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 by DNA glycosylase or by spontaneous depurination. AP sites are repaired by AP endonucleases during the process of base excision repair; however, an imbalance in this DNA repair system may cause mutations as well as cell death. We have established a sensitive and convenient slot-blot method to detect AP sites in genomic DNA using a novel aldehyde reactive probe (ARP), which reacts with the aldehydic group of ring-opened AP sites. The reaction of 1 mM of ARP with 15 microg of genomic DNA containing 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 enables detection of 2.4 AP sites per 10(7) bases. By using this ARP-slot-blot assay, the rate of spontaneous depurination of calf thymus DNA was determined. Under physiological conditions, AP sites were increased at 1.54 AP sites/10(6) nucleotides/day (9000 AP sites/cell/day). This highly sensitive assay allows us to determine the endogenous level of AP sites in genomic DNA, as well as to investigate whether DNA-damaging agents cause imbalances of base excision/AP endonuclease repair in vivo and in vitro.
Nakamura
J
J
Department of Environmental Sciences and Engineering, The University of North Carolina, Chapel Hill 27599-7400, USA.
Walker
V E
VE
Upton
P B
PB
Chiang
S Y
SY
Kow
Y W
YW
Swenberg
J A
JA
D001402:B-Lymphocytes / Q000378:metabolism; D001665:Binding Sites; D001681:Biological Assay; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects*; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D008741:Methyl Methanesulfonate; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods; D011687:Purines / Q000737:chemistry* / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity
D018736:DNA Adducts; D006023:Glycoproteins; D011687:Purines; D011743:Pyrimidines; D004247:DNA; D008741:Methyl Methanesulfonate