The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.

中文翻译：

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与 RPA 的关键交互使 XPA 在 NER 复合物中定向。

XPA 蛋白与单链 DNA (ssDNA) 结合蛋白 RPA 一起作为中心支架发挥作用，以确保修复因子在多蛋白核苷酸切除修复 (NER) 机器中的正确定位。我们之前确定了与 RPA32C 域结合的无序 XPA N 末端的短基序的结构。然而，XPA DNA 结合域 (XPA DBD) 和 RPA70AB 串联 ssDNA 结合域之间的第二次接触，这可能会影响 XPA 和 RPA 在受损 DNA 底物上的方向，仍然很难表征。NMR 用于绘制 XPA DBD 和 RPA70AB 的结合界面。将 NMR 和 X 射线散射数据与全面的对接和细化相结合，揭示了 XPA DBD 和 RPA70AB 如何在模型 NER DNA 底物上定向。该结构模型能够设计抑制与 RPA70AB 相互作用的 XPA 突变。这些突变降低了基于细胞的 NER 测定中的活性，证明了 XPA DBD-RPA70AB 相互作用的功能重要性。我们的研究结果为关于 XPA 在 NER 气泡中的结合位置提供了持续的争议，为疾病相关的 XPA 错义突变故障的分子基础提供了结构性见解，并有助于理解 NER 机制的结构和机械作用。