Báo cáo khoa học: The multi-replication protein A (RPA) system – a new perspective ppt

REVIEW ARTICLE
The multi-replication protein A (RPA) system – a new
perspective
Kengo Sakaguchi, Toyotaka Ishibashi*, Yukinobu Uchiyama and Kazuki Iwabata
Department of Applied Biological Science, Tokyo University of Science, Chiba, Japan
Replication protein A (RPA) is a single-stranded DNA
(ssDNA)-binding protein complex comprising a hetero-
trimeric combination of a large (70 kDa), middle
(32 kDa) and small (14 kDa) subunit [1,2]. Function-
ally, RPA corresponds to an alternative form of a
bacterial ssDNA-binding protein (SSB). Until 2005,
only one copy of the RPA complex was thought to be
present in eukaryotes [1–9]. Indeed, preliminary
analysis of the genomes of mammals and yeast
indicated that they encoded a single copy of each
subunit of the RPA complex [1,2]. However, we
recently found that higher plants have at least three
different species of complex (types A, B and C), each
displaying a different biological function [10–12]. Orig-
inally, we intended to investigate the plant repair
system [13–43], but during the course of this study we
Keywords
convergent evolution; DNA polymerases;
eukaryotic DNA metabolism; meiotic pairing
and recombination; multi-RPA system;
O. sativa and A. thaliana; paralog ⁄ ortholog/
analog/heterolog; Rad51 ⁄ DMC1 ⁄ Lim15;
replication protein A; RPA subunits (70, 32
and 14 kDa)
Correspondence
K. Sakaguchi, Department of Applied

Biological Science, Faculty of Science and
Technology, Tokyo University of Science,
2641 Yamazaki, Noda, Chiba 278 8510,
Japan
Fax: +81 471 23 9767
Tel: +81 471 24 1501 (ext. 3409)
E-mail:
*Present address
Department of Biochemistry and
Microbiology, University of Victoria, Victoria,
Canada
(Received 11 September 2008, revised 26
November 2008, accepted 5 December
2008)
doi:10.1111/j.1742-4658.2008.06841.x
Replication protein A (RPA) complex has been shown, using both in vivo
and in vitro approaches, to be required for most aspects of eukaryotic
DNA metabolism: replication, repair, telomere maintenance and homolo-
gous recombination. Here, we review recent data concerning the function
and biological importance of the multi-RPA complex. There are distinct
complexes of RPA found in the biological kingdoms, although for a long
time only one type of RPA complex was believed to be present in eukary-
otes. Each complex probably serves a different role. In higher plants, three
distinct large and medium subunits are present, but only one species of the
smallest subunit. Each of these protein subunits forms stable complexes
with their respective partners. They are paralogs as complex. Humans pos-
sess two paralogs and one analog of RPA. The multi-RPA system can be
regarded as universal in eukaryotes. Among eukaryotic kingdoms, para-
logs, orthologs, analogs and heterologs of many DNA synthesis-related
factors, including RPA, are ubiquitous. Convergent evolution seems to be

ubiquitous in these processes. Using recent findings, we review the compo-
sition and biological functions of RPA complexes.
Abbreviations
ATR, ataxia telangiectasia mutated and Rad3-related; dsDNA, double-stranded DNA; MMS, methyl methanesulfonate; NER, nucleotide
excision repair; PCNA, proliferating cell nuclear antigen; pol a, DNA polymerase a; RPA, replication protein A; SC, synaptinemal complex;
SSB, single-stranded DNA-binding protein; ssDNA, single-stranded DNA.
FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS 943
serendipitously discovered the involvement of RPA
[10–12]. Interestingly, RPAs are not necessarily com-
pletely independent complexes. Only one copy of the
small subunit was found, whereas there were three sets
of the large and middle subunits [10–12]. The mode of
action of these RPA complexes seems to be universal,
at least in Plantae. Each RPA complex must be inde-
pendently related to various DNA synthetic events
within the plant. Because DNA replication and repair
are generally very similar between animals and plants
[13,44–66], the role of the RPA complex should be
reconsidered in the light of this new finding. Therefore,
we retrospectively searched reports about screening for
RPA homologs in animals and fungi. Humans carry
two homologs of the middle subunit (HsRPA2 and
HsRPA4) [67–69]. Moreover, Richard et al. recently
reported that the two human SSB homologs (hSSB1
and hSSB2) possess a domain organization that is
closer to archaeal SSB than to RPA [70]. Although the
genetic and biochemical characteristics of hSSB1 are
totally different from those of human RPA, both are
critical for genomic stability [70]. Thus, like Plantae,
the human DNA repair enzymes also function as a

