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. 2008 Nov 14;283(46):31649-56.
doi: 10.1074/jbc.M806155200. Epub 2008 Sep 18.

The TREX1 double-stranded DNA degradation activity is defective in dominant mutations associated with autoimmune disease

Duane A Lehtinen  1 , Scott Harvey, Matthew J Mulcahy, Thomas Hollis, Fred W Perrino
Affiliations

The TREX1 double-stranded DNA degradation activity is defective in dominant mutations associated with autoimmune disease

Duane A Lehtinen et al. J Biol Chem. .

Abstract

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 approximately 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.

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Figures

FIGURE 1.
FIGURE 1.
The TREX1 dimer with bound ssDNA. The TREX1 protomer backbones (orange and green) are shown. The Asp-18 (blue), Asp-200 (magenta), Arg-114 (red), and Arg-128 (gray) residue side chains (shown as sticks) are indicated in each of the TREX1 protomers. The active sites (highlighted circles) and dimer interface (brackets) are indicated. The Ca2+ divalent ions (cyan) coordinated by D18 and D200 (black dotted lines) and 4-mer ssDNA (as sticks) are shown bound in the active sites. The β3 strands and α4 helices at the dimer interface are shown as cartoons. The hydrogen bonding at the dimer interface (black dotted lines) is indicated. The Arg-114 of each protomer hydrogen bonds to the backbone carbonyl oxygens of Gln-98 and Arg-99 in the opposing protomer. The figure was prepared using the program PyMol (Delano Scientific).
FIGURE 2.
FIGURE 2.
The ssDNA exonuclease activities of TREX1WT and D200N variants. Standard exonuclease reactions (30 μl) were prepared with a fluorescein-labeled 30-mer oligonucleotide and dilutions of the recombinant wild type (TREX1WT), mutant (TREX1D200N/D200N) homodimer, and (TREX1WT/D200N) heterodimer were prepared at 10 times the final concentrations. Samples (3 μl) containing the TREX1 enzymes to yield the final indicated concentrations were added to reactions. The incubations were 20 min at 25 °C. The reaction products were subjected to electrophoresis on 23% urea-polyacrylamide gels (A) and quantified (B) as described under “Experimental Procedures.” The position of migration of the 30-mer is indicated. Relative activities of TREX1D200N/D200N and TREX1WT/D200N are compared with TREX1WT dimers. The relative activity was calculated as relative activity = 100 × [(fmol of dNMP released/s/fmol of mutant enzyme)/(fmol of dNMP released/s/fmol WT enzyme)].
FIGURE 3.
FIGURE 3.
The ssDNA exonuclease activities of TREX1WT and R114H variants. The standard exonuclease reactions (30 μl) were prepared with a fluorescein-labeled 30-mer oligonucleotide, and dilutions of the recombinant wild type (TREX1WT), mutant (TREX1R114H/R114H) homodimer, and (TREX1WT/R114H) heterodimer were prepared at 10 times the final concentrations. Samples (3 μl) containing the TREX1 enzymes to yield the final indicated concentrations were added to reactions. The incubations were 20 min at 25 °C. The reaction products were subjected to electrophoresis on 23% urea-polyacrylamide gels (A) and quantified (B) as described under “Experimental Procedures.” The position of migration of the 30-mer is indicated. Relative activities of TREX1R114H/R114H and TREX1WT/R114H are compared with TREX1WT dimers. The relative activity was calculated as relative activity = 100 × [(fmol of dNMP released/s/fmol of mutant enzyme)/(fmol of dNMP released/s/fmol WT enzyme)].
FIGURE 4.
FIGURE 4.
