Fluorescence anisotropy study of radiation-induced DNA damage clustering based on FRET
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RESEARCH PAPER
Fluorescence anisotropy study of radiation-induced DNA damage clustering based on FRET Ken Akamatsu 1 & Naoya Shikazono 1 & Takeshi Saito 2 Received: 11 September 2020 / Revised: 1 November 2020 / Accepted: 18 November 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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 FRET . Fluorescence anisotropy . Clustered DNA damage . Ionizing radiation
Introduction A clustered DNA damage site (cluster) or a multiply damaged site, where more than two lesions exist within a few helical turns, is believed to cause irreversible biological effects such as cell death and carcinogenesis because the DNA cannot be repaired [1–3]. Densely ionizing radiation such as a heavy ion beam and a low-energy electron [4, 5] should produce a cluster and is responsible for mutagenesis [6–10]. In the past few decades, numerous experimental and computational studies have examined damage localization and the influence of different types of ionizing radiations (reviewed in [11–13]). The * Ken Akamatsu [email protected] 1
DNA Damage Chemistry Research Group, Institute for Quantum Life Science, National Institutes for Quantum and Radiological Science and Technology (QST), 8-1-7 Umemidai, Kizugawa, 619-0215 Kyoto, Japan
2
Division of Radiation Life Science, Institute for Integrated Radiation and Nuclear Science, Kyoto University, 2 Asashiro-Nishi, Kumatori, Sennan, Osaka 590-0494, Japan
experimental approaches have mainly estimated single- and double-strand break yields using electrophoretic analyses of the irradiated plasmid, phage DNA [14–17], and genomic DNA extracted from the irradiated mammalian cells [18–20]. Moreover, the direct visualization i
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