PCNA directs type 2 RNase H activity on DNA replication and repair substrates

Abstract

Ribonuclease H2 is the major nuclear enzyme degrading cellular RNA/DNA hybrids in eukaryotes and the sole nuclease known to be able to hydrolyze ribonucleotides misincorporated during genomic replication. Mutation in RNASEH2 causes Aicardi-Goutières syndrome, an auto-inflammatory disorder that may arise from nucleic acid byproducts generated during DNA replication. Here, we report the crystal structures of Archaeoglobus fulgidus RNase HII in complex with PCNA, and human PCNA bound to a C-terminal peptide of RNASEH2B. In the archaeal structure, three binding modes are observed as the enzyme rotates about a flexible hinge while anchored to PCNA by its PIP-box motif. PCNA binding promotes RNase HII activity in a hinge-dependent manner. It enhances both cleavage of ribonucleotides misincorporated in DNA duplexes, and the comprehensive hydrolysis of RNA primers formed during Okazaki fragment maturation. In addition, PCNA imposes strand specificity on enzyme function, and by localizing RNase H2 and not RNase H1 to nuclear replication foci in vivo it ensures that RNase H2 is the dominant RNase H activity during nuclear replication. Our findings provide insights into how type 2 RNase H activity is directed during genome replication and repair, and suggest a mechanism by which RNase H2 may suppress generation of immunostimulatory nucleic acids.

Figure 1.

Structure of the Afu PCNA:RNase HII complex. (A) Three RNase HII molecules (cyan, tan and red) bind the PCNA homotrimeric ring (grey) in unique orientations. Two binding modes (cyan and tan) extend away from the center of the ring, while the third (red) obstructs the opening. On the right the complex is rotated 90° about the plane of the PCNA ring. (B–D) The three orientations of RNase HII observed in the complex with PCNA are shown independently from the same viewpoint. Active site residues (D6, E7, D101 and D129) are shown as yellow sticks. RNase HII is anchored to PCNA by its C-terminal PIP-box motif (residues 199–205) and rotates about a flexible hinge (black arrow). For each binding mode, a close-up of the hinge region (residues 195–198) reveals conformational changes in RNase HII and PCNA (right panel). PCNA residues R241, S244 and D150, and RNase HII residues S195 and R198 are labeled. The final refined model is shown in simulated annealing omit maps calculated for models in which residues corresponding to the hinge are removed (B, 3.0σ; C, 1.5σ; D, 4.0σ). (E) Schematic illustrating the range of motion between the three observed conformations. The hinge point (black sphere) marks the average Cα coordinate of PIP-box residue Q199. Cylinders are coloured according to the above models and yellow spheres highlight the average Cα coordinate of their respective active site residues. The PCNA ring is shown for reference (grey semi-transparent surface). (F) Close-up of the hinge region for the two RNase HII chains in which the entire sequence (¹⁹⁵SNLR¹⁹⁸) is resolved. These and all other molecular graphics were generated using PyMOL (DeLano Scientific LLC).

Figure 2.

PCNA enhances RNase HII activity on certain substrates containing single ribonucleotides. (A) Superposition of the three RNase HII conformations (tan, cyan and red) with the apo RNase HII (grey) crystal structure (PDB ID: 1I39) highlighting the hinge region (residues ₁₉₅SNLR₁₉₈) and PIP-box motif (residues ₁₉₉QKTLDDF₂₀₅). Active sites residues are in yellow. (B) The RNase HII-PCNA interaction requires the PIP-box, but not the hinge region: GST-tagged wild-type, hinge deletion mutant (ΔSNLR), or PIP-box deletion mutant (ΔPIP) RNase HII was bound to glutathione beads and incubated with PCNA. Bound and unbound proteins were separated by SDS-PAGE and Coomassie stained. (M = Molecular weight marker) (C) Substrates used in panels D–F and G, respectively: 5′-labeled oligonucleotides (*) with a single ribonucleotide were annealed to a complementary DNA 18-mer. RNA bases in lower case (red); DNA bases in uppercase (black); cleavage positions indicated by arrows. (D) PCNA enhances RNase HII activity: Substrate D₁₄R₁D₃:D₁₈ was incubated with Afu RNase HII and/or PCNA and separated by denaturing PAGE. (E) Enhancement of activity was lost when the RNase HII PIP-box was deleted (ΔPIP), (F) or when four hinge residues were removed (ΔSNLR). (G) PCNA inhibits RNase HII activity on substrate containing a ribonucleotide nearer the 5′-end (D₅R₁D₁₂:D₁₈). Contaminating nuclease activity in the recombinant proteins was excluded as neither PCNA alone, nor the RNase HII D101N active site mutant (data not shown) showed any substrate cleavage. All protein concentrations are indicated in nM; all assays were performed with 250 nM of substrate. Reactions incubated for 20 min at 30°C, except F (120 min).

Figure 3.

