线粒体 DNA 中 G4 DNA 形成的表征及其在线粒体基因组不稳定性中的潜在作用,The FEBS Journal

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线粒体 DNA 中 G4 DNA 形成的表征及其在线粒体基因组不稳定性中的潜在作用
The FEBS Journal ( IF 5.5 ) Pub Date : 2021-07-06 , DOI: 10.1111/febs.16113
Sumedha Dahal ₁ , Humaira Siddiqua ₁ , Vijeth K. Katapadi ₁ , Divyaanka Iyer ₁ , Sathees C. Raghavan ₁

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线粒体拥有自己的基因组，可以独立于核 DNA 进行复制。线粒体是细胞产生活性氧的动力源，因此线粒体基因组经常受到氧化损伤。先前的研究已经证明线粒体缺失与衰老和人类疾病有关。许多这些缺失都与非 B DNA 结构相邻。因此，我们研究了与线粒体基因组不稳定性相关的非规范结构。电脑模拟研究表明，线粒体 DNA (mtDNA) 中存在 > 100 个 G-四链体基序（其中 5 个有可能形成 3 板 G4 DNA）、23 个反向重复和 3 个镜像重复。进一步的分析表明，在线粒体疾病患者的缺失断点中，大多数位于 G4 DNA 基序。有趣的是，约 50% 的缺失发生在碱基对位置 8271-8281，约 35% 是由于 12362-12384 的缺失，约 12% 是由于 15516-15545 的缺失。使用电动迁移测定、圆二色性 (CD) 和 Taq 聚合酶终止测定来表征线粒体脆弱区域 3 板 G-四链体 DNA 结构的形成。所有 5 个区域都可以以 KCl 依赖性方式折叠成分子内和分子间 G-四链体结构。G4 DNA形成平行方向，在 LiCl 的存在下被废除。G4 DNA 的形成影响复制和转录。最后，BG4 与 MitoTracker 的免疫定位证实了线粒体基因组中 G-四链体的形成。因此，我们表征了人类线粒体区域中 5 种不同 G-四链体结构的形成，这可能有助于线粒体缺失的形成。

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Characterization of G4 DNA formation in mitochondrial DNA and their potential role in mitochondrial genome instability

Mitochondria possess their own genome which can be replicated independently of nuclear DNA. Mitochondria being the powerhouse of the cell produce reactive oxygen species, due to which the mitochondrial genome is frequently exposed to oxidative damage. Previous studies have demonstrated an association of mitochondrial deletions to aging and human disorders. Many of these deletions were present adjacent to non-B DNA structures. Thus, we investigate noncanonical structures associated with instability in mitochondrial genome. In silico studies revealed the presence of > 100 G-quadruplex motifs (of which 5 have the potential to form 3-plate G4 DNA), 23 inverted repeats, and 3 mirror repeats in the mitochondrial DNA (mtDNA). Further analysis revealed that among the deletion breakpoints from patients with mitochondrial disorders, majority are located at G4 DNA motifs. Interestingly, ~ 50% of the deletions were at base-pair positions 8271–8281, ~ 35% were due to deletion at 12362–12384, and ~ 12% due to deletion at 15516–15545. Formation of 3-plate G-quadruplex DNA structures at mitochondrial fragile regions was characterized using electromobility shift assay, circular dichroism (CD), and Taq polymerase stop assay. All 5 regions could fold into both intramolecular and intermolecular G-quadruplex structures in a KCl-dependent manner. G4 DNA formation was in parallel orientation, which was abolished in the presence of LiCl. The formation of G4 DNA affected both replication and transcription. Finally, immunolocalization of BG4 with MitoTracker confirmed the formation of G-quadruplex in mitochondrial genome. Thus, we characterize the formation of 5 different G-quadruplex structures in human mitochondrial region, which may contribute toward formation of mitochondrial deletions.

更新日期：2021-07-06

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