Mitochondrial DNA Damage Initiates a Cell Cycle Arrest by a Chk2-associated Mechanism in Mammalian Cells

Christopher A Koczor,Inna N Shokolenko,Amy K Boyd,Shawn P Balk,Glenn L Wilson,Susan P Ledoux

doi:10.1074/jbc.m109.036020

Christopher A Koczor, Inna N Shokolenko + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.036020

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Abstract

Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.

Highlights

The mammalian mitochondrion contains 2–10 copies of mitochondrial DNA (mtDNA), a circular double-stranded DNA molecule that encodes 13 proteins, 22 tRNAs, and 2 rRNAs [7]
Because of the importance of mtDNA in ATP production and the production of reactive oxygen species (ROS) that can occur from aberrant electron transport, it is likely that mitochondria exert some level of control over cellular proliferation
MtDNA damage was observed at menadione concentrations that were lower than the concentrations at which cell death had been observed previously [4]

Summary

Introduction

The mammalian mitochondrion contains 2–10 copies of mtDNA, a circular double-stranded DNA molecule that encodes 13 proteins, 22 tRNAs, and 2 rRNAs [7]. Experiments utilized menadione to produce ROS in HeLa cells to determine whether there is a link between mtDNA integrity and cell cycle arrest. Protein Isolation—Cells were exposed to menadione as described, and following exposure, the cells were trypsinized mtDNA Damage and Cell Cycle Arrest

Results

Conclusion