Communication between DNA polymerases and Replication Protein A within the archaeal replisome

Abstract

Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrate that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveil how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA is shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.

Fig. 1. Winged-helix domains in the archaeal replisome.

a Schematic representation of the archaeal core replisome and RPA binding single-stranded DNA on the lagging strand. b Genes from Pyrococcus abyssi str. GE5 that encode DNA replication factors composing the replisome. Genes encoding a protein that contains a winged-helix domain are highlighted in green.

Fig. 2. The C-terminal region of Rpa2 contains a conserved WH domain.

a Cartoon representation of P. abyssi RPA (PDB ID 8AAJ). b Focus on Rpa2^WH, for which a representative structure from the NMR ensemble is shown. c NMR structural ensemble of Rpa2^WH, color coded with S² values from red to blue denoting high and restricted amplitude motions respectively (gray: no value). d Schematic representation of the rpa locus. All the primers used to construct mutants are indicated, as well as the number of transformation assays and screened clones for both expected mutants: deletion of the three rpa genes (top) and the WH domain of Rpa2 (bottom).

Fig. 3. Rpa2^WH connects RPA to the archaeal replicative DNA polymerases.

a–h Biolayer Interferometry (BLI) results for (a) specific binding of immobilized histidine-tagged PriSL at 50 nM to 10 µM wild-type RPA (teal) and to 10 µM RPA^ΔWH (purple); b specific binding of immobilized biotin-tagged nucleoprotein RPA filaments to 12.5 nM PriSL (teal) and immobilized biotin-tagged nucleoprotein RPA^ΔWH filaments to 12.5 nM PriSL (purple); c specific binding of immobilized histidine-tagged PolD at 50 nM to 1 µM wild-type RPA (teal) and to 1 µM RPA^ΔWH (purple); d specific binding of immobilized biotin-tagged nucleoprotein RPA filaments to 250 nM PolD (teal) and immobilized biotin-tagged nucleoprotein RPA^ΔWH filaments to 250 nM PolD (purple). e Specific binding of PriSL (500, 250, 125, 62.5, 31.25, 15.62, 7.81 nM, n = 3) to immobilized histidine-tagged Rpa2 C-terminal winged-domain. f Specific binding of PriSL (100, 50, 25, 12.5, 6.25, 3.12, 1.56 nM, n = 3 biological replicates) to immobilized biotin-tagged nucleoprotein RPA filaments. g Specific binding of PolD (1000, 500, 250, 125, 62.5, 31.25, 15.62 nM, n = 3 biological replicates) to immobilized histidine-tagged Rpa2^WH. h Specific binding of PolD (1000, 500, 250, 125, 62.5, 31.25, 15.62 nM, n = 3) to immobilized biotin-tagged nucleoprotein RPA filaments. Steady-state analyses were performed using the average signal measured at the end of the association steps. Data are represented as mean value ± standard deviations (error bars). Raw data are provided in the source data file. i–l Identification of the Rpa2^WH binding surface to the DNA polymerases by NMR. i, j NMR chemical shift perturbations (CSP) and peak intensity ratios I_cplx/I_free (log₁₀ scale) on Rpa2^WH induced by PriSL^ΔCTD and PolD, respectively. The dotted blue and red lines correspond to I_cplx/I_free ratios of 0.4 and 0.25 for the complex with PriSL^ΔCTD and 0.4 and 0.17 for the complex with PolD, as used for the color coding in (k, l). Error bars in the I_cplx/I_free histograms represent the noise standard deviation in the spectra (see section “Methods”). For clarity, the most affected regions in terms of CSP and/or intensity ratio are highlighted by light red boxes. Secondary structure elements are indicated at the top. k, l Mapping of the intensity ratio I_cplx/I_free on Rpa2^WH color coded from black (no attenuation), blue (weak attenuation) to red (large attenuation) as indicated. Residues in gray denote missing data (proline or overlapping signals). The unfolded residues 200-205 that transiently contact PriSL^ΔCTD are highlighted in light violet. The residues that are most severely affected by the binding (I_cplx/I_free < 0.29 for PriSL^ΔCTD and <0.26 for PolD) are depicted as transparent spheres and delineate the respective binding surfaces to the polymerases.

Fig. 4. Impact of RPA binding on PolD and PriSL primer extension activity.

