Repair of Unusual DNA Structures

描述（由申请人提供）：核苷酸切除修复（NER）途径负责识别和修复多种螺旋扭曲DNA损伤，因此在维持基因组稳定性方面至关重要。 研究表明，NER可以进一步细分为快速转录偶联修复（TCR）途径，其优先修复转录基因，以及较慢的全局基因组修复（GGR）途径，其修复基因组的其余部分。 随着序列特异性三链体形成寡核苷酸（TFO）可以作为第三链与双链体DNA结合以产生非规范螺旋结构的发现，引入螺旋扭曲损伤以监测基因组中特定位点的修复的方法成为可能。 该提案的长期目标是使用产生位点特异性螺旋改变的技术来了解TCR和GGR途径在修复改变的DNA结构中的相对贡献。 该提案的具体目标是：1.比较TCR途径与GGR途径在修复异常DNA结构中的作用。2.研究不同螺旋扭曲分子的修复和识别。 将使用多种位点特异性DNA结合分子、源自人类修复缺陷细胞系的无细胞提取物以及DNA修复因子和纯化蛋白质的特异性抗体的组合进行旨在监测不寻常DNA结构的识别和修复的实验。 具体而言，GGR蛋白，XPC，和TCR蛋白，CSB，在识别和修复改变的DNA结构的作用将被检查和比较的NER蛋白，XPA的作用。

DESCRIPTION (provided by applicant): The nucleotide excision repair (NER) pathway is responsible for the recognition and repair of a diverse array of helix distorting DNA lesions and is therefore crucial in the maintenance of genomic stability. Studies have revealed that NER can be further subdivided into the fast transcription coupled repair (TCR) pathway, which preferentially repairs transcribed genes, and the slower global genome repair (GGR) pathway, which repairs the remainder of the genome. With the discovery that sequence-specific triplex forming oligonucleotides (TFOs) could bind as a third strand to duplex DNA to create a non-canonical helical structure, a method of introducing helix distorting lesions to monitor repair at specific sites in the genome became possible. The long-term objective of this proposal is to use the technology of producing site-specific helical alterations to understand the relative contributions of the TCR and GGR pathways in the repair of altered DNA structures. The specific aims of this proposal are: 1. To compare the roles of the TCR pathway versus the GGR pathway in the repair of unusual DNA structures. 2. To examine the repair and recognition of different helix distorting molecules. Experiments designed to monitor recognition and repair of unusual DNA structures will be performed using combinations of a variety of site-specific DNA binding molecules, cell-free extracts derived from human repair deficient cell lines, and specific antibodies to DNA repair factors and purified proteins. Specifically, the roles of the GGR protein, XPC, and the TCR protein, CSB, in recognition and repair of altered DNA structures will be examined and compared to the role of the NER protein, XPA.