A quantitative assay for assessing the effects of DNA lesions on transcription
Changjun You1, Xiaoxia Dai, Bifeng Yuan
1Department of Chemistry, University of California-Riverside, Riverside, CA, USA
Abstract:
Most mammalian cells in nature are quiescent but actively transcribing mRNA for normal physiological processes; thus, it is important to investigate how endogenous and exogenous DNA damage compromises transcription in cells. Here we describe a new competitive transcription and adduct bypass (CTAB) assay to determine the effects of DNA lesions on the fidelity and efficiency of transcription. Using this strategy, we demonstrate that the oxidatively induced lesions 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) and the methylglyoxal-induced lesion N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) strongly inhibited transcription in vitro and in mammalian cells. In addition, cdA and cdG, but not N(2)-CEdG, induced transcriptional mutagenesis in vitro and in vivo. Furthermore, when located on the template DNA strand, all examined lesions were primarily repaired by transcription-coupled nucleotide excision repair in mammalian cells. This newly developed CTAB assay should be generally applicable for quantitatively assessing how other DNA lesions affect DNA transcription in vitro and in cells.
Insights
DNA damage significantly inhibits transcription and can cause mutations. A new assay reveals how specific DNA lesions impact transcription and are repaired by transcription-coupled nucleotide excision repair in cells.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Mammalian cells transcribe mRNA for physiological processes, but DNA damage can disrupt this.
- Understanding how DNA lesions affect transcription is crucial for cell health.
Purpose of the Study:
- To develop and utilize a novel assay to assess the impact of DNA damage on transcription.
- To investigate the effects of specific DNA lesions on transcription fidelity and efficiency.
Main Methods:
- Development of the competitive transcription and adduct bypass (CTAB) assay.
- In vitro and in vivo experiments using mammalian cells to analyze DNA lesion effects.
Main Results:
- Oxidative lesions (cdA, cdG) and methylglyoxal-induced lesion (N(2)-CEdG) inhibited transcription.
- cdA and cdG induced transcriptional mutagenesis, while N(2)-CEdG did not.
- All tested lesions were repaired by transcription-coupled nucleotide excision repair when on the template strand.
Conclusions:
- The CTAB assay is a valuable tool for quantifying DNA lesion effects on transcription.
- Specific DNA lesions differentially affect transcription and mutagenesis.
- Transcription-coupled nucleotide excision repair plays a key role in mitigating DNA damage during transcription.
