Replication Protein A, the Main Eukaryotic Single-Stranded DNA Binding Protein, a Focal Point in Cellular DNA Metabolism

Abstract

Replication protein A (RPA) is a heterotrimeric protein complex and the main single-stranded DNA (ssDNA)-binding protein in eukaryotes. RPA has key functions in most of the DNA-associated metabolic pathways and DNA damage signalling. Its high affinity for ssDNA helps to stabilise ssDNA structures and protect the DNA sequence from nuclease attacks. RPA consists of multiple DNA-binding domains which are oligonucleotide/oligosaccharide-binding (OB)-folds that are responsible for DNA binding and interactions with proteins. These RPA-ssDNA and RPA-protein interactions are crucial for DNA replication, DNA repair, DNA damage signalling, and the conservation of the genetic information of cells. Proteins such as ATR use RPA to locate to regions of DNA damage for DNA damage signalling. The recruitment of nucleases and DNA exchange factors to sites of double-strand breaks are also an important RPA function to ensure effective DNA recombination to correct these DNA lesions. Due to its high affinity to ssDNA, RPA's removal from ssDNA is of central importance to allow these metabolic pathways to proceed, and processes to exchange RPA against downstream factors are established in all eukaryotes. These faceted and multi-layered functions of RPA as well as its role in a variety of human diseases will be discussed.

Keywords: DNA binding; DNA damage signalling; DNA repair; DNA replication; homologous recombination; protein interactions; replication protein A.

Figure 1

Structure of RPA. (A) The bars represent the three RPA subunits with DNA-binding domains (DBDs) DBD-A to DBD-F highlighted and their borders indicated by amino acid numbering of the human protein sequences [14,22]. Each DBD (colour code: RPA70: F (light brown), A (wheat), B and C (green); RPA32: D (light blue); RPA14: E (light orange)) contains a highly conserved OB-fold. The structure of one of the DBDs is highlighted in the bubble within the panel. The DBD domains A to D are able to bind to ssDNA. In contrast, the unstructured linker between DBD-F and A is shown in yellow, and the winged helix domain (WH) of RPA32 in light cyan are mainly protein–protein interaction sites. DBD-C contains the zinc-binding motifs represented by the protruding Zn. The N-terminus of RPA32, shown in dark blue and with the letter P, has recognition sites for multiple protein kinases including CDKs and PIKKs [23]. The lines between the three subunits represent the points of interaction between the subunits through the heterotrimerisation core (adapted from [12,24]). In the panel, the human RPA aa sequence is used for the numbering. (B) The experimental RPA structure presents an image of the RPA heterotrimer including RPA70 (with DBD-A in wheat plus DBD-B and C in green, the associated Zn atom is shown in red), RPA32 in marine blue, RPA14 in light orange, and oligo(dT) ssDNA in orange with the 5′- and 3′-end marked (structure derived from PDB 4gop is in the 30 nt-binding mode [13] and presented using Pymol (Schrödinger Inc. (USA)). In panel (C), human RPA32 and STN1 OB-fold structures were predicted by AlphaFold [25] and are shown in yellow light and magenta, respectively. STN1 is a subunit of the RPA-like CST complex and is involved in the initiation of G strand synthesis at telomeres ([20,21], see also Section 5). Both human structures were aligned to the experimental Ustilago maydis RPA (structure derived from 4gop) using Pymol (for a better overview, aa N93 to T124 of STN1, which do not align to any structure, were omitted). The loops containing D157 of STN1, D151 of human RPA32, and D155 of Ustilago maydis RPA32 are shown in red. Panels (D,E) present images of the RPA trimer predicted by AlphaFold [25] without DNA and in a similar position as Ustilago maydis RPA in panels (B,C). DBD-F, which is lacking in the Ustilago maydis RPA structure, and DBD-A are in light green and light brown, respectively. The linker between the two domains is coloured in yellow. DBD-B, DBD-C and the short linker between the two domains are in green. The WH domain of RPA32, which is not shown in the Ustilago maydis RPA structure, is presented in cyan. DBD-D plus the linker between DBD-D and WH are shown in light blue. The loop in DBD-D containing the conserved D151 is called the D loop (see also panels (B,C)) and is shown in red. It is important to note that the linker regions of RPA70 and RPA32 plus the N-terminal phosphorylation sequences of RPA32 have a very low per-residue model confidence score (pLDDT) and might be unstructured. To allow a better presentation of the RPA complex, the N-terminus of RPA32, aa 1–33, was omitted in panels (D,E) but is shown in the Supplemental Figure S1. In panel (E), the OB-fold of STN1 shown in light pink is aligned to human RPA using PyMol (for a better overview, aa N93 to T124 of STN1, which did not align to any structure, were omitted in the presentation). The loop containing the conserved D157 (D loop) is presented in red. The RPA domains are marked with blue letters, A to F (DBD-A to F) and WH in the structures.

