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Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells
Published on: February 24, 2014
Sequence specific mutations induced by N-nitrosodimethylamine at two marker loci in metabolically competent human
K L Dobo1, D A Eastmond, A J Grosovsky
1Environmental Toxicology Graduate Program, University of California, Riverside 92521, USA.
Abstract:
N-Nitrosodimethylamine (NDMA) is a potent mutagen and animal carcinogen to which many people are exposed through the consumption of contaminated food and the use of tobacco products. Although the mutational specificity of NDMA has been studied in bacteria, little is known about the specific types of mutations induced by NDMA in the human genome. Knowledge of the mutational spectrum of NDMA in human genes may help to substantiate the role of NDMA in the etiology of human cancers. In the current study, the mutational spectrum of NDMA was characterized at the tk and hprt loci, in human lymphoblastoid cells capable of metabolically activating NDMA. A number of patterns were observed among NDMA-induced mutations. At both marker loci, G:C-->A:T transitions dominated the mutational spectrum of NDMA, which were indicative of the mutagenicity of the O6meG lesion. In addition, the majority of G:C-->A:T mutations occurred at guanines 3' to another guanine. Almost all of these mutations originated on the non-transcribed strand, which suggests that transcription-coupled repair influenced the distribution of G:C-->A:T transitions at the tk and hprt loci. Furthermore, the observation of hotspots for G:C-->A:T mutations, within both loci, suggests that differential repair kinetics may exist, and consequently affect the distribution of mutations. Finally, a comparison of the site specificity of G:C-->A:T mutations at the tk and hprt loci, indicated that the gene used for mutational analysis influenced the site specificity of NDMA-induced mutations, and possibly reflects the number of 5'-GG-3' sites in the tk and hprt loci that when mutated would yield a mutant phenotype.
Insights
N-Nitrosodimethylamine (NDMA) induces G:C to A:T mutations in human cells, primarily at guanine sites. Transcription-coupled repair and gene-specific mutation hotspots influence the observed mutational spectrum.
Area of Science:
- Toxicology
- Genetics
- Molecular Biology
Background:
- N-Nitrosodimethylamine (NDMA) is a known mutagen and carcinogen found in food and tobacco.
- Its mutagenic effects are well-studied in bacteria, but its impact on the human genome is less understood.
- Understanding NDMA's mutational spectrum in humans is crucial for cancer etiology research.
Purpose of the Study:
- To characterize the mutational spectrum of NDMA in human genes.
- To investigate the types and patterns of mutations induced by NDMA.
- To explore the influence of DNA repair mechanisms and gene context on mutation distribution.
Main Methods:
- NDMA-induced mutations were analyzed at the tk and hprt loci in human lymphoblastoid cells.
- Cells were metabolically activated to process NDMA.
- Mutational spectra were determined by sequencing the affected gene loci.
Main Results:
- G:C to A:T transitions were the predominant mutation type induced by NDMA.
- These transitions often occurred at guanine bases positioned 3' to another guanine.
- Mutations predominantly arose on the non-transcribed DNA strand, suggesting transcription-coupled repair.
- Hotspots for G:C to A:T mutations were identified, indicating differential repair kinetics.
- The specific gene locus analyzed influenced the site-specificity of NDMA-induced mutations.
Conclusions:
- NDMA primarily induces G:C to A:T transitions in human cells, consistent with O6-methylguanine lesions.
- Transcription-coupled repair significantly shapes the distribution of these mutations.
- Gene-specific factors and repair mechanisms contribute to mutation hotspots and patterns.
- The findings provide insights into NDMA's role in human carcinogenesis.
