Review
J. Radiat. Res., 50, 19–26 (2009)
Generation, Biological Consequences and Repair Mechanisms of
Cytosine Deamination in DNA#
Shin-Ichiro YONEKURA, Nobuya NAKAMURA, Shuji YONEI
and Qiu-Mei ZHANG-AKIYAMA*
Deamination of Cytosine/Uracil/Transition Mutations/Base Excision Repair/Uracil-DNA Glycosylase/
dUTPase.
Base moieties in DNA are spontaneously threatened by naturally occurring chemical reactions such
as deamination, hydrolysis and oxidation. These DNA modifications have been considered to be major
causes of cell death, mutations and cancer induction in organisms. Organisms have developed the DNA
base excision repair pathway as a defense mechanism to protect them from these threats. DNA glycosylases, the key enzyme in the base excision repair pathway, are highly conserved in evolution. Uracil
constantly occurs in DNA. Uracil in DNA arises by spontaneous deamination of cytosine to generate promutagenic U:G mispairs. Uracil in DNA is also produced by the incorporation of dUMP during DNA
replication. Uracil-DNA glycosylase (UNG) acts as a major repair enzyme that protects DNA from the
deleterious consequences of uracil. The first UNG activity was discovered in E. coli in 1974. This was
also the first discovery of base excision repair. The sequence encoded by the ung gene demonstrates that
the E. coli UNG is highly conserved in viruses, bacteria, archaea, yeast, mice and humans. In this review,
we will focus on central and recent findings on the generation, biological consequences and repair
mechanisms of uracil in DNA and on the biological significance of uracil-DNA glycosylase.
GENERATION, BIOLOGICAL CONSEQUENCES
OF CYTOSINE DEAMINATION
DNA reacts continually with H2O (hydrolysis) and reactive
oxygen species, resulting in multiple spontaneous DNA
modifications.1–6) Four bases normally present in DNA contain
exocyclic amino groups. The loss of these amino groups
(deamination) in cytosine, adenine, guanine and 5methylcytosine occurs spontaneously and results in the
conversion to uracil, hypoxanthine, xanthine and thymine,
respectively.3,4,6) The deamination of cytosine (Fig. 1) is
estimated to result in 100–500 uracil residues per mammalian
genome per day.1,4–6) Cytosine deamination in single-stranded
DNA is reported to be 200–300 times faster than that in doublestranded DNA.1,3–6) Single-stranded regions in replication
forks and transcription bubbles are not protected from deam*Corresponding author: Phone: +075-753-4097,
Fax: +075-753-4087,
E-mail: [email protected]
Department of Biological Sciences, Graduate School of Science, Kyoto
University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
#
Foot-note: Translated and modified from Radiat. Biol. Res. Comm. Vol.
42; 132–146 (2007, in Japanese).
doi:10.1269/jrr.08080
ination by the complementary strand.1,4) Pro-mutagenic G:U
mispairs cause G:C to A:T transition mutations, if not repaired
prior to the next round of DNA replication.1,3–5,7) Deamination
of cytosine in cyclobutane dimers is markedly faster than that
of cytosine in other sequences,1,4,6) and has been implicated in
UV-mutagenesis in both bacteria and mammalian cells.1,8,9)
Uracil also occurs in DNA through incorporation of
dUMP instead of dTMP by DNA polymerases during replication.1,3–5,10,11) Incorporated dUMP forms U:A base pairs that
are not directly mutagenic, but may be cytotoxic.1,3–5,12)
Substitution of thymine with uracil is a major source of
endogenous abasic (AP) sites,12) which may mediate the cytotoxicity of uracil in DNA. dUTP is a normally occurring inter-
Fig. 1. Deamination of the 4 position of cytosine generates uracil.
J. Radiat. Res., Vol. 50, No. 1 (2009); http://jrr.jstage.jst.go.jp
20
S.-I. Yonekura et al.
