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：
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
项目类别：
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

项目摘要

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： DNA 代谢过程中发生的生理性 DNA 断裂和病理性 DNA 断裂应对各种压力，导致人类基因组不稳定。 DNA脆弱性由替代 DNA 二级结构产生的 DNA 是许多人类疾病的已知原因，并且也会发生在正常的 DNA 过程中。这些结构的形成可以由单链 DNA 产生，当 DNA 双链体在复制和转录等 DNA 过程中解开，因此可能受到以下因素的影响细胞活动、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展开时有可能形成稳定的二级结构，可用于作为拓扑异构酶 II (TOP2) 识别和切割的信号，并导致去除过量的超螺旋。我们对整个可用的人类基因组序列进行了计算评估以获得将单链序列折叠成多发夹结构的最佳能力，并确定了高度整个基因组中稳定的 DNA 二级结构。我们将测量 TOP2 介导的 DNA 断裂这些位点根据细胞活动引起的 DNA 超螺旋变化，然后分析 TOP2 切割位点以识别结构特征，并检查 DNA 二级结构是否影响 TOP2 切割的去除复合物。 TOP2 介导的断裂通常也与由于使用 TOP2 造成的病理损伤有关抑制剂作为抗癌药物。许多 DNA 二级结构丰富且脆弱的区域位于癌症内部特定易位参与基因区域，包括急性髓系白血病 (AML) 重排区域。我们将确定这些区域的 DNA 脆弱性是否可以作为评估 DNA 脆弱性的生物标志物。致癌重排的潜在发展。我们首先测试基因区域的DNA是否脆弱 AML 重排对各种化疗药物敏感，如果这种敏感性导致人类细胞中 AML 重排的形成。然后，测试这种敏感性是否可以预测重排患者形成后，我们将检查 AML 患者正常细胞中这些区域的 DNA 断裂情况与正常个体相比，AML 重排作为评估个体易感性的一种手段到反洗钱。这些实验将促进利用DNA脆性作为生物标志物的临床应用。和考虑到个性化医疗，我们将评估自然发生的序列变异对易断裂和 AML 易位参与基因区域的脆弱性，以进一步鉴定导致 DNA 脆弱性的结构特征，并揭示了未利用的后果非编码变体。我们的初步结果表明序列变异可以影响 DNA 断裂该区域的频率通过改变二级结构形成能力的程度或类型来实现。这个提议将阐明DNA结构驱动的脆弱性的机制和功能特征并提供基础用于未来在疾病诊断中脆弱部位破损的临床应用。