Genotoxic agents not mutagenic to mitochondria

Science Spotlight

Genotoxic agents not mutagenic to mitochondria

Oct. 17, 2016

Fluorescent staining of mitochondrial networks in cultured human cells.
Figure provided by BJ Valente

Cancer and neurodegeneration are common ailments of aging populations. In many instances these diseases are correlated with increased rates and burden of DNA mutation. While many researchers focus on these mutations in nuclear DNA (nDNA), pathogenic mutations are also observed in the other source of human DNA – mitochondrial DNA (mtDNA). Inherited defects in mitochondrial genes cause or are linked to diseases involving energy metabolism while mutations or deletions in mitochondrial genes acquired through life are linked to neurodegeneration and cancer. Understanding causes and mechanisms of DNA mutation is particularly important in mtDNA because evolutionary studies have demonstrated it is nearly 10 times more prone to mutation than nDNA. One prevailing theory for this increased mutation rate in mtDNA is that this genome may have an increased susceptibility to damaging agents including reactive oxygen species and carcinogens. After exposing cells to genotoxic agents mtDNA exhibits far more DNA damage than nDNA; however, scientists have struggled to directly observe a corresponding elevation in mutations to mtDNA. In their recent Nucleic Acids Research publication, BJ Valente and his colleagues in the Bielas Laboratory (Public Health Sciences and Human Biology Divisions) developed a reliable mutation detection approach to show that DNA damaging agents do not induce mutations in mtDNA.

Standard sequencing technologies are sufficient to study the changes in mtDNA over generations; however, these approaches fail to give clear results on highly damaged DNA and can induce new 'mutations'. To overcome this challenge, researchers combined an old approach with new, high-throughput technologies. Instead of directly sequencing mtDNA it was digested with the restriction endonuclease, TaqI. This enzyme recognizes a somewhat common 4 bp DNA sequence, 5-TCGA-3' that was present at multiple sites in mouse mtDNA. Researchers then designed Taqman probes that flank the TaqI sites. If mtDNA is mutated at the TaqI site it will not be digested, and thus the Taqman probe will anneal, generating fluorescent signal during PCR amplification. However, a normal, non-mutated TaqI site in the mtDNA will be digested, PCR cannot proceed and no fluorescence is detected. This allowed researchers to effectively 'count' how many mutations were present in mouse mtDNA. This technique was termed Digital Random Mutation Capture (dRMC) or Digital Deletion Detection (3D) depending on if probes were designed for mutations or deletions. Mutation detection by 3D and dRMC are not reliant upon polymerase fidelity and thus sidestep issues of erroneous sequence reads caused by amplification of damaged DNA.

Using this approach scientists tested the effects of prolonged exposure to two DNA-damaging agents, benzo[a]pyrene (B[a]P) and N-ethyl-N-nitrosourea (ENU), on mtDNA in mice. Subjects were exposed to sub-chronic doses of either agent for 28 days and then mtDNA was extracted from liver and marrow. These tissues were chosen because of their high exposure to orally dosed compounds and their proliferative nature, which increases the chance of encountering mutations. Surprisingly, neither compound increased the incidence of point mutation or deletion in mtDNA isolated from either tissue. Importantly, the compounds were active in cells as they caused mutations in nuclear DNA. Despite detecting no difference in the sequence of mtDNA of untreated and exposed animals, the researchers verified that B[a]P induced DNA damage in the mtDNA of exposed animals. To measure lesions caused by B[a]P the mice were treated for a single day and mtDNA was again harvested from liver and marrow. Rather than 3D or dRMC, researches used quantitative long-range PCR to test for bulky B[a]P adducts that prevent DNA polymerization.

These experiments revealed that DNA-damaging agents cause lesions within mtDNA yet do not appear to generate point mutations or deletions. The authors suggest a few possibilities for this unexpected finding, one being that mtDNA mutations are the result of errors in DNA synthesis rather than a product DNA damage. Increased mtDNA mutation rates have been observed in mice expressing functional mutants of the polymerase specific for mtDNA, Pol γ. These results opened more questions than they answered, "Follow-up on this study really focuses on the next questions: Why don’t mitochondrial genomes mutate with damage?; what’s the mechanism that recognizes mtDNA damage?; and what happens to a damaged mitochondrial genome? The answers to these questions may have direct implications for our understanding of human health conditions where mtDNA mutations and mitochondrial dysfunction are implicated, as in pathologies of aging, cancer, and neurodegenerative diseases," said Valente. Using 3D and dRMC as well as other novel approaches the members of the Bielas Lab are sure to provide answers to some of these new questions.


Funding for this research was provided by the National Institutes of Health.

Valente BJ, Ericson NG, Long AS, White PA, Marchetti F, Bielas JH. 2016. Mitochondrial DNA exhibits resistance to induced point and deletion mutations. Nucleic Acids Res. Epub ahead of print.

This work has also been featured by Fred Hutch Center News