mediate in nucleotide metabolism, but its level is kept low by
an efficient dUTPase (DUT). The pyrophosphatase activity of
dUTPase hydrolyzes dUTP to dUMP and PPi, which prevents
the incorporation of dUMP into DNA.1,3–5) Uracil incorporation into DNA is presumed to occur on an average of one per
2,000–3,000 nucleotides in the dut mutant deficient in
dUTPase. Tye et al. (1978) reported that E. coli mutants defective in both the dut and ung genes accumulate more uracil in
their DNA.10) Lari et al. (2006) determined the steady-state
levels of uracil residues in DNA using the Ung-ARP assay.13)
The uracil levels are 3,000–8,000/106 nucleotides in the ung
dut mutant of E. coli, in contract to about 1/106 nucleotides in
the wild-type strain (ung+ dut+).12,13)
The frequency of spontaneous G:C to A:T transitions
significantly increases in E. coli and S. cerevisiae ung
mutants deficient in UNG.1,3–5,14–16) Increased spontaneous
mutations in the ung mutants were also observed using λ
bacteriophage.16) When λ phage is treated with bisulfite, an
agent known to cause cytosine deamination in DNA, the
frequency of clear-plaque mutations is increased an
additional 20-fold when the phage is grown on E. coli ung
mutant rather than on ung+ cells.16) On the other hand, the
frequency of bisulfite-induced mutations in E. coli ung
mutant is almost equal to that in the wild-type strain. The
authors suggested that cellular uracil DNA glycosylase
might be inactivated by the bisulfite treatment.16)
URACIL-DNA GLYCOSYLASES IN BACTERIA,
YEAST AND NEMATODE
Endogenous DNA damage is mainly repaired by the base
excision repair pathway. The repair pathway is initiated by
monofunctional DNA glycosylases that only remove the
altered base (e.g., uracil-DNA glycosylase) or bifunctional
glycosylases that in addition to base removal incise the
resulting apurinic/apyrimidinic (AP) site by an associated
AP lyase activity (e.g., endonuclease III).1,3,4,17–19) The uracil
N-glycosylase activity (UNG) of E. coli was the first DNA
glycosylase to be discovered in a search for activities that
would repair uracil in DNA.20,21) UNG recognizes uracil in
DNA and removes it from the DNA backbone as the first
step of the base excision repair pathway for uracil.20) The
open reading frame of the ung gene in E. coli encodes a protein of 229 amino acids. The purified enzyme from E. coil
hydrolyzes the N-glycosylic bond between uracil and deoxyribose, thus releasing a free uracil and leaving an AP site
in DNA.3–5) AP endonucleases recognize the resultant AP
site and cleave the phosphodiester backbone 5’ to the AP
site. This step is followed by DNA repair synthesis by DNA
polymerases, and the DNA nick is finally rejoined by the
action of DNA ligase.2–5) UNG of E. coli can remove roughly
800 uracil residues from DNA per minute and is present at
about 300 molecules per cell.4)
UNG of E. coli removes uracil from both the U:G and
U:A mispairs in DNA.1,3–5,18) Far more uracil residues than
normal are incorporated into DNA in the dut mutant cells,
as described above. The increased levels of uracil in DNA
are also reduced by UNG. UNG does not possess associated
AP lyase activity, and the resultant AP sites are processed by
AP endonucleases.1,3–5,22) E. coli cells deficient in dUTPase
(dut) and exonuclease III (the major AP endonuclease in E.
coli, xthA) are therefore not viable.15)
In E. coli, there are some uracil-removing enzymatic
activities other than UNG. For example, mismatch-specific
uracil-DNA glycosylase (MUG) also removes uracil from
U:G mismatches.1,3-5) MUG has been thought to be a backup
enzyme of UNG.3–5,23) MUG can act on mutagenic alkylated
bases and 3,N4-ethenocytosine.23)
Endonuclease V (Nfi) in E. coli selectively cleaves
untreated single-stranded DNA and duplex DNA containing
uracil, hypoxanthine and mismatches.24–26) When the nfi
mutant is treated with nitrous acid, it exhibits high frequencies
of A:T to G:C and G:C to A:T transition mutations.11,24–26)
Nitrous acid causes deamination of adenine and converts
adenine to hypoxanthine.26) The resulting deoxyinosine
(hypoxanthine deoxyribonucleoside) induces A:T to G:C
transitions through pairing with cytosine, resulting in possible
targets for Nfi.27) The Nfi enzyme was originally identified
as a deoxyinosine 3’-endonuclease that cleaves DNA near
dIMP residues, mismatched bases, urea residues and AP
sites.24–26) While nitrous acid causes deamination of cytosine
to generate uracil, it does not play the causative role in
increased G:C to T:A transitions in the nfi mutant cells,
because the ung mutant does not affect the transition frequency induced by nitrous acid. Schounten and Weiss
(1999) suggested a role for endonuclease V in the removal
of deaminated guanine (i.e., xanthine) from DNA.26)
Bacteriophage PBS2 normally contains uracil instead of
thymine in its DNA. Bacillus subtilis is the natural host for
this bacteriophage. As indicated by the high level of UNG
activity present in extracts from B. subtilis, PBS2 would be
easily inactivated in B. subtilis. Following infection, the
phage induces the expression of PBS2 DNA encoded uracilDNA glycosylase inhibitor (Ugi).28,29) Ugi forms a stable 1:1
complex with Ung and inactivates it. Ugi specifically inhibits the uracil-DNA glycosylase activity of UNG family-1
protein.3–5,28,29) Therefore, Ugi is often used to examine
whether or not the biological effect of uracil-removing activities is due to the family 1 uracil-DNA glycosylase.3,30)
In yeast, UNG homologs function in both the nucleus and
mitochondria. The ung-1 mutant of S. cerevisiae shows a
high frequency of spontaneous mutations in the nuclear
SUP4-o gene31) and mitochondrial petite loci, compared
with the isogenic wild-type cells.32) Deletion of the dut-1
gene that encodes dUTPase is lethal in S. cerevisiae owing
to cell cycle arrest.4,33,34) However, the viability of the dut-1
strain is restored when the ung gene is disrupted simultaneously or exogenous dTMP is supplied.4,33,34) Guillet et al.
