Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay

Abstract

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.

Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress

“…␤-Pol null cells are hypersensitive to the cytotoxic effect of MMS exposure (15,16) and therefore more MMS-induced lesions may persist in genomic DNA of ␤-pol null cells than wild-type cells. To test this idea, QPCR was used to measure MMS-induced DNA polymerase blocking lesions (e.g., N3-methyl adenine, AP sites, and single-strand breaks) in a segment of genomic DNA (23,(27)(28)(29)(30)(31)(32)(33)(34)(35). Wild-type and ␤-pol null cells were examined with and without a repair incubation after termination of MMS exposure.…”

DNA damage by anti-cancer agents resolved at the nucleotide level of a single copy gene: evidence for a novel binding site for cisplatin in cells

“…It can be used with a wide range of agents since it is not limited to those whose lesion can be converted into a strand break. It should be possible to use sslig-PCR with many types of chemical carcinogens and we have used it to detect UV induced lesions (data not shown) which are also known to block taq polymerase (11). Therefore studies are now possible at the cellular level to determine sites of lesions and to reveal, for example, if there is a correlation between adduct formation and the positions of base mutations in particular genes.…”

“…The lack of techniques to measure precise DNA binding within intact cells has also meant that the sequence selectivity of repair of individual lesions is a largely unexplored area. Southern blotting and PCR based methods currently available for the measurement of DNA repair in the intact cell can do so at the level of the gene (10-20 kb) (9,10) and regions within the gene (500-2000 bp) (11)(12)(13) respectively, but cannot give information at the level of the individual base.…”

Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells

“…These conditions must be met by keeping the PCR in the exponential phase. The first step towards this criterion is to perform cycle tests to determine quantitative conditions for the gene of interest [3]. This can be accomplished using a non- 5 Because of the long run time of our PCR programs, we add BSA (100 ng/μL final concentration) to the PCR mix to increase the stability of the polymerase [6].…”