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3-01
Damage Spectrum of DNA Strand Break Termini
Induced by 4 He 2+ Ion Beam Compared with that
by 60 Co -rays
K. Akamatsu and K. Saito
Division of Environment and Radiation Sciences, NSED, JAEA
1. Introduction
Chemical structures of DNA lesions and their
distribution are quite important to clarify following
biological repair procedures. In case of ionizing radiations,
especially, it is expected that three-dimensional distribution
of DNA damages is diversified as well as their chemical
structures, and the diversity makes investigations of
physicochemical processes in radiation effects difficult. In
fact, double-strand breaks produced by high linear energy
transfer (LET) alpha particles are more difficult to rejoin in
living cells than those produced by low LET 137 1)
Cs -rays .
So far, we have investigated difference of DNA damage
spectrum between radiations using existing and/or newly
developed analytical method 2) . Here, we reveal the results
of strand break termini induced by 4 He 2+ (He) ion beam in
comparison with that by 60 Co -rays (7cell, Takasaki). The
ion beam used was obtained from TC in TIARA.
2. Experiments
Sample preparation and irradiation
Linear formed pUC19 plasmid DNA digested by Sma I,
(pUC19/Sma I) was used. pUC19/Sma I aqueous solution
was mounted on a silicon plate and dried thoroughly in
vacuum under P 2O 5. For -irradiation, the dried DNA
sample on a plate was put into a glass tube sealed in vacuum
to achieve secondary electron equilibrium. Each sample
was irradiated at a dose (dose rate) of 40 (0.333), 100
(0.833), 400 (3.33), 900 (7.50), and 1,800 kGy (15.0 kGy/h)
at r.t., respectively. For He ion beam, the DNA- mounted
plate was fixed in a cylindrical chamber (39 mm, height:
22 mm) with a Kapton ® window (8 m) under argon gas.
Each sample was irradiated with ~ 67 keV/m of the beam,
which was controlled using a depth-tunable cell irradiation
equipment, was irradiated at doses of 50, 100, 200, 400, and
Fig. 1 Protocol for quantification of DNA strand
break termini with phosphate.
JAEA-Review 2010-065
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Table 1 Spectra of strand break termini in DNA
irradiated with 4 He 2+ ion beam and 60 Co -rays.
900 kGy at r.t.. The irradiated DNA samples were
recovered by water at 0 °C. The aqueous solutions were
lyophilized and kept at -20 °C until use.
Analytical methods
To obtain strand break terminal spectra, SVPD method 2)
and terminal phosphate analysis method 3) were used. The
latter protocol was illustrated in Fig. 1. This method is
bases on a known quantification technique of free phosphate
using colorimetry of malachite green. As CIAP can
hydrolyze terminal phosphate groups both at 3’ and 5’ ends,
all strand break termini with phosphate can be quantified by
the method in principle.
3. Results and Discussion
The results of these analyses of DNA irradiated with He
ion beam and 60 Co -rays are summarized in Table 1 (3’X
means 3’termini neither with OH nor phosphate). At a
glance, the yields of all categorized termini for He ion beam
were around 2-3 times larger than those for -rays except for
the yields for 3’OH. This result suggests that high-LET
radiation such as He ion beam is likely to decompose sugar
moieties in DNA compared with low-LET one at least in
case of the direct radiation effects.
4. Acknowledgments
We would like to gratefully thank Dr. Y. Sugo for
operating the TC line, and also thank Drs. M. Kikuchi and
Y. Kobayashi for -ray irradiation experiments.
References
1) C. M. deLala et al., Radiat. Res. 144 (1995) 43.
2) K. Akamatsu, Anal. Biochem. 362 (2007) 22.
3) K. Akamatsu, Radiat. Chem. 89 (2010) 3.