Oxidative DNA damage processing; role in human pathology and aging

DNA氧化损伤处理；

• 批准号: 7861977
• 负责人: PHILIP COURTLAND HANAWALT
• 金额: $ 33.56万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7861977
• 关键词: Age; Aging; Apoptosis; Apoptotic; Base Excision Repairs; Behavior; Biochemical; Biological Assay; Biological Models; Bypass; Cancer Etiology; Cell Death; Cell physiology; Cells; Cellular Structures; Cessation of life; Cockayne Syndrome; DNA; DNA Damage; DNA lesion; DNA-Directed RNA Polymerase; Defect; Development; Disease; Distal; Electrophoresis; Employee Strikes; Event; Excision; Exhibits; Fluorescent in Situ Hybridization; Free Radicals; Genes; Genetic Transcription; Genetic Vectors; Genome; Genomics; Growth Factor; Hereditary Disease; Human; Human Genetics; Human Pathology; In Vitro; Incidence; Learning; Lesion; Malignant Neoplasms; Mammalian Cell; Measures; Mediating; Metabolic; Modeling; Mutagenesis; Mutation; Neurologic; Normal Cell; Nucleotide Excision Repair; Oxidants; Pathology; Pathway interactions; Patients; Plasmids; Positioning Attribute; Premature aging syndrome; Process; Progeria; RNA Interference; Reaction; Reactive Oxygen Species; Recruitment Activity; Reporter Genes; Reporting; Resistance; Role; Site; Skin Cancer; Source; Sunburn; Sunlight; Symptoms; Syndrome; System; Telomere Shortening; Therapeutic Intervention; Transcript; Transcription-Coupled Repair; Transfection; UV induced DNA damage; UV sensitive syndrome; Xeroderma Pigmentosum; attenuation; base; carcinogenesis; chemical carcinogen; genome sequencing; human disease; in vivo; nervous system disorder; novel; novel strategies; oxidative DNA damage; promoter; public health relevance; repair enzyme; repaired; response; sunlight-induced; tumor growth; vector

DESCRIPTION (provided by applicant): Our overall objective is to understand how processing of damaged DNA relates to human genetic disease, cancer and aging. Having pioneered the discovery of nucleotide excision repair (NER), we are elucidating the sub-pathways of global genomic repair (GGR) and transcription-coupled repair (TCR). The TCR-deficient diseases, Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS), present indistinguishable biochemical responses to UV; UVSS patients have only superficial consequences of sunburn while those with CS suffer severe neurological/developmental defects, segmental progeria and early death. Notably, no cancers of any type have been reported for patients with these syndromes. We hypothesize that the severe features of CS are due to apoptosis triggered by prolonged transcription arrest as a consequence of defective TCR of oxidative base damage or to defective transcriptional bypass of such damage generated by endogenous reactive oxygen species, while UVSS cells are normal with respect to processing base damage in expressed genes. In support of this model we find that CS cells are hypersensitive to oxidants, and that UVSS but not CS cells are proficient in host cell reactivation of plasmids containing oxidized bases. However, definitive biochemical evidence for TCR of oxidized bases is lacking. Our model for TCR postulates that an arrested RNA polymerase (RNAP) recruits repair enzymes to transcription- blocking lesions. Reported studies with an oxidized base positioned at a unique site in the DNA template strand indicate that RNAP can bypass, transiently pause or arrest at these lesions. We propose to use a novel transcription assay with multiple randomly-positioned lesions induced in the template, to let the transcription system tell us which lesions and which sequence contexts are most relevant for further analysis. After determining the types and positions of the lesions that cause arrest in vitro, we will construct single-lesion vectors for transfection into human cells to measure in vivo transcription rates upstream and downstream of the lesion; sequencing the transcripts will reveal transcriptional mutagenesis. We propose to develop the sensitive Comet-FISH approach with gene-specific probes to comparatively quantify low levels of particular oxidative lesions and their removal from transcribed or silent sequences and from the genome overall. Cells with missing or reduced base excision repair or NER activities will be employed to investigate processing of oxidative lesions. Specific enhancement of 8oxoG in DNA will be achieved by interference RNA-mediated MTH1 knockdown, to eliminate complications of other lesions and other oxidative effects. We will focus on differences between CS and UVSS as a model system to elucidate the role of processing of oxidative base damage in aging, disease and neurological degeneration, as well as the underlying cause of the cancer-resistance of these syndromes. PUBLIC HEALTH RELEVANCE: Free radicals from endogenous and environmental sources are a constant threat to genomic integrity. The induced damage can arrest DNA and RNA polymerases, events that can unleash irreversible apoptotic pathways or mutagenicity. We propose novel approaches for elucidation of the effects of oxidative DNA lesions on transcription, and for the analysis of repair of physiologically relevant levels of these lesions in transcriptionally active or silent genomic domains and in the genome overall, using the Comet- FISH assay. Results from the project will advance our understanding of cellular processes leading to carcinogenesis, aging, and other pathologies. They will also further the development of effective strategies for therapeutic intervention in human disease.

