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 quantitati on of abasic (AP) sites in DNA, in which the biotinylated reagent, cal led the aldehyde reactive probe (ARP) specifically reacts with aldehyd e 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 feas ibility and the sensitivity of ARP assay. For immobilization of DNA, a protamine sulfate-coated plate was used instead of the conventional U V-irradiated plate to enhance DNA binding. As the result. the time for immobilization was shortened to 1 h without any loss of signal. The a mount of [H-3]-labeled DNA bound to the plate was proportional to the DNA concentration employed. When DNA containing AP sites was treated w ith ARP in solution prior to coating the protamine-plate, the sensitiv ity of the assay was greatly increased. A linear relationship between the DNA concentration and the signal intensity was also observed. Thus , similar to 0.1 fmol of AP sites (0.5 sites per 10(5) nt) could be de tected in DNA isolated from HeLa cells after treatment with a subletha l dose (0.5 mM) of methylmethanesulfonate (MMS). Using this system, th e number of total methylpurines generated by MMS in the cellular DNA w as estimated after heat treatment, which converted methylated base les ions to AP sites. It was shown that the number of AP sites was about 1 40 sites per 10(4) nt with 25 mM MMS and 10% of total methylated bases were already released without heat depurination. (C) 1998 Elsevier Sc ience B.V.