荧光成像线粒体 DNA 碱基切除修复揭示氧化应激反应的动态变化。
Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses.
机构信息
Department of Chemistry, ChEM-H Institute, and Stanford Cancer Institute, Stanford University, Stanford, CA, 94305, USA.
Department of Neurosurgery, Department of Neurology and Neurological Sciences, and Wu Tsai Neuroscience institute, Stanford University School of Medicine, Stanford, CA, 94305, USA.
出版信息
Angew Chem Int Ed Engl. 2022 Feb 1;61(6):e202111829. doi: 10.1002/anie.202111829. Epub 2021 Dec 22.
Mitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. We employ a molecular approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe (UBER) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.
随着年龄的增长和神经退行性变，细胞中的线粒体功能下降，这在很大程度上是由于线粒体 DNA（mtDNA）积累的突变，这些突变是由于 DNA 修复缺陷引起的。然而，测量这种修复活性具有挑战性。我们采用一种分子方法，通过使用荧光探针（UBER）来可视化线粒体碱基切除修复（BER）活性，该探针与 BER 活性产生的 AP 位点快速反应。将探针施用于培养细胞显示出定位于线粒体的信号，从而能够选择性地观察 mtDNA BER 中间体。该探针在氧化应激下显示出升高的 DNA 修复活性，并对糖苷酶活性的抑制做出反应。此外，该探针揭示了氧化应激开始和 BER 初始步骤之间的时间滞后。细胞中缺乏 MTH1 会导致在延长的氧化应激下 BER 活性的需求增加，而 OGG1 活性的缺乏会限制糖基化能力。
Zotero 插件