Department of Pharmacological Sciences, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
Department of Environment and Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Int J Mol Sci. 2023 Aug 12;24(16):12715. doi: 10.3390/ijms241612715.
After cellular differentiation, nuclear DNA is no longer replicated, and many of the associated proteins are downregulated accordingly. These include the structure-specific endonucleases Fen1 and DNA2, which are implicated in repairing mitochondrial DNA (mtDNA). Two more such endonucleases, named MGME1 and ExoG, have been discovered in mitochondria. This category of nuclease is required for so-called "long-patch" (multinucleotide) base excision DNA repair (BER), which is necessary to process certain oxidative lesions, prompting the question of how differentiation affects the availability and use of these enzymes in mitochondria. In this study, we demonstrate that Fen1 and DNA2 are indeed strongly downregulated after differentiation of neuronal precursors (Cath.a-differentiated cells) or mouse myotubes, while the expression levels of MGME1 and ExoG showed minimal changes. The total flap excision activity in mitochondrial extracts of these cells was moderately decreased upon differentiation, with MGME1 as the predominant flap endonuclease and ExoG playing a lesser role. Unexpectedly, both differentiated cell types appeared to accumulate less oxidative or alkylation damage in mtDNA than did their proliferating progenitors. Finally, the overall of mtDNA repair was not significantly different between proliferating and differentiated cells. Taken together, these results indicate that neuronal cells maintain mtDNA repair upon differentiation, evidently relying on mitochondria-specific enzymes for long-patch BER.
细胞分化后，核 DNA 不再复制，相应地，许多相关蛋白也下调。这些蛋白包括结构特异性内切酶 Fen1 和 DNA2，它们与线粒体 DNA（mtDNA）的修复有关。在线粒体中还发现了另外两种这样的内切酶，命名为 MGME1 和 ExoG。这类核酸酶需要所谓的“长补丁”（多核苷酸）碱基切除 DNA 修复（BER），这对于处理某些氧化损伤是必要的，这就提出了一个问题，即分化如何影响这些酶在线粒体中的可用性和使用。在这项研究中，我们证明 Fen1 和 DNA2 在神经元前体（Cath.a-分化细胞）或小鼠肌管分化后确实强烈下调，而 MGME1 和 ExoG 的表达水平变化极小。这些细胞的线粒体提取物中的总切口消除活性在分化后适度降低，MGME1 是主要的切口内切酶，ExoG 作用较小。出乎意料的是，与增殖的祖细胞相比，两种分化的细胞类型似乎在 mtDNA 中积累的氧化或烷基化损伤更少。最后，增殖细胞和分化细胞之间的 mtDNA 修复的整体速度没有显著差异。总之，这些结果表明神经元细胞在分化后维持 mtDNA 修复，显然依赖于线粒体特异性酶进行长补丁 BER。
