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Properties and applications of human DNA repair genes
1Biomedical Sciences Division, Lawrence Livermore National Laboratory, CA 94550.
Abstract:
The importance of understanding DNA repair processes is discussed in terms of the origins of human cancer. Several human repair genes have been mapped to specific human chromosomes using somatic cell hybrids. It is noteworthy that 3 of these genes lie in the same region of chromosome 19: genes ERCC1 and ERCC2, which are involved in nucleotide excision repair, and XRCC1, which is involved in the repair of strand breaks. The genes XRCC1 and ERCC2 were cloned from cosmid libraries prepared from DNA transformants of the CHO mutants EM9 and UV5, respectively. Analysis of the cDNA sequence of ERCC2 showed that the protein encoded by this gene is highly homologous (73%) to the RAD3 repair protein in the yeast Saccharomyces cerevisiae. Thus, the known properties of RAD3 combined with the high homology provide the first insight about the biochemical role of a human repair protein involved in the incision step of nucleotide excision repair. So far XRCC1 is the only cloned mammalian gene involved in repairing damage from ionizing radiation. The UV5 mutant line was also applied to problems in environmental mutagenesis by introducing the mouse cytochrome P(3)450 (P450IA2 subfamily) gene for metabolic activation of aromatic amines. We show in a rapid differential cytotoxicity assay with 2 compounds found in cooked beef (IQ, 2-amino-3-methylimidazo[4,5-f]quinoline and PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) that this gene is efficiently expressed in the transformed UV5P3 cells. Reversion of the repair deficiency in these cells will give a matched pair of cell lines that are metabolically proficient and repair deficient. Such lines will provide a rapid assay for genotoxic heterocyclic amines requiring activation.
Insights
Understanding DNA repair is crucial for cancer origins. Researchers cloned human repair genes ERCC1, ERCC2, and XRCC1, revealing insights into DNA repair mechanisms and aiding environmental mutagenesis studies.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- DNA repair mechanisms are vital for preventing mutations that lead to cancer.
- Human repair genes are being mapped to identify their roles in DNA damage response.
- Chromosome 19 harbors key genes involved in nucleotide excision repair and strand break repair.
Purpose of the Study:
- To understand the origins of human cancer by studying DNA repair processes.
- To clone and characterize human DNA repair genes, specifically ERCC1, ERCC2, and XRCC1.
- To develop novel cell lines for assessing the genotoxicity of environmental mutagens.
Main Methods:
- Somatic cell hybridization was used to map human repair genes to chromosomes.
- Cosmid libraries were employed to clone the XRCC1 and ERCC2 genes.
- cDNA sequencing and protein homology analysis were performed to understand ERCC2's function.
- Transfection of a mouse cytochrome P450 gene into a repair-deficient cell line (UV5) was utilized.
- Differential cytotoxicity assays were conducted to test the genotoxicity of heterocyclic amines.
Main Results:
- Three human repair genes (ERCC1, ERCC2, XRCC1) were localized to chromosome 19.
- ERCC2 shares high homology with yeast RAD3, suggesting a role in nucleotide excision repair.
- XRCC1 was identified as the first cloned mammalian gene for ionizing radiation repair.
- Transformed UV5P3 cells efficiently expressed the mouse P450 gene, enabling metabolic activation of aromatic amines.
- A rapid assay for genotoxic heterocyclic amines was developed using repair-deficient and metabolically proficient cell lines.
Conclusions:
- The study provides insights into the biochemical function of human repair proteins.
- Cloned genes and developed cell lines offer valuable tools for cancer research and environmental mutagenesis.
- Understanding DNA repair pathways is essential for developing strategies to combat cancer and environmental toxins.