Crystal structure of the heterotrimeric RNase H2 complex. A, ribbon diagram representation of human RNase H2 reveals a linear arrangement of subunits where the core of the catalytic domain (RNASEH2A; blue) is stacked on the interwoven auxiliary RNASEH2B (green) and RNASEH2C (red) subunits. The C terminus of RNASEH2A, residues 284–299 (blue arrow), extends from the core domain and bridges the other two subunits. The first residue of the extension, RNASEH2A:284, and the last residue of the core domain, RNASEH2A:257, are indicated by gray and blue asterisks, respectively. The C terminus of RNASEH2C (red arrow) forms a kinked α-helix at the intersubunit interface. B, electrostatic surface of human RNase H2 indicates two positively charged patches: one in the catalytic active site groove (gray arrow) and the second on the interwoven RNASEH2BC heterodimer (black arrow). Electrostatic surface potential was calculated using PDB2PQR (34) and colored by potential (+9kT/e to −9 kT/e; electropositive in blue and electronegative in red) on the solvent accessible surface using the APBS tool (35). These and all other molecular graphics were generated using PyMOL (DeLano Scientific LLC).

The RNASEH2C C terminus, but not residues 91–116, are essential for complex formation and enzyme activity. A, RNASEH2C (red and pink ribbons), together with RNASEH2A (blue ribbons) and RNASEH2B (green ribbons), form a triple β-barrel. End points of the disordered loop in RNASEH2C (residues Thr-86 and Arg-118) are indicated. The chain is continuous from residue Arg-118 to Gln-160 (pink ribbon). B, Coomassie Blue-stained SDS-PAGE gels show purification of GST-tagged RNASEH2B (GST-B) and associated proteins. Affinity purification after coexpression of GST-RNASEH2B and RNASEH2A with either RNASEH2C^ΔC (left panel) or RNASEH2C^Δ91–116 (right panel). C, RNASEH2ABC^Δ91–116 shows the same activity as wild-type RNase H2 (RNASEH2ABC). In contrast, RNASEH2ABC^ΔC is more than 100-fold less active. Point mutations D34A/D169A (RNASEH2A^catBC) abolish all detectable RNase H2 activity. D, sequence alignment for human (Hs), Mus musculus (Mm), Canis familiaris (Cf), and Bos taurus (Bt) RNASEH2C. Secondary structure elements for human RNASEH2C are shown in red (arrows for β-strands; spirals for α helices). The magenta box marks amino acids deleted in RNASEH2C^Δ91–116; the vertical black arrow marks where RNASEH2C^ΔC was truncated; * mark residues mutated in AGS.

The C terminus of RNASEH2A is essential and sufficient for its interaction with RNASEH2B and -C. A, surface representation of the interface between human RNASEH2B (green), RNASEH2C (red) and the C terminus of RNASEHA:284–299 (blue ribbon). Amino acids found to be essential for binding in the peptide array (Tyr-287, Phe-288, Arg-291, Leu-293, Ala-296) are shown as sticks. B, Coomassie Blue stained SDS-PAGE gels show purification of GST-tagged RNASEH2B (GST-B) and associated proteins. Schematics illustrate subunit compositions of expressed complexes. Complexes composed of full-length subunits (RNASEH2ABC or RNASEH2BC) or with RNASEH2A truncated after residue 247 (RNASEH2A^ΔCBC) were bound to glutathione-Sepharose (bound) and released by cleavage with Precision Protease (cleaved). C, reconstitution of enzyme activity after mixing of RNASEH2A and RNASEH2BC in vitro. RNA₁₈:DNA₁₈ substrate was cleaved with increasing concentrations of purified proteins. + BC (dashed lines) indicates addition of RNASEH2BC heterodimer; RNASEH2A^cat and RNASEH2A^catBC indicate D34A and D169A point mutations in RNASEH2A (catalytically inactive); RNASEH2A^ΔC indicates truncated RNASEH2A:1–247; mock indicates background activity from the cleaved fraction of a GST-only purification. D, affinity purification after co-expression of human GST-RNASEH2B and RNASEH2C with either murine RNASEH2A (RNASEH2mmA-hsBC), A. fulgidus RNaseHII (RNASEH2Afu-hsBC) or Afu RNaseHII fused to residues 248–299 of human RNASEH2A (RNASEH2Afu-hsA^C-BC). E, cleavage of D₁₄R₁D₃:D₁₈ substrate by chimeric RNase H2 complexes described under D, as well as wild-type human RNase H2 (RNASEH2ABC), murine RNase H2 (mmRNASEH2ABC) and A. fulgidus RNaseHII (Afu RNASEH2). F, fluorescence anisotropy of a fluorescein-labeled peptide (RNASEH2A:283–299) as a function of increasing concentrations of RNASEH2BC. G, peptide array corresponding to RNASEH2A residues 275–299 probed with His-tagged RNASEH2BC and anti-His-HRP. Alanine point mutations are printed in red; RNASEH2A residues essential for binding are indicated in bold; “antibody only” shows background binding of anti-His-HRP to the array. H, sequence alignment for human (Hs) and Mus musculus (Mm) RNASEH2A with RNase HII from Archeoglobus fulgidus (Afu_rnhB). Secondary structure elements for human RNASEH2A are shown in blue (arrows for β-strands; loops for α-helices). The vertical black arrow marks where RNASEH2A^ΔC was truncated; dark blue boxes highlight peptides sufficient for binding RNASEH2BC; dark blue arrowheads indicate residues essential for binding in the peptide arrays described above; * mark residues mutated in AGS. This and all other sequence alignments were generated using ALINE (36).

AGS mutations at the RNASEH2A/RNASEH2C interface lead to reduced protein stability and enzyme activity. A, crystal structure of human RNase H2 is shown as ribbons and colored as in Fig. 1. Residues mutated in AGS are shown as yellow sticks. Nitrogen and oxygen atoms of highlighted residues are blue and red, respectively. B, close-up showing AGS affected mutations in the vicinity of the RNASEH2C C-terminal kinked helix. The left panel highlights residues in our human RNase H2 crystal structure while the right panel shows the corresponding residues for our re-refined murine model. (Arrows indicate the direction of the helix dipole running from positive to negative). C, fluorescence-based thermal stability assay was used to measure the melting temperatures of recombinant AGS mutant complexes. All mutations that cluster at the heterotrimer interface destabilize RNase H2. Error bars indicate the standard deviation of 10 independent measurements. D, most of these AGS mutations also cause reduced enzyme activity. Cleavage of D₁₄R₁D₃:D₁₈ substrate with increasing amounts of RNase H2 was measured in triplicate.