Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 48 (2015)

Localization of Rad51 and phosphorylated DNA-PKsc after
heavy-ion irradiation in mammalian cells
M. Izumi*1 and T. Abe*1
Accelerated heavy-ion particles with high linear
energy transfer (LET) induce complex clustered DNA
damage, which is considered an obstacle to efficient
repair and causes different biological effects compared with
low-LET radiation such as X-ray. However, the biologicaleffects of heavy-ion irradiation are not fully understood at
the molecular level. To analyze the repair mechanism for
DNA double-strand breaks (DSBs) caused by heavy ions,
we have investigated cell sensitivity to heavy ions using thewild-type CHO-AA8 cells and two CHO mutant lines
deficient in homologous recombination (HR)1) or
non-homologous end-joining (NHEJ)2) in a previous study3)
and found that HR, but not NHEJ, is primarily involved in
the repair pathway induced by high-LET ionizing radiation.
In this study, we investigated the localization of
Rad51 and the phosphorylated form of the catalytic subunitof the DNA-dependent kinase (DNA-PKcs) in CHO cells
afterX-rayor
heavy-ionirradiation
withimmunoflorescence staining. Rad51 is essential for HRand is involved in strand transfer during homologouspairing,4) whereas DNA-PKcs is involved in NHEJ andphosphorylated upon DNA damage on threonin-2609.5)
One hour after X-ray irradiation, hundreds of Rad51or phosphorylated DNA-PKcs foci were observed in nuclei(Fig. 1A), although they were not colocalized with eachother. The number of foci started to decrease 4 h afterirradiation and fractions of Rad51 and phosphorylatedDNA-PKcs foci were colocalized. A small fraction ofDSBs was not repaired within 24 h (data not shown), andthe foci remaining at 16 h after irradiation contained both
Rad51 and phosphorylated DNA-PKcs, suggesting that
irreparable DSBs recruite repair proteins of the NHEJ andHR pathways.
On the other hand, large fractions of Rad51 andphosphorylated DNA-PKcs foci were colocalized 1 h afteriron-ion irradiation (LET = 1000 keV/µm) (Fig. 1B),
suggesting that the majority of DSBs have differentstructures in nature. Although our previous results suggestthat NHEJ is not involved in the repair pathway after
heavy-ion irradiation,3) we observed that phosphorylatedDNA-PKcs was recruited to DSBs. These results suggestthat repair proteins of both pathways are recruited to DSBs
induced by heavy ions, and finally HR is selected fordamage repair.
Currently, we are investigating how the cell cycle andLET affect the repair kinetics and colocalization of Rad51
and phosphorylated DNA-PKcs. We are also planning toexamine the localization of 53BP1 and Rif1 after heavy-ion
irradiation, which are involved in the selection of repair
pathways for DSB repair.6,7)

Fig. 1 Representative images of CHO nuclei (blue) withRad51 (green) and phosphorylated DNA-PKsc (red) foci.
The cells were irradiated with 5 Gy of X-ray (A) or iron
ions (B) and fixed with 4% paraformaldehyde at indicatedtime points after irradiation. Foci formation of Rad51 andphosphorylated
DNA-PKcs
wasdetected
byimmunofluorescence staining.
References*1 RIKEN Nishina Center1) R.S. Tebbs et al.: Proc. Natl. Acad. Sci. USA 92, 6354 (1995)
2) S.R. Peterson et al.: Proc. Natl. Acad. Sci. USA 92, 3171
(1995)
3) M. Izumi and T. Abe.: RIKEN Accel. Prog. Rep. 47, in press(2014)
4) A. Shinohara et al: 69, 457 (1992)
5) P. Douglas et al.: Biochem. J. 368, 243 (2002).
6) C. Escribano-Diaz et al.: Mol. Cell 49, 872 (2013)7) J. R. Chapman et al.: Mol. Cell 49, 858 (2013)- 301 -完全版2014_本文.indd 301

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