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QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME
Nakamura, J., S. Asakura, S D. Hester, I. M. Jones, K. W. Caldecott, AND J. A. Swenberg. QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME. NUCLEIC ACIDS RESEARCH. Oxford University Press, Cary, NC, 31(17):e104, (2003).
Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.
DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or during the base excision repair pathways. Here we established a new real-time assay to assess an imbalance of DNA SSB repair by indirectly measuring PARP activation through the depletion of intracellular NAD(P)H. A water-soluble tetrazolium salt is used to monitor the amount of NAD(P)H in living cells through its reduction to a yellow colored water-soluble formazan dye. While this assay is not a direct method, it does not require DNA extraction or alkaline treatment, both of which could potentially cause an artifactual induction of SSBs . In addition it takes only four hours and requires less than a half million cells to perform this measurement. Using this assay, we demonstrated that exposure to an alkylating agent caused a significant variation in the decrement of NAD(P)H in human lymphoblastoid cell lines, established from eight different healthy individuals. These results suggest that human individuals may have different capacities to repair SSBs. This convenient and reproducible assay can be a useful tool for the screening of human populations who may have a diminished capacity for either single strand breaks or base excision repair.