A clustered DNA damage site (cluster), in which two or more lesions exist within a few helical turns, is believed to be a key factor determining the fate of a living cell exposed to a DNA damaging agent such as ionizing radiation. However, the structural details of a cluster such as the number of included lesions and their proximity are unknown. Herein, we develop a method to characterize a cluster by fluorescence anisotropy measurements based on Förster resonance energy transfer (homo-FRET). Plasmid DNA (pUC19) was irradiated with 2.0 and 0.52 MeV/u 4He2+, or 0.37 MeV/u 12C5+ ion beams (linear energy transfer: ~ 70, ~ 150, ~ 760 keV/μm, respectively) and 60Co γ-rays as a standard (~ 0.2 keV/μm) in the solid state. The irradiated DNA was labeled with an aminooxyl fluorophore (Alexa Fluor 488) to the aldehyde/ketone moieties such as apurinic/apyrimidinic sites. Homo-FRET analyses provided the apparent base separation values between lesions in a cluster produced by each ion beam track as 21.1, 19.4, and 18.7 base pairs. The production frequency of a cluster increases with increasing linear energy transfer of radiation. Our results demonstrate that homo-FRET analysis has the potential to discover the qualitative and the quantitative differences of the clusters produced not only by a variety of ionizing radiation but also by other DNA damaging agents.
Keywords: Clustered DNA damage; FRET; Fluorescence anisotropy; Ionizing radiation.