Human replication protein A induces dynamic changes in single-stranded DNA and RNA structures
Human replication protein A induces dynamic changes in single-stranded DNA and RNA structures
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DOI：
10.1074/jbc.ra119.009737
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发表时间：
2019-07
期刊：
影响因子：
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通讯作者：
Qing-Man Wang;Yan-Tao Yang;Yi-Ran Wang;B. Gao;X. Xi;Xi-Miao Hou
中科院分区：
文献类型：
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作者：
Qing-Man Wang;Yan-Tao Yang;Yi-Ran Wang;B. Gao;X. Xi;Xi-Miao Hou
复制蛋白A(RPA)是真核生物中主要的单链DNA结合蛋白，在基因组维持中具有重要作用。RPA通过多种方式与单链DNA结合，最近的研究表明RPA与单链DNA的相互作用是动态的。然而，RPA如何在不同的结合模式之间交替并在这种动态相互作用中改变单链DNA结构仍是未知的。在这里，我们使用单分子FRET系统地研究了人RPA与单链DNA之间的相互作用。我们发现RPA可以与不同长度的单链DNA采用不同类型的结合络合物，导致单链DNA的伸直或弯曲，这取决于单链DNA底物的长度和结构以及RPA的浓度。重要的是，我们注意到一些复合体是高度动态的，而另一些则看起来相对静态。在上述观察的基础上，我们提出了一个解释RPA如何与单链DNA动态结合的模型。值得注意的是，荧光各向异性表明RPA也可以与RNA结合，但结合亲和力低于与单链DNA的结合。在单分子水平上，我们观察到RPA正在经历快速和重复的与RNA的结合和解离。这项研究可能为RPA与单链DNA和RNA结合的丰富动力学提供新的见解。
Replication protein A (RPA) is the major eukaryotic ssDNA-binding protein and has essential roles in genome maintenance. RPA binds to ssDNA through multiple modes, and recent studies have suggested that the RPA–ssDNA interaction is dynamic. However, how RPA alternates between different binding modes and modifies ssDNA structures in this dynamic interaction remains unknown. Here, we used single-molecule FRET to systematically investigate the interaction between human RPA and ssDNA. We show that RPA can adopt different types of binding complexes with ssDNAs of different lengths, leading to the straightening or bending of the ssDNAs, depending on both the length and structure of the ssDNA substrate and the RPA concentration. Importantly, we noted that some of the complexes are highly dynamic, whereas others appear relatively static. On the basis of the above observations, we propose a model explaining how RPA dynamically engages with ssDNA. Of note, fluorescence anisotropy indicated that RPA can also associate with RNA but with a lower binding affinity than with ssDNA. At the single-molecule level, we observed that RPA is undergoing rapid and repetitive associations with and dissociation from the RNA. This study may provide new insights into the rich dynamics of RPA binding to ssDNA and RNA.