The structure of the mammalian RNase H2 complex provides insight into RNA.NA hybrid processing to prevent immune dysfunction

Abstract

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA.DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi-Goutières syndrome. We have determined the crystal structure of the three-protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein-protein interaction motifs that could allow for efficient participation in multiple cellular functions. We have identified four conserved acidic residues in the active site that are necessary for activity and suggest a two-metal ion mechanism of catalysis for RNase H2. An Okazaki fragment has been modeled into the RNase H2 nucleic acid binding site providing insight into the recognition of RNA.DNA junctions by the RNase H2. Further structural and biochemical analyses show that some RNase H2 disease-causing mutations likely result in aberrant protein-protein interactions while the RNase H2A subunit-G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification.

FIGURE 1.

Ribbon structure of the mouse RNase H2 enzyme complex consisting of the RNase H2A (green), RNase H2B (gold), and RNase H2C (blue) proteins. The elongated complex is stabilized by the weaving together of the H2B and H2C proteins to form a triple-barrel motif that provides a platform for binding the H2A protein. The active site of the catalytic H2A protein is located near one end of the complex with no physical contributions from the H2B or H2C protein.

FIGURE 2.

Interactions within RNase H2 triple-barrel motif. A, eighteen strands contributed from the H2C (blue) and N-terminal H2B (gold) proteins interact in parallel and antiparallel fashion to form three perpendicular barrels (numbered). The end and side of barrel 1 form a platform for the stable interaction with the H2A protein (green). B, a superposition of the triple-barrel motifs from the H2B and H2C proteins on the eukaryotic transcription factor IIF (TFIIF) (magenta) reveals a similar architecture. The structural similarity between the two complexes suggests a role for DNA binding and bending as well as protein interaction for H2B/H2C as seen in TFIIF. C, schematic of triple-barrel topology formed in the mammalian RNase H2. Strands from the H2C protein are in blue, H2B in gold, and H2A in green.

FIGURE 3.

The mammalian RNase H2A protein has N- and C-terminal domains not present in bacterial RNase H2 proteins. The N-terminal domain (yellow) of mouse RNase H2A forms the first strand of the central β-sheet that forms the core of the protein. A disulfide bond (Cys-24 to Cys-29) helps anchor the strand in place. The resulting exposed edge of the β-sheet strongly suggests an additional protein-protein interface for the RNase H2 complex.

FIGURE 4.

Model of RNA·DNA hybrid binding to the RNase H2 complex. A, a model Okazaki fragment was docked into the active site of the mammalian RNase H2 by a superposition of the bacterial RNase H in complex with a double strand RNA·DNA duplex (PDB ID: 1ZBI; see text for details). The three-protein RNase H2 complex is shown as an electrostatic surface representation. The RNA·DNA duplex was modified to contain an RNA·DNA junction on the ribonucleotide-containing strand. The minor groove of the double strand oligonucleotide straddles the β6–α6 loop of the protein, which may play a role in substrate recognition. B, the model is positioned in the active site to simulate hydrolysis between the last two 3′ ribonucleotides. The four conserved acidic residues (Asp-35, Glu-35, Asp-142, and Asp-170) likely coordinate two divalent metal ions (blue spheres) for catalysis. Several positively charged amino acids are positioned to make favorable electrostatic interactions with the bound oligonucleotide, including Lys-168, which may serve as a sensor of the RNA·DNA junction by interacting with the 2′-hydroxyl group of the ribonucleotides. To illustrate the position of the RNA·DNA junction, ribonucleotides are shown in yellow and deoxyribonucleotides are shown in magenta.

FIGURE 5.

AGS mutations in RNase H2. A, mutation of residue Lys-162 (red) to threonine in the H2B protein or residue Arg-69 (red) to tryptophan in the H2C protein results in catalytically active enzyme. The structure indicates mutation of K162T may disrupt packing of the helix against the core of the protein that could destabilize potential protein-protein interactions with the distal end of the complex. Likewise, mutation of R69W could potentially disrupt protein interactions with any partner binding the exposed β-sheet of the H2A protein. Residues Ala-177, Val-185, and Tyr-219 are in disordered region shown at the top of the complex. B, residue Gly-37 (red) of the H2A protein lies within the active site near the catalytic Asp-34 and Glu-35 amino acids.

FIGURE 6.

Activity of RNase H2 on substrate containing a single ribonucleotide. A, sequence of oligonucleotide substrate with ribonucleotide indicated in lowercase and deoxyribonucleotides in uppercase. B, RNase H2 WT (lane 2, 0.4 nm; lane 3, 4 nm; and lane 4, 40 nm) or G37S mutant (lane 6, 0.4 nm; lane 7, 4 nm; and lane 8, 40 nm) enzyme complex was incubated with oligonucleotide. Lanes 1 and 5 contain no RNase H2. Quantification of the RNase H2A activity indicates that RNase H2 WT generates the products at 67 nm/nmol enzyme compared with 2.2 nm/nmol for the RNase H2 G37S mutant enzyme indicating a 30-fold reduction in activity.