I. HIGHLIGHTS OF THE YEARRIKEN Accel. Prog. Rep. 54 (2021)

RIKEN Accel. Prog. Rep. 54 (2021)

Kinetics of Rad51 foci in G2 phase after heavy-ion irradiation inmammalian cells
M. Izumi∗1 and T. Abe∗1Ratio of Rad51/γH2AX

*

*

1.0
0.8
0.6
0.4
0.2
0X

CArFig. 1. Ratio of Rad51/phosphorylated histone H2AX(γH2AX) foci 1 h after irradiation in HeLa cells. Aphidicolin (10 µM) was added 30 min before irradiation, andcells were irradiated with 2 Gy of X-rays, carbon ions
(LET = 80 keV/µm), or argon ions (LET = 300 keV/µm)
and incubated in the presence of aphidicolin for 1 h afterirradiation. The foci formation of Rad51 and γH2AX wasdetected by immunostaining. Student’t t test: ∗ P < 0.01.
Error bars represent SD.
∗1RIKEN Nishina Center

X-ray
Number of γH2AX or Rad51
foci/cell (%)

DNA double-strand breaks (DSBs) are the most lethal
type of damage caused by ionizing irradiation and arerepaired mainly by non-homologous end joining (NHEJ)
or homologous recombination (HR) in mammalian cells;
alternative NHEJ and/or single-strand annealing work
only when both NHEJ and HR are impaired. Accelerated heavy-ion particles with high linear energy transfer(LET) induce complex and fragmented DNA damageaﬀecting the pathway choice and the eﬃciency of DSB
repair.
Several published results of survival assay using Chinese hamster mutant cell lines deﬁcient in NHEJ orHR suggest that NHEJ is inhibited after heavy-ion irradiation.1,2) In contrast, studies using inhibitors andmouse mutant cell lines suggest that NHEJ is a major repair pathway after heavy-ion irradiation, although
HR is more important for higher-LET radiation.3,4) It
is also reported that clustered DNA damage enhances
end resection, which could promote HR, alt-NHEJ, andSSA.5,6) Therefore, the DNA repair mechanism after
heavy-ion irradiation is still controversial in higher eukaryotes.
Our previous study using human ﬁbroblast and a speciﬁc inhibitor against NHEJ or HR suggests that NHEJ
and HR work competitively and compensate for eachother after X-irradiation.7) On the other hand, NHEJ
is the major repair pathway after heavy-ion irradiation,and HR does not seem to compensate for NHEJ. How-Carbon(LET = 80 keV/μm)

Argon(LET = 300 keV/μm)

γH2AX
Rad51

γH2AX
Rad51

100
806040
γH2AX
Rad51

200135813581358

Time from irradiation (h)Fig. 2. Time course of phosphorylated histone H2AX
(γH2AX) and Rad51 foci after irradiation in G2 cells.
The percentage of foci per cell was plotted by normalizing the numbers at the maximum time point as 100%after irradiation.

ever, it is unknown whether HR eﬃciently repairs DSBs
caused by heavy ions or whether the DNA damage checkpoint delays the entry into the S/G2 phase, where HRoccurs.
In this study, we examined the DNA repair kinetics
as well as the repair pathway usage in the G2 phase.
Exponentially growing HeLa cells were irradiated withX-rays, carbon ions, or argon ions and incubated in thepresence of aphidicolin to arrest S-phase progression andinhibit transition from the S to the G2 phase. The repair eﬃciency and pathway usage were estimated by thekinetics of the phosphorylated histone H2AX foci andRad51 foci, which reﬂect the DSBs and HR, respectively.
S-phase cells were identiﬁed by the pan-nucleic stainingof the phosphorylated histone H2AX and excluded fromanalysis.
The ratio of Rad51/phosphorylated histone H2AX
foci induced by heavy-ion irradiation was higher thanthat induced by X-rays (Fig. 1). The number of Rad51
foci decreased more slowly after heavy-ion irradiation
than after X-ray irradiation, with the kinetics similar to
those of phosphorylated histone H2AX (Fig. 2). Theseresults suggest that HR is favored after heavy-ion irradiation, although HR repairs DSBs less eﬃciently after
heavy-ion irradiation.
References

1) H. Wang et al., DNA Repair 7, 725 (2008).
2) S. C. Genet et al., Oncol. Rep. 28, 1591 (2012).
3) H. Ma et al., Radiat. Oncol. 10, 225 (2015).
4) A. Gerelchuluun et al., Rad. Res. 183, 345 (2015).
5) H. Yajima et al., DNA Repair 12, 936 (2013).
6) A. Shibata et al., EMBO J. 30, 1079 (2011).
7) M. Izumi, T. Abe, RIKEN Accel. Prog. Rep. 53, 24 (2020).

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