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Identification of cellular defect in UVS1, a UV-sensitive Chinese hamster ovary mutant cell line
1Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai, Japan.
Abstract:
UVS1 is an intermediately UV-sensitive Chinese hamster ovary mutant originally isolated by its hypersensitivity to an anticancer drug, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride. By cell fusion analysis, UVS1 complemented the UV sensitivity of the mouse lymphoma cell line US31 from the eighth complementation group of UV-sensitive rodent cell lines. By enzyme-linked immunosorbent assay we found that within 3 h after UV irradiation both pyrimidine dimers and (6-4)photoproducts in UVS1 were not removed from chromosomal DNA in UVS1 at all. Twenty-four h after UV irradiation the removal rate of (6-4)photoproducts was intermediate between CHO9, the parental cell line, and 43-3B, a UV-hypersensitive Chinese hamster ovary mutant of the complementation group 1, whereas the pyrimidine dimers in UVS1 were removed less efficiently as 43-3B. Alkaline elution assay showed that the incising activity to damaged DNA after UV irradiation of UVS1 was as low as that of 43-3B. The number of 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride-induced DNA interstrand cross-links of UVS1 was almost equal to that of 43-3B and about 1.5 times more than that of CHO9, suggesting that the gene products defective in UVS1 and 43-3B are essential for the excision repair of DNA damages produced by 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride.
Insights
The UVS1 Chinese hamster ovary mutant shows intermediate UV sensitivity and impaired DNA repair. This defect affects the removal of UV-induced DNA damage and cross-links, impacting DNA repair mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair
Background:
- UVS1 is a Chinese hamster ovary mutant exhibiting intermediate UV sensitivity.
- It was initially identified due to hypersensitivity to an anticancer drug, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea hydrochloride.
- UVS1 complements the UV sensitivity of the mouse lymphoma cell line US31, placing it in the eighth complementation group of UV-sensitive rodent cell lines.
Purpose of the Study:
- To investigate the DNA repair characteristics of the UVS1 mutant.
- To determine the efficiency of removing UV-induced DNA lesions, specifically pyrimidine dimers and (6-4)photoproducts.
- To assess the role of the defective gene product in DNA interstrand cross-link repair.
Main Methods:
- Cell fusion analysis to determine complementation groups.
- Enzyme-linked immunosorbent assay (ELISA) to quantify DNA photoproducts after UV irradiation.
- Alkaline elution assay to measure DNA incising activity and interstrand cross-links.
Main Results:
- UVS1 showed no removal of pyrimidine dimers and (6-4)photoproducts within 3 hours post-UV irradiation.
- At 24 hours, (6-4)photoproduct removal was intermediate, while pyrimidine dimer removal was less efficient than the parental CHO9 cell line.
- Incising activity in UVS1 was as low as the highly UV-sensitive 43-3B mutant, and it accumulated significant DNA interstrand cross-links.
Conclusions:
- The UVS1 mutant exhibits significant defects in DNA excision repair pathways.
- The gene product defective in UVS1 is crucial for repairing DNA damage induced by UV radiation and specific anticancer drugs.
- These findings contribute to understanding the complex mechanisms of DNA repair in mammalian cells.