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: 10533315
• 负责人: YUH-HWA WANG
• 金额: $ 32.98万
• 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 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10533315
• 关键词: Acute Myelocytic Leukemia; Affect; Antineoplastic Agents; Base Sequence; Benign; Binding; Binding Proteins; Binding Sites; Biological Assay; Biological Markers; Cancer Etiology; Cell physiology; Cells; Characteristics; Chemical Agents; Chemical Exposure; Chemotherapy-Oncologic Procedure; Chromosome Fragile Sites; Chromosomes; Clinical; Complex; DNA; DNA Maintenance; DNA Structure; DNA-Directed RNA Polymerase; Data; Development; Disease susceptibility; Dose; Environmental Exposure; Evaluation; Excision; Exposure to; Foundations; Frequencies; Future; Gene Rearrangement; Generations; Genes; Genetic Transcription; Genome; Genomic Instability; Genotype; Hematopoietic stem cells; Human; Human Genome; In Vitro; Individual; Ligation; Malignant Neoplasms; Maps; Measures; Mediating; Metabolism; Mind; Modeling; Molecular; Monitor; Mutation; Normal Cell; Outcome; Pathologic; Patients; Pharmaceutical Preparations; Physiological; Population; Predisposition; Process; Residual state; Risk Assessment; Role; Signal Transduction; Single Nucleotide Polymorphism; Single-Stranded DNA; Site; Stress; Structure; Superhelical DNA; Testing; Therapeutic; Therapy-Related Acute Myeloid Leukemia; Time; Topoisomerase II; Topoisomerase-II Inhibitor; Transcription Initiation Site; Untranslated RNA; Variant; Work; Yeasts; acute myeloid leukemia cell; cancer biomarkers; chemotherapeutic agent; clinical application; disease diagnostic; enzyme activity; experimental study; genome-wide; high risk population; human disease; insertion/deletion mutation; novel diagnostics; personalized medicine; promoter; response; screening; stem

Project Summary/Abstract: Both physiological DNA breaks occurring during DNA metabolic processes and pathological DNA breaks responding to a wide range of stresses, contribute to the outcome of human genome instability. DNA fragility generated by alternative DNA secondary structures is a known cause of many human diseases, and also occurs in normal DNA processes. Formation of these structures can arise from single-stranded DNA when the DNA duplex is unwound during DNA processes such as replication and transcription, and thus can be affected by cellular activities, nucleotide sequences, and chemical exposures. Here we will examine if DNA regions having potential to form stable secondary structures when unwound during cell processes, can serve as signals for topoisomerase II (TOP2) to recognize and cleave, and lead to the removal of the excessive supercoiling. We have carried out a computational evaluation of the entire available human genome sequence for optimal ability to fold single-stranded sequences into multiple-hairpin structures, and identified sites of highly stable DNA secondary structures throughout the genome. We will measure TOP2-mediated DNA breaks at these sites upon changes in DNA supercoiling from cell activities, then analyze TOP2-cleaved sites to identify structural features, and examine if DNA secondary structures influence the removal of TOP2 cleavage complexes. TOP2-mediated breaks are also often associated with pathological damage due to the use of TOP2 inhibitors as anticancer drugs. Many DNA secondary structure-rich and fragile regions are located within cancer- specific translocation-participating gene regions, including acute myeloid leukemia (AML)-rearranged regions. We will determine whether DNA fragility at these regions can serve as a biomarker for assessing the potential development of cancer-causing rearrangements. We will first test if DNA fragility at gene regions of AML rearrangements is sensitive to various chemotherapeutic agents, and if this sensitivity leads to the formation of AML rearrangements in human cells. Then, to test if this sensitivity can predict the rearrangement formation in patients, we will examine DNA breakage at these regions in normal cells of AML patients with the AML rearrangements, compared to that of normal individuals, as a means to evaluate individual susceptibility to AML. These experiments will facilitate the clinical application of using DNA fragility as a biomarker. With personalized medicine in mind, we will evaluate the effect of naturally occurring sequence variants on the fragility of the break-prone and AML translocation-participating gene regions, to further identify structure characteristics contributing to DNA fragility, and to reveal an unexploited consequence of non-coding variants. Our preliminary results suggest that sequence variants can influence DNA break frequency of the region by changing the extent or the type of secondary structure forming ability. This proposal will elucidate the mechanistic and functional features of DNA structure-driven fragility and provide a foundation for future clinical use of fragile site breakage in disease diagnostics.

项目摘要/摘要： 在DNA代谢过程和病理DNA断裂过程中，两个生理DNA断裂发生 应对广泛的压力，有助于人类基因组不稳定性的结果。 DNA脆弱性 替代DNA二级结构产生的是许多人类疾病的已知原因，也发生 在正常的DNA过程中。当DNA时，这些结构的形成可能是由单链DNA产生的 在复制和转录等DNA过程中，双链体是解开的，因此可能会受到 细胞活性，核苷酸序列和化学暴露。在这里，我们将检查DNA区域是否 在细胞过程中解开时，有可能形成稳定的二级结构，可以服务 作为拓扑异构酶II（TOP2）识别和裂解的信号，并导致去除过多 超螺旋。我们已经对整个可用的人类基因组序列进行了计算评估 为了最佳能力将单链序列折叠到多发蛋白结构中，并确定了高度的位点 整个基因组中稳定的DNA二级结构。我们将测量TOP2介导的DNA断裂 这些站点会随着细胞活性的DNA超串联而变化，然后分析Top2旋转的位点以识别 结构特征，并检查DNA二级结构是否影响去除Top2裂解 复合物。 TOP2介导的断裂也经常因使用TOP2而与病理损害有关 抑制剂作为抗癌药物。许多DNA二级结构富含和脆弱的区域都位于癌症中 特定的易位参与基因区域，包括急性骨髓性白血病（AML）重新定位区域。 我们将确定这些区域的DNA脆弱性是否可以作为评估的生物标志物 导致癌症重排的潜在发展。我们将首先测试DNA脆弱性在基因区域 AML的重排对各种化学治疗剂敏感，如果这种敏感性导致 人类细胞中AML重排的形成。然后，要测试这种灵敏度是否可以预测重排 在患者中形成，我们将检查AML患者正常细胞中这些区域的DNA断裂 与普通个体相比，AML重排作为评估个体易感性的手段 到AML。这些实验将促进使用DNA脆性作为生物标志物的临床应用。和 考虑到个性化医学，我们将评估自然发生的序列变体对 突破性和AML易位的基因区域的脆弱性，以进一步识别 有助于DNA脆弱性的结构特征，并揭示了未开发的结果 非编码变体。我们的初步结果表明序列变体会影响DNA断裂 通过更改二级结构形成能力的程度或类型来实现区域的频率。这个建议 将阐明DNA结构驱动脆弱性的机械和功能特征，并提供基础 用于将来的临床用途，可在疾病诊断中脆弱的部位断裂。