Fig 1.
X-ray data collection and refinement statistics for TREX1-DNA complexes.
dI, deoxyinosine; dsDNA, double-stranded DNA; Mg2+, magnesium ion; r.m.s, root-mean-square; ssDNA, single-stranded DNA; TREX1, three prime repair exonuclease 1.
Fig 2.
(A) Schematic representation of the DNA/RNA products generated by Endo V. (B) The nuclease activities of TREX1 in digesting bubbled DNAs containing a hypoxanthine base (also named dI), including dI-bubbled DNA and dI-bubbled DNA with 5′-overhang. The concentration of all substrates was 0.5 μM. (C) An overview of the TREX1-dI-ssDNA structure. The upper panel shows the dI-ssDNA molecule in the TREX1-dI-ssDNA structure. The omitted electron density map (black) is contoured at 2.0 σ. Scissile phosphate, Mg2+, and water molecules are shown in orange, blue, and light blue balls, respectively. The hydrogen bonds between DNA, TREX1, water, and Mg2+ are marked with blue dotted lines. dI, deoxyinosine; Endo V, endonuclease V; Mg2+, magnesium ion; ssDNA, single-stranded DNA; TREX1, three prime repair exonuclease 1.
Fig 3.
The TREX1-dI-T-dsDNA structure.
(A) An overview of the TREX1-dI-T-dsDNA structure. The phosphate atoms of dsDNA in the TREX1-dI-T-dsDNA structure are shown as orange balls. The metal ions and active site residues in the TREX1-dI-ssDNA and TREX1-dI-T-dsDNA structures are colored in gray and blue, respectively. (B) Schematic representation for the Leu24-Pro25-Ser26 cluster wedging in the duplex end of dI-T-dsDNA. The scissile and nonscissile strands are colored in red and pink, respectively. dI, deoxyinosine; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; TREX1, three prime repair exonuclease 1.
Fig 4.
The TREX1-L-structural dsDNA structure.
(A) The nuclease activities of TREX1 in digesting duplex DNAs with 4-nt-long 3′-overhang. (B) The asymmetric unit in the crystal contained 1 TREX1 dimer and 2 ssDNA molecules. The parts shown with a transparent mode depict the symmetry of TREX1 and DNA. The DNA duplex was formed by the ssDNAs bound to 2 TREX1 molecules in separate dimers. The 2 3′-ends of this duplex were 1 nt and 4 nt long and formed an L-like structure at each 3′-terminal, and they are referred to as 1-nt-L-DNA and 4-nt-L-DNA, respectively. (C) The 5′-ends of the duplex region in 1-nt-L-DNA were blocked by Leu24-Pro25-Ser26 cluster. (D) Schematic representation of the 2 modes for TREX1 binding with 1-nt-L-DNA and 4-nt-L-DNA. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; TREX1, three prime repair exonuclease 1.
Fig 5.
The TREX1-Y-structural dsDNA structure.
(A) The nuclease activities of TREX1 in digesting Y-structural DNA. (B) An overview of the TREX1-Y-structural dsDNA structure. The last 2 phosphates in the 5′-end are displayed as orange balls and labeled as “P.” Arg128 interacted with the G4 and G5 bases via hydrogen bonding. (C) Schematic comparison of the TREX1-Y-structural dsDNA and the TREX1-L-structural dsDNA structures (1-nt-L-DNA). The cutting sites are labeled by scissors. dsDNA, double-stranded DNA; TREX1, three prime repair exonuclease 1.
Fig 6.
The terminal unwinding activity of TREX1 empowered by the Leu24-Pro25-Ser26 cluster.
(A) Structure comparison of the loop region between the highly conserved β1 and β2 of TREX1, TREX2, and other classical DEDDh exonucleases. The PDB structures used in the structural alignment are mouse TREX1 (PDB accession code: 5YWU), human TREX2 (PDB accession code: 1Y97), Caenorhabditis elegans CRN-4 (PDB accession code: 3CG7), E. coli RNase T (PDB accession code: 4KA0), E. coli ExoX (PDB accession code: 4FZX), human ISG20 (PDB accession code: 31WLJ), and human 3′Hexo (PDB accession code: 4QOZ). In this region, only TREX1 and TREX2 contain a small helix that starts the Leu24-Pro25-Ser26 cluster. (B) The nuclease activities of TREX2 and RNase T in digesting duplex DNAs with 3′-overhang. The concentration of duplex DNA was 0.5 μM. TREX2 exhibited activities in digesting the duplex regions of DNA. RNase T at 500 nM only removed the 3′-overhang in duplex DNA and produced a duplex DNA with 1- or 0-nucleotide 3′-overhang. When the concentration of RNase T was increased by 20-fold to 10,000 nM, the double-strand structure of the substrates still persisted. (C) The nuclease activities of wild-type TREX1 in digesting ssDNA, dsDNA, and a PCR product (A linear 708 bp dsDNA). The concentration of ssDNA and dsDNA were 0.5 μM. The amount of the PCR product was 300 ng, and the concentration of EDTA was 5 mM. (D)(E)(F) The nuclease activities of 3 TREX1 mutants (L24W, S26W, and L24W/P25W/S26W) in digesting ssDNA and dsDNA substrates. dsDNA, double-stranded DNA; PDB, Protein Data Bank; ssDNA, single-stranded DNA; TREX1, three prime repair exonuclease 1.
Fig 7.
Structure comparison for the binding modes of TREX1 with various DNA substrates.
(A) Superposition of the three structures of TREX1 in complexing with a duplex DNA with a long 3′-overhang (≥4-nt), including 4-nt-long 3′-overhang (4-nt-L-DNA) in the TREX1-L-structural dsDNA structure and the tight and loose conformations in the previous structures of TREX1-dsDNA complex (PDB accession code: 4YNQ). The colors and schematic diagrams of 3 duplex DNAs are displayed in the bottom panel. The right panel shows a close look for the difference between the scissile strands in the 3 duplex DNAs. The relative positions of the scissile strand of the duplex DNA in these 3 structures are different, only the positions of the last 2 nucleotides at the 3′-end of the scissile strand can fit well with each other. (B) Superposition of the three structures of TREX1 in complexing with a duplex DNA with a short 3′-overhang (<4-nt), including 1-nt-long 3′-overhang in the TREX1-L-structural dsDNA structure (1-nt-L-DNA), Y-structural DNA with 2-nt-long 3′-overhang in the TREX1-Y-structural dsDNA structure, and dI-containing dsDNA in the TREX1-dI-T-dsDNA structure. Comparison of these structures indicates the similarity of the binding regions between TREX1 and the 3′-end and 5′-end of the three types of dsDNA substrates. (C) Binding mode 1 is TREX1 with ssDNA substrates. (D) Binding mode 2 is TREX1 with duplexes of a long 3′-overhang, schematic representations in Boxes 1, 2, and 3. The PDB accession codes of these structures are 5YWT (4-nt-long L-structural dsDNA), 4YNQ (tight conformation), and 4YNQ (loose conformation). Binding mode 3 is TREX1 with a duplex of a short 3′-overhang, schematic representations in Boxes 4, 5, and 6. The PDB accession codes of these structures are 5YWT (1-nt-long L-structural dsDNA), 5YWS (2-nt-long Y-structural dsDNA), and 5YWU (dI-T-dsDNA). The scissile and nonscissile strands are colored in red and pink, respectively. α7 is the seventh α-helix (152–162 a.a). dI, deoxyinosine; dsDNA, double-stranded DNA; PDB, Protein Data Bank; ssDNA, single-stranded DNA; ssRNA, single-stranded RNA; TREX1, three prime repair exonuclease 1.