的发现，在这种发现中，与其他染色体相比，蚜虫蛋白诱导的所有脆弱位点均显示出更高的倾向，可将其折叠成稳定的二级结构。这项工作还将完善当前的细胞遗传学定义的大型脆弱位点，并在非脆弱区域定义其他脆弱的位点。目的是编译具有具有很高潜力的基因列表，这些基因区域将其折叠成一个二级结构。这些区域将在体外和细胞中的二级结构形成和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，为了在DNA诊断测试中临床应用DNA脆弱性，我们将开发高通量DNA断裂映射策略，以识别和定量二级结构富含二级结构和转运的基因区域内的断裂。我们使用离子洪流的个人基因组机进行了结合结合介导的PCR断裂映射，并进行了大规模平行的DNA测序。最后，我们将研究环境和治疗剂是否在这些二次结构和癌症特异性易位参与基因区域会产生DNA断裂。这些实验将为在诊断中使用脆弱的位点破裂的临床应用铺平道路。该建议将为结构研究生成有用的工具，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。