ROLE OF 5-METHYLCYTOSINE IN THE PHOTOCHEMISTRY OF DNA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 众所周知，紫外线被活细胞吸收后会引起一系列影响。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是由细胞基因组DNA中形成的光产物介导的。随着UVB光(290-320 nm)在生物环境中的重要性日益增加，由于地球臭氧层的耗尽，紫外线诱导的DNA损伤在诱发皮肤癌和其他有害的生物效应方面可能发挥越来越大的作用。 这个项目中描述的工作的最终结果将是增加对哺乳动物和植物DNA吸收UVB光后发生的化学变化的理解。特别是，该提案侧重于次要碱基5-甲基胞嘧啶(M5C)在调节UVB光吸收所致损伤中的作用。本项目有两个组成部分的目标。第一个目标是分离和鉴定一些我们认为与实现对UVB诱导的DNA损伤的化学理解相关的光产物。这些化合物是c，S和t，S混合环丁烷二聚体，(6-4)M5C与胸腺嘧啶和胞嘧啶的(6-4)加合物和杜瓦加合物，以碱基和核苷的形式存在。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来制备这些化合物。高效液相色谱(HPLC)将被用来分离各种纯状态的化合物。然后，将使用质子和碳-13核磁共振光谱、紫外光谱、电喷雾电离和MALDI质谱学以及适当的圆二色谱对它们进行结构表征。还将测定它们在DNA消化条件下的化学稳定性。 第二个目的是确定在与目标1相关的实验中分离出的各种产物是否也是在DNA的背景下和在细胞核环境中形成的。将重点介绍四种类型的系统，即小牛胸腺DNA和核以及小麦DNA和核。前者的M5C含量与人类DNA相似(约5%的DNA胞嘧啶被甲基化)；后者的DNA约有30%的胞嘧啶甲基化。用于检测辐照DNA中的这些产物的方案包括以下步骤。辐射后，蛋白质将被去除，DNA将被温和的化学核酸酶消化。摘要将由高效液相色谱检查，以确定是否存在与感兴趣的M5C产品的真品具有相同保留时间的产品；为此，将使用紫外光、荧光和质谱检测来验证高效液相色谱法色谱图中的感兴趣的产品与真品具有相同的性质。各种产物的产率将被测定，并与涉及其他嘧啶碱基配对的相应光产物的公布产率进行比较。还将进行实验，以确定在这些UVB照射的系统中是否形成了M5C的脲-丙烯腈和丙烯酰胺类化合物，例如以前在我的实验室分离的化合物。

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.