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

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A Correction to this article was published on 13 March 2021

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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.

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Funding

This work was supported by a Grants-in-aid for Scientific Research (for Scientific Research (C): 16K00551) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Authors and Affiliations

  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

    Ken Akamatsu & Naoya Shikazono

  2. Division of Radiation Life Science, Institute for Integrated Radiation and Nuclear Science, Kyoto University, 2 Asashiro-Nishi, Kumatori, Sennan, Osaka, 590-0494, Japan

    Takeshi Saito

Authors
  1. Ken Akamatsu
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  2. Naoya Shikazono
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  3. Takeshi Saito
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Contributions

K. Akamatsu. designed the research, performed the experiments, and wrote the manuscript. K. Akamatsu. and N. Shikazono analyzed and interpreted the data. T. Saito supported 60Co γ-ray irradiation experiments. All authors discussed the results and gave a lot of comments and suggestions for preparing the article.

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Correspondence to Ken Akamatsu.

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Akamatsu, K., Shikazono, N. & Saito, T. Fluorescence anisotropy study of radiation-induced DNA damage clustering based on FRET. Anal Bioanal Chem 413, 1185–1192 (2021). https://doi.org/10.1007/s00216-020-03082-w

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  • DOI: https://doi.org/10.1007/s00216-020-03082-w

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