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Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.

ABSTRACT: In this chapter, we describe a gene-specific quantitative PCR (QPCR)-based assay for the measurement of DNA damage, using amplification of long DNA targets. This assay has been used extensively to measure the integrity of both nuclear and mitochondrial genomes exposed to different genotoxins and has proven to be particularly valuable in identifying reactive oxygen species-mediated mitochondrial DNA damage. QPCR can be used to quantify both the formation of DNA damage as well as the kinetics of damage removal. One of the main strengths of the assay is that it permits monitoring the integrity of mtDNA directly from total cellular DNA without the need for isolating mitochondria or a separate step of mitochondrial DNA purification. Here we discuss advantages and limitations of using QPCR to assay DNA damage in mammalian cells. In addition, we give a detailed protocol of the QPCR assay that helps facilitate its successful deployment in any molecular biology laboratory.

SUBMITTER: Furda A

PROVIDER: S-EPMC4407362 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.

Furda Amy A Santos Janine H JH Meyer Joel N JN Van Houten Bennett B

Methods in molecular biology (Clifton, N.J.) 20140101

In this chapter, we describe a gene-specific quantitative PCR (QPCR)-based assay for the measurement of DNA damage, using amplification of long DNA targets. This assay has been used extensively to measure the integrity of both nuclear and mitochondrial genomes exposed to different genotoxins and has proven to be particularly valuable in identifying reactive oxygen species-mediated mitochondrial DNA damage. QPCR can be used to quantify both the formation of DNA damage as well as the kinetics of d ...[more]

PMID: 24623245

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Project description:DNA-protein crosslinks (DPCs) are complex DNA lesions that induce mutagenesis and cell death. DPCs are created by common antitumor drugs, reactive oxygen species, and endogenous aldehydes. Since these agents create other types of DNA damage in addition to DPCs, identification of the mechanisms of DPC repair is challenging. In this study, we created plasmid substrates containing site-specific DPC lesions, as well as plasmids harboring lesions that are selectively repaired by the base excision or nucleotide excision repair (NER) pathways. These substrates were transfected into mammalian cells and a quantitative real-time PCR assay employed to study their repair. This assay revealed that DPC lesions were rapidly repaired in wild-type human and Chinese hamster derived cells, as were plasmids harboring an oxoguanine residue (base excision repair substrate) or cholesterol lesion (NER substrate). Interestingly, the DPC substrate was repaired in human cells nearly three times as efficiently as in Chinese hamster cells (>75% vs ?25% repair at 8?h post-transfection), while there was no significant species-specific difference in the efficiency with which the cholesterol lesion was repaired (?60% repair). Experiments revealed that both human and hamster cells deficient in NER due to mutations in the xeroderma pigmentosum A or D genes were five to ten-fold less able to repair the cholesterol and DPC lesions than were wild-type control clones, and that both the global genome and transcription-coupled sub-pathways of NER were capable of repairing DPCs. In addition, analyses using this PCR-based assay revealed that a 4?kDa peptide DNA crosslink was repaired nearly twice as efficiently as was a ?38?kDa DPC, suggesting that proteolytic degradation of crosslinked proteins occurs during DPC repair. These results highlight the utility of this PCR-based assay to study DNA repair and indicate that the NER machinery rapidly and efficiently repairs plasmid DPC lesions in mammalian cells.

Dataset Information

Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.

Publications

Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.

Similar Datasets

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