Hatano Y., Katsumura Y., Mozumder A. (Eds.) Charged Particle and Photon Interactions with Matter - Recent Advances, Applications, and Interfaces
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540 Charged Particle and Photon Interactions with Matter
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543
20
Spectroscopic Study
of
Radiation-Induced
DNA
Lesions and
Their
Susceptibility
toEnzymatic
Repair
Akinari Yokoya
Japan Atomic Energy Agency
Tokai,
Japan
Kentaro Fujii
Japan Atomic Energy Agency
Tokai,
Japan
Naoya Shikazono
Japan Atomic Energy Agency
Tokai,
Japan
Masatoshi Ukai
Tokyo University of Agriculture and Technology
Tokyo,
Japan
Contents
20.1 Introduction ..........................................................................................................................544
20.2 Direct and Indirect Effects on Induction of DNA Strand Breaks
andNucleobaseLesions...............................................................................................545
20.3 Clustered
DNA Damage and Its Susceptibility to Enzymatic Repair.................................. 547
20.4
Biological
Relevance of Clustered Damage ......................................................................... 552
20.5
Intermediate Species and Degradation Processes of DNA Revealed
bySynchrotronSoft
X-Rays as Probes................................................................................. 555
20.6
Role
of Water Molecules Surrounding DNA and Photoelectron Spectroscopy
forDNASolutions ................................................................................................................565
20.7 Summary .............................................................................................................................. 569
References...................................................................................................................................... 570
544 Charged Particle and Photon Interactions with Matter
20.1 introduCtion
Ionizing radiation induces chemically stable molecular changes in cellular DNA (DNA damage).
The biological effects of ionizing radiation, such as cell killing, induction of mutations, chromo-
some aberrations, transformation, and the adaptive responses of irradiated cells, are thought to arise
from the formation of such DNA damage. However, it has been gradually recognized that living
cells can effectively repair DNA damage through several enzymatic pathways. DNA double-strand
breaks (DSBs), for instance, are thought to be one of the main types of DNA damage that induce
the cell-killing effect. Most DSBs (80%–90%) initially produced in a cell can be rejoined even in
cell extracts (de Lara etal., 1995), but the boundary line that divides repairable and unrepairable
DNA damage has not yet been claried. A long-standing and important question remains as to
which kinds of DNA damage slip through the defenses of the enzymatic repair system in a living
cell, ultimately causing deleterious radiation effects. It has been suggested that the susceptibility
of DNA damage to repair strongly depends on the track structure of the radiation that induced the
damage, represented by a linear energy transfer (LET) value. LET is dened as the ratio of dE to dl
(dE/dl), where dE is the mean energy and dl is the distance traversed by the charged particle (ICRU,
1970). Monte Carlo track simulation studies have predicted that radiation with higher LET has a
greater tendency to produce clusters of isolated DNA lesions by dense ionization/excitation within
a distance of a few nanometers than radiation with lower LET (Nikjoo etal., 1994, 1999). These
clustered sites of damage can be categorized into two groups: DSB and non-DSB types of damage.
DSB damage consists of two or more single-strand breaks (SSBs) produced within about six base
pairs’ separation in DNA (Hanai et al., 1998). Non-DSB damage, on the other hand, comprising two
or more lesions, including nucleobase lesions, abasic sites (AP sites), and SSBs formed within about
10 base pairs’ separation by a single radiation track, has been proposed to be more biologically rel-
evant (Ward, 1988; Goodhead, 1994a). It is becoming clear that clustered sites of DNA damage are
less readily repaired than isolated nucleobase lesions, SSBs, or AP sites and, therefore, may induce
serious genetic changes in cells. In order to understand the initial processes of induction of DNA
damage, including the site of clustering of isolated lesions, and the susceptibility of this damage to
repair, spectroscopic techniques combined with biochemical assays have been used. In Chapter 19,
the molecular mechanisms of radiation damage to DNA are extensively presented in terms of their
physicochemical aspects. In this chapter, recent studies aiming at clarifying the nature of DNA dam-
age in terms of its biological reparability, particularly by base-excision repair (BER) proteins, are
highlighted. New approaches using spectroscopic techniques combined with a brilliant synchrotron
radiation source to reveal the role of the photoelectric effect on DNA induction are also introduced.
