Transcriptional Mutagenesis Induced by 8-Oxoguanine in Mammalian Cells

Abstract

Most of the somatic cells of adult metazoans, including mammals, do not undergo continuous cycles of replication. Instead, they are quiescent and devote most of their metabolic activity to gene expression. The mutagenic consequences of exposure to DNA–damaging agents are well documented, but less is known about the impact of DNA lesions on transcription. To investigate this impact, we developed a luciferase-based expression system. This system consists of two types of construct composed of a DNA template containing an 8-oxoguanine, paired either with a thymine or a cytosine, placed at defined positions along the transcribed strand of the reporter gene. Analyses of luciferase gene expression from the two types of construct showed that efficient but error-prone transcriptional bypass of 8-oxoguanine occurred in vivo, and that this lesion was not repaired by the transcription-coupled repair machinery in mammalian cells. The analysis of luciferase activity expressed from 8OG:T-containing constructs indicated that the magnitude of erroneous transcription events involving 8-oxoguanine depended on the sequence contexts surrounding the lesion. Additionally, sequencing of the transcript population expressed from these constructs showed that RNA polymerase II mostly inserted an adenine opposite to 8-oxoguanine. Analysis of luciferase expression from 8OG:C-containing constructs showed that the generated aberrant mRNAs led to the production of mutant proteins with the potential to induce a long-term phenotypical change. These findings reveal that erroneous transcription over DNA lesions may induce phenotypical changes with the potential to alter the fate of non-replicating cells.

Author Summary

The DNA molecule is used as a template for duplication, to transmit genetic information to the progeny of a given cell, but also as a template for the transcription machinery. This machinery converts genetic information from the DNA form to the RNA form used for protein synthesis. Chemical alterations of the DNA molecule caused by endogenous or environmental stresses are responsible for the generation of mutations. Indeed, these lesions can induce replication errors when DNA is duplicated during cell division. These mutations have been shown to be responsible for many genetic diseases and other sporadic diseases, such as cancer. However, less is known about their effects on transcription. We report here that a specific DNA lesion may lead to erroneous transcription events, ultimately leading to the production of aberrant proteins. The magnitude of these errors seems to depend largely on the DNA sequences surrounding the lesion and the capacity of the cell to repair this lesion. We also show that the production of aberrant protein from the erroneous transcription products may affect the phenotype of the cells concerned. Lesion-induced transcription errors may also play a role in the development of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases.