Effects of UV Irradiation on DNA Structure-induced Genetic Instability

描述（由申请人提供）：虽然大多数DNA以标准的B-DNA形式存在，但重复的DNA序列可以形成替代结构的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大量存在，通常与脆性X综合征、亨廷顿氏病和肌强直性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诱变性，并且经常与染色体断裂热点共定位，例如在Burkitt淋巴瘤中定位易位断点热点的c-MYC基因启动子区域。此外，诱变的H-DNA和z -DNA形成序列刺激DNA双链断裂（DSBs），导致缺失和易位，暗示它们与易位相关的疾病病因有关。除了它们固有的不稳定性外，已经证明来自外源的DNA损伤可能富集在非b形成序列中，这种损伤可能难以修复。来自太阳的紫外线辐射是一种无处不在的环境致癌物，我们每天都接触到它，它与黑色素瘤等癌症有关。在B-DNA中，紫外线照射导致6-4光产物（6-4PPs）和环丁烷嘧啶二聚体（CPDs）等DNA损伤的形成。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、稳定性和致突变潜力尚不清楚。我们的长期目标是了解紫外线照射如何影响人类疾病中非b DNA结构诱导的遗传不稳定性。本提案的直接目标是验证非b DNA区域比b DNA更容易受到紫外线照射损伤和更难修复的假设。我们将使用哺乳动物细胞系统和我们实验室先前开发的突变报告试验来验证这一假设。特异性目标1解决了紫外线照射后非b DNA序列的致突变潜力。我们的突变报告实验将在哺乳动物COS-7细胞中测试紫外线照射后非b DNA的致突变潜力。特异性目标2解决了非b dna形成序列和结构内部和周围紫外线损伤形成的可能性。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。特异性Aim 3描述了利用缺乏不同修复蛋白的细胞系处理非b DNA结构的紫外线损伤的修复途径。

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.