Research Group for Radiation and Biomolecular Sciences
Group Leader : Akinari Yokoya
Members : Kentaro Fujii, Miho Noguchi, Ayumi Urushibara, Toshitaka Oka, Yuki Sugaya,
Iyo Shiraishi, Takuya Shiina

In vitro study of repairability of 8-oxoG/single strand breakcontaining clusters depends on their relative positions
The biological consequences of clusters DNA damage site
containing a single strand break (SSB) and nucleobase lesion(s)remain largely unknown. We examined the repairbility of two- andthree-lesion clustered damage sites containing a 1-nucleotide gap(GAP) and 8-oxo-7,8-dihydroguanine(s) (8-oxoG(s)) by a baseexcision repair enzyme, Fpg, in vitro [1]. Fpg speciﬁcally excises
purine base lesions such as 8-oxoG residues from DNA backbone
via its AP lyase activity, and subsequently cleaves the DNA. The
processing of the tandem cluster by Fpg leads to the generation ofan additional band with a slightly larger size (18 mer OH termini)
than that observed after excision of a single 8-oxoG (Fig. 1).

the additional 8-oxoG on the complementary strand does not inhibit
the action of Fpg at the 8-oxoG placed with a GAP in tandem.
LET dependence of the yield of DNA damage induced infully hydrated DNA films by ion particle-irradiation
In order to clarify the characteristics of DNA damage inducedby high LET charged particle-radiation, the yield of DNA damage
induced in closed-circular plasmid DNA (pUC18) were measured
after exposing to various kinds of radiation (He, Ne and C ions;
2 to 900 keV/μm) using the JAEA-TIARA and NIRS-HIMAC
facilities [2]. Hydrated DNA samples were used together withbase excision repair enzymes, Fpg (see above) and EndoIII,
which mainly excises pyrimidine base lesions, to detect thelesions as enzyme sensitive sites (ESSs). The results show that 1)
the yields of ESS decrease drastically with increasing LET andvery few ESSs are induced >100 keV/μm, althoguh the yield ofSSB does not depend signiﬁcantly on LET of the ions (Fig. 2), 2)
the yield of duble strand breaks (DSBs) increases with increasing
LET, however, 3) those of clustered damage sites visualized asadditional DSBs by enzymatic treatment decrease with increasing
LET, and 4) C and Ne ions induce less nucleobase lesions than Heions for comparable LET values. These results indicate that theyields of clusters containing nucleobase lesions, which are lessreadily processed by the base excision repair enzymes, depend
not only on LET but also on the ion particle used.

Group Activities

The research objective of the research group is to fully
characterize molecular processes by which ionizing radiation
alters the chemical structure of DNA in cells. Two approaches
were proposed to address the main objective: 1) Obtain
experimental evidence of repairability of artificial clustered
DNA damage in vitro. 2) Determine the yields of DNA lesions
in a simple model DNA molecule, in terms of LET (LinearEnergy Transfer) as an index of charged particle-radiation.

Fig. 1 Gel image of the excision of an 8-oxoG by Fpg placed intandem with a GAP. (A) The shematic diagram of a Cy5labeled double stranded oligonucleotide containing a GAP andan 8-oxoG tamdem clsuter. (B) 5’ Cy5-labeled tandem cluster(8G-2, GAP+1) was incubated with increasing amounts(0-10 units) of Fpg. The conﬁguration of the tandem cluster isindicated above the gel. 5’ Cy5-labeled duplex oligonucleotides
containing a single 8-oxoG (8G-2) incubated with Fpg and asingle GAP (GAP+1) are shown for comparison.
The activity of the enzyme causes successive β- andδ-elimination reactions and generates a 1-nt gap. We treated thecluster with 0.1M NaOH, as an alkaline treatment is known toconvert the β-elimination product to δ-elimination product atthe 3’ end. The result that the additional band disappeared after
the alkaline treatment indicates that the band corresponds to afragment generated by β-elimination. Further, to look at the effectof an additional 8-oxoG on the opposite strand for the excision of
the 8-oxoG placed with a GAP in tandem, a three-lesion cluster(two 8-oxoGs and one GAP) was treated with Fpg. The 8-oxoG
in tandem with a GAP in the three-lesion cluster is excised in afashion similar to the excision of the 8-oxoG with a GAP in thetandem two-lesion cluster, forming two bands. This indicates thatFig. 2 Dependence of the yield of SSB or n(ESS or SSB) on LET for
fully hydrated DNA irradiated with 4He2+ ions (A), 12C5+,6+ (B)and 20Ne8+,10+ (C) at 5.6 °C ( ○ ), or following post-irradiation
incubation for 30 min at 37 °C in the absence ( ● ) or presenceof either Nth ( △ ), Fpg ( □ ), or both Nth and Fpg ( ◇ ).
The yields of isolated nucleobase lesions visualized by the
enzymatic treatment decrease drastically with increasing LET.
References[1] M. Noguchi et al., Mutat. Res. 732, 34 (2012).
[2] T. Ushigome et al., Radiat. Res. 177, 614 (2012).

ASRC 18