Abstract
Apurinic/apyrimidinic (AP) site, an intermediate in base excision repair, is the most common DNA lesion that is lethal and premutagenic. We have developed a method for rapid detection and quantitation of AP sites in DNA with the Aldehyde Reactive Probe (ARP). In this study, we have developed a novel method for in situ analysis of intracellular AP sites using fluorescein-conjugated ARP, FARP-1 (5-[N'-(2-Aminooxyacetamidoethyl) thioureido] fluorescein). It is conceivable that the long patch sub-pathway is relatively dominant in S phase. Using synchronized HeLa cells, we examined the number of apparent MMS-induced AP sites in G1 and S phase cells by ARP method. The numbers of AP sites were not different significantly in both phases. By the FARP-1 method, we successfully analyzed AP sites in MMS-treated and propydium iodide-stained cell nuclei using a flow cytometer. The reaction of FARP-1 was specific, since the signal was suppressed by the pre-treatment of cell nuclei with methoxyamine. More AP sites were detected in S phase after MMS treatment, suggesting that both G1 and S phase cells efficiently repair the methylated bases by BER, since more DNA is included in a single S phase cell than in a G1 cell.
