The Structural and Biochemical Characterization of Human RNase H2 Complex Reveals the Molecular Basis for Substrate Recognition and Aicardi-Goutières Syndrome Defects

Figiel, Małgorzata; Chon, Hyongi; Cerritelli, Susana M.; Cybulska, Magdalena; Crouch, Robert J.; Nowotny, Marcin

doi:10.1074/jbc.m110.181974

Cited by 59 publications

(70 citation statements)

References 29 publications

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“…The RNase H2A Arg-108 is located on a unique loop that interfaces with the H2C subunit. A recent structure of the human RNase H2 complex (PDB ID 3P56) (32,33) confirms the Arg-108 interaction with the H2C subunit and indicates specific contacts with the H2C Leu-134 and Glu-135 residues. Also, the human RNase H2 structure locates RNase H2A Arg-291 more than 50 Å removed from the active site positioned to interface with the H2B and H2C subunits.…”

Section: Discussionmentioning

confidence: 99%

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

Coffin

Hollis

Perrino

2011

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

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

Coffin

Hollis

Perrino

2011

Journal of Biological Chemistry

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“…(C) RNase H2 residues (yellow sticks) affected by missense mutations identified in SLE patients, mapped on the structure of the human enzyme (PDB ID, 3PUF; ref. 63 (27). Hypomorphic variants of RNASEH2 genes are associated with SLE.…”

Section: Carriers Of Ags Mutations Frequently Develop Sle-associated mentioning

confidence: 99%

Defective removal of ribonucleotides from DNA promotes systemic autoimmunity

Günther¹,

Kind²,

Reijns³

et al. 2014

J. Clin. Invest.

196

178

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Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE-and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.

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“…Not only does the GRG motif largely relate to the enzyme's activity, but it also functions in substrate recognition and binding [13]. As shown in the crystal structure of the complex between bacterial RNase H2 and nucleic acid, the 5' phosphate is located at the active site and there are interactions between the 2'-OH substituent of the RNA with the conserved GRG motif [14][15][16].…”

mentioning

confidence: 99%

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

Shen

Zheng

et al. 2015

Open Life Sciences

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RNase H2 consists of three subunits (H2A, H2B and H2C) and is involved in the hydrolysis of RNA/DNA hybrids. The GRG motif in RNase H2 is highly conserved and recognizes the ribonucleotides misincorporated into dsDNA. The mutant G37S in the GRG motif of human RNase H2A was found to be correlated with Aicardi-Goutièressyndrome (AGS). In this study, 4 mutants (G37S, G37A, R38A and G39A) were prepared and their biochemical properties of secondary structure, activity and binding affinity with substrate were studied in order to explore the function of the GRG motif. The activity assay showed that the mutations resulted in significantly decreased RNase activity. The binding efficiency assay demonstrated that binding affinities between 4 mutants and 1-ribo FAMlabeled substrate were significantly weakened. These results all indicated that the GRG motif contributes directly to substrate binding and is closely related to the enzyme activity.

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The Structural and Biochemical Characterization of Human RNase H2 Complex Reveals the Molecular Basis for Substrate Recognition and Aicardi-Goutières Syndrome Defects

Cited by 59 publications

References 29 publications

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

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

Defective removal of ribonucleotides from DNA promotes systemic autoimmunity

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

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