J. Radiat. Res., Vol. 50, No. 1 (2009); http://jrr.jstage.jst.go.jp
Repair Mechanisms of Cytosine Deamination in DNA.
(2006) identified a viable allele (dut1-1) of the DUT gene in
S. cerevisiae.34) The mutant shows growth delay and cell
cycle abnormalities and exhibits a potent spontaneous mutator phenotype. All of the phenotypes of the dut1-1 mutant
are suppressed by the simultaneous inactivation of the UNG.
These results suggest that single-strand breaks are accumulated in the DNA as a result of the UNG-mediated removal
of incorporated dUMP from DNA.33) Interestingly, the
viability of the dut1-1 mutant is strongly impaired by
simultaneous deficiency of the AP endonuclease.34)
The anticancer drug 5-fluorouracil affects the level of dUTP
in the nucleotide pool and increases incorporation of dUMP
into DNA. This is one of the mechanisms of 5-fluorouracil
cytotoxicity.11) Ung gene disruption in S. cerevisiae has
protective effects against the lethality of 5-fluorouracil.
These findings suggest a new strategy by which we might
reduce the adverse side effects of anticancer drugs such as
5-fluorouracil.35)
Recently, we cloned the ung-1 gene of the nematode C.
elegans, whose which product (CeUng-1) shares 49% identity and 69 % similarity with E. coli UNG.30) The ung-1 gene
of C. elegans is located on chromosome III: 13.69 ± 0.087
cM and has 3 exons, encoding a 282 amino acid protein (32
KDa). Structural element analysis indicates that this protein,
potentially belonging to the UNG family, has eight conserved DNA-contacting elements in its sequence:α1~α8 and
β1~β4.3,30) These domains are highly conserved among the
known UNG sequences, including CeUng-1, in the database
(Fig. 2). Purified CeUng-1 removes uracil from both U:G
and U:A in double-stranded oligonucleotides. The activity of
CeUng-1 is inhibited by Ugi, a specific inhibitor of the
family-1 UNG,28,29) indicating that CeUng-1 is a member of
21
the family-1 UNG group.
The ung-1 mutant of C. elegans36) is viable and fertile, and
lays eggs at the normal rate. We further compared the life
span of the ung-1 mutant worms with that of the wildtype
N2 strain. There is no difference between the life span profiles of the two strains.30) These results suggest the existence
of backup enzyme(s). However, as shown in Fig. 3, residual
uracil excision activity could not be detected in the extract
from the ung-1 mutant.30)
URACIL-DNA GLYCOSYLASES IN
MAMMALIAN CELLS
In mammals, there is much more production of uracil
derived from cytosine deamination than in bacteria. However,
it is uncertain whether UNG suppresses spontaneous mutation in mammals. As shown in Fig. 2, human UNG shows
strikingly high identity (40–55%) to UNG glycosylase of
other organisms.1,3–5,37,38) Furthermore, the exogenous introduction of human UNG suppresses the high frequency of
spontaneous mutation in the E. coli ung mutants.38)
In human cells, transgenic Ugi-expressing cells exhibit a
high frequency of spontaneous mutations due to increased
G:C to A:T transitions.39) These facts indicate that human
UNG and E. coli UNG possess the same function; that is,
these enzymes remove uracil incorporated into DNA during
replication and/or produced by deamination of cytosine in
DNA. The human UNG is gene located at 12q24.1 of chromosome 12 and spans ~13.8 Kb.4,5,40,41) This gene encodes
both mitochondrial UNG1 and nuclear UNG2 isoforms.42,43)
Mitochondrial UNG1 and nuclear UNG2 are the major
uracil-DNA glycosylases in human cells.3–5,41–43) These two
Fig. 2. Partial amino acid sequence alignment for C. elegans Ung homolog with H. sapiens, M. musculus and E. coli Ung. Conserved amino acid residues are shown in bold. Portions of the active-site motif A and B of the family 1 Ung are underlined. The
highly conserved Gly121-Gln122, Tyr125, Phe136, Apn182, His247, Leu251 residues in CeUng-1 are marked with plus symbols.
