ROLE OF 5-METHYLCYTOSINE IN THE PHOTOCHEMISTRY OF DNA

5-甲基胞嘧啶在 DNA 光化学中的作用

• 批准号: 7369070
• 负责人: MARTIN D SHETLAR
• 金额: $ 0.05万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369070
• 关键词: ROLE; METHYLCYTOSINE; PHOTOCHEMISTRY; DNA

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ultraviolet (UV) light is known to cause a number of effects after absorption by living cells. Among these are genetic mutation, cell death and cellular transformation. These latter types of damage are mediated, at least in part, by photoproducts formed in the genomic DNA of the cell. With the increasing importance of UVB light (between 290 and 320 nm) in the environment of living organisms, due to depletion of the ozone layer of the Earth, UV-induced lesions in DNA could play an increasing role in induction of skin cancer and other unwanted biological effects. The end result of the work described in this project would be increased understanding of what happens chemically to mammalian and plant DNA after it absorbs UVB light. In particular, the proposal focuses on the role of the minor base 5-methylcytosine (m5C) in mediating damage induced by absorption of UVB light. The present project has two component aims. The first aim is directed towards isolating and characterizing a number of photoproducts that we believe are relevant to achieving a chemical understanding of UVB-induced DNA damage. These compounds are the c,s and t,s mixed cyclobutane dimers , (6-4) adducts and Dewar adducts of m5C with thymine and cytosine, both in nucleobase and nucleoside form. To achieve this aim, the compounds will be prepared utilizing procedures that avoid conditions under which these compounds are unstable. High performance liquid chromatography (HPLC) will be used to isolate the various compounds in a pure state. They will then be structurally characterized using proton and carbon-13 NMR spectroscopy, UV spectroscopy, electrospray ionization and MALDI mass spectrometry and, where appropriate, circular dichroic spectroscopy. Their chemical stability under conditions of DNA digestion will also be determined. The second Aim is to determine if the various products isolated in experiments related to Aim 1 are also formed within the context of DNA and in the environment of a cell nucleus. Four types of system will be focussed on, namely calf thymus DNA and nuclei and wheat DNA and nuclei. The former DNA has a m5C content similar to human DNA (about 5% of DNA cytosines are methylated); the latter DNA has about 30% methylated cytosines. The protocols used for detection of these products in irradiated DNA include the following steps. After irradiation, protein will be removed and the DNA will be digested with gentle chemical nucleases. The digest will be examined by HPLC, to determine whether products are present that have identical retention times as those of authentic samples of the m5C products of interest; for this purpose, UV, fluorescence and mass spectral detection will be used to verify that the products of interest in the HPLC chromatogram have identical properties to those of authentic product. Yields of the various products will be determined and compared to published yields of corresponding photoproducts involving other pyrimidine base pairings. Experiments will also be conducted to determine if ureidoacrylonitrile and acrylamidine type compounds of m5C, such as isolated previously in my laboratory, are formed in these UVB irradiated systems.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。已知紫外线（UV）光在活细胞吸收后会引起许多影响。其中包括遗传突变，细胞死亡和细胞转化。这些后一种类型的损伤至少部分是由细胞基因组DNA中形成的光产物介导的。由于地球臭氧层的耗尽，在活生物体环境中，UVB光的重要性（在290 nm之间）的重要性越来越多，紫外线诱导的DNA病变可能在诱导皮肤癌和其他不必要的生物学作用中起着越来越多的作用。 该项目描述的工作的最终结果将是对哺乳动物和植物DNA吸收UVB光后化学发生的情况的了解。特别是，该提案的重点是次要基碱5-甲基胞嘧啶（M5C）在吸收UVB光引起的损害中的作用。本项目具有两个组成目标。第一个目的是针对隔离和表征许多我们认为与对UVB诱导的DNA损伤的化学理解有关的光产物。这些化合物是C，S和T，S混合环丁烷二聚体，（6-4）的M5C与胸腺氨酸和胞嘧啶的加合物合并，均以核苷酶和核苷形式形式。为了实现这一目标，将使用避免这些化合物不稳定的条件的程序准备好化合物。高性能液相色谱（HPLC）将用于隔离纯态的各种化合物。然后，它们将在结构上使用质子和碳-13 NMR光谱，紫外光谱，电喷雾电离和MALDI质谱法以及适当的圆形二分色谱法进行结构表征。在DNA消化条件下，它们的化学稳定性也将得到确定。 第二个目的是确定在与AIM 1相关的实验中分离的各种产物是否也在DNA的背景下以及在细胞核的环境中形成。四种类型的系统将集中在小腿胸腺DNA，核以及小麦DNA和核。前DNA具有类似于人DNA的M5C含量（大约5％的DNA胞嘧啶是甲基化的）；后一种DNA的甲基化胞嘧啶约为30％。用于在辐照的DNA中检测这些产品的方案包括以下步骤。辐照后，将去除蛋白质，并用温和的化学核酸酶消化DNA。 HPLC将检查摘要，以确定存在保留时间是否与感兴趣的M5C产品的真实样品相同的产品；为此，将使用紫外线，荧光和质谱检测来验证HPLC色谱图中感兴趣的产物是否具有与真实产物相同的特性。将确定各种产品的产量，并将其与涉及其他嘧啶基碱基配对的相应光产物的已发表的产量进行比较。还将进行实验，以确定M5C的尿素丙烯腈和丙烯酰胺型化合物（例如先前在我的实验室中分离出来）是否在这些UVB辐照系统中形成。