Repair of Unusual DNA Structures

描述(申请人提供)：核苷酸切除修复(NER)途径负责识别和修复一系列不同的螺旋扭曲的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可进一步细分为快速转录偶联修复(TCR)途径和较慢的全球基因组修复(GGR)途径，前者优先修复转录基因，后者修复基因组的其余部分。随着序列特异性三链形成寡核苷酸(TFOS)可以作为第三链与双链DNA结合以创建非规范螺旋结构的发现，一种引入螺旋扭曲损伤来监测基因组特定位置修复的方法成为可能。这项建议的长期目标是利用产生位点特异性螺旋改变的技术来了解TCR和GGR通路在修复改变的DNA结构中的相对贡献。这个建议的具体目的是：1.比较TCR通路和GGR通路在异常DNA结构修复中的作用。2.检测不同螺旋扭曲分子的修复和识别能力。旨在监测异常DNA结构识别和修复的实验将使用各种位点特异性DNA结合分子、来自人类修复缺陷细胞系的无细胞提取物以及针对DNA修复因子和纯化蛋白的特异性抗体的组合来进行。具体地说，我们将研究GGR蛋白XPC和TCR蛋白CSB在识别和修复改变的DNA结构中的作用，并与NER蛋白XPA的作用进行比较。

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.