Functional Consequences of the RNase H2A Subunit Mutations That Cause Aicardi-Goutières Syndrome

Abstract

Mutations in the three genes encoding the heterotrimeric RNase H2 complex cause Aicardi-Goutières Syndrome (AGS). Our mouse RNase H2 structure revealed that the catalytic RNase H2A subunit interfaces mostly with the RNase H2C subunit that is intricately interwoven with the RNase H2B subunit. We mapped the positions of AGS-causing RNase H2A mutations using the mouse RNase H2 structure and proposed that these mutations cause varied effects on catalytic potential. To determine the functional consequences of these mutations, heterotrimeric human RNase H2 complexes containing the RNase H2A subunit mutations were prepared, and catalytic efficiencies and nucleic acid binding properties were compared with the wild-type (WT) complex. These analyses reveal a dramatic range of effects with mutations at conserved positions G37S, R186W, and R235Q, reducing enzymatic activities and substrate binding affinities by as much as a 1000-fold, whereas mutations at non-conserved positions R108W, N212I, F230L, T240M, and R291H reduced activities and binding modestly or not at all. All mutants purify as three-subunit complexes, further supporting the required heterotrimeric structure in eukaryotic RNase H2. These kinetic properties reveal varied functional consequences of AGS-causing mutations in the catalytic RNase H2A subunit and reflect the complex mechanisms of nuclease dysfunction that include catalytic deficiencies and altered protein-nucleic acid interactions relevant in AGS.Aicardi-Goutières Syndrome (AGS) 2 is a rare genetic neurological disorder that mimics congenital viral infection with evidence of high interferon-␣ levels in serum and cerebrospinal fluid (1, 2). Mutations in the five genes RNASEH2A, RNASEH2B, RNASEH2C, TREX1, and SAMHD1 are known to cause AGS (3-5). The RNASEH2A, RNASEH2B, and RNASEH2C genes encode the RNase H2 heterotrimeric complex that recognizes and cleaves ribonucleotides present in RNA/DNA duplexes (4, 6). The TREX1 gene encodes a homodimeric 3Ј 3 5Ј DNA exonuclease (7-12), and the SAMHD1 gene encodes an enzyme of unknown function. The identification of disease-causing mutations in genes encoding the nucleases RNase H2 and TREX1 provides evidence for the concept that failure to properly process endogenous nucleic acids triggers an interferon-mediated immune response, helping to explain the phenotypic overlap of AGS with congenital infection (13). Furthermore, there is emerging evidence for a clinical, biochemical, and genetic connection between AGS and related autoimmune disorders including systemic lupus erythematosus, indicating that dysfunctional nucleic acid metabolism leads to immune activation and an autoimmune phenotype (14).RNase H2 provides the majority of ribonuclease H activity in human cells (15). All ribonuclease H enzymes specifically recognize RNA/DNA hybrids and hydrolyze the ribonucleotidecontaining polynucleotide strand. These enzymes are divided into two major families, type 1 and type 2, based on amino acid sequence similarities and distinguishing biochemical properties that sugge...

Mutation of the conserved GRG motif and decreasing activity of human RNase H2

“…This phenomenon strongly suggested that the GRG motif mutations in RNase H2A affect the binding affinity tremendously, which in turn affected the enzyme's activity. This was consistent with a previous report by Perrino et al which demonstrated that G37S activity decreases with decreasing substrate binding affinity [10]. The mutation of the GRG motif may affect the dihedral angle of the loop and the surrounding residues.…”

“…The K d values are shown in Table 2. The wild type RNase H2 complex bound to FAM-labeled duplex DNA with a K d value at 26.1 nM, which is comparable to that reported in literature [10]. K d values of the 4 mutants (G37A, G37S, R38A, and G39A) are 1009.5 ± 91.41 nM, 100.2 ± 8.09 nM, 554.5 ± 43.88 nM, and 765.6 ± 79.04 nM, respectively.…”

“…Gly37 in the GRG motif is very close to the active site, and thus is involved in the interaction with 2'-OH in the junction [19][20][21][22]. It was reported that G37S mutation of human RNase H2A impaired substrate cleavage and led to AGS [10]. In this study, the 4 mutants of the GRG motif in RNase H2A were cloned and purified in order to test the importance of the GRG motif, specifically the 3 mutants (G37A, R38A and G39A) and the AGS-related mutant (G37S) ( Figure 1B).…”

“…Because Ca 2+ cannot activate RNase H2, Ca 2+ was substituted for Mg 2+ to prevent catalysis in the experiments [6,10,11,16]. 1 nM FAM-labeled DNA 15 …”

“…The carboxyfluorescein (FAM) labeled-oligonucleotides DNA 15 -RNA 1 -DNA 14 (5'-FAM-ACCTATCTAACAACACT CAcTCCATCATAT-3') and the complementary DNA (5'-ATATGATGGAGTGAGTGTTGTTAGATAGGT-3') [10] were purchased from Takara (Japan). The ribonucleotide is indicated with the lowercase letter.…”