J. Radiat. Res., Vol. 50, No. 1 (2009); http://jrr.jstage.jst.go.jp
22
S.-I. Yonekura et al.
Fig. 3. Cleavage activity for uracil-containing double-stranded
oligonucleotide in extract from C. elegans wild-type and ung-1
mutant in the presence or absence of Ugi. The extracts were prepared from wild-type N2 (w, lanes 2, 3, 8 and 9) and ung-1 mutant
TM2862 (m, lanes 4, 5, 10 and 11) of C. elegans. 32P-labeled U:G
or U:A (20 fmol) was incubated at 26°C for 5 min with the extract
in the presence (lanes 3, 5, 9 and 11) or absence (lanes 2, 4, 8, and
10) of 1 unit of Ugi. Lanes 1–6, U:G; lanes 7–12, U:A. Lanes 1 and
7, no enzyme; lanes 6 and 12; 9-mer marker.
UNGs share a common catalytic center but possess different
N-terminal amino acids that are derived from two different
transcripts by utilizing promoters PA and PB. Exon 1A of
UNG2 is transcribed from promoter PA, and exon 1B of
UNG1 from promoter PB.4,44) Moreover, alternative splicing
of the mRNA occurs for each transcript.4,44)
UNG2 co-localizes with PCNA and replication protein A
(RPA) in replication foci.4,45) UNG2 functions in postreplicative removal of uracil incorporated into DNA in
mammalian cells.4,45) The specific role of UNG2 in the
removal of misincorporated uracil has been demonstrated by
the inhibition of immediate post-replicative removal of
incorporated uracil in isolated nuclei by neutralizing antiUNG antibodies,45) and by the slow removal of incorporated
uracil in nuclei from UNG–/– mice.46) UNG2 also removes
uracil from U:G mispairs produced by cytosine deamination
in DNA.1,4,5,41–43) It is therefore reasonable to speculate that
the UNG2 dysfunction results in an increased level of uracil
in the DNA of UNG null mice, as described above.46)
Although uracil is the main substrate of UNG1 and
UNG2, these enzymes also remove the oxidized cytosine
derivatives isodialuric acid, alloxan and 5-hydroxyuracil.47)
Furthermore, Akbari et al. (2007) found that mRNA for
UNG1, but not UNG2, is increased after exposure to H2O2,
indicating regulatory effects of oxidative stress on mitochondrial base excision repair.48) It is likely that UNG1 has an
important role in the repair of oxidized pyrimidines.47,48)
Recent studies have revealed that UNG has a role in antibody diversification related to the activation induced
cytosine deaminase (AID) and class-switch recombination
via formation of AP sites.49–53) Enzymatic deamination of
cytosine in DNA is catalyzed by AID in activated B cells.51–53)
DNA diversification of functional immunoglobulin genes is
triggered by AID-mediated deamination of cytosine residues
at the immunoglobulin locus, followed by the removal of
uracil from U:G lesions by UNG.51–54) Expression of AID in
E. coli gives a mutator phenotype that yields G:C to A:T
transitions, and such expression in ung mutant cells yields a
much higher mutation frequency than the sum of their independent mutation frequencies.53) Nilsen et al. (2000) reported
that UNG knockout mice remained tumor-free and showed
no pathological phenotypes for up to 12 months, but beyond
18 months, they showed a higher morbidity and greatly
increased incidence of B-cell lymphomas in older mice.46)
That deficiency in UNG activity affected somatic hypermutation and class-switch recombination.49) In humans, UNG
deficiency was observed in patients with high-IgM
syndrome. The mutation of phase IA is dominant, as somatic
hypermutation is deficient in UNG mutant cells.53,54)
Proteins required for the S-S recombination, including
DNA-PK, ATM, Mre11, Rad50-Nbs1, γH2AX, Mdc1 and
XRCC4-ligase IV, are important for faithful joining of S
regions.55)
STRUCTURAL ASPECTS OF URACIL-DNA
GLYCOSYLASES
As shown in Fig. 2, CeUng-1 contains certain conserved
residues that have counterparts in E. coli and human UNG.30)
The CeUng-1 polypeptide has highly conserved active sites
A and B.3,19,30,56–60) Active site A is present between residues
116~137 in CeUng-1, 58~77 in E. coli UNG and 138~159
in human UNG.19,30,57–59) Active site B is also highly conserved between residues 247 and 253 in CeUng-1 (Fig. 1).
