Journal: The Journal of Biological Chemistry

Article Title: Dominant Mutations of the TREX1 Exonuclease Gene in Lupus and Aicardi-Gouti?res Syndrome *

doi: 10.1074/jbc.M111.276287

Figure Lengend Snippet: The ssDNA exonuclease activities of TREX1 D200H variants. Standard exonuclease reactions (30 μl) were prepared with a fluorescein-labeled 30-mer oligonucleotide, and dilutions of the recombinant TREX1WT, TREX1D200H/D200H, and TREX1WT/D200H 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 reactions were incubated for 30 min at 25 °C. A and B, the reaction products were subjected to electrophoresis on 23% urea-polyacrylamide gels (A) and quantified as described under “Experimental Procedures.” B, to precisely quantify results, the relative exonuclease activities of TREX1WT and TREX1WT/D200H were assayed in triplicate at 38, 57, and 76 pm as described above. Plots of activity versus enzyme concentrations were used to confirm the linearity of the assay and to generate the enzyme activity values. The average activities and standard errors were determined by regression analysis using SigmaPlot 8.02 (SPSS Science, Inc.). 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)). The position of migration of the 30-mer is indicated.

Article Snippet: The average activities and standard errors were determined by regression analysis using SigmaPlot 8.02 (SPSS Science, Inc.). b 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)). c dsDNA exonuclease activity assays were performed at enzyme concentrations linear during the time course.

Techniques: Labeling, Recombinant, Incubation, Electrophoresis, Activity Assay, Mutagenesis, Migration