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A Quantitative Study of DNA Double-strand Breaks
Induced by Heavy-ion Beams: a Problem on the
Conventional DNA-sample Preparation
Y. Yokota, T. Funayama, Y. Mutou, T. Sakashita, M. Suzuki, M. Kikuchi and Y. Kobayashi
Linear energy transfer (LET) is an important factor of
radiation quality. Biological effects (e.g. chromosomal
aberration and cell death) per absorbed dose are greater in
high-LET heavy ions than in low-LET radiation such as
1)
X-rays and -rays . It is a general consensus of the
underlying mechanism that heavy ions induce irreparable
DNA damage in irradiated cells more frequently than
low-LET radiation. In computer simulation studies, the
interaction between the high-density ionization along a
particle track of heavy ions and higher-order chromosomal
structure is predicted to induce clustered DNA damage in
irradiated cells.
DNA double-strand breaks (DSBs) are believed to be the
most serious DNA damage induced in irradiated cells
because DSBs make a chromosome into fragments and
destroy the genome information of organisms. Since 1990s,
DSBs have been quantified with pulsed-field gel
electrophoresis (PFGE) technology. PFGE can separate
DNA fragments with sizes up to several mega base pairs
(bp) by changing the direction of electric field periodically 2) .
In conventional studies, genome DNA preparation from
irradiated cells was performed in agarose plugs for avoiding
excess DNA fragmentation during experimental
procedures 3) .
The length and the number of radiation-induced DNA
fragments are measured to determine the space of
neighboring DSBs on a chromosome and the number of
induced DSBs, respectively. However, this DNA fragment
analysis could not measure the DNA fragments shorter than
4)
10 kbp (similar to gene sizes) correctly . So,
heavy-ion-induced DNA fragmentation in irradiated cells is
not totally understood. The purpose of our study is to
quantify the DNA fragments shorter than 10 kbp in the cells
irradiated with heavy ions.
In the last year, we have found that DNA fragments are
partly lost from the agarose plug into surrounding buffer
during the conventional DNA preparation procedure (Fig. 1).
In our experiment, DNA molecular size standards (mixture
of 500 bp and 5 kbp ladders) were used as a substitute of
genome DNA. They were suspended in 0.75% high-purity
agarose and 1.0 × 0.5 × 0.15 mm3 agarose plugs were casted
with a plug mold. Then, agarose plugs were incubated in
0.5 M EDTA for 24 h and in 0.5 × TBE buffer for 2 h before
PFGE assay (a mock procedure of the conventional DNA
preparation). After PFGE, electrophoresis gel was
incubated in SYBR Green I solution to stain DNA with
fluorescent dye. DNA band pattern was visualized on a
UV transilluminator (Fig. 1A). Gel image was recorded
JAEA-Review 2010-065
Radiation-Applied Biology Division, QuBS, JAEA
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with a cooled CCD video camera. DNA content of each
band was measured based on the fluorescence intensity and
normalized to that of the agarose plug (Fig. 1B). The
smaller the bands compared between both lanes were, the
larger the difference in normalized DNA content became.
This means that the loss of DNA fragments from the agarose
plug is dependent on band sizes. This phenomenon makes
difficult to reveal the total number of DNA fragments in
irradiated cells.
In future, we will develop the new method for collecting
DNA fragments totally from irradiated cells to characterize
heavy-ion-induced DSBs. An expectant approach is to lyse
irradiated cells in a test tube and capture the various sizes of
DNA fragments with an anion-exchange resin.
References
1) Y. Yokota et al., Int. J. Radiat. Biol. 79 (2003) 681-685.
2) D. C. Schwartz et al., Cell 37 (1984) 67-75.
3) Y. Yokota et al., Radiat. Res. 163 (2005) 520-525.
4) Y. Yokota et al., Radiat. Res. 167 (2007) 94-101.
Fig. 1 Loss of DNA fragments from agarose plugs is
dependent on size. (A) DNA molecular size
standards are embedded in agarose plugs and run in
PFGE. Left lane: no treatment, right lane: the plug
was incubated in 0.5 M EDTA buffer for 24 h and in
0.5 × TBE buffer for 2 h (a mock procedure of the
conventional DNA preparation) before PFGE. (B)
DNA content of each band was measured and
normalized to that of the plug.