The position of a conserved Apn182 is involved in catalysis
and conserved for distinguishing cytosine and uracil, which
plays a major role in establishing the high degree of base
specificity displayed by UNG enzyme.3,19,30,56–60) Aromatic
residue, Tyr125, Phe136 and His247 are involved in the
stacking interaction with uracil.19,30,58) The sequence
Gly121-Gln1223,19,56–60) is conserved together with Leu251
in CeUng-1.30)
OTHER ENZYMATIC ACTIVITIES FOR
REMOVAL OF URACIL FROM DNA
UNG is categorized as a family-1 uracil-DNA glycosylase
and its homologous genes are highly conserved in S.
cerevisiae, S. pombe and mammals.3,4,61) Recent studies
revealed that mammalian cells contain at least three
additional families of uracil removing enzymes, classified
according to the differences in their substrate recognition
and amino acid sequence (Table 1). They are the MUG
(mismatch-specific uracil-DNA glycosylase)/TDG (thymine
J. Radiat. Res., Vol. 50, No. 1 (2009); http://jrr.jstage.jst.go.jp
Repair Mechanisms of Cytosine Deamination in DNA.
Table 1.
Family
23
DNA glycosylase families
DNA glycosylase E. coli S. cerevisiae S. Pombe C. elegans Mammals
Family 1
UNG
+
+
+
+
+
Family 2
MUG/TDG
+
–
+
?
+
Family 3
SMUG1
–
–
–
–
+
Family 4
MBD4
–
–
+
–
+
DNA glycosylase) family (family-2), the SMUG (single
strand- specific monofunctional uracil-DNA glycosylase)
family (family-3) and the MBD4 (methyl-binding domain)
family (family-4).3–5,19,48,62,63)
SMUG1 possesses a uracil-removing activity and functions as a backup enzyme for UNG. SMUG1 is identified as
a new uracil-DNA glycosylase family and found in Xenopus
laevis.64) SMUG1 prefers single-stranded DNA as its substrate, but also removes uracil from double-stranded DNA.
UNG-knockout mice (UNG–/–) are fertile and develop
normally to adulthood with no apparent changes in phenotype.46,65) Unexpectedly, UNG–/– mice show only slightly
elevated spontaneous mutation frequencies. There is an
increase in the mutation frequency in the thymus (1.3-fold)
and in the spleen (1.4-fold) in the UNG-deficient mice.46,65)
In these organs, residual uracil removing activities are the
lowest among the organs tested in the mice. However, uracil
residues in the genome DNA are increased about 100-fold
in the UNG null mice.
It is reasonable to speculate that, because of the presence
of residual uracil removing activity catalyzed by SMUG1,
UNG null mice do not exhibit a greatly increased mutation,
at least overall in the whole genome. Furthermore, UNG and
SMUG1 function in different pathways.1,3,47) SMUG1
removes uracil in potent mutagenic U:G base pairs produced
by cytosine deamination in DNA.1,3–5) On the other hand,
UNG has a central function for the removal of uracil from
misincorporated dUMP residues, which are not directly
mutagenic.45,46) An et al. (2005) reported that stable siRNAmediated knockdown of SMUG1 in mouse embryonic fibroblasts has a mutator phenotype.66) An additive 10-fold
increase in the frequency of spontaneous G:C to A:T transitions is observed in cells deficient in both SMUG1 and
UNG.4,66) These results demonstrate that these enzymes have
distinct and non-redundant roles in preventing mutations.
