ROLE OF 5-METHYLCYTOSINE IN THE PHOTOCHEMISTRY OF DNA

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 已知紫外（UV）光在被活细胞吸收后会引起许多效应。其中包括基因突变、细胞死亡和细胞转化。这些后一种类型的损伤至少部分地由细胞基因组DNA中形成的光产物介导。随着UVB光（290至320 nm之间）在生物体环境中的重要性日益增加，由于地球臭氧层的消耗，UV诱导的DNA损伤在诱导皮肤癌和其他有害生物效应方面可能发挥越来越大的作用。 该项目中描述的工作的最终结果将增加对哺乳动物和植物DNA在吸收UVB光后发生的化学反应的理解。特别是，该提案侧重于次要碱基5-甲基胞嘧啶（m5 C）在介导由UVB光吸收引起的损伤中的作用。本项目有两个目标。第一个目标是针对隔离和表征的一些photoproducts，我们认为是相关的实现化学的理解UVB诱导的DNA损伤。这些化合物是c，s和t，s混合环丁烷二聚体，（6-4）加合物和杜瓦加合物的m5 C与胸腺嘧啶和胞嘧啶，无论是在核碱基和核苷的形式。为了实现该目的，将利用避免这些化合物不稳定的条件的程序来制备化合物。将使用高效液相色谱法（HPLC）分离纯态的各种化合物。然后，使用质子和碳-13 NMR光谱、UV光谱、电喷雾电离和MALDI质谱以及适当的圆二色性光谱对其进行结构表征。还将测定它们在DNA消化条件下的化学稳定性。 第二个目标是确定在与目标1相关的实验中分离的各种产物是否也在DNA的背景下和细胞核的环境中形成。重点介绍了小牛胸腺DNA和细胞核、小麦DNA和细胞核四种体系。前一种DNA的m5 C含量与人类DNA相似（约5%的DNA胞嘧啶被甲基化）;后一种DNA约有30%的胞嘧啶被甲基化。用于检测辐照DNA中这些产物的方案包括以下步骤。照射后，蛋白质将被去除，DNA将被温和的化学核酸酶消化。将通过HPLC检查消化物，以确定是否存在与目标m5 C产品真实样品具有相同保留时间的产品;为此，将使用UV、荧光和质谱检测来验证HPLC色谱图中的目标产品与真实产品具有相同特性。将测定各种产物的产率，并将其与涉及其他嘧啶碱基配对的相应光产物的公开产率进行比较。还将进行实验以确定在这些UVB照射的系统中是否形成脲基丙烯腈和丙烯脒型m5 C化合物，例如先前在我的实验室中分离的化合物。

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.