HIGHLY SENSITIVE APURINIC APYRIMIDINIC SITE ASSAY CAN DETECT SPONTANEOUS AND CHEMICALLY-INDUCED DEPURINATION UNDER PHYSIOLOGICAL CONDITIONS/

One of the most prevalent lesions in DNA is the apurinic/apyrimidinic (AP) site, which is derived from the cleavage of the N-glycosyl bond b y DNA glycosylase or by spontaneous depurination. AP sites are repaire d by AP endonucleases during the process of base excision repair; howe ver, an imbalance in this DNA repair system may cause mutations as wel l as cell death. We have established a sensitive and convenient slot-b lot method to detect AP sites in genomic DNA using a novel aldehyde re active probe (ARP), which reacts with the aldehydic group of ring-open ed AP sites. The reaction of 1 mM of ARP with 15 mu g of genomic DNA c ontaining AP sites at 37 degrees C was completed within 1 min. The AP site-ARP complex was remarkably stable during incubation in TE buffer, even at 100 degrees C for 60 min. The sensitivity of this assay enabl es detection of 2.4 AP sites per 10(7) bases. By using this ARP-slot-b lot assay, the rate of spontaneous depurination of calf thymus DNA was determined. Under physiological conditions, AP sites were increased a t 1.54 AP sites/10(6) nucleotides/day (9000 AP sites/cell/day). This h ighly sensitive assay allows us to determine the endogenous level of A P sites in genomic DNA, as well as to investigate whether DNA-damaging agents cause imbalances of base excision/AP endonuclease repair in vi vo and in vitro.