描述（由申请人提供）：我们的总体目标是了解受损DNA的处理如何与人类遗传疾病，癌症和衰老相关。在率先发现核苷酸切除修复（NER）之后，我们正在阐明全局基因组修复（GGR）和转录偶联修复（TCR）的子途径。TCR缺陷性疾病，Cockayne综合征（CS）和紫外线敏感综合征（UVSS），目前难以区分的生化反应，紫外线; UVSS患者只有表面的晒伤的后果，而与CS遭受严重的神经/发育缺陷，节段性早衰症和早期死亡。值得注意的是，没有任何类型的癌症已报告的患者与这些综合征。 我们假设CS的严重特征是由于细胞凋亡引发的长期转录停滞作为结果的缺陷TCR的氧化性碱基损伤或缺陷的转录旁路的内源性活性氧产生的这种损伤，而UVSS细胞是正常的相对于在表达的基因中处理碱基损伤。在这个模型的支持，我们发现CS细胞是氧化剂过敏，而不是CS细胞的UVSS是精通宿主细胞中的质粒含有氧化的碱基的再活化。然而，缺乏氧化碱基TCR的明确生物化学证据。我们的TCR模型假设RNA聚合酶（RNAP）被抑制，募集修复酶到转录阻断损伤。在DNA模板链中的独特位点定位氧化碱基的报告的研究表明，RNAP可以绕过，短暂暂停或停止在这些病变。我们建议使用一种新的转录检测方法，在模板中诱导多个随机定位的病变，让转录系统告诉我们哪些病变和哪些序列背景最相关，以进行进一步分析。在确定导致体外停滞的病变的类型和位置后，我们将构建用于转染到人类细胞中的单病变载体，以测量病变上游和下游的体内转录速率;对转录物进行测序将揭示转录突变。 我们建议开发敏感的彗星-FISH方法与基因特异性探针，以相对定量低水平的特定氧化损伤和它们从转录或沉默序列和基因组整体的去除。缺失或减少碱基切除修复或NER活性的细胞将用于研究氧化损伤的处理。DNA中8 oxoG的特异性增强将通过干扰RNA介导的MTH 1敲减来实现，以消除其他病变和其他氧化作用的并发症。我们将专注于CS和UVSS之间的差异作为一个模型系统，以阐明氧化性碱损伤的处理在衰老，疾病和神经退行性变中的作用，以及这些综合征的抗癌性的根本原因。 公共卫生相关性：来自内源性和环境来源的自由基是对基因组完整性的持续威胁。诱导的损伤可以阻止DNA和RNA聚合酶，这些事件可以释放不可逆的凋亡途径或致突变性。 我们提出了新的方法来阐明氧化DNA损伤对转录的影响，并分析这些损伤在转录活性或沉默的基因组结构域和整个基因组中的生理相关水平的修复，使用彗星- FISH测定。 该项目的结果将促进我们对导致致癌，衰老和其他病理的细胞过程的理解。它们还将进一步发展人类疾病治疗干预的有效战略。