Research Group for Radiation and Biomolecular Science
Group Leader : Akinari Yokoya
Members : Kentaro Fujii, Miho Noguchi, Ayumi Urushibara, Ayumi Narita, Yuki Sugaya,
Iyo Shiraishi, Takuya Shiina, Kiichi Kaminaga, Yukiko Kanari, Yuka Sakamoto

The research objectives of Radiation and Biomolecular Science
Group is to elucidate a molecular and cellular process of transgenerational effects by radiation. In JFY2012, we have (1)deterimined the yields of DNA lesions by ion-particle or X-ray
irradiations, (2) observed a chromosomal instability induced byDNA damage of non-double-strand-break (non-DSB) type.
Yield of AP sites and other lesions induced in DNA by ionand X-ray irradiation
In order to obtain the yields of DNA lesions such as singlestrand breaks (SSBs), base lesions, and abasic (AP) sites arisingin a plasmid DNA molecule irradiated with various ionizing
radiations, plasmid DNA (pUC18) was exposed to carbon ions
(LET: 13 keV/m and 60 keV/m) or X-rays (4 keV/m) insolutions [1]. Concentrations of Tris in the sample were changed
from 0.66 to 200 mM to examine the effect of scavenging
capacity. Irradiated samples were then analysed using anenzymatic probe method. We used Nfo, Fpg and Nth proteins toconvert the AP sites, purine base lesions, and pyrimidine baselesions into detectable SSBs. At higher concentrations of Tris,
the yields of single base lesions were significantly higher thanthose of single AP sites (Fig. 1). The relative yields of single APsites were less than 10 % of the total damage produced at ascavenger capacity mimicking that in cells (3×108 /s). The
dependence of the yield of AP sites on scavenging capacity wassimilar to that of SSBs. The ratios of the yield of isolated AP
sites to that of SSBs induced by carbon ion or X-ray irradiation
were relatively constant over the tested range of scavenger
capacity. These results indicate that the reaction of waterradiolysis products, presumably OH radicals, with the sugarphosphate moieties in the DNA backbone induces both AP sites
and SSB with a constant ratio.

Induction of chromosomal instability by non-DSB damage
When base lesions, AP sites or SSBs are proximately arising
within two DNA helical turns, this site has been called asclustered damage known to be one of the most harmful damage
in a cell. The clustered damage sites are categorized into twotypes, namely DSB and non-DSB type. There have been little
evidences how the non-DSB clustered damage is involved in theinduction of trans-generational effects so far. To elucidate therole of non-DSB damage, particularly composed of base lesions,in the induction of trans-generational effect, we investigatedinstability of human chromosome with base lesions transferred
into non-damaged mouse cells [3].
A human chromosome exposed to ultra-violet-A (UV-A) lightwhich efficiently induces base lesions such as oxidative guanine
was transferred into un-irradiated mouse recipient cells by
microcell fusion. We examined the frequency of chromosome
aberration induced in the transferred human chromosome using
a whole chromosome painting fluorescence in situ hybridization(WCP-FISH) method (Fig. 2). The human and mouse
chromosome were stained in red and in blue, respectively. The
transfer of un-irradiated human chromosome did not cause anyeffects for both the transferred and recipient mousechromosomes (Fig. 2c and 2d). In contrast, the transfer of theUV-A irradiated human chromosome enhanced the number ofchromosome aberration (Fig. 2b), and chromosome aberrations
were observed not only in the human chromosome but also in
unirradiated mouse chromosomes (Fig. 2e and 2f). These results
suggest that non-DSB damage could induce chromosomal
instability even in the un-irradiated recipient mouse cells.
b; 4000 kJ/m2a; 0 kJ/m2-8

Yield of SSB or base lesions(/Gy/Da)

10SSB
Pyrimidine base lesions-9c10defPurine base lesions
Normal Chromosome

AP sites-10106107108101091010

Scavenger capacity(/s)
Fig. 1 Dependence of the yield of prompt SSBs or single baselesions on scavenging capacity in the plasmid DNA
solution irradiated with C6+ ions (13 keV/m) at 5.6 °C (●:prompt SSB) or after a post-irradiation incubation for 30min at 37 °C in the presence of Nth (▲: pyrimidine baselesions), Fpg (◆ : purine base lesions), or Nfo (■: APsites). The dotted lines indicate previously reported data
[2] shown for comparison.

Chromosome aberration

Fig. 2 Mouse chromosomes with un-irradiated human chromosome (a).
Normal mouse (c) and human (d) are indicated. Mouse
chromosomes with UV-A irradiated (4000 kJ/m2) humanchromosome (b). Delayed chromosome aberrations wereobserved such as rejoining of the irradiated human and a mouse
chromosome (e), or rejoining of the two human chromosomes(f).

References[1] T. Shiina et al., Radiat. Environ. Biophys. 52, 99 (2013).
[2] T. Ushigome et al., Radiat. Res. 177, 614 (2012).
[3] A. Urushibara et al., Mut. Res., submitted.