Department of Molecular Biology and Genetics, Aarhus University, DK-8000, Aarhus, Denmark.
Department of Biochemistry and Molecular Biology, Life sciences Sector, Federal University of Paraná, 81531-990, Curitiba, Puerto Rico, Brazil.
Physiol Plant. 2019 Jun;166(2):494-512. doi: 10.1111/ppl.12801. Epub 2018 Aug 31.
Mitochondria are one of the major sites of reactive oxygen species (ROS) production in the plant cell. ROS can damage DNA, and this damage is in many organisms mainly repaired by the base excision repair (BER) pathway. We know very little about DNA repair in plants especially in the mitochondria. Combining proteomics, bioinformatics, western blot and enzyme assays, we here demonstrate that the complete BER pathway is found in mitochondria isolated from potato (Solanum tuberosum) tubers. The enzyme activities of three DNA glycosylases and an apurinic/apyrimidinic (AP) endonuclease (APE) were characterized with respect to Mg dependence and, in the case of the APE, temperature sensitivity. Evidence for the presence of the DNA polymerase and the DNA ligase, which complete the repair pathway by replacing the excised base and closing the gap, was also obtained. We tested the effect of oxidative stress on the mitochondrial BER pathway by incubating potato tubers under hypoxia. Protein carbonylation increased significantly in hypoxic tuber mitochondria indicative of increased oxidative stress. The activity of two BER enzymes increased significantly in response to this oxidative stress consistent with the role of the BER pathway in the repair of oxidative damage to mitochondrial DNA.
线粒体是植物细胞中活性氧(ROS)产生的主要部位之一。ROS 会损害 DNA,这种损害在许多生物体中主要通过碱基切除修复(BER)途径来修复。我们对植物中的 DNA 修复知之甚少,尤其是在线粒体中。通过蛋白质组学、生物信息学、western blot 和酶测定相结合,我们在此证明了从马铃薯(Solanum tuberosum)块茎中分离的线粒体中存在完整的 BER 途径。研究了三种 DNA 糖苷酶和一种无嘌呤/无嘧啶(AP)内切酶(APE)的酶活性与镁依赖性的关系,并且在 APE 的情况下,还研究了其对温度敏感性的关系。还获得了证明聚合酶和连接酶存在的证据,这两种酶通过替换切除的碱基并封闭缺口完成修复途径。我们通过在缺氧条件下孵育马铃薯块茎来测试氧化应激对线粒体 BER 途径的影响。缺氧块茎线粒体中蛋白质羰基化明显增加,表明氧化应激增加。两种 BER 酶的活性显著增加,这与 BER 途径在修复线粒体 DNA 氧化损伤中的作用一致。