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

Abstract

The TREX1 enzyme processes DNA ends as the major 3 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 AicardiGoutières 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.Processing of DNA ends is an important step in many DNA metabolic pathways such as replication, repair, and recombination. The 3Ј 3 5Ј exonucleases play a critical role in correcting fragmented, modified, mispaired, or even normal nucleotides to generate 3Ј termini suitable for downstream events. The drastic consequences that result from impaired 3Ј exonuclease activities underscore the importance of these enzymes for cell survival. Proofreading of DNA synthesis by 3Ј exonucleases is one of the major determinants of mutagenesis and genome stability and cells lacking this ability show a high incidence of cancers (1-3) (for review, see Ref. 4). Cells with defects in proteins containing 3Ј exonuclease activity, such as the Werner syndrome protein, MRE11, APE1, and p53 proteins display chromosomal instability, cell cycle checkpoint defects, and sensitivity to ionizing radiation (5-9).The major 3Ј 3 5Ј exonuclease activity detected in human cell extracts is catalyzed by the TREX1 enzyme. The genes encoding the TREX1 and closely related TREX2 proteins have been identified and cloned (10, 11), and the recombinant proteins confirm the robust catalytic nature of these enzymes (12, 13). Amino acid sequence analysis reveals the TREX proteins belong to the DnaQ family of 3Ј 3 5Ј exonucleases; a structurally conserved group of exonucleases that span Archaea and bacteria to humans and includes such proteins as the exonuclease domains of Werner syndrome protein, the bacterial ⑀ subunit of DNA polymerase III (⑀ subunit), and exonuclease I (Exo I) 2 (14 -17). A hallmark of the DnaQ family exonucleases is three conserved sequence motifs known as Exo I, II, and III. These motifs contain four ...

Dominant Mutations of the TREX1 Exonuclease Gene in Lupus and Aicardi-Goutières Syndrome

“…Structural studies of the TREX1-DNA complex provide direct evidence for ssDNA binding of at least four nucleotides in length in the active sites ( Fig. 1) (21). Amino acid residues Asp-18 and Asp-200 are two of the divalent metal ion Mg 2ϩ -coordinating aspartates in the TREX1 active site that contribute to DNA binding and are required for catalysis.…”

“…The TREX1 D18N, D200N, and D200H homodimers are catalytically inactive with respect to ssDNA and dsDNA degradation of polynucleotides. However, this complete loss of TREX1 DNA degradation activity alone does not sufficiently explain the dominant genetics of the D18N, D200N, and D200H alleles because there are other TREX1 AGS alleles, such as the insertion mutations of aspartate at position 201 (D201ins) and alanine at position 124 (A124ins), that result in elimination of TREX1 DNA degradation activities but exhibit recessive genetics (14,21). The dominant genetic phenotypes exhibited by the TREX1 D18N, D200N, and D200H alleles likely result from the generation of TREX1 enzymes with dysfunctional catalytic potential and fully functional nicked dsDNA binding properties.…”

“…The TREX1 AGS-causing mutations locate predominantly to the catalytic core region with a few notable exceptions positioned in the C-terminal hydrophobic region (22). Some TREX1 AGS-causing mutants exhibit dramatically lower levels of catalytic function, whereas others show more modest effects on the ssDNA degradation activities, yet all yield similar human pathologies (21,23). The TREX1 systemic lupus erythematosus-associated mutations are located mostly in the C-terminal region with a few variants positioned in the catalytic core.…”

“…Our structural studies of the TREX1 catalytic domain with bound DNA reveal the protein-polynucleotide interactions that explain the requirement for ssDNA in the active site and highlight the extensive interface contacts in the remarkably stable dimeric enzyme (21). The TREX1 AGS-causing mutations locate predominantly to the catalytic core region with a few notable exceptions positioned in the C-terminal hydrophobic region (22).…”

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

“…The TREX1 N-terminal 242 amino acids contain the catalytic domain and the C-terminal region contains a proposed transmembrane helix that is not found in TREX2. TREX1 and TREX2 have similar homodimeric structures with distinct elements that point to different biological roles (19,20). Deletion of the TREX2 gene in mice increases susceptibility to induced skin carcinogenesis (21), contrasting the autoimmune phenotype of the TREX1 knock-out mouse.…”