Temporal and Spatial Relationships of Proteins in Yeast NER

描述（由申请人提供）：紫外线阳光会引起DNA中的化学反应，如果任由其积累，会引起皮肤细胞的突变，最终导致皮肤癌的发展。针对紫外线对皮肤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，可以消除对DNA的损伤，包括紫外线引起的病变。NER的分子基础正在被阐明，但关于事件的顺序，特别是NER进展的步骤的机制仍然存在许多问题。本研究的具体目的是确定在进化保守的真核生物模型系统酵母酿酒酵母中：(1)NER是否发生在细胞核内的空间局部区域，以及Radio、Rad14和Rad23蛋白募集的时间和顺序；(2) DNA损伤识别蛋白因子Rad14和可能的Rad4/Rad23复合体在下游蛋白Radio参与之前是必需的；(3) Rad1蛋白的关键氨基酸残基是否需要Rad1/Radio复合物募集到NER位点。这些问题将使用一种新的技术来回答，这种技术是在荧光显微镜的帮助下在活酵母细胞中标记和跟踪蛋白质。细胞将暴露在紫外线下，通过比较标记蛋白和突变基因的适当组合来监测蛋白质向DNA修复中心的募集情况，并将其作为时间的函数来回答上述问题。

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.