20.2 direCt and indireCt eFFeCts on induCtion
o
Fdnas
trand
b
reaks
and n
uCleobase
l
esions
DNA damage is thought to be induced by both direct energy deposition on DNA (direct effect)
(see review by Bernhard and Close, 2003) and reactions with diffusible water radicals (indirect
effect) (see review by O’Neill and Fielden, 1993). Most mechanistic studies to date (von Sonntag,
1987; O’Neill, 2001) have focused on the indirect effects using dilute, aqueous solutions contain-
ing DNA. The results indicated that the hydroxyl radical (OH
•
) is the main water radiolysis species
that induces SSBs and DSBs in DNA, whereas hydrated electrons, H atoms, and hydroxyl radicals
induce DNA nucleobase lesions. Experimental (de Lara etal., 1995) and theoretical (Nikjoo etal.,
2002) studies have indicated that in living cells or under highly scavenging conditions similar to
those involved in OH
•
scavenging in the cell, ∼40% of the lesions induced in DNA by low-LET
radiation could be ascribed to direct effects, increasing to ∼70% for high-LET α-particles. In the
pioneering work by Krisch etal. (1991), the yields of SSBs and DSBs by both direct and indirect
effects were determined using SV40 DNA-irradiated γ-rays under conditions of greatly varying rad-
ical scavenger concentration. They also concluded that in high scavenging conditions, DSBs from
Spectroscopic Study of Radiation-Induced DNA Lesions 545
indirect effects are produced predominantly by local clusters of OH
•
from single energy deposition
events. The experimentally obtained ratios between direct and indirect effects examined by various
biological
end points have been summarized by Becker and Sevilla (1993).
When
comparing the yield of DNA damage among various irradiation experiments, one should
focus on both the track structure of radiation and the scavenging capacity of the sample. In addition
to the SSB and DSB yields, heat-labile site and base lesions also contribute to total damage yields. In
order to gure out the nature of various radiations that induce DNA damage, a few studies have been
performed using the same DNA sample and the same scavenger condition as those used in previous
studies. The yield of DNA damage induced by x-rays (150kVp) has been measured using closed
circular plasmid (pUC18) DNA (Yokoya etal., 2006) to compare the yield induced by lower-LET
γ-rays or higher-LET Al
K
soft x-rays (1.8keV) (Fulford etal., 2000, 2001). The common experi-
mental method for detecting DNA damage using plasmid DNA is shown in Figure 20.1. Several
DNA solutions with three kinds of radical scavenger capacities and fully hydrated DNA samples
were irradiated to determine the indirect contribution by the reaction of diffusible water radicals,
such as OH
•
, with the direct action of secondary electrons. The yield of prompt SSBs summarized
in Table 20.1 decreased with increasing levels of scavenging capacity (Figure 20.2), indicating that
the fraction of damage attributable to direct effects under these cell-mimetic conditions was about
30%. Heat-labile sites were hardly detected under the two conditions of higher scavenging capacity.
The yields of oxidative nucleobase lesions revealed by the treatments of Nth or Fpg proteins (experi-
mental details are described below) are summarized in Table 20.2. The yields of nucleobase lesions
decreased with increasing levels of scavenging capacity (Figure 20.3), although a large fraction of
direct effects was obtained under the cell-mimetic conditions: 60% for treatment with Nth and 40%
for
treatment with Fpg.
In
order to know how the damage yields depend on the radiation track structure, the data is
compared with those obtained by irradiation with lower-LET γ-rays and higher-LET soft x-rays
(1.8keV) and shown in Figure 20.2. The three lines have different slopes and intersect around the
cell-mimetic conditions. The extrapolated values of the 150kVp soft x-ray data under the condi-
tion of highest scavenging capacity (hydration condition) are larger than those for the other two
Radiation
Closed circular
plasmid DNA
(pUC18)
Prompt SSB
Prompt DSB
Open circular
Linear
+ Base-excision
repair enzyme
37°C, 30 min
Enzymatically
induced SSB
Enzymatically induced
DSB=clustered damage
B*
ssb
+ Enzyme
Nth (Endo III)
AP site, DHT, thymine glycol
Fpg
AP site, 8-oxoG, Fapy-guanine
ssb
B*
DNA
B* : Oxidative base lesion
Base lesion cluster
Figure 20.1 Schematic illustration of the experimental method for detection of DNA damage using BER
proteins
and closed circular plasmid DNA as the substrate of the enzymes.
