Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay.
The polymerase chain reaction (PCR) represents an alternative to the current methods for investigating DNA damage and repair in specific genomic segments. In theory, any DNA lesion which blocks Taq polymerase can be measured by this assay. We used quantitative PCR (QPCR) to determine the lesion frequencies produced by cisplatin and ultraviolet light (UV) in a 2.3 kilobase (kb) segment of mitochondrial DNA and a 2.6 kb segment of the DHFR gene in mouse leukemia L1210 cells. The frequency of UV-induced lesions increased linearly with dose, and was 0.58 lesions/10 kb/10 J/m2 in the mitochondrial DNA, and 0.37 lesions/10 kb/10 J/m2 in the DHFR gene. With cisplatin, the lesion frequency also increased linearly with dose, and was 0.17 lesions/10 kb/10 microM in the DHFR gene, and 0.07 lesions/10 kb/10 microM in mitochondrial DNA. This result is contrary to that of Murata et al., 1990 (1), in which mitochondrial DNA received greater cisplatin damage than did nuclear DNA. Using PCR to measure the repair of UV-induced lesions in the DHFR gene segment, we observed that less than 10% of the lesions were removed by 4 h, but over 70% of the lesions were removed by 8 h. Repair of 43% of UV-induced lesions in mitochondrial DNA was also observed during a 24 h period.
Kalinowski
D P
DP
Department of Pathology, University of Vermont, Burlington 05405.
Illenye
S
S
Van Houten
B
B
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002945:Cisplatin / Q000633:toxicity*; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000187:drug effects* / Q000235:genetics / Q000528:radiation effects*; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D007939:Leukemia L1210; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays / Q000009:adverse effects
D004272:DNA, Mitochondrial; D013762:Tetrahydrofolate Dehydrogenase; D002945:Cisplatin