Oxidative DNA damage processing; role in human pathology and aging

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病、癌症和衰老有关。在发现核苷酸切除修复(NER)之后，我们正在阐明全球基因组修复(GGR)和转录偶联修复(TCR)的子途径。Cockayne综合征(CS)和紫外线敏感综合征(UVSS)是TCR缺乏的疾病，对紫外线的生化反应不明显；UVSS患者只有表面的晒伤后果，而CS患者则出现严重的神经/发育缺陷、节段性早衰和过早死亡。值得注意的是，没有关于这些综合征患者的任何类型癌症的报道。我们推测，CS的严重特征是由于氧化碱基损伤的TCR缺陷或内源性活性氧物种对这种损伤的转录旁路缺陷而导致的长时间转录停滞引发的细胞凋亡，而UVSS细胞在处理表达的基因中的碱基损伤方面是正常的。为了支持这一模型，我们发现CS细胞对氧化剂高度敏感，而UVSS而不是CS细胞擅长宿主细胞重新激活含有氧化碱基的质粒。然而，氧化碱基的TCR缺乏确凿的生化证据。我们的TCR模型假设，受阻的RNA聚合酶(RNAP)将修复酶招募到转录阻断的病变中。报道的研究表明，RNAP可以绕过、短暂暂停或停止在这些病变处。我们建议使用一种新的转录实验，在模板中诱导多个随机定位的损伤，让转录系统告诉我们哪些损伤和哪些序列上下文最相关，以便进一步分析。在确定了在体外导致停滞的病变的类型和位置后，我们将构建单个病变载体，用于转染人类细胞，以测量病变上游和下游的体内转录速率；对转录产物进行测序将揭示转录突变。我们建议开发具有基因特异性探针的敏感的彗星-FISH方法，以相对定量低水平的特定氧化损伤及其从转录或沉默序列和整个基因组中的移除。碱基切除修复或NER活性缺失或降低的细胞将被用于研究氧化损伤的处理。通过干扰RNA介导的MTH1基因敲除，将实现DNA中8oxoG的特异性增强，以消除其他损伤的并发症和其他氧化效应。我们将集中讨论CS和UVSS之间的差异作为模型系统，以阐明氧化碱基损伤的处理在衰老、疾病和神经退行性变中的作用，以及这些综合征抗癌的潜在原因。 公共卫生相关性：来自内源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，这两个事件可以释放不可逆转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，并使用彗星-FISH分析来分析这些损伤在转录活跃或沉默的基因组区域以及整个基因组中生理相关水平的修复。该项目的结果将促进我们对导致癌症发生、衰老和其他病理的细胞过程的理解。它们还将进一步制定对人类疾病进行治疗干预的有效战略。

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.