The XPA scaffold protein in Nucleotide Excision Repair

核苷酸切除修复（NER）通过去除由DNA形成的有害DNA加合物来保护人类细胞。 环境毒素和太阳紫外线辐射。人类NER缺陷导致DNA修复障碍 着色性干皮病，其特征在于易患皮肤癌、神经系统癌、 异常和过早死亡。NER对受损DNA的修复也有一个缺点-它有助于 显著地促进肿瘤对用抗肿瘤剂，特别是顺式- 和卡铂，两种在肿瘤学中使用最广泛的处方药。 我们建议利用Scharer和Chazin实验室在化学方面的综合专业知识， 生物化学，细胞生物学，结构生物学和小分子发现，以阐明支架蛋白如何 XPA协调NER切口复合体的组装和组织。尽管规模不大（273 XPA作为NER复合物的中心支架，也与4种关键的NER蛋白相互作用 作为DNA然而，XPA如何被招募到损伤部位并通过其各种因素定位其他因素， 相互作用仍然知之甚少。此外，鉴于其在组织和协调 NER复合物的轨迹，XPA是一个有吸引力的潜在阿基里斯之踵，以抑制NER为目标。 目的1将检验TFIIH通过拟议的相互作用将XPA招募到UV损伤部位的假设 涉及XPA的C-末端区域和TFIIH的p8亚基的界面。我们将从生物化学和 从结构上描述这种相互作用，并确定如何在干扰这种相互作用的界面突变， 在体外和体内相互作用影响NER。取消XPA和TFIIH之间的交互也将解决 长期存在的问题是，损伤识别后的步骤对于全球基因组是否相同 和转录偶联NER -TFIIH和XPA的到达是两种途径共同的第一步。 TFIIH对受损DNA的作用产生了一个开放的“NER气泡”，为XPA提供了一个着陆平台 和RPA，并且是NER切口所需的。XPA的支架功能依赖于其与 结合未受损链的RPA。目标2将确定分子基础和功能的影响 XPA和RPA的协调行动。结构、生物化学和生物物理方法， 细胞分析将检验XPA和RPA之间的两个相互作用位点同时 以合作的方式参与并促进NER。基于这些结果，我们在碎片方面的专业知识 基于分子的发现将用于开发和验证靶向XPA-RPA界面的初始抑制剂。 这些研究预计将提供：（i）戏剧性的新机制的见解XPA的核心作用， 组装和协调NER机制;（ii）XPA相互作用表面的身份，这是至关重要的 （iii）XPA和其他NER因素之间的接口是适用于以下目标的原则证明： 评估通过抑制NER克服肿瘤对DNA损伤疗法的抗性的潜力。

Nucleotide excision repair (NER) protects human cells by removing harmful DNA adducts formed by environmental toxins and solar UV irradiation. Defects in NER in humans lead to the DNA repair disorder xeroderma pigmentosum, which is characterized by high predisposition to skin cancer, neurological abnormalities and premature death. The repair of damaged DNA by NER also has a downside – it contributes significantly to the development of resistance of tumors to treatment with antitumor agents, in particular cis- and carboplatin, two of the most widely prescribed drugs in oncology. We propose to leverage the combined expertise of the Scharer and Chazin laboratories in chemistry, biochemistry, cell biology, structural biology, and small molecule discovery to elucidate how the scaffold protein XPA coordinates the assembly and organization of NER incision complexes. Despite its modest size (273 residues), XPA functions as the central scaffold of NER complexes, interacting with 4 key NER proteins as well as DNA. However, how XPA is recruited to the site of damage and positions other factors through its various interactions remains poorly understood. Moreover, given its essential role in organizing and orchestrating the trajectory of NER complexes, XPA is an attractive potential Achilles Heel to target for suppressing NER. Aim 1 will test the hypothesis that TFIIH recruits XPA to sites of UV damage through a proposed interaction interface involving the C-terminal region of XPA and the p8 subunit of TFIIH. We will biochemically and structurally characterize this interaction and determine how mutations in the interface that disturb this interaction affect NER in vitro and in vivo. Abolishing the interaction between XPA and TFIIH will also address the long-standing question of whether the steps following damage recognition are the same for global genome and transcription-coupled NER – the arrival of TFIIH and XPA is the first step common to both pathways. The action of TFIIH on damaged DNA creates an open “NER bubble” that provides a landing platform for XPA and RPA and is required for NER incision. The scaffolding function of XPA is reliant on its coordination with RPA, which binds the undamaged strand. Aim 2 will determine the molecular basis and functional implications of the coordinated action of XPA and RPA. Structural, biochemical, and biophysical approaches combined with cellular assays will test the hypothesis that the two interaction sites between XPA and RPA are simultaneously engaged and contribute to NER in a cooperative fashion. Based on these results, our expertise in fragment based molecular discovery will be used to develop and validate initial inhibitors targeting XPA-RPA interfaces. These studies are expected to provide: (i) dramatic new mechanistic insights into the central role of XPA in assembling and coordinating the NER machinery; (ii) the identity of XPA interaction surfaces that are critical to NER; (iii) proof of principle that interfaces between XPA and other NER factors are suitable targets for evaluating the potential of overcoming tumor resistance to DNA damaging therapies by suppressing NER.