Effects of UV Irradiation on DNA Structure-induced Genetic Instability

描述(申请人提供)：虽然大多数DNA以标准的B-DNA形式存在，但重复的DNA序列可以形成另一种结构的DNA(或非B-DNA)，如H-DNA和Z-DNA。这些非B-DNA形成序列在哺乳动物基因组中含量丰富，经常与脆性X综合征、亨廷顿病和强直性肌营养不良等疾病有关。我们发现H-DNA和Z-DNA形成序列在哺乳动物细胞和小鼠中具有高度的突变性，并且经常与染色体断裂热点共定位，例如在Burkitt淋巴瘤中映射到易位断裂点热点的c-myc基因的启动子区域。此外，突变的H-DNA和Z-DNA形成序列刺激DNA双链断裂(DSB)，导致缺失和易位，提示它们与易位相关的疾病病因学有关。除了它们固有的不稳定性外，已经证明来自外源的DNA损伤可以被富含在非B形成序列中，并且这种损伤可能是难以修复的。来自太阳的紫外线辐射是一种普遍存在的环境致癌物质，我们每天都会接触到这种物质，并与黑色素瘤等癌症有关。在B-DNA中，紫外线照射会导致DNA损伤，如6-4个光产物(6-4pps)和环丁烷嘧啶二聚体(CPDS)。然而，外源DNA损伤剂如紫外线照射如何影响H-DNA和Z-DNA结构的形成、稳定性和诱变潜力还不是很清楚。我们的长期目标是了解紫外线辐射如何影响非B DNA结构导致的人类疾病的遗传不稳定性。这项提议的直接目标是检验这样一个假设，即非B DNA区域将比B-DNA更容易受到紫外线照射的损伤，并且更难修复。我们将使用哺乳动物细胞系统和我们实验室之前开发的突变报告测试来验证这一假设。特定目标1解决紫外线照射后非B DNA序列的诱变潜力。我们将使用我们的突变报告试验在哺乳动物COS-7细胞中测试紫外线照射后非B DNA的诱变潜力。具体目标2涉及在非B DNA形成序列和结构内及其周围形成紫外线损伤的可能性。我们将确定非B DNA结构如何影响病变形成，以及病变形成如何影响非B DNA结构的形成。特定目的3描述了使用缺乏不同修复蛋白的细胞系在非B DNA结构上处理紫外线损伤所涉及的修复途径。

DESCRIPTION (provided by applicant): While most DNA exists in the canonical B-DNA form, repetitive DNA sequences can form alternatively structured DNA (or non-B-DNA) such as H-DNA and Z-DNA. These non-B DNA-forming sequences are abundant in mammalian genomes and are often associated with diseases such as Fragile X Syndrome, Huntington's disease and myotonic dystrophy. We have found that H-DNA and Z-DNA-forming sequences are highly mutagenic in mammalian cells and in mice, and often co-localize with chromosome breakage hotspots, such as in the promoter region of the c-MYC gene that maps to a translocation breakpoint hotspot in Burkitt's lymphoma. Furthermore, the mutagenic H-DNA and Z-DNA-forming sequences stimulate DNA double-strand breaks (DSBs), leading to deletions and translocations, implicating them in translocation-related disease etiology. In addition to their intrinsic instability, it has been demonstrated that DNA damage from exogenous sources may be enriched in non-B-forming sequences and this damage may be refractory to repair. UV irradiation from the sun is a ubiquitous environmental carcinogen that we are exposed to on a daily basis and has been associated with cancers such as melanoma. In B-DNA, UV irradiation causes the formation of DNA lesions such as 6-4 photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). However, how exogenous DNA damaging agents such as UV irradiation affect H-DNA and Z-DNA structure formation, stability, and mutagenic potential is not well understood. Our long-term objectives are to understand how UV irradiation affects non-B DNA structure-induced genetic instability in human disease. The immediate objectives of this proposal are to test the hypothesis that non-B DNA regions will be more prone to UV irradiation damage, and more refractory to repair than B-DNA. We will test this hypothesis using a mammalian cell system and a mutation reporter assay our lab has previously developed. Specific Aim 1 addresses the mutagenic potential of non-B DNA sequences after UV exposure. The mutagenic potential of non-B DNA after UV irradiation will be tested in mammalian COS-7 cells using our mutation reporter assay. Specific Aim 2 addresses the potential of UV lesion formation within and surrounding non-B DNA-forming sequences and structures. We will determine how non-B DNA structures affect lesion formation and how lesion formation affects the formation of non-B DNA structures. Specific Aim 3 characterizes the repair pathways involved in processing UV lesions at the non-B DNA structures using cell lines deficient in different repair proteins.