Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, North Carolina 27157, USA.
J Biol Chem. 2011 Sep 16;286(37):32373-82. doi: 10.1074/jbc.M111.276287. Epub 2011 Aug 1.
TREX1 is a potent 3' → 5' exonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit dominant immune dysfunction phenotypes. Failure to appropriately disassemble genomic DNA during normal cell death processes could lead to persistent DNA signals that trigger the innate immune response and autoimmunity. We tested this concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease locates 3' termini generated by endonucleases and degrades the nicked DNA polynucleotide. A competition assay was designed using TREX1 dominant mutants and variants to demonstrate that an intact DNA binding process, coupled with dysfunctional chemistry in the active sites, explains the dominant phenotypes in TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175 positioned adjacent to the active sites act with the Arg-128 residues positioned in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for entry into the active sites. Metal-dependent ssDNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to DNA 3' termini by active TREX1 enzyme. Thus, the dominant disease genetics exhibited by the TREX1 D18N, D200N, and D200H alleles parallel precisely the biochemical properties of these TREX1 dimers during dsDNA degradation of plasmid and chromatin DNA in vitro. These results support the concept that failure to degrade genomic dsDNA is a principal pathway of immune activation in TREX1-mediated autoimmune disease.
TREX1 是一种有效的 3'→5'外切核酸酶，可降解单链和双链 DNA（ssDNA 和 dsDNA）。家族性寒冷性蕈样肉芽肿病和 Aicardi-Goutières 综合征中位于天冬氨酸 18 位和天冬氨酸 200 位的 TREX1 突变引发显性免疫功能障碍表型。在正常细胞死亡过程中，如果不能适当地将基因组 DNA 进行分段，可能会导致持续的 DNA 信号触发先天免疫反应和自身免疫。我们使用 dsDNA 质粒和染色质对此概念进行了测试，并表明 TREX1 外切核酸酶定位在内切酶产生的 3'末端，并降解缺口 DNA 多核苷酸。设计了一种竞争测定法，使用 TREX1 显性突变体和变体来证明完整的 DNA 结合过程，加上活性位点的功能失调化学，解释了 TREX1 D18N、D200N 和 D200H 等位基因中的显性表型。位于活性位点附近的 TREX1 残基精氨酸 174 和赖氨酸 175 与位于催化核心的精氨酸 128 残基协同作用，促进 dsDNA 的熔解，并产生 ssDNA 进入活性位点。无活性的显性 TREX1 突变体中活性位点的金属依赖性 ssDNA 结合有助于 DNA 保留，并阻止活性 TREX1 酶进入 DNA 3'末端。因此，TREX1 D18N、D200N 和 D200H 等位基因表现出的显性疾病遗传学与这些 TREX1 二聚体在体外质粒和染色质 DNA 的 dsDNA 降解过程中的生化特性完全一致。这些结果支持了这样一种概念，即不能降解基因组 dsDNA 是 TREX1 介导的自身免疫性疾病中免疫激活的主要途径。