Figure 1

Structure of RPA. (A) The bars represent the three RPA subunits with DNA-binding domains (DBDs) DBD-A to DBD-F highlighted and their borders indicated by amino acid numbering of the human protein sequences [14,22]. Each DBD (colour code: RPA70: F (light brown), A (wheat), B and C (green); RPA32: D (light blue); RPA14: E (light orange)) contains a highly conserved OB-fold. The structure of one of the DBDs is highlighted in the bubble within the panel. The DBD domains A to D are able to bind to ssDNA. In contrast, the unstructured linker between DBD-F and A is shown in yellow, and the winged helix domain (WH) of RPA32 in light cyan are mainly protein–protein interaction sites. DBD-C contains the zinc-binding motifs represented by the protruding Zn. The N-terminus of RPA32, shown in dark blue and with the letter P, has recognition sites for multiple protein kinases including CDKs and PIKKs [23]. The lines between the three subunits represent the points of interaction between the subunits through the heterotrimerisation core (adapted from [12,24]). In the panel, the human RPA aa sequence is used for the numbering. (B) The experimental RPA structure presents an image of the RPA heterotrimer including RPA70 (with DBD-A in wheat plus DBD-B and C in green, the associated Zn atom is shown in red), RPA32 in marine blue, RPA14 in light orange, and oligo(dT) ssDNA in orange with the 5′- and 3′-end marked (structure derived from PDB 4gop is in the 30 nt-binding mode [13] and presented using Pymol (Schrödinger Inc. (USA)). In panel (C), human RPA32 and STN1 OB-fold structures were predicted by AlphaFold [25] and are shown in yellow light and magenta, respectively. STN1 is a subunit of the RPA-like CST complex and is involved in the initiation of G strand synthesis at telomeres ([20,21], see also Section 5). Both human structures were aligned to the experimental Ustilago maydis RPA (structure derived from 4gop) using Pymol (for a better overview, aa N93 to T124 of STN1, which do not align to any structure, were omitted). The loops containing D157 of STN1, D151 of human RPA32, and D155 of Ustilago maydis RPA32 are shown in red. Panels (D,E) present images of the RPA trimer predicted by AlphaFold [25] without DNA and in a similar position as Ustilago maydis RPA in panels (B,C). DBD-F, which is lacking in the Ustilago maydis RPA structure, and DBD-A are in light green and light brown, respectively. The linker between the two domains is coloured in yellow. DBD-B, DBD-C and the short linker between the two domains are in green. The WH domain of RPA32, which is not shown in the Ustilago maydis RPA structure, is presented in cyan. DBD-D plus the linker between DBD-D and WH are shown in light blue. The loop in DBD-D containing the conserved D151 is called the D loop (see also panels (B,C)) and is shown in red. It is important to note that the linker regions of RPA70 and RPA32 plus the N-terminal phosphorylation sequences of RPA32 have a very low per-residue model confidence score (pLDDT) and might be unstructured. To allow a better presentation of the RPA complex, the N-terminus of RPA32, aa 1–33, was omitted in panels (D,E) but is shown in the Supplemental Figure S1. In panel (E), the OB-fold of STN1 shown in light pink is aligned to human RPA using PyMol (for a better overview, aa N93 to T124 of STN1, which did not align to any structure, were omitted in the presentation). The loop containing the conserved D157 (D loop) is presented in red. The RPA domains are marked with blue letters, A to F (DBD-A to F) and WH in the structures.

