Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Using cryo electron microscopy we obtained a 3D reconstruction of the RPA trimerisation core bound with ssDNA (∼55 kDa) at ∼4.7 Å resolution and a dimeric RPA assembly on ssDNA. FRET-based solution studies reveal dynamic rearrangements of DBDs during coordinated RPA binding and this activity is regulated by phosphorylation at S178 in RPA70. We present a structural model on how dynamic DBDs promote the cooperative assembly of multiple RPAs on long ssDNA.

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-strand DNA (ssDNA) intermediates, activating the DNA damage response and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA yet also allow RPA displacement by lower affinity proteins. We generated fluorescent versions of Saccharomyces cerevisiae RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA while RPA remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. These findings reveal how RPA interacting proteins with lower ssDNA binding affinities can access the occluded ssDNA and remodel individual DBDs to replace RPA.

The heterotrimeric eukaryotic Replication protein A (RPA) is a master regulator of numerous DNA metabolic processes. For a long time it has been viewed as an inert protector of ssDNA and a platform for assembly of various genome maintenance and signaling machines. Later, modular organization of the RPA DNA binding domains suggested a possibility for a dynamic interaction with ssDNA. This modular organization has inspired several models for the RPA-ssDNA interaction that aimed to explain how RPA, the high affinity ssDNA binding protein, is replaced by the downstream players in DNA replication, recombination and repair that bind ssDNA with much lower affinity. Recent studies, and in particular single-molecule observations of RPA-ssDNA interactions, led to the development of a new model for the ssDNA handoff from RPA to a specific downstream factor where not only stability and structural rearrangements, but also RPA conformational dynamics guide the ssDNA handoff. Here we will review the current knowledge of the RPA structure, its dynamic interaction with ssDNA, and how RPA conformational dynamics may be influenced by posttranslational modification and proteins that interact with RPA, as well as how RPA dynamics may be harnessed in cellular decision making.

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing singlestrand DNA (ssDNA) intermediates, activating the DNA damage response, and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA, but also allow RPA displacement by lower affinity proteins. We have made fluorescent versions of RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA, while RPA as a whole remains bound to ssDNA.The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. This demonstrates how RPA interacting proteins, with lower ssDNA binding affinity, can access the occluded ssDNA and remodel individual DBDs to replace RPA. Main Text:In every eukaryotic cell, RPA binds to transiently exposed ssDNA and serves as a hub protein to coordinate essential DNA metabolic processes including replication, recombination, repair, and telomere maintenance 1,2 . Cellular functions of RPA rely on its high affinity ssDNA binding, its ability to physically interact with over two dozen DNA processing enzymes, and to correctly position these enzymes on complex DNA structures. The precise mechanisms through which RPA functions in many contexts; and more importantly, how it differentiates between multiple DNA metabolic events (DNA replication, repair or recombination) is a long-standing puzzle 1,3 .RPA is heterotrimeric, flexible, and modular in structure. It is composed of three subunits:RPA70, RPA32 and RPA14 (Figs.1a, b). The subunits harbor six 3 oligonucleotide/oligosaccharide binding folds (OB-folds; labeled A through F). We refer to the DNA binding OB-folds as DNA binding domains (DBDs; Fig. 1a). RPA binds to ssDNA with high, sub-nanomolar affinity, but can be displaced by DNA binding proteins with much lower DNA binding affinity. Recent studies have suggested that the RPA-ssDNA complex is relatively dynamic 4,5,6, positing a selective dissociative mechanism where not all DBDs are stably bound to the DNA at the same time and microscopic dissociation of individual DNA binding domains occurs.In all existing models for RPA function, DBDs A and B are assigned as high affinity binding domains. Purified DBD-A, DBD-B and DBD-A/DBD-B constructs bind ssDNA with KD values 2 µM, 20 µM and 50 nM, respectively 7-9 . The trimerization core made up of DBD-C (RPA70), DBD-D (RPA32) and DBD-E (RPA14) is considered to have a weaker ssDNA binding affinity (Kd>5μM) 10 . Additionally, mutational analysis of individual aromatic residues that interact with the ssDNA in either DBD-C or DBD-D show minimal perturbations on ssDNA binding affinity 6 . Paradoxically, in the recently solved crystal structure of the RPA-ssDNA complex (Fig. 1b), the interactions of all four DBD's with ssDNA are similar with DBD-C having more contacts with ssDNA bases than DBD-A, DBD-B or DBD-D 11 . Thus, the exact nature of the contributions from...

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