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，我们正在阐明全球基因组修复（GGR）和转录偶联修复（TCR）的子通路。tcr缺陷疾病，Cockayne综合征（CS）和UV敏感综合征（UVSS），对UV的生化反应没有区别；UVSS患者只有晒伤的表面后果，而CS患者则有严重的神经/发育缺陷、节段性早衰和早期死亡。值得注意的是，没有任何类型的癌症报告的患者与这些综合征。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导致的长时间转录停滞引发的细胞凋亡，而UVSS细胞在表达基因的加工碱基损伤方面是正常的。为了支持这一模型，我们发现CS细胞对氧化剂过敏，并且UVSS而不是CS细胞精通宿主细胞对含有氧化碱基的质粒的再激活。然而，氧化碱的TCR的明确的生化证据是缺乏的。我们的TCR模型假设阻滞RNA聚合酶（RNAP）招募修复酶到转录阻断病变。有报道称，在DNA模板链的独特位置上有一个氧化碱基的研究表明，RNAP可以绕过、暂时停止或阻止这些损伤。我们建议使用一种新的转录试验，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。在体外确定引起阻滞的病变类型和位置后，我们将构建单病变载体转染到人细胞中，以测量病变上游和下游的体内转录率；转录本测序将揭示转录突变。我们建议开发敏感的Comet-FISH方法，使用基因特异性探针来相对量化低水平的特定氧化损伤，并从转录或沉默序列以及整个基因组中去除它们。缺失或减少碱基切除修复或NER活性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敲低，可以实现DNA中8oxoG的特异性增强，从而消除其他病变并发症和其他氧化效应。我们将重点关注CS和UVSS之间的差异，作为一个模型系统来阐明氧化基损伤在衰老、疾病和神经退行性变中的作用，以及这些综合征耐癌的潜在原因。

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.