Increases in mitochondrial DNA content and 4977-bp deletion upon ATM/Chk2 checkpoint activation in HeLa cells.
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.
Chemical Genetics Laboratory, RIKEN Advanced Science Institute, and CREST, JST, Wako-shi, Saitama, Japan.
Rong
R
Niu
Minoru
M
Yoshida
Feng
F
Ling
D064007:Ataxia Telangiectasia Mutated Proteins; D020029:Base Pairing / Q000235:genetics*; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004265:DNA Helicases / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D018628:Gene Dosage / Q000235:genetics; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D024101:Mitochondrial Proteins / Q000378:metabolism; D008954:Models, Biological; D059645:Mutation Rate; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics
D018797:Cell Cycle Proteins; D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; C066470:MCM3 protein, human; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; D017382:Reactive Oxygen Species; C499631:TFAM protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C084859:MCM2 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; C508765:PIF1 protein, human