Temporal and Spatial Relationships of Proteins in Yeast NER

紫外线阳光诱导DMA的化学反应，如果允许积累，会引起皮肤突变 细胞，并最终导致皮肤癌的发展。生物学上主要的防御手段之一是 紫外线对皮肤的致癌作用是核苷酸切除修复（NER），这是一种生物途径， 去除DNA损伤，包括紫外线引起的损伤。NER的分子基础正在被 阐明，但许多问题仍然关于事件的顺序，特别是， 净入学率进步的步骤。这项建议的具体目的是以一种渐进的方式确定 保守真核生物模型系统、酵母、S.酿酒酵母：1）NER是否发生在空间定位 细胞核内的区域以及蛋白质Radio、Rad 14和Rad 23的募集的时间和顺序， 2)DNA损伤识别蛋白因子Rad 14和可能的Rad 4/Rad 23复合物是 参与之前所需的下游蛋白质的放射性，和3）是否关键的氨基酸残基， Rad 1蛋白是将Rad 1/ Radio复合物募集到NER位点所必需的。这些 这些问题将使用一种新的技术来回答，在这种技术中，蛋白质被标记并在活体中被跟踪。 借助荧光显微镜观察酵母细胞。细胞将暴露于紫外线下， 将通过比较适当的蛋白质与DNA修复中心之间的关系来监测蛋白质与DNA修复中心之间的关系。 标记蛋白和突变基因的组合来回答上述问题。初步 在实验中，其中一种蛋白质，无线电，已经被荧光标记并测试了它的作用， NER;结果表明，NER发生在核内的空间局部区域。这些实验 正在进行中的S.酿酒酵母模型系统而不是人类细胞。 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日益严重的健康问题，这是由于人类癌症的增加。 暴露在有害的紫外线阳光下。癌症临床预防和治疗的新进展是 解决这一日益严重的问题的关键。更详细地了解生物化学， 细胞修复DNA将有助于为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.