Scott Harvey

Mutations in the gene encoding the 3′-5′ DNA exonuclease TREX1 are associated with systemic lupus erythematosus

TREX1 acts in concert with the SET complex in granzyme A–mediated apoptosis, and mutations in TREX1 cause Aicardi-Goutières syndrome and familial chilblain lupus. Here, we report monoallelic frameshift or missense mutations and one 3′ UTR variant of TREX1 present in 9/417 individuals with systemic lupus erythematosus but absent in 1,712 controls (P = 4.1 × 10−7). We demonstrate that two mutant TREX1 alleles alter subcellular targeting. Our findings implicate TREX1 in the pathogenesis of SLE.

A mutation in TREX1 that impairs susceptibility to granzyme A-mediated cell death underlies familial chilblain lupus

We recently described a novel autosomal-dominant genodermatosis, termed familial chilblain lupus, and mapped its genetic locus to chromosome 3p21. Familial chilblain lupus manifests in early childhood with ulcerating acral skin lesions and is associated with arthralgias and circulating antinuclear antibodies. In this study, we report the identification of a heterozygous missense mutation (D18N) in TREX1 encoding the 3′-5′repair exonuclease 1 in affected individuals of the family with chilblain lupus. The homodimeric TREX1 is the most abundant intracellular DNase in mammalian cells. We have recently shown that TREX1 plays a role in apoptotic single-stranded DNA damage induced by the killer lymphocyte protease granzyme A. D18N affects a highly conserved amino acid residue critical for catalytic activity. Recombinant mutant TREX1 homodimers are enzymatically inactive, while wild type/mutant heterodimers show residual exonucleolytic activity, suggesting a heterozygous loss of function. Lymphoblastoid cells carrying the D18N mutation are significantly less sensitive to granzyme A-mediated cell death, suggesting a novel role for this caspase-independent form of apoptosis in the pathogenesis of familial chilblain lupus. Our findings also warrant further investigation of TREX1 in common forms of lupus erythematosus.

The Crystal Structure of TREX1 Explains the 3' Nucleotide Specificity and Reveals a Polyproline II Helix for Protein Partnering.

The TREX1 enzyme processes DNA ends as the major 3′ → 5′ exonuclease activity in human cells. Mutations in the TREX1 gene are an underlying cause of the neurological brain disease Aicardi-Goutières syndrome implicating TREX1 dysfunction in an aberrant immune response. TREX1 action during apoptosis likely prevents autoimmune reaction to DNA that would otherwise persist. To understand the impact of TREX1 mutations identified in patients with Aicardi-Goutières syndrome on structure and activity we determined the x-ray crystal structure of the dimeric mouse TREX1 protein in substrate and product complexes containing single-stranded DNA and deoxyadenosine monophosphate, respectively. The structures show the specific interactions between the bound nucleotides and the residues lining the binding pocket of the 3′ terminal nucleotide within the enzyme active site that account for specificity, and provide the molecular basis for understanding mutations that lead to disease. Three mutant forms of TREX1 protein identified in patients with Aicardi-Goutières syndrome were prepared and the measured activities show that these specific mutations reduce enzyme activity by 4–35,000-fold. The structure also reveals an 8-amino acid polyproline II helix within the TREX1 enzyme that suggests a mechanism for interactions of this exonuclease with other protein complexes.

The TREX1 Double-stranded DNA Degradation Activity Is Defective in Dominant Mutations Associated with Autoimmune Disease

Mutations in TREX1 have been linked to a spectrum of human autoimmune diseases including Aicardi-Goutières syndrome (AGS), familial chilblain lupus (FCL), systemic lupus erythematosus, and retinal vasculopathy and cerebral leukodystrophy. A common feature in these conditions is the frequent detection of antibodies to double-stranded DNA (dsDNA). TREX1 participates in a cell death process implicating this major 3′ → 5′ exonuclease in genomic DNA degradation to minimize potential immune activation by persistent self DNA. The TREX1 D200N and D18N dominant heterozygous mutations were identified in AGS and FCL, respectively. TREX1 enzymes containing the D200N and D18N mutations were compared using nicked dsDNA and single-stranded DNA (ssDNA) degradation assays. The TREX1WT/D200N and TREX1WT/D18N heterodimers are completely deficient at degrading dsDNA and degrade ssDNA at an expected ∼2-fold lower rate than TREX1WT enzyme. Further, the D200N- and D18N-containing TREX1 homo- and heterodimers inhibit the dsDNA degradation activity of TREX1WT enzyme, providing a likely explanation for the dominant phenotype of these TREX1 mutant alleles in AGS and FCL. By comparison, the TREX1 R114H homozygous mutation causes AGS and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1R114H/R114H homodimer has dysfunctional dsDNA and ssDNA degradation activities and does not detectibly inhibit the TREX1WT enzyme, whereas the TREX1WT/R114H heterodimer has a functional dsDNA degradation activity, supporting the recessive genetics of TREX1 R114H in AGS. The dysfunctional dsDNA degradation activities of these disease-related TREX1 mutants could account for persistent dsDNA from dying cells leading to an aberrant immune response in these clinically related disorders.

