A structural and dynamic model for the assembly of Replication Protein A on single-stranded DNA

Luke A Yates,Xiaodong Zhang,Nilisha Pokhrel,Rajika L Perera,Ricardo J Aramayo,Maria Spies,Colleen C Caldwell,Joshua A Kaplan,Edwin Antony

doi:10.1038/s41467-018-07883-7

Luke A Yates, Xiaodong Zhang + Show 7 more

Open Access

https://doi.org/10.1038/s41467-018-07883-7

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Abstract

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.

Highlights

Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed single-stranded DNA (ssDNA) to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players
Structural, biochemical, and biophysical studies have shown that RPA can associate with ssDNA in different modalities; a low affinity mode which binds 8–12 nucleotides, with structural studies showing that DNA binding domains (DBDs)-A and DBD-B can bind and cover 8 nts ssDNA13,18–20, and a high-affinity compact mode involving all four major DBDs (A–D) that bends a 28 nts ssDNA tract in a horse-shoe shape configuration[13,21]
Recombinant ScRPA was purified to homogeneity as judged by SDS–PAGE and gel filtration analysis coupled with inline multi-angle laser light scattering (SEC-MALS), suggesting intact complexes (Supplementary Fig. 1a, b)

Summary

Introduction

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. The smallest subunit Rfa[3] has a single OB-fold (DBD-E) and forms part of the trimerisation core (Tri-C) consisting of DBD-C of Rfa[1], DBD-D of Rfa[2], and DBD-E of Rfa[3] Both DBD-F and DBD-E have been shown to weakly interact with DNA, DBD-A, DBD-B, DBD-C, and DBD-D are primarily responsible for RPA’s ssDNA-binding activities[7,8,9]. Based on these studies it was thought that the Tri-C, which contains DBD-C and DBD-D, has a weaker association with ssDNA compared to DBD-A and DBD-B and is only involved in binding to longer ssDNA in the compact mode[9,13,18,22]

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