Human replication protein A induces dynamic changes in single-stranded DNA and RNA structures.

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.