Biochemical analysis of H2O2-induced mutation spectra revealed that multiple damages were involved in the mutational
Tomohiko Sugiyama1, Mahima R Sanyal1
1Department of Biological Sciences, Ohio University, Athens, OH 45701, USA; Molecular and Cellular Biology Graduate Program, Ohio University, Athens, OH 45701, USA.
Abstract:
Reactive oxygen species (ROS) are a major threat to genomic integrity and believed to be one of the etiologies of cancers. Here we developed a cell-free system to analyze ROS-induced mutagenesis, in which DNA was exposed to H2O2 and then subjected to translesion DNA synthesis by various DNA polymerases. Then, frequencies of mutations on the DNA products were determined by using next-generation sequencing technology. The majority of observed mutations were either C>A or G>A, caused by dAMP insertion at G and C residues, respectively. These mutations showed similar spectra to COSMIC cancer mutational signature 18 and 36, which are proposed to be caused by ROS. The in vitro mutations can be produced by replicative DNA polymerases (yeast DNA polymerase δ and ε), suggesting that ordinary DNA replication is sufficient to produce them. Very little G>A mutation was observed immediately after exposure to H2O2, but the frequency was increased during the 24 h after the ROS was removed, indicating that the initial oxidation product of cytosine needs to be maturated into a mutagenic lesion. Glycosylase-sensitivities of these mutations suggest that the C>A were made on 8-oxoguanine or Fapy-guanine, and that G>A were most likely made on 5-hydroxycytosine modification.
Insights
Reactive oxygen species (ROS) cause DNA mutations linked to cancer. Our cell-free system reveals how ROS-induced DNA damage leads to specific mutations during DNA replication, mimicking cancer signatures.
Area of Science:
- Genomics
- Molecular Biology
- Cancer Research
Background:
- Reactive oxygen species (ROS) are implicated in genomic instability and cancer development.
- Understanding ROS-induced DNA damage is crucial for cancer etiology research.
Purpose of the Study:
- To develop a cell-free system for analyzing ROS-induced mutagenesis.
- To characterize the mutation spectra resulting from ROS exposure and subsequent DNA synthesis.
Main Methods:
- DNA was exposed to hydrogen peroxide (H2O2) in a cell-free system.
- Translesion DNA synthesis was performed using various DNA polymerases.
- Next-generation sequencing (NGS) was used to determine mutation frequencies.
Main Results:
- The majority of mutations were C>A and G>A, consistent with COSMIC cancer signatures 18 and 36.
- Replicative DNA polymerases (yeast Pol δ and ε) produced these mutations.
- G>A mutation frequency increased 24 hours post-ROS exposure, indicating lesion maturation.
Conclusions:
- The cell-free system effectively models ROS-induced mutagenesis.
- ROS-induced DNA damage can lead to cancer-associated mutational signatures via standard DNA replication.
- Specific oxidative DNA lesions, like 5-hydroxycytosine, are likely responsible for observed G>A mutations.
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