Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76798, USA.
Department of Biology, New York University, New York, NY 10003, USA.
Nucleic Acids Res. 2020 Dec 2;48(21):12348-12364. doi: 10.1093/nar/gkaa909.
XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively 'opening' these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a 'kinetic gating' mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how 'opening' is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed 'open' undamaged DNA in solution and that such 'opening' can largely occur without one or the other of the β-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound 'open' DNA adopts multiple conformations in solution notwithstanding the DNA's original structure or the β-hairpins. Molecular dynamics simulations reveal compensatory roles of the β-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA 'opening' of undamaged sites and the dynamic nature of 'open' DNA may shed light on how the protein functions within and beyond nucleotide excision repair in cells.
XPC/Rad4 通过选择性地“打开”这些位点，同时沿未受损的 DNA 快速扩散，从而启动真核生物核苷酸切除修复，针对结构多样的螺旋不稳定/扭曲 DNA 损伤。先前的结构研究表明，当 Rad4 与 DNA 连接时，也可以打开未受损的 DNA，这表明存在一种“动力学门控”机制，即通过有效打开与扩散来区分损伤。然而，缺乏支持这种机制的溶液研究，以及如何实现“打开”的机制仍不清楚。在这里，我们通过与 DNA 连接的复合物的晶体结构和荧光构象分析，展示了 Rad4 确实可以在溶液中“打开”未受损的 DNA，并且这种“打开”在很大程度上可以在 BHD2 或 BHD3 结构域中的一个或另一个β发夹结构缺失的情况下发生。值得注意的是，尽管 DNA 具有原始结构或β发夹，但是 Rad4 结合的“打开”DNA 在溶液中可以采用多种构象。分子动力学模拟揭示了β发夹的补偿作用，这可能使处理和打开各种损伤的能力具有稳健性。我们的研究展示了如何使用基于荧光的研究来获取与整体结构研究互补的信息。未受损部位的束缚促进的 DNA“打开”以及“打开”DNA 的动态性质可能阐明了该蛋白在细胞内和细胞外核苷酸切除修复中的作用机制。
