Fluorescence anisotropy study of radiation-induced DNA damage clustering based on FRET

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 Forster resonance energy transfer (homo-FRET). Plasmid DNA (pUC19) was irradiated with 2.0 and 0.52 MeV/u He-4(2+), or 0.37 MeV/u C-12(5+) ion beams (linear energy transfer: similar to 70, similar to 150, similar to 760 keV/mu m, respectively) and Co-60 gamma-rays as a standard (similar to 0.2 keV/mu m) 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.

Automated summary

This study characterized clustered DNA damage sites through fluorescence anisotropy measurements, revealing an increase in the apparent base separation values between lesions in a cluster with higher linear energy transfer of radiation. This method has the potential to discover qualitative and quantitative differences in clustered DNA damage produced by various ionizing radiation and other damaging agents.