Highly sensitive assay of DNA abasic sites in mammalian cells-optimization of the aldehyde reactive probe method

We have recently developed a novel method for detection and quantitation of abasic (AP) sites in DNA, in which the biotinylated reagent, called the aldehyde reactive probe (ARP) specifically reacts with aldehyde groups of AP sites and biotin-tagged damage is detected by an ELISA-like assay. The present study has been carried out to improve the feasibility and the sensitivity of ARP assay. For immobilization of DNA, a protamine sulfate-coated plate was used instead of the conventional UV-irradiated plate to enhance DNA binding. As the result, the time for immobilization was shortened to 1 h without any loss of signal. The amount of [3H]-labeled DNA bound to the plate was proportional to the DNA concentration employed. When DNA containing AP sites was treated with ARP in solution prior to coating the protamine-plate, the sensitivity of the assay was greatly increased. A linear relationship between the DNA concentration and the signal intensity was also observed. Thus, ∼0.1 fmol of AP sites (0.5 sites per 105 nt) could be detected in DNA isolated from HeLa cells after treatment with a sublethal dose (0.5 mM) of methylmethanesulfonate (MMS). Using this system, the number of total methylpurines generated by MMS in the cellular DNA was estimated after heat treatment, which converted methylated base lesions to AP sites. It was shown that the number of AP sites was about 140 sites per 104 nt with 25 mM MMS and 10% of total methylated bases were already released without heat depurination.