Figure 2

DNA synthesis at a eukaryotic replication fork. This schematic drawing of a eukaryotic replication fork shows CMG helicase (orange) unwinding dsDNA into the leading plus lagging strand templates and the replication proteins involved in DNA synthesis at the fork. Heterotrimeric RPA binds the resulting ssDNAs. The navy blue DNA strands are the parental strands. Pol ε (grey) newly synthesises the leading strand (royal blue DNA) in the 5′ to 3′ direction as indicated by the dashed arrow, and it is associated with the CMG. In contrast, the homotrimeric AND-1-CTF4-WDHD1 protein complex (dark grey, named AND-1) links CMG to the Pol α complex. The primer (red) synthesised by the primase function of Pol α allows for replication synthesis initiation by the DNA polymerase activity of Pol ⍺ (pink) in a 5′–3′ direction to start Okazaki fragment synthesis for lagging strand synthesis. PCNA (purple) and RFC (turquoise) replace Pol ⍺, making a landing site for Pol δ (maroon) to bind to the RNA–DNA and PCNA and elongate it until this complex reaches the next Okazaki on the parent strand, allowing the maturation of the Okazaki fragments to occur (adapted from [49,52,60,61]). The solid arrows represent incoming and leaving proteins.

Figure 3

Nucleotide excision repair. The schematic drawing depicts the global genomic NER (ggNER) pathway as the most prominent NER pathway in cells [97,98,99]. Eukaryotic cells also repair bulky lesions via the transcription-coupled NER (tcNER) pathway, which only occurs in a transcription-dependent manner on the transcribed strand [97,98,99]. The tcNER pathway feeds into the presented pathway but was omitted for simplification reasons. (A) DNA damage can result in bulky DNA lesions. (B) RAD23B and XPC (pink) recognise these damaged sites in dsDNA. (C) They recruit the multi-subunit protein complex TFIIH (transcription facto II H, light green) that contains XPB in purple and XPD in dark green to verify and extend the opening of the dsDNA. (D) XPA, in orange, and the multi-coloured RPA are recruited to the DNA damage site to bind and protect newly unwound ssDNA. The positioning of RPA and XPA ensures the correct localisation of XPF and XPG, in darker pink colours, to undertake their incision functions as shown in the panel. (E–G) DNA elongation occurs after binding of RFC (grey) and loading of PCNA (purple) and DNA polymerase (dark grey) recruitment (panel G). (H) Ligase (purple) binds the newly synthesised DNA at the incision site and ligates the nick in the dsDNA to yield repaired dsDNA (adapted from Schärer, 2013 [99]).

Figure 4

Homologous recombination. Double-strand breaks (DSBs) are highly toxic for cells and occur in DNA after high-energy radiation or cellular processes such as antibody development. Following DSB, the binding of MRN and chromatin modifications including the ubiquitination of cellular proteins such as RPA70 and RPA32 allows HR to occur (for more details, see the text). Then, BLM helicase (orange) unwinds the dsDNA in an ATP-dependent fashion and RPA binds to ssDNA. DNA2 (purple) interacts with RPA and degrades one DNA strand in the 5′–3′ direction controlled by RPA. The phosphorylation of RPA70 NTD alters the RPA–BLM interactions, causing a reduction in BLM activity [105]. The mediator BRCA2 via its partner DSS1 interacts with the resulting RPA–ssDNA filaments and exchanges RPA toward RAD51 [106]. The RAD51–ssDNA filament then starts a homology search of these overhangs (adapted from [103]).