The TREX1 3′ exonuclease has dsDNA degradation activity. The supercoiled dsDNA plasmids 1 (A, lane 2) and 2 (B, lane 8) were nicked by incubating with Nt.BbvCI restriction enzyme (A, lane 3; and B, lane 9) and purified as described under “Experimental Procedures.” The plasmid 2 was linearized by incubating with EcoRI (C, lane 2) or SacI (D, lane 8) and purified. Exonuclease time course reactions (100 μl) were prepared containing nicked dsDNA plasmid 1 (A) or 2 (B) or linearized dsDNA plasmid 2 (C and D) and 7.6 nm TREX1WT. The samples (20 μl) were removed after incubation for the indicated times (A, lanes 4–7; and B, lanes 10–13). The reaction products were subjected to electrophoresis on agarose gels, and the amounts of dNMP excised by TREX1WT were estimated as described under “Experimental Procedures.” The positions of migration of Form I supercoiled dsDNA (dsDNA), Form II nicked dsDNA (Nicked dsDNA), Form III linear dsDNA (linear dsDNA), and circular ssDNA (ssDNA) are indicated. Lane 1 contains the 1-kb ladder (Invitrogen).
FIGURE 5.
FIGURE 5.
The TREX1WT/D200N and TREX1WT/D18N heterodimers have defective dsDNA degradation activities. The exonuclease reactions (20 μl) were prepared containing nicked dsDNA plasmid 1 (10 μg/ml = 1.6 nm nicks) and no enzyme (lanes 2, 8, and 14) or the indicated increased concentrations (in nm) of TREX1WT (lanes 3–7), TREX1WT/D200N (lanes 9–13), and TREX1WT/D18N (lanes 15–19). The reactions were 30 min, and the products were subjected to electrophoresis on agarose gels. Lane 1 (ds) contains the supercoiled dsDNA plasmid 1. The positions of migration of Form I supercoiled dsDNA (dsDNA), Form II nicked dsDNA (Nicked dsDNA), and circular ssDNA (ssDNA) are indicated.
FIGURE 6.
FIGURE 6.
The D200N- and D18N-containing TREX1 mutant enzymes inhibit the TREX1WT dsDNA degradation activity. Exonuclease reactions (20 μl) were prepared containing nicked dsDNA plasmid 1 (10 μg/ml = 1.6 nm nicks) and no enzyme (A and B, lane 2), the indicated concentration of TREX1WT only (A and B, lane 3), or a mixture of TREX1WT with the indicated increased concentrations of TREX1D200N/D200N (A, lanes 4–8), TREX1WT/D200N (A, lanes 9–13), TREX1D18N/D18N (B, lanes 4–8), and TREX1WT/D18N (lanes 9–13). The reactions were 30 min, and the products were subjected to electrophoresis on agarose gels. Lane 1 contains the supercoiled dsDNA plasmid 1. The positions of migration of Form I supercoiled dsDNA (dsDNA), Form II nicked dsDNA (Nicked dsDNA), and circular ssDNA (ssDNA) are indicated.
FIGURE 7.
FIGURE 7.
The TREX1R114H/R114H dsDNA degradation activity is more dysfunctional than the ssDNA exonuclease activity. Exonuclease time course reactions (200 μl) were prepared containing nicked dsDNA plasmid 1 (10 μg/ml = 1.6 nm nicks) and 7.6 nm TREX1WT (A) and 260 nm TREX1R114H/R114H (B). The samples (20 μl) were removed prior to enzyme addition (lanes 2 and 5) and after incubation for the indicated times (A, lanes 3 and 4; and B, lanes 7–12). The reaction products were subjected to electrophoresis on agarose gels. Lanes 1 and 5 (ds) contain the supercoiled dsDNA plasmid 1. The positions of migration of Form I supercoiled dsDNA (dsDNA), Form II nicked dsDNA (Nicked dsDNA), and circular ssDNA (ssDNA) are indicated.
FIGURE 8.
FIGURE 8.
The TREX1R114H/R114H and TREX1WT/R114H enzymes do not inhibit the TREX1MWT dsDNA degradation activity. Exonuclease reactions (20 μl) were prepared containing nicked dsDNA plasmid 1 (10 μg/ml = 1.6 nm nicks) and no enzyme (lane 2), the indicated concentration of TREX1WT only (lane 3), or a mixture of TREX1WT with the indicated increased concentrations of TREX1R114H/R114H (lanes 4–8), and TREX1WT/R114H (lanes 9–13). The reactions were 30 min, and the products were subjected to electrophoresis on agarose gels. Lane 1 contains the supercoiled dsDNA plasmid 1. The positions of migration of Form I supercoiled dsDNA (dsDNA), Form II nicked dsDNA (Nicked dsDNA), and circular ssDNA (ssDNA) are indicated.
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