The XPA scaffold protein in Nucleotide Excision Repair

核苷酸切除修复(NER)通过去除有害的DNA加合物保护人类细胞 环境毒素和太阳紫外线辐射。人类NER缺陷导致DNA修复障碍 色素性干皮病，其特点是易患皮肤癌、神经系统疾病 异常和过早死亡。NER修复受损的DNA也有一个不利的方面--它有助于 对于肿瘤对抗肿瘤药物治疗的耐药性的发展，特别是顺式- 以及卡铂，这是肿瘤科最广泛使用的两种处方药。 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来阐明支架蛋白是如何 XPA协调NER切口复合体的组装和组织。尽管其规模不大(273 残基)，XPA作为NER复合体的中心支架，还与4个关键的NER蛋白相互作用 作为DNA。然而，XPA是如何被招募到受损地点并通过其各种不同的因素定位其他因素的 相互作用仍然知之甚少。此外，鉴于其在组织和协调 在NER复合体的轨道上，XPA是一个有吸引力的潜在的抑制NER的靶点。 目标1将测试TFIIH通过提议的相互作用将XPA招募到紫外线损伤部位的假设 涉及XPA的C-末端区域和TFIIH的p8亚基的界面。我们将通过生物化学和 从结构上描述这种相互作用，并确定界面上的突变如何干扰这种相互作用 在体外和体内，相互作用影响NER。取消XPA和TFIIH之间的互动也将解决 长期存在的问题是，损伤识别之后的步骤是否适用于全球基因组 转录偶联的NER-TFIIH和XPA的到来是这两条途径共同的第一步。 TFIIH对受损DNA的作用创造了一个开放的“NER泡泡”，为XPA提供了一个着陆平台 和RPA，是NER切开所必需的。XPA的支架功能依赖于它与 RPA，它结合未受损的链。目标2将确定分子基础和功能含义 XPA和RPA的协调行动。结构、生化和生物物理方法结合 细胞分析将检验XPA和RPA之间的两个相互作用位点同时存在的假设 以合作的方式参与并为NER做出贡献。基于这些结果，我们在碎片方面的专业知识 基于分子发现将用于开发和验证针对XPA-RPA界面的初始抑制剂。 这些研究有望提供：(I)对XPA在 组装和协调NER机制；(2)对以下方面至关重要的XPA相互作用面的身份 NER；(3)证明XPA和其他NER因素之间的接口是适用于 评估通过抑制NER来克服肿瘤对DNA损伤治疗的耐药性的潜力。

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.