Temporal and Spatial Relationships of Proteins in Yeast NER

描述（由申请人提供）：紫外线阳光诱导DNA中的化学反应，如果允许其积累，会引起皮肤细胞突变，最终导致皮肤癌的发展。针对紫外线对皮肤的致癌作用的主要生物防御之一是核苷酸切除修复（NER），这是一种生物途径，用于消除对DNA的损伤，包括紫外线诱导的病变。NER的分子基础正在阐明，但许多问题仍然存在关于事件的顺序，特别是NER进展步骤的机制。该建议的具体目标是在进化保守的真核模型系统中确定酵母S。酿酒酵母：（1）NER是否发生在核内的空间定位区域，以及蛋白质Radio、Rad 14和Rad 23的募集的时间和顺序;（2）在下游蛋白质Radio参与之前需要DNA损伤识别蛋白因子Rad 14和可能的Rad 4/Rad 23复合物;以及（3）Rad 1蛋白的关键氨基酸残基是否是将Rad 1/Radio复合物募集到NER位点所必需的。这些问题将使用一种新的技术来回答，在这种技术中，蛋白质在荧光显微镜的帮助下在活酵母细胞中被标记和跟踪。将细胞暴露于紫外线下，通过比较标记蛋白质和突变基因的适当组合来回答上述问题，将蛋白质向DNA修复中心的募集作为时间的函数进行监测。 在初步实验中，其中一种蛋白质Radio已经被荧光标记并测试了其在NER中的作用;结果表明NER发生在细胞核内的空间定位区域。这些实验是在美国进行的。酿酒酵母模型系统而不是人类细胞。然而，由于NER通路的进化保守性，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，这是由于人类暴露于有害的紫外线阳光的水平增加。临床癌症预防和治疗的新进展是解决这一日益严重的问题的重要组成部分。更详细地了解细胞修复DNA的生物化学将有助于为药物寻找新的潜在药物靶点，从而最大限度地降低皮肤癌风险。

DESCRIPTION (provided by applicant): UV sunlight induces chemical reactions in DNA 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 the U.S. 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.