546 Charged Particle and Photon Interactions with Matter
irradiations, and the data points for the hydrated sample show the expected SSB yields. The SSB
yields
demonstrate a rather complicated relationship:
SSB
γ-rays
> SSB
150kVp
> SSB
1.8keV
under dilute conditions
SSB
γ-rays
∼ SSB
150kVp
> SSB
1.8keV
under cell-mimetic conditions
SSB
γ-rays
> SSB
150kVp
∼ SSB
1.8keV
under conditions in which the direct effect is dominant
10
–8
10
–9
10
–10
10
–7
10
5
SSB (Gy/Da)
10
6
10
7
10
8
10
9
10
10
10
11
Scavenging capacity(s
–1
)
γ-rays, Fulford(2000)
Al-K x-rays(1.8 keV),
Fulford et al. (2000)
γ-rays, Yokoya et al. (2002)
Cell-mimetic condition
70%
30%
Soft x-rays(150 kVp),
Yokoya et al.(2006)
Figure 20.2 Dependence of the yield of prompt SSBs induced in pUC18 plasmid DNA by 150 kVp soft x-ray
irradiation on the scavenging capacity at 277K under an aerobic condition. The data points represented as closed
circles are cited from our previous report (Yokoya etal., 2006). Previous data for γ-irradiation (Fulford, 2000) and
Al-K soft x-ray (1.8 keV) irradiation (Fulford etal., 2001) are shown by dotted lines and are compared with our data
for 150kVp soft x-ray irradiation (closed circles). The yield of a hydrated pUC18 sample irradiated with γ-rays at
277K under an aerobic condition is shown in open circles for comparison, assuming that the scavenging capacity
of the hydrated sample was 1 × 10
10
s
−1
.
table 20.1
yields
of s
trand
b
reaks
i
nduced
in p
uC18
p
lasmid
dna i
rradiated
with
s
oft
x
-rays
under v
arious
s
cavenging
Conditions
tris Concentration
(m
m)
a
scavenging
Capacity
(s
−1
) ssb (gy/da) dsb (gy/da) ssb/dsb
0.2 3
× 10
5
2.28 × 10
−8
ND —
2.5 4
× 10
6
5.02 × 10
−9
3.04 × 10
−10
17
50 7
× 10
7
9.61 × 10
−10
6.61 × 10
−11
19
Hydrated
b
— 1.48 × 10
−10
1.82 × 10
−11
8
SSB, single-strand
breaks; DSB, double-strand breaks.
a
Concentrations of other solutes in the sample solution: 50 μg/mL pUC18 DNA;
1 mM EDTA (Yokoya etal., 2006).
b
Fully hydrated samples were prepared by exposing dry pUC18 DNA lm to a condition
of 97% relative humidity over 16h. The hydration level was estimated to be 35 water
molecules
per nucleotide (Yokoya etal., 2002).
Spectroscopic Study of Radiation-Induced DNA Lesions 547
This should be kept in mind when the yield of DNA damage is compared with that induced by other
radiations, because the yield strongly depends on the radical scavenging conditions during irradiation.
20.3 Clustered dna damage and itssusCeptibility
to
e
nzymatiC r
epair
The fact that additional DSBs are generated after exposure of cells to ionizing radiation has been
known for over 30 years (Dugle etal., 1976; Ahnstrom and Bryant, 1982), and this is now considered a
strong piece of evidence supporting the in vivo processing of clustered damage. Blaisdell and Wallace
table 20.2
yields
of h
eat-labile
s
ites
and n
ucleobase
l
esions
v
isualized
by
e
nzymatic
t
reatment
under v
arious
s
cavenging
Conditions
tris Concentration (mm)
a
heat-labile sites nth-sensitive sites Fpg-sensitive sites
0.2 1.03
× 10
−8
2.31 × 10
−8
2.99 × 10
−8
2.5 4.80 × 10
−10
4.11 × 10
−9
3.69 × 10
−9
50 ND 8.33 × 10
−10
8.13 × 10
−10
Hydrated
b
ND 5.19 × 10
−10
3.16 × 10
−10
a
Concentrations of other solutes in the sample solution: 50 μg/mL pUC18 DNA; 1 mM EDTA
(Yokoya
etal., 2006).
b
Fully hydrated samples were prepared by exposing dry pUC18 DNA lm to a condition of 97%
relative humidity over 16 h. The hydration level was estimated to be 35 water molecules per
nucleotide
(Yokoya etal., 2002).
