FIGURE 4.

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.