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

Abstract

The 3 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 ϳ100-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 ϳ10 5 -fold, confirming their catalytic role.During the multistep processes of DNA replication, repair, and recombination DNA ends are often remodeled by enzymes containing 3Ј 3 5Ј-exonuclease activities to remove mispaired, modified, fragmented, and normal nucleotides from DNA 3Ј termini. The major 3Ј 3 5Ј-exonuclease activity detected in mammalian cell extracts is catalyzed by the TREX1 1 enzyme (1-3). The gene encoding TREX1 and the closely related TREX2 gene were cloned (4, 5), and the 3Ј 3 5Ј-exonuclease activities of the recombinant proteins confirmed the catalytically robust nature of these enzymes (6, 7). In addition to the TREX enzymes, several other proteins containing 3Ј 3 5Ј-exonuclease activities have recently been identified in human cells (for recent reviews, see Refs. 8 and 9). The sequences and structures of these enzymes indicate a diverse collection that includes multidomain proteins such as the Werner syndrome (WRN) (10 -12) and p53 proteins (13) and the "proofreading" DNA polymerases ␦, ⑀, and ␥. The 3Ј 3 5Ј-exonuclease activities have been detected in the hMRE11 subunit of a doublestrand break DNA repair complex (14, 15) and the base excision repair apurinic/apyrimidinic endonuclease, .The presence of 3Ј 3 5Ј-exonuclease activities in a multitude of proteins likely reflects the importance of this activity in the proper maintenance of DNA 3Ј-ends.Defective 3Ј 3 5Ј-exonucleases impair critical DNA metabolic pathways and elicit devastating consequences in cells and animals. The Trex1Ϫ/Ϫ mice exhibit dramatically reduced survival resulting from inflammatory myocarditis leading to cardiomyopathy and circulatory failure (21). Deficiencies in the WRN protein result in the premature aging associated with Werner syndrome patients (22), and mice deficient in p53 or in the proofreading exonuclease of DNA polymerase ␦ show high incidences of cancers (23-25). Eliminating both APE alleles in mice results in early embryonic lethality (26), and defects in the hMRE11 complex have been associated with an ataxiatelangiectasia-like disorder and the Nijmegen breakage syndrome characterized by chrom...

Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis

“…Furthermore, the conserved Asn-6140 residue proved to be essential for activity. The available structure information (24,33) leads us to suggest that this Asn residue might contribute to substrate binding by forming a hydrogen bond with the ribose oxygen of the penultimate nucleotide.…”

“…Given that the various DEDD family members use a similar but not identical set of residues to coordinate the two metal ions and to orient the attacking hydroxide ion and its nucleotide substrate (24,25,27,30,33), more experimentation and structure information will be required to understand the precise functional and structural role of individual ExoN residues and the other subdomains present in nidovirus nsp14 proteins. In this context, the role of the putative Zn-finger structure, which uniquely separates the Exo I and II motifs in CoV and torovirus ExoNs (13), will be of major interest.…”

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.…”

“…DimerArg-114 in TREX1 (Arg-107 in TREX2) is conserved in the mammalian dimeric 3Ј exonucleases (19,20). TREX1 mutants R114A and R114K were generated to further test the requirement for arginine at this position.…”

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

“…The crystal structure of the human TREX2 protein revealed the unique dimeric nature of the TREX family exonucleases and provided the initial picture of a human DnaQ family member (27). Symmetry in the dimer positions the active sites of each monomer on opposite edges providing open access for DNA interactions and suggested a mechanism for its non-processive catalysis.…”