RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

Abstract

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

Figure 1

Schematic representation of the RPA complex. The OB folds A-F are shown as light blue rectangles, the winged-helix domain of RPA32 is in red. OB folds A-D (DBDs A-D) support the ssDNA-binding activity of the RPA complex. Heterotrimerization of the RPA complex is mediated by OB folds C of RPA70, D of RPA32 and E of RPA14, which are brought together through a triple helix bundle (dashed arrows). The phosphorylated residues found in the N-terminus of RPA32 are represented in bold along with their respective kinases. Solid and dashed arrows indicate major or minor contributions to specific phosphorylation events by the various kinases. SUMOylated residues in RPA70 are represented as orange circles. This figure was inspired by Fanning et al..

Figure 2

DNA damage signaling on RPA-ssDNA. RPA-ssDNA elicits DNA damage signaling by recruiting the ATR-ATRIP kinase and the PRP19 complex to trigger a phosphorylation-ubiquitination feed-forward loop that activates ATR-ATRIP. The kinase activity of ATR is required for efficient recruitment of the PRP19 complex onto RPA-ssDNA. PRP19 then exerts its ubiquitin ligase activity on the RPA-ssDNA platform to facilitate further recruitment of ATR-ATRIP and autophosphorylation of ATR. In parallel, RPA-ssDNA directs the loading of the 9-1-1 clamp by the Rad17-RFC2-5 complex onto adjacent ssDNA-dsDNA junctions. This allows the recruitment of the ATR activator TopBP1 and of Rhino, which potentiate ATR-ATRIP kinase activity to protect replication forks and maintain genomic stability. Phosphorylation and ubiquitination events are depicted as green and white circles, respectively.