A methodology for estimating localization of apurinic/apyrimidinic sites in DNA using fluorescence resonance energy transfer

Abstract

We have developed a methodology for estimating localization of lesions on double-stranded DNA using fluorescence resonance energy transfer (FRET). We focused on apurinic/apyrimidinic (AP) sites, which are typical DNA lesions induced by radiation and chemicals and produced spontaneously under physiological conditions. Donor-acceptor fluorescent probes with O-amino groups (Alexa Fluor 350-Alexa Fluor 488 dye pair) were used for selectively labeling AP sites. pUC19 plasmid subjected to heat treatment (pH 5.2, 70 °C) was used as a model double-stranded DNA containing AP sites. The results of both FRET analysis and theoretical study enabled us to prove that AP sites induced by the heat treatment are distributed almost randomly along the DNA molecule. This methodology will be useful for estimating the risk of ionizing radiation and chemicals (e.g., pollutants and anticancer agents) based on the probability of producing "clustered DNA damage sites," which are considered to be less easily repairable and, therefore, more harmful to living systems.