Temporal and Spatial Relationships of Proteins in Yeast NER

紫外线会在皮肤中引发化学反应，如果允许累积，就会导致皮肤突变 并最终导致皮肤癌的发展。针对这种病毒的主要生物防御之一 紫外线对皮肤的致癌作用是核苷酸切除修复(NER)，这是一种进化到 去除DNA损伤，包括紫外线引起的损伤。NER的分子基础是 得到了澄清，但关于事件的顺序，特别是通过什么机制 NER的步骤是进步的。这项提案的具体目标是在进化上确定一种 保守的真核模型系统，酵母，酿酒酵母：1)NER是否存在于空间定域化 核内区域和蛋白质Radio、Rad14和Rad23的招募时间和顺序， 2)DNA损伤识别蛋白因子Rad14和可能的Rad4/RAD23复合体是 在参与下游蛋白质无线电之前需要，以及3)关键氨基酸残基是否 Rad1蛋白，是将Rad1/Radio复合体招募到NER位点所必需的。这些 问题将使用一种新技术来回答，在这种技术中，蛋白质被标记并在体内进行跟踪 在荧光显微镜的帮助下培养酵母细胞。细胞会暴露在紫外光下并进行募集 蛋白质到DNA修复中心的比例将作为时间的函数进行监测，通过比较适当的 结合标记蛋白和突变基因来回答上述问题。在预赛中 实验中，其中一种蛋白质，Radio，已经被荧光标记并测试其在 NER；结果表明，NER存在于核内的空间定域区。这些实验 出于技术原因，是在酿酒酵母模型系统而不是人类细胞中进行的。 然而，由于NER途径的进化保守性，这些发现将转移到 人类系统。皮肤癌是美国社会中一个日益严重的健康问题，因为人类 暴露在破坏性紫外线阳光下的水平。临床癌症预防和治疗的新进展 这是解决这一日益严重的问题的关键部分。对生物化学有更详细的了解 细胞修复DNA将有助于为可能最小化皮肤的药物寻找新的潜在药物靶点 癌症风险。

UV sunlight induces chemical reactions in DMA which, if allowed to accumulate, give rise to mutations in skin cells and eventually lead to the development of skin cancer. One of the chief biological defenses against the carcinogenic effects of UV light on skin is Nucleotide Excision Repair (NER), a biologic pathway evolved to remove damage to DNA including UV light-induced lesions. The molecular underpinnings of NER are being elucidated, but many questions remain regarding the order of events, particularly, the mechanisms by which the steps of NER progress. The specific aims of this proposal are to determine in an evolutionarily conserved eukaryotic model system, the yeast, S. cerevisiae: 1) whether NER occurs in spatially localized regions within the nucleus and the timing and order of recruitment of the proteins Radio, Rad14 and Rad23, 2) that the DNA damage recognition protein factor Rad14 and perhaps the Rad4 / Rad23 complex are required prior to participation of the downstream protein Radio, and 3) whether key amino acid residues of the Rad1 protein, are required for recruitment of the Rad1 / Radio complex to an NER site. These questions will be answered using a novel technique in which the proteins are labeled and tracked in live yeast cells with the aid of a fluorescence microscope. Cells will be exposed to UV light and the recruitment of the proteins to DNA repair centers will be monitored as a function of time by comparing appropriate combinations of labeled proteins and mutant genes to answer the above questions. In preliminary experiments, one of the proteins, Radio, has already been fluorescently labeled and tested for its action in NER; results suggest that NER occurs in spatially localized regions within the nucleus. These experiments are being carried out in the S. cerevisiae model system rather than human cells for technical reasons. However, due to the evolutionary conservation of the NER pathway, the findings will be transferable to the human system. Skin cancer is a growing health problem in US society as a result of increased human exposure levels to damaging UV sunlight. New advances in clinical cancer prevention and treatment are a vital part of addressing this growing problem. A more detailed understanding of the biochemistry by which cells repair DNA will aid in finding new potential drug targets for Pharmaceuticals that might minimize skin cancer risk.