A 17 nucleotide-long primer labeled with a 5′ Cy5 fluorophore annealed to a 87 nucleotide-long template was used as substrate in reactions with all deoxyribonucleotides and ribonucleotides at physiological concentrations (sequences are shown in Supplementary Table 4). Reactions were incubated for 10 min at 55 °C with PolD or PriSL, and one of several RPA constructs. Experiments were repeated n = 4 times, band integration was performed in all 4 biological replicates (87 nt band for PolD gels, >70 nt bands for PriSL gels) to derive standard deviation. Each bar shows the mean value, standard deviation is represented as error bars, and individual measurements are shown as white dots. Uncropped gels are provided as a Source Data file. a–d Impact of RPA binding on PolD primer-extension activity. PolD was incubated at a concentration of 0.25 µM, with increasing amounts of different RPA constructs ranging from 0.05 to 0.8 µM (lanes 4-8). Lanes 1 and 2 contain an oligonucleotide ladder of 87-nt and a negative control experiment without proteins, respectively. Lane 3 contains PolD (0.25 µM) in the absence of RPA. a primer extension assay by PolD in the presence of RPA, b of Rpa2^WH, c of RPA^ΔWH and d RPA^ΔWH + Rpa2^WH. e Impact of RPA binding on PriSL primer-extension activity. The primer-template substrate was the same as in (a). PriSL (0.2 µM) was incubated with increasing amounts of different RPA constructs ranging from 0.05 to 0.8 µM (lanes 3–7). Lane 1 is the negative control without proteins. Lane 2 contains PriSL (0.2 µM) without RPA. Lane 8 contains oligonucleotide ladders (87 nt and 57-nt). From the left to the right panels: e primer extension assay by PriSL in the presence of RPA, f of Rpa2^WH, g of RPA^ΔWH and h RPA^ΔWH + Rpa2^WH. The short ~20 nt PriSL primer extension products (black arrowheads) and the PolD exonuclease digestion products (*) are highlighted.

Fig. 5. Crystal structure of the Primase-Rpa2^WH complex.

a–c Electron density map and model of the Primase-Rpa2^WH complex crystal structure. The 2mFo-DFc electron density map is contoured at σ = 1.5. c PriS-Rpa2^WH interface, with Rpa2^WH residues colored according to the intensity ratio I_cplx/I_free from blue (weak attenuation) to red (large attenuation) as in Fig. 3k. d–e Detailed view of the interface between PriS and Rpa2^WH. f Surface of Primase and Rpa2^WH colored according to their Coulomb potential, calculated in ChimeraX v1.7; and multiple sequence alignment of the linker between the trimeric core helix of Rpa2 and the WH domain in Thermococcales showing the conservation of acidic residues.

Fig. 6. Conserved protein-protein interaction interfaces on winged-helix domains.

Superposition of a Pyrococcus abyssi Rpa2^WH in complex with PriS with b human Rpa2^WH in complex with SMARCAL1^N-ter (PDB ID 4MQV) and c human Stn1^WH in complex with Polα (PDB ID 8D0K).

Fig. 7. Cryo-EM structure of PolD in complex with Rpa2^WH.

a Final composite map of PolD-Rpa2^WH at 2.9 Å global average resolution. b PolD-Rpa2^WH interface, with Rpa2^WH residues colored according to the intensity ratio I_cplx/I_free from blue (weak attenuation) to red (large attenuation) as in Fig. 3l. c Superposition of the two different classes that resulted from a focused 3D classification with a soft mask around Rpa2^WH, and d, e a detailed view of the interface between PolD and Rpa2^WH for each class. f Schematic drawing of Rpa2^WH with the secondary structure elements that comprise the PolD interface colored in blue. g, h Comparison of the rotational displacement difference between 3D classes 1 and 2, and alignments of the Primase-Rpa2^WH structure to Rpa2^WH in the PolD-Rpa2^WH structure, showing the respective volumes of the resulting clashes in red.

Fig. 8. Proposed model for the interaction of Primase and RPA during primer elongation.

a Superposition of the PriSL^ΔCTD-Rpa2^WH crystal structure with DNA-bound PrimPol (PDB ID 5L2X), aligned to our previously reported structure of ssDNA-bound RPA trimeric core (PDB ID 8AAS). The PrimPol-DNA ternary structure was used to model the DNA substrate in the PriSL active site. The RPA-ssDNA structure was oriented so that the C-terminal of the Rpa2 core would face the N-terminal of the Rpa2^WH, while matching the 5’ and 3’ ends of the DNA template. b Schematic diagram of our proposed ‘WH-bait’ mechanism for WH domain-dependent PriSL stimulation by RPA. A potential polymerase switch event mediated by the PolD binding site in Rpa2^WH is shown in step 4.