10
–10
10
–9
10
–8
10
–7
10
11
10
10
10
9
10
8
10
7
10
6
10
5
Scavenging capacity(s
–1
)
Yield of damage (Gy/Da)
Cell-mimetic condition
Heat-labile site
Nth-sensitive site
Fpg-sensitive site
Figure 20.3 Dependence of the yields of heat-labile and enzyme-sensitive sites in pUC18 plasmid DNA
induced by soft x-ray irradiation on the scavenging capacity at 277K under an aerobic condition. (
⬛
) Heat-
labile sites; (
△
) Nth-sensitive sites; (▽) Fpg-sensitive sites. (Data from Yokoya, A. etal., Radiat. Prot. Dosim.,
122, 86, 2006.)
548 Charged Particle and Photon Interactions with Matter
(2001) were the rst to demonstrate that in Escherichia coli cells, the level of additional DSBs gener-
ated after irradiation was dependent on the amount of glycosylase (Fpg) and, thus, was related to the
amount of clustered damage. Yang etal. further demonstrated that the induction of DSBs, mutations,
and lethality all strongly correlated with the expression levels of glycosylases (hOGG1 and hNTH1)
after exposure of human B lymphoblastoid cells to 3Gy of γ-rays (Yang etal., 2004, 2006). It is impor-
tant to note that in these studies, substantial amounts of additional DSBs were observed at high doses
(∼500Gy) or at high levels of glycosylases. On the other hand, at sublethal doses, only a fraction (∼10%)
of the clustered DNA damage sites was converted to DSBs in nonhomologous end-joining (NHEJ)-
decient CHO xrs-5 cells after irradiation with γ-rays (Gulston etal., 2004), and DSB-repair-procient
Chinese hamster V79 cells and human hematopoietic cells did not generate additional DSBs after
postirradiation by γ-rays (Georgakilas etal., 2004; Gulston etal., 2004). Collectively, these results sug-
gest that (1) an abortive enzymatic repair of clusters predominates under high doses or high levels of
glycosylases in vivo and (2) at sublethal doses, cells have mechanisms to avoid the formation of DSBs
or to quickly rejoin de novo DSBs through NHEJ repair and alternative pathways. It is important to
note that enzymatic processing of clustered lesions has not been observed after irradiation with high-
LET radiation. In the case of wild-type Chinese hamster V79 cells, the amount of DSBs remained
essentially constant after α-irradiation (Gulston etal., 2004).
Recently, BER proteins have been widely used as enzymatic probes to detect the nucleobase
lesions induced by various radiation sources (Melvin etal., 1998; Prise etal., 1999; Milligan etal.,
2000; Sutherland etal., 2000). Endonuclease III (Nth) and formamidopyrimidine-DNA glycosyl-
ase (Fpg) are typical enzymes used for these studies. The Nth protein excises mainly pyrimidine
base lesions including ring-saturated pyrimidines, such as 5,6-dihydrothymine (DHT), thymine
glycol, and AP sites (Demple and Linn, 1980; Breimer and Lindahl, 1984; Dizdaroglu etal., 1993;
Hatahet etal., 1994). The Fpg protein, on the other hand, excises mainly purine base lesions, such
as 2,6-diamino-4-hydroxyl-5-N-methylformamidopyrimidine, 7,8-dihydro-8-oxo-2′deoxyguanine
(8-oxoG), and AP sites (Chetsanga and Lindahl, 1979; Tchou etal., 1991; Boiteux etal., 1992). These
enzymatic probes convert nucleobase lesions into readily detectable SSBs. Using this method, it was
revealed that nucleobase lesions were produced considerably more frequently by irradiation than
strand breaks, that is, the yield of total nucleobase lesions induced in dry DNA lms was about
threefold
larger than that of SSBs (Yokoya etal., 2002).