Exonuclease TREX1 degrades double-stranded DNA to prevent spontaneous lupus-like inflammatory disease.

Significance The TREX1 enzyme degrades DNA, and mutations in the TREX1 gene cause autoimmune diseases. The TREX1 D18N mutation causes a form of lupus called familial chilblain lupus. We solved the structure of TREX1 D18N bound to dsDNA, showing how the enzyme interacts with dsDNA. We also replaced the TREX1 WT gene in mice with the TREX1 D18N mutated gene and showed how this mutation causes a lupus-like disease. Together, the TREX1 D18N–dsDNA structure and the spontaneous disease exhibited in the TREX1 D18N mouse help to define how TREX1 degrades dsDNA to prevent this molecule from acting as an autoantigen in the mouse and, most likely, in humans to promote autoimmune disease. The TREX1 gene encodes a potent DNA exonuclease, and mutations in TREX1 cause a spectrum of lupus-like autoimmune diseases. Most lupus patients develop autoantibodies to double-stranded DNA (dsDNA), but the source of DNA antigen is unknown. The TREX1 D18N mutation causes a monogenic, cutaneous form of lupus called familial chilblain lupus, and the TREX1 D18N enzyme exhibits dysfunctional dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune disease. We determined the structure of the TREX1 D18N protein in complex with dsDNA, revealing how this exonuclease uses a novel DNA-unwinding mechanism to separate the polynucleotide strands for single-stranded DNA (ssDNA) loading into the active site. The TREX1 D18N dsDNA interactions coupled with catalytic deficiency explain how this mutant nuclease prevents dsDNA degradation. We tested the effects of TREX1 D18N in vivo by replacing the TREX1 WT gene in mice with the TREX1 D18N allele. The TREX1 D18N mice exhibit systemic inflammation, lymphoid hyperplasia, vasculitis, and kidney disease. The observed lupus-like inflammatory disease is associated with immune activation, production of autoantibodies to dsDNA, and deposition of immune complexes in the kidney. Thus, dysfunctional dsDNA degradation by TREX1 D18N induces disease in mice that recapitulates many characteristics of human lupus. Failure to clear DNA has long been linked to lupus in humans, and these data point to dsDNA as a key substrate for TREX1 and a major antigen source in mice with dysfunctional TREX1 enzyme.

The Structure of the Mammalian RNase H2 Complex Provides Insight into RNA·DNA Hybrid Processing to Prevent Immune Dysfunction

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.

The Human TREX2 3′ → 5′-Exonuclease Structure Suggests a Mechanism for Efficient Nonprocessive DNA Catalysis

The 3′ → 5′-exonucleases process DNA ends in many DNA repair pathways of human cells. Determination of the human TREX2 structure is the first of a dimeric 3′-deoxyribonuclease and indicates how this highly efficient nonprocessive enzyme removes nucleotides at DNA 3′ termini. Symmetry in the TREX2 dimer positions the active sites at opposite outer edges providing open access for the DNA. Adjacent to each active site is a flexible region containing three arginines positioned appropriately to bind DNA and to control its entry into the active site. Mutation of these three arginines to alanines reduces the DNA binding capacity by ∼100-fold with no effect on catalysis. The human TREX2 catalytic residues overlay with the bacterial DnaQ family of 3′-exonucleases confirming the structural conservation of the catalytic sites despite limited sequence identity, and mutations of these residues decrease the still measurable activity by ∼105-fold, confirming their catalytic role.

