JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

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Molecular Analysis of Carbon Ion Induced Mutations in
Yeast Saccharomyces cerevisiae Cells
K. Shimizu a) , Y. Matuo a, b) , Y. Izumi b) , Y. Hase c) , S. Nozawa c) ,
A. N. Sakamoto c) and I. Narumi c)
a) Radioisotope Research Center, Osaka University, b) Research Institute of Nuclear Engineering,
University of Fukui, c) Radiation-Applied Biology Division, QuBS, JAEA
1. Introduction
This study is intended to elucidate the molecular
mechanism of the mutagenesis caused by ion beam
irradiation. Ion beam irradiation is expected to increase
mutation frequency and spectrum, since it has a high linear
energy transfer (LET). However, the detailed molecular
mechanism of its action has not been proven.
Recently, we reported that the main mutations induced by
high-LET carbon-ion irradiation were GC to TA
transversions 1) . DNA adducts such as 8-oxodG, which are
produced by interactions of reactive oxygen species (ROS)
with these molecules, can create various pairing formations
in DNA molecules, for example, pairing with dA in the syn
conformation to produce a GC to TA mutation 2) . In this
study, we used the yeast mutant strains ogg1 and msh2,
which are deficient in mismatch repair mechanisms.
Mutations involving 8-oxodGs were caused by ion beam
irradiation in the ogg1 and msh2 mutant strains.
2. Materials and methods
2.1. Yeast strains
S. cerevisiae haploid strains used in this study were S288c
(MATα, RAD), rad52 (MATα, G160/2b), ogg1 (MATα,
OGG1) and msh2 (MATα, msh2).
2.2. Irradiation methods
To determine the biological effectiveness of high LET
ion beam irradiation, yeast cells were exposed to heavy ions
accelerated at the AVF cyclotron (TIARA; JAEA Takasaki,
Takasaki, Japan).
As for the physical properties of the carbon ions, the
JAEA-Review 2010-065
mean LET in the yeast was estimated to be 107 keV/μm
water equivalent. This LET value was calculated by means
of the ELOSS code program, which uses the elementary
composition and the density of the target to calculate the
energy loss of ion particles.
3. Results and Discussion
One of the most common consequences of ROS exposure
is the incorporation of oxidized nucleotides such as 8-oxodG
into the genome. Mismatch repair has been shown to
repair oxidative damage by lowering the level of 8-oxodG
that is incorporated into the genome, presumably by
recognizing mismatches different from those recognized by
base excision repair.
The distribution of the mutations induced by carbon ion
beam irradiation is shown in Fig. 1. In the ogg1 mutant
strain, there were minor hot spots at positions 330, 515, 602,
and 745 (Fig. 1 (C)). The mutations in msh2 mutant were
distributed evenly for base substitution, except for a minor
hot spot at position 345. These results suggest that the
incorporation of damaged nucleotides was not uniform.
In comparison with the surrounding sequence context of
mutational base sites, the C residues in the 5’- (A/T) C (A/T)
-3’ sequence were found to be easily mutated (data not
shown). These observations are in agreement with results
obtained in wild type cells.
References
1) Y. Matuo et al., Mutat. Res. 602 (2006) 7.
2) M. Inoue et al., J. Biol. Chem. 273 (1998) 11069.
Fig. 1 The mutation spectra induced by carbon ion beam irradiation of yeast. White triangles: base substitute
mutations, inverted black triangles: deletion/insertions. (A) nucleotide position number of URA3 gene; (B)
mutation spectrum of wild type; (C) ogg1; (D) msh2.
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