DNA damage by anticancer agents and its repair: mapping in cells at the subgene level with quantitative polymerase...

The quantitative polymerase chain reaction (QPCR)-based assay was used to measure DNA damage and repair to a small (523 bp) fragment of the single-copy human N-ras gene in K562 cells. Compared with previous methods DNA preparation from treated cells and the subsequent detection of the radioactive product were considerably simplified. The results demonstrated that QPCR can be used to measure damage in a small gene segment, caused by cisplatin, nitrogen, and quinacrine mustards. Drug-DNA adducts produced by two novel minor groove binding, sequence-specific molecules (AT-486 and DSB-120) could be detected at physiologically relevant concentrations of drug. For both cisplatin and nitrogen mustard the concentrations required to cause damage in cells were higher than those needed to cause equivalent damage in isolated DNA. In contrast both AT-486 and quinacrine mustard caused more damage at equimolar concentrations in cells than in isolated DNA. DSB-120, which is closely related to AT-486, was found to be 15-fold less effective than the latter at causing damage in treated cells despite similar reactivity with isolated DNA. Repair of damage caused by quinacrine mustard to the same small gene fragment was found to proceed at a constant rate over 24 h. The QPCR assay presented here is a simple quantitative method to measure damage and repair in subgene functional units such as promoters, introns, and exons.