The
yields of the nucleobase lesions induced in a diluted solution showed similar values for Nth-
and Fpg-treatment (Yokoya etal., 2008a, Figure 20.3). On the other hand, these nucleobase lesions
were induced with different efciencies in the hydrated sample (see Table 20.3). These results indi-
cate that the direct energy deposition of soft x-ray irradiation induced damage in the plasmid DNA
through
different mechanisms from that induced by reaction with water radicals.
The
yields of clustered damage, revealed as the yields of the additional DSBs induced by Nth- and
Fpg-treatment, were similar to those for spontaneous DSBs under conditions of lower scavenging
capacity, although they were signicantly larger than those under conditions of higher scavenging
capacity (Figure 20.4). At the highest scavenging capacity (fully hydrated sample), these yields
were nearly twice of those for DSBs (see Table 20.3). These results show that direct effects might
efciently induce oxidative nucleobase lesions and clustered damage sites (including nucleobase
lesions) rather than DSBs. A theoretical study has also predicted that most SSBs are induced con-
comitantly with nucleobase lesions (Nikjoo etal., 2001). The hydrating water shell surrounding a
DNA molecule helps in inducing oxidative nucleobase lesions, but does not play a signicant role in
inducing SSBs (Yokoya etal., 2002). Thus, direct energy deposited onto the hydration shell might
be transferred to DNA, inducing a large amount of nucleobase lesions as well as base lesion clusters.
In order to elucidate the precursors of DNA damage, a low-temperature electron paramagnetic
resonance (EPR) method has been applied to crystalline or dry samples of DNA and its components
to investigate free radicals induced by irradiation. These studies have shown that base and sugar radi-
cals were induced by electron trapping (radical anions) or hole transfer (radical cations) (see review by
Bernhard and Close, 2004); however, much less is known about the mechanisms of the direct effect,
Spectroscopic Study of Radiation-Induced DNA Lesions 549
table 20.3
yields
of dsb
s
and e
nzymatically
v
isualized
Clustered
d
amageunder
v
arious
s
cavenging
Conditions
tris Concentration (mm)
a
prompt
dsb
s
prompt dsbs +
h
eat-labile dsbs
nth-induced
dsb
s
Fpg-
induced
dsb
s
0.2 ND 3.33
× 10
−9
1.77 × 10
−9
2.99 × 10
−9
2.5 3.04 × 10
−10
2.32 × 10
−10
2.22 × 10
−10
2.41 × 10
−10
50 6.61 × 10
−11
3.15 × 10
−11
6.47 × 10
−11
1.09 × 10
−10
Hydrated
b
1.82 × 10
−11
1.60 × 10
−11
3.42 × 10
−11
3.29 × 10
−11
DSB, double-strand breaks.
a
Concentrations of other solutes in the sample solution: 50μg/mL pUC18 DNA; 1mM EDTA
(Yokoya
etal., 2006).
b
Fully hydrated samples were prepared by exposing dry pUC18 DNA lm to a condition of 97%
relative humidity over 16 h. The hydration level was estimated to be 35 water molecules per nucleo-
tide
(Yokoya etal., 2002).
10
–11
10
–10
10
–9
10
–8
2
3
4
5
6
7
2
3
4
5
6
7
2
3
4
5
6
7
10
11
10
10
10
9
10
8
10
7
10
6
10
5
Scavenging capacity(s
–1
)
DSB and enzymatically induced DSB (Gy/Da)
Cell-mimetic condition
Prompt DSBs
Prompt+ heat-labile DSBs
Nth-induced DSB
Fpg-induced DSB
Figure 20.4 Dependence of the yields of DSBs and enzymatically induced DSBs in pUC18 plasmid DNA
irradiated by soft x-ray on the scavenging capacity at 277K under an aerobic condition. (⚫) Prompt DSBs; (○)
Prompt + heat-labile DSBs; (△) Nth-induced DSBs; (▽) Fpg-induced DSBs. (Reproduced from Yokoya, A.
etal.,
Radiat. Phys. Chem., 77, 1280, 2008b. With permission.)