Interestingly, cells with mutations in the SMUG1 and UNG
genes are hypersensitive to ionizing radiation, indicating a
critical role of the enzymes they encode in the repair of DNA
damage generated by the oxidation of cytosine. SMUG1 is
present only in insects and vertebrates,4) and has not been
identified in C. elegans.30)
Both UNG2 and SMUG1 are important for the prevention
of mutations caused by cytosine deamination, and their
functions are non-redundant. Furthermore, SMUG1 has a
DNA glycosylase activity to remove thymine whose methyl
group has been oxidized, 5-hydroxymethyluracil and 5formyluracil from DNA.66–69)
In C. elegans, UNG removes uracil from both U:G and
U:A base pairs in the absence of SMUG.30) However, it is
not likely that C. elegans has any uracil-removing enzymes
belonging to such other known families, because the C.
elegans database does not reveal any homolog of other family Ung enzymes, including Mug, SMUG1, TDG and
GBD4.30)
Two other DNA glycosylases, TDG and MBD4, have
been identified in mammals.63,70,71) TDG and MBD4 are
strictly specific for double-stranded DNA, and have very low
turnover numbers. TDG and MBD4 can repair uracil, thymine, and oxidized pyrimidine in methylated CpG sites.63,71–73)
The roles of MBD4 may be limited to repair of mismatched
uracil, thymine and some damaged pyrimidines in doublestranded DNA, particularly in CpG and 5-methyl-CpG contexts.73) Deficiency of MBD4 in mice enhances mutations at
CpG sites and alters apoptosis in response to DNA damage.73) MBD4 interacts strongly with MLH.63,71–73)
Uracil in DNA has been thought to occur in different positions, and in addition, the sequence context where it occurs
may be different. It seems likely that the type of uracil- DNA
glycosylase and the mechanism of repair in the downstream
steps will depend on these factors.4) Uracil in the replication
fork may be generated by cytosine deamination, and may be
removed by any of the four uracil-DNA glycosylases, UNG,
SMUG1, TGD or MBD4. TDG and MBD4 mainly operate
in the CpG context, while UNG and SMUG1 operate in any
sequence context.4,73) UNG is a major DNA glycosylase for
removing uracil from U:G mispairs, as is as SMUG1.4)
PERSPECTIVES
Cytosine deamination and the resultant mutations were
thought to be inevitable disasters for cells when people first
started to study cytosine deamination. It has clearly been
demonstrated that dUTPase removes dUTP from the nucleotide pool and there are several uracil-removing enzymes,
which constitute the of UNG superfamily. Furthermore, the
generation of uracil in DNA and its repair mechanism have
been found to be deeply involved in antigen production
processes. The following subjects are key for clarifying the
J. Radiat. Res., Vol. 50, No. 1 (2009); http://jrr.jstage.jst.go.jp
24
S.-I. Yonekura et al.
mechanisms of the biological effects and repair of cytosine
deamination in DNA at the biochemical and molecular
levels: the binding and regulation of various base excision
repair enzymes at the damage site; regulation of the expression of uracil DNA glycosylase; physical and functional
interactions between UNG and other proteins involved in
base excision repair; functional roles of the different
glycosylases and coupling to downstream steps in the base
excision repair pathway; identification of novel DNA glycosylases and proteins associated with them. For overviews,
the reader should consult previous excellent reviews written
by Krokan et al.,1,4) Friedberg et al.,3) Barnes and Lindahl5)
and Pearl.19)
ACKNOWLEDGEMENTS
We would like to thank the editor-in-chief of the Journal
of Radiation Research, Dr. Yoshiya Furusawa, for giving us
the opportunity to write this review article. We also wish to
thank Dr. Grigory L. Dianov, Oxford University, for his
helpful discussion. The studies done in our laboratory were
financially supported in part by the Global Center of
Excellence Program [Formation of a Strategic Base for Biodiversity and Evolutionary Research (A06): from Genome to
Ecosystem] and Grants-in-Aid for Scientific Research
19510056 from the Ministry of Education, Culture, Sports
and Technology, Japan (to QM. Z.). We are grateful to the
Central Research Institute of Electric Power Industry
(Tokyo) for supporting Q-M. Zhang-Akiyama.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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Received on July 15, 2008
Revision received on August 26, 2008
Accepted on August 28, 2008
J-STAGE Advance Publication Date: November 6, 2008
J. Radiat. Res., Vol. 50, No. 1 (2009); http://jrr.jstage.jst.go.jp