Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 56 (2023)
RIKEN
Accel. Prog. Rep. 56 (2023)

Heavy-ion irradiation enhanced the post-transcriptional modiﬁcationof CtIP
M. Izumi,∗1 T. Tsukada,∗1 and T. Abe∗1
Among DNA damages caused by ionizing radiation,
DNA double strand breaks (DSBs) are the most lethal
as misrepaired or unrepaired DSBs can cause a lossof genetic information, canceration, and cell death.
Mammalian cells have four pathways to repair DSBs:
canonical non-homologous end joining (NHEJ), homologous recombination (HR), alternative NHEJ (altNHEJ), and single strand annealing (SSA). Accelerated heavy-ion particles with high linear energy transfer (LET) induce complex clustered DNA damage including DSBs, single strand breaks, and base damages
within one or two helical turns of DNA. The clustered
DNA damage is considered as an obstacle to eﬃcient
repair and aﬀects the pathway choice for DSB repair.
After exposure to low-LET radiation, NHEJ is thedominant repair pathway throughout the cell cycle,
whereas HR works only in the late S/G2 phase. AltNHEJ and SSA are considered to be functional only
when both the NHEJ and HR are impaired.1) However, the repair mechanism after heavy-ion irradiation
has not been fully understood. Our previous study
using mammalian cells and speciﬁc inhibitors against
NHEJ or HR suggests that NHEJ is the major repair
pathway after 2 Gy of heavy-ion irradiation.2) In addition, we have shown that HR is favored after heavyion irradiation in G2-phase, although HR repairs DSBs
less eﬃciently after heavy-ion irradiation than after Xray irradiation.3) Moreover, recruitment of Rad51 (HRfactor) to DSB is suppressed by high dose (>15 Gy)
heavy-ion irradiation,4) suggesting that the pathway
choice is dependent on the cell cycle, LET, and dose.
In this study, we examined the modiﬁcation ofCTBP-interacting protein (CtIP) after heavy-ion irradiation. CtIP is involved in end-resection of DSBs
for generating 3′ -single strand DNA, which promotes
HR, SSA, and alt-NHEJ.5) CtIP is phosphorylatedby active ATM as well as cyclin-dependent kinase inS/G2 phase. Phosphorylation of CtIP facilitates its
interaction with BRCA1 and Mre11/Rad50/Nbs1 nuclease complex. Recent studies suggest that the balance between 53BP1-Rif1 and CtIP-BRCA1 controls
the pathway choice. Thus, the choice between NHEJ
and HR/alt-NHEJ/SSA is determined by which among
end-joining or end resection occurs ﬁrst.6)
Exponentially growing HeLa cells were irradiatedwith argon ions (LET = 300 keV/µm) of diﬀerent doses (2–30 Gy), and chromatin fractions wereobtained and subjected to an immunoblot analysis(Fig. 1). In unirradiated cell extracts (at 0 hour), both
phosphorylated and non-phosphorylated forms of CtIP
∗1RIKEN Nishina Center- 196 -X
2 Gy

CtIP
DNA-PKRad51
γH2AX
lamin b1

5 Gy

15 Gy

30 Gy

0 1 3 8 1 3 8 1 3 8 1 3 8 (h)phosphorylated
unphosphorylated

Ar
2 Gy

5 Gy

15 Gy

30 Gy

0 1 3 8 1 3 8 1 3 8 1 3 8 (h)CtIPphosphorylated
unphosphorylated

DNA-PKRad51
γH2AX
lamin b1
Fig. 1. Immunoblot analysis of chromatin-bound repair
proteins after irradiation. HeLa cells were irradiatedwith indicated doses of X ray or Ar ions. Tritoninsoluble fractions (chromatin fractions) were preparedat indicated time points following irradiation and subjected to immunoblot. CtIP, phosphorylated DNA-PK,
Rad51, and phosphorylated histone H2AX (γH2AX)
were detected. Arrowheads indicate the retarded modiﬁed forms, which were absent in X-ray irradiated extracts. Lamin B1 was detected as a loading control.

were detected. The level of phosphorylated forms increased after both 15 and 30 Gy of X-ray irradiation.
Argon-ion irradiation induced phosphorylation at thelower dose (2–5 Gy), suggesting that the end-resection
is enhanced after heavy-ion irradiation. In addition,
we found a more retarded form of CtIP after high dose(>15 Gy) of argon-ion irradiation. The retarded form
may be hyperphosphorylated or modiﬁed by small proteins such as ubiquitin or SUMO. Moreover, we observed the same form after the carbon-ion (LET =80 keV/µm) irradiation (data not shown).
We also detected phosphorylated DNA-PK (NHEJfactor) and Rad51 (Fig. 1). The amount of phosphorylated DNA-PK was dependent on the dose, whereasthat of Rad51 increased up to 5 Gy after argonirradiation and decreased at 15 Gy irradiation as previously reported.4) These results suggest that CtIP
can promote alt-NHEJ and/or SSA at high doses(>15 Gy).

Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 56 (2023)

References
1) R. Ceccaldi et al., Trends Cell Biol. 26, 52 (2016).
2) M. Izumi et al., RIKEN Accel. Prog. Rep. 53, 24 (2020).
3) M. Izumi et al., RIKEN Accel. Prog. Rep. 54, 33 (2021).
4) M. Izumi et al., RIKEN Accel. Prog. Rep. 55, 174(2022).
5) P. Cejka et al., Annu. Rev. Genet. 55, 285 (2021).
6) C. Escribano-Diaz et al., Mol. Cell 49, 872 (2013).

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