Temporal and Spatial Relationships of Proteins in Yeast NER

描述(申请人提供)：紫外线阳光会在DNA中引发化学反应，如果允许积累，就会导致皮肤细胞突变，最终导致皮肤癌的发展。防止紫外线对皮肤致癌作用的主要生物防御措施之一是核苷酸切除修复(NER)，这是一种进化来消除DNA损伤的生物途径，包括紫外线诱导的损伤。NER的分子基础正在被阐明，但关于事件的顺序，特别是NER步骤进展的机制，仍然存在许多问题。本建议的具体目的是在进化保守的真核模型系统中确定酵母酿酒酵母：(1)NER是否发生在细胞核内的空间定位区域，以及蛋白质Radio、Rad14和RAD23的招募时间和顺序；(2)DNA损伤识别蛋白因子Rad14，可能还有Rad4/RAD23复合体在下游蛋白质Radio参与之前是必需的；以及(3)Rad1蛋白的关键氨基酸残基是否需要将Rad1/Radio复合体招募到NER位点。这些问题将用一种新的技术来回答，在荧光显微镜的帮助下，在活的酵母细胞中标记和跟踪蛋白质。细胞将暴露在紫外线下，通过比较标记蛋白质和突变基因的适当组合来回答上述问题，将作为时间的函数来监测蛋白质向DNA修复中心的招募。 在初步实验中，已经对其中一种蛋白质Radio进行了荧光标记，并对其在NER中的作用进行了测试；结果表明，NER存在于细胞核内的空间定位区域。由于技术原因，这些实验在酿酒酵母模型系统中进行，而不是在人类细胞中进行。然而，由于NER途径的进化保守性，这些发现将可以转移到人类系统。皮肤癌是美国社会日益严重的健康问题，原因是人体暴露在破坏性紫外线阳光下的水平增加。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关键部分。更详细地了解细胞修复DNA的生物化学将有助于为可能将皮肤癌风险降至最低的药物寻找新的潜在药物靶点。

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.