Wrapping of single-stranded DNA by Replication Protein A and modulation through phosphorylation.
Wrapping of single-stranded DNA by Replication Protein A and modulation through phosphorylation.
复制标题
复制蛋白 A 包裹单链 DNA 并通过磷酸化进行调节。
DOI：
10.1101/2024.03.28.587234
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Antony,Edwin
中科院分区：
文献类型：
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作者：
Chadda,Rahul;Kaushik,Vikas;Ahmad,IramMunir;Deveryshetty,Jaigeeth;Holehouse,Alex;Sigurdsson,SnorriThD;Bothner,Brian;Dastvan,Reza;Origanti,Sofia;Antony,Edwin
DNA代谢过程中出现的单链DNA（ssDNA）中间体被复制蛋白A（RPA）屏蔽。RPA与ssDNA结合并充当看门人，以特殊的特异性将ssDNA引导至下游DNA代谢途径。理解这种RPA依赖性特异性的机制基础需要全面理解ssDNA与RPA结合时的结构构象。以前的研究表明RPA对ssDNA的拉伸。然而，结构研究发现RPA周围有ssDNA的部分包裹。因此，为了调和模型，在这项研究中，我们使用单分子FRET和双电子-电子共振（DEER）光谱测量了游离ssDNA和RPA-ssDNA复合物的端到端距离，发现RPA结合后ssDNA的端到端距离只有一个小的系统性增加。这种变化不符合线性拉伸模型，而是支持RPA DNA结合结构域轮廓周围ssDNA的部分包裹。此外，我们揭示了如何在RPA 70亚基中的关键Ser-384位点的磷酸化通过重塑DNA结合结构域来提供对包裹的ssDNA的访问。这些发现为RPA结合的ssDNA建立了精确的结构模型，为RPA如何促进ssDNA的重塑以用于后续下游过程提供了有价值的见解。
Single-stranded DNA (ssDNA) intermediates, which emerge during DNA metabolic processes are shielded by Replication Protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper, directing the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent specificity requires a comprehensive understanding of the structural conformation of ssDNA when bound to RPA. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and Double Electron-Electron Resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.