Chemical Genetics Laboratory, RIKEN Advanced Science Institute, and CREST, JST, Wako-shi, Saitama, Japan.
PLoS One. 2012;7(7):e40572. doi: 10.1371/journal.pone.0040572. Epub 2012 Jul 10.
Activation of the Mec1/Rad53 damage checkpoint pathway influences mitochondrial DNA (mtDNA) content and point mutagenesis in Saccharomyces cerevisiae. The effects of this conserved checkpoint pathway on mitochondrial genomes in human cells remain largely unknown. Here, we report that knockdown of the human DNA helicase RRM3 enhances phosphorylation of the cell cycle arrest kinase Chk2, indicating activation of the checkpoint via the ATM/Chk2 pathway, and increases mtDNA content independently of TFAM, a regulator of mtDNA copy number. Cell-cycle arrest did not have a consistent effect on mtDNA level: knockdown of cell cycle regulators PLK1 (polo-like kinase), MCM2, or MCM3 gave rise, respectively, to decreased, increased, or almost unchanged mtDNA levels. Therefore, we concluded that the mtDNA content increase upon RRM3 knockdown is not a response to delay of cell cycle progression. Also, we observed that RRM3 knockdown increased the levels of reactive oxygen species (ROS); two ROS scavengers, N-acetyl cysteine and vitamin C, suppressed the mtDNA content increase. On the other hand, in RRM3 knockdown cells, we detected an increase in the frequency of the common 4977-bp mtDNA deletion, a major mtDNA deletion that can be induced by abnormal ROS generation, and is associated with a decline in mitochondrial genome integrity, aging, and various mtDNA-related disorders in humans. These results suggest that increase of the mitochondrial genome by TFAM-independent mtDNA replication is connected, via oxidative stress, with the ATM/Chk2 checkpoint activation in response to DNA damage, and is accompanied by generation of the common 4977-bp deletion.
Mec1/Rad53 损伤检查点途径的激活会影响酿酒酵母中的线粒体 DNA（mtDNA）含量和点突变。该保守检查点途径对人细胞中线粒体基因组的影响在很大程度上尚不清楚。在这里，我们报告说，人 DNA 解旋酶 RRM3 的敲低增强了细胞周期阻滞激酶 Chk2 的磷酸化，表明通过 ATM/Chk2 途径激活了检查点，并且独立于 TFAM（mtDNA 拷贝数的调节剂）增加 mtDNA 含量。细胞周期停滞对 mtDNA 水平没有一致的影响：细胞周期调节剂 PLK1（丝氨酸/苏氨酸激酶 polo 样激酶）、MCM2 或 MCM3 的敲低分别导致 mtDNA 水平降低、增加或几乎不变。因此，我们得出结论，RRM3 敲低后 mtDNA 含量的增加不是对细胞周期进程延迟的反应。此外，我们观察到 RRM3 敲低会增加活性氧物种（ROS）的水平；两种 ROS 清除剂，N-乙酰半胱氨酸和维生素 C，抑制了 mtDNA 含量的增加。另一方面，在 RRM3 敲低细胞中，我们检测到常见的 4977-bp mtDNA 缺失的频率增加，这是一种主要的 mtDNA 缺失，可由异常 ROS 生成诱导，与线粒体基因组完整性下降、衰老和各种与 mtDNA 相关的人类疾病有关。这些结果表明，TFAM 独立的 mtDNA 复制引起的线粒体基因组增加，通过氧化应激，与 ATM/Chk2 检查点激活有关，该激活是对 DNA 损伤的反应，并且伴随着常见的 4977-bp 缺失的产生。