PCNA promotes efficient cleavage of Okazaki-like substrates by RNase HII. (A) Sequences of Okazaki-like fragments used in B–F (RNA bases in lower case, grey; DNA bases in upper case, black). *R₁₀D₁₃:D₄₀ represents newly primed lagging strand DNA, while D₁₇ + *R₁₀D₁₃:D₄₀ corresponds to an unprocessed Okazaki junction. Arrows indicate cleavage sites with the size of the arrow indicating the extent of cleavage in the presence of PCNA. Each 5′-labeled substrate (2.5 nM) was incubated with Afu RNase HII and/or PCNA and separated by denaturing PAGE. (B) RNase HII generates smaller fragments from an unprocessed Okazaki junction. (C,D) PCNA enhances comprehensive cleavage of the RNA primer at a fully formed Okazaki junction, but not on primase-primed DNA. (E,F) 3′ labelling of R₁₀D₁₃ (0.5 nM) demonstrates that PCNA enhances cleavage at the junctional ribonucleotide of a fully formed Okazaki junction. All protein concentrations are given in nM. In vitro Afu PCNA homotrimer formation is inefficient at low concentrations, and consequently high concentrations of PCNA were used to supply sufficient PCNA trimeric rings for stimulation of RNase HII activity.

Figure 4.

Model of RNA/DNA hybrid binding to the PCNA:RNase HII complex. An RNA/DNA hybrid was docked into the active site of Afu RNase HII, based on conserved metal coordination geometry (see ‘Materials and Methods’ section). The hybrid was extended through the PCNA ring with B-form DNA duplex. The three panels show molecular surfaces of the three RNase HII conformations (cyan, tan and red) from the same orientation on a single PCNA ring (grey). In the closed conformation (red), the duplex could not be modelled through the PCNA ring without significant steric clashes. The active site residues are shown in yellow and the binding groove (K143, R46, Q43, G161, S162, G163 and Y164) in blue. The color of bases along the RNA/DNA hybrid indicate the observed pattern of PCNA stimulation (green) and inhibition (red) on RNase HII cleavage. Arrows indicate the 5′- and 3′-ends of the RNA strand.

Figure 5.

Crystal structure of a human PCNA:RNASEH2B PIP-box peptide complex. (A) A peptide corresponding to the C-terminal 18 residues of the RNASEH2B subunit (green ribbon) binds to each of the three PCNA monomers (light blue ribbon). (B) A view of the PIP-box interface shows the C-terminal component of the peptide (sticks, carbon atoms green) anchored into the hydrophobic pocket of PCNA (semi-transparent surface and light blue ribbon), while the N-terminal region forms hydrogen bonds (black dashes) with the backbone of the PCNA C-terminus. Residues F300 and F301 of RNASEH2B are labeled. Nitrogen and oxygen atoms in the interface are blue and red, respectively. (C) To compare Cα traces of the human RNASEH2B peptide (green) and the PIP-box of each of the Afu RNase HII conformations (cyan, tan and red), PCNA components of the Afu (grey ribbon) and human (light blue ribbon) complexes were superimposed.

Figure 6.

PCNA is sufficient to localize human RNase H2 to replication foci. (A) EGFP-RNASEH2B, the PIP-box mutant EGFP-RNASEH2B (FF > AA) and RNASEH1-EGFP (without mitochondrial localization signal) (5) localize to the nucleus in non-extracted COS7 cells. (B) Detergent extracted cells: EGFP-RNASEH2B requires an intact PIP-box to co-localize with PCNA at replication foci. RNASEH1-EGFP does not localize to replication foci. (C) EGFP-RNASEH2B co-localizes with PCNA through early-, mid- and late S-phase EGFP (green), PCNA (red), DAPI (blue); scale bars are 10 µm. (D) Quantification of co-localization between EGFP-RNASEH2B and PCNA in cells represented in panel B. Categories represent the percentage of foci with co-localizing signals in a cell. Graph shows average values of three independent experiments (100 cells per experiment); error bars indicate standard deviations. (E) RNase H2 interacts with PCNA in COS7 cells in a PIP-box dependent manner. EGFP proteins were affinity purified using GFP-Trap (Chromotek) from cell lysates. Bound/unbound proteins were detected by Western analysis.

Figure 7.

Models for PCNA:RNase H2 function in DNA repair and replication. (A) PCNA promotes type 2 RNase H recognition and cleavage of ribonucleotides that may become misincorporated during DNA synthesis. (B) Okazaki fragment maturation can occur through cleavage of 5′–3′-flaps by FEN1 or DNA2. Cleavage generates single stranded 5′-ppp RNA-DNA chimeras that have the potential to be highly immunogenic. (C) Fold-backs in raised flaps prevent resolution of these flaps by FEN1 (57). PCNA:RNase H2 cannot degrade the RNA primers in such hairpin structures, as PCNA obscures the primer. (D) PCNA:RNase H2 can efficiently remove RNA primers from Okazaki fragment junctions prior to flap generation by Pol δ, preventing formation of 5′-ppp nucleic acids.