RNaseH2 mutants that cause Aicardi–Goutieres syndrome are active nucleases

Mutations in the genes encoding the RNaseH2 and TREX1 nucleases have been identified in patients with Aicardi–Goutieres syndrome (AGS). To determine if the AGS RNaseH2 mutations result in the loss of nuclease activity, the human wild-type RNaseH2 and four mutant complexes that constitute the majority of mutations identified in AGS patients have been prepared and tested for ribonuclease H activity. The heterotrimeric structures of the mutant RNaseH2 complexes are intact. Furthermore, the ribonuclease H activities of the mutant complexes are indistinguishable from the wild-type enzyme with the exception of the RNaseH2 subunit A (Gly37Ser) mutant, which exhibits some evidence of altered nuclease specificity. These data indicate that the mechanism of RNaseH2 dysfunction in AGS cannot be simply explained by loss of ribonuclease H activity and points to a more complex mechanism perhaps mediated through altered interactions with as yet identified nucleic acids or protein partners.

Exonucleases and the Incorporation of Aranucleotides into DNA

The polymerization of nucleotide analogs into DNA is a common strategy used to inhibit DNA synthesis in rapidly dividing tumor cells and viruses. The mammalian DNA polymerases catalyze the insertion of the arabinofuranosyl analogs of dNTPs (aranucleotides) into DNA efficiently, but elongate from the 3′ aranucleotides poorly. Slow elongation provides an opportunity for exonucleases to remove aranucleotides. The exonuclease activity associated with DNA polymerase δ removes araCMP from 3′ termini with the same efficiency that it removes a paired 3′ deoxycytosine suggesting that the proofreading exonucleases associated with DNA polymerases might remove aranucleotides inefficiently. A separate 30 kDa exonuclease has been purified from mammalian cells that removes araCMP from 3′ termini. The activity of this enzyme in the cell could remove aranucleotides from 3′ termini of DNA and decrease the efficacy of the analogs. Inhibition analysis of the purified exonuclease shows that this enzyme is inhibited by thioinosine monophosphate (TIMP) with aKi=17 μM. When high TIMP levels are generated in HL-60 cells, incorporation of araC in DNA is increased about 16-fold relative to total DNA synthesis. This increased araC in DNA is likely a result of exonuclease inhibition in the cell. Thus, exonucleases in cells might play an important role in removing aranucleotides inserted by DNA polymerases.

The TREX1 Exonuclease R114H Mutation in Aicardi-Goutières Syndrome and Lupus Reveals Dimeric Structure Requirements for DNA Degradation Activity

Background: Mutations in the TREX1 exonuclease gene cause a spectrum of autoimmune diseases. Results: The TREX1 Arg-114 residue acts across the stable dimer interface. Conclusion: TREX1 residues in one protomer contribute to DNA degradation catalyzed in the opposing protomer. Significance: These data help to explain the heterozygous disease condition. Mutations in the TREX1 gene cause Aicardi-Goutières syndrome (AGS) and are linked to the autoimmune disease systemic lupus erythematosus. The TREX1 protein is a dimeric 3′ DNA exonuclease that degrades DNA to prevent inappropriate immune activation. One of the most common TREX1 mutations, R114H, causes AGS as a homozygous and compound heterozygous mutation and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1 proteins containing R114H and the insertion mutations aspartate at position 201 (D201ins) and alanine at position 124 (A124ins), found in compound heterozygous AGS with R114H, were prepared and the DNA degradation activities were tested. The homodimer TREX1R114H/R114H exhibits a 23-fold reduced single-stranded DNA (ssDNA) exonuclease activity relative to TREX1WT. The TREX1D201ins/D201ins and TREX1A124ins/A124ins exhibit more than 10,000-fold reduced ssDNA degradation activities. However, the TREX1R114H/D201ins and TREX1R114H/A124ins compound heterodimers exhibit activities 10-fold greater than the TREX1R114H/R114H homodimer during ssDNA and double-stranded DNA (dsDNA) degradation. These higher levels of activities measured in the TREX1R114H/D201ins and TREX1R114H/A124ins compound heterodimers are attributed to Arg-114 residues of TREX1D201ins and TREX1A124ins positioned at the dimer interface contributing to the active sites of the opposing TREX1R114H protomer. This interpretation is further supported by exonuclease activities measured for TREX1 enzymes containing R114A and R114K mutations. These biochemical data provide direct evidence for TREX1 residues in one protomer contributing to DNA degradation catalyzed in the opposing protomer and help to explain the dimeric TREX1 structure required for full catalytic competency.