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 的处理与人类遗传疾病、癌症和衰老之间的关系。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，科凯恩综合征 (CS) 和紫外线敏感综合征 (UVSS)，对紫外线表现出难以区分的生化反应； UVSS 患者仅遭受晒伤的表面后果，而 CS 患者则遭受严重的神经/发育缺陷、节段性早衰和过早死亡。值得注意的是，尚未报告患有这些综合征的患者罹患任何类型的癌症。 我们假设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产生的这种损伤的转录旁路缺陷造成的长期转录停滞引发的细胞凋亡，而UVSS细胞在表达基因中处理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，并且UVSS而非CS细胞能够熟练地重新激活含有氧化碱基的质粒的宿主细胞。然而，缺乏氧化碱基 TCR 的明确生化证据。我们的 TCR 模型假设，停滞的 RNA 聚合酶 (RNAP) 会招募修复酶来修复转录阻断病变。报道的研究表明，氧化碱基位于 DNA 模板链的独特位点，RNAP 可以绕过、短暂暂停或阻止这些病变。我们建议使用一种新颖的转录测定法，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与进一步分析最相关。在确定体外引起阻滞的病灶类型和位置后，我们将构建单病灶载体转染至人体细胞中，测量病灶上下游的体内转录率；对转录本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彗星 FISH 方法，以相对量化低水平的特定氧化损伤及其从转录或沉默序列以及整个基因组中的去除。碱基切除修复或 NER 活性缺失或减少的细胞将用于研究氧化损伤的处理。 DNA中8oxoG的特异性增强将通过干扰RNA介导的MTH1敲低来实现，以消除其他病变的并发症和其他氧化效应。我们将重点关注CS和UVSS作为模型系统之间的差异，以阐明氧化碱损伤处理在衰老、疾病和神经变性中的作用，以及这些综合征抗癌的根本原因。 公共健康相关性：来自内源性和环境来源的自由基对基因组完整性构成持续威胁。诱导的损伤可以抑制 DNA 和 RNA 聚合酶，从而释放不可逆的细胞凋亡途径或致突变性。 我们提出了新的方法来阐明氧化性 DNA 损伤对转录的影响，并使用 Comet-FISH 分析分析转录活性或沉默基因组结构域以及整个基因组中这些损伤的生理相关水平的修复。 该项目的结果将增进我们对导致致癌、衰老和其他病理的细胞过程的理解。他们还将进一步开发治疗干预人类疾病的有效策略。

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.