multiple system. Furthermore, the multi-RPA or SSB–
RPA mixed system is presumably universal in eukary-
otes. Here, in the light of these recent discoveries, we
review the function and structure of the RPA com-
plexes.
There are many reports in the literature concerning
the role of RPAs. RPA is ubiquitous and essential for
a wide variety of DNA metabolic processes, including
DNA replication, repair and recombination [1]. In par-
ticular, RPA is required for cross-over during meiosis
[71–74]. According to a recent report [75], the large
and middle subunits of human RPA may act as an
independent prognostic indicator of colon cancer, as
well as therapeutic targets for regulation by tumor sup-
pressors involved in the control of cell proliferation.
Thus, despite the previous studies on RPA, there are
many new areas of research involving this complex
that still need to be addressed.
History of RPA studies
We begin this review by summarizing studies that first
identified RPA as a factor necessary for SV40 replica-
tion in vitro [76–79]. RPA is required for activation of
the pre-replication complex to form the initiation com-
plex, and for the ordered loading of essential initiator
functions, such as DNA polymerase a–primase (pol a)
complex, to the origins of replication [76–79]. The gen-
eral role of RPA has been studied in great detail in
mammals and yeasts [1,2]. It was originally thought
that the RPA complex was evolutionarily conserved
throughout eukaryotes and that the function is funda-

mental irrespective of DNA synthesis. Many data were
obtained on the assumption that there is just one RPA
copy. RPA accumulates along stretches of ssDNA gen-
erated during DNA replication and repair (Fig. 1A)
[1,5–8,79–87]. RPA also plays an essential role in
DNA repair and is required for nucleotide excision
A
B
Fig. 1. (A) RPA in the DNA replication. (B) The role of RPA in NER.
The multi-replication protein A system K. Sakaguchi et al.
944 FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS
repair (NER) [88–90]. During strand elongation in
DNA replication ⁄ repair, RPA stimulates the action of
DNA polymerases such as pol a, pol d, pol e, pol k
and pol j [5–8,80,81,85–87]. Conversely, pol f is not
under the influence of RPA, suggesting that RPA-
dependent ssDNA stretching is not always essential for
DNA polymerization [88]. RPA interacts with XPA at
sites of DNA damage, stimulating XPA–DNA contact
and recruiting the incision proteins ERCC1 ⁄ XPF and
XPG to the damaged site (Fig. 1B) [89–91]. These pro-
cesses include damage detection and signaling, tran-
scriptional responses, DNA damage checkpoints and
apoptosis [4,7]. RPA is known to interact specifically
with numerous transcription, replication and repair
proteins including T antigen, the tumor suppressor
p53, the transcription factors Gal4 and VP16, DDB,
uracil DNA glycosylase, recombinases and the DNA
helicases, Bloom’s and Werner’s proteins.
RPA is also a checkpoint protein that has been iden-

tified by the generation of a mutant in the large sub-
unit in yeast [92]. In addition, RPA was found to be
necessary for the removal of oxidized base lesions from
genomic DNA in long-patch base excision repair
[93,94]. RPA also interacts with Rad51 and Rad52,
thereby playing a role in initiating homologous recom-
bination events [95–111]. In the repair of double-strand
breaks by homologous recombination in Saccharomy-
ces cerevisiae, RPA stimulates DNA strand exchange
by Rad51 protein, provided that RPA is added to a
pre-existing complex of Rad51 protein and ssDNA.
RPA is also implicated in forming the meiotic recom-
bination nodules [112–118]. Furthermore, RPA has a
specific interaction with the tumor suppressor p53
[119–121] and promotes DNA binding and chromatin
association of ataxia telangiectasia mutated and Rad3-
related (ATR) in vitro via ATR interacting protein
[122]. RPA is also required to recruit and activate
Rad17 complexes for checkpoint signaling in vivo
[123]. Thus, the functions of RPA are surprisingly
ambiguous. Namely, RPA functions in a wide range of
systems from DNA replication to DNA damage and
stress responses (biochemical and cell biological) as
well as cross-over in meiosis [1,2].
It is thought that the major interaction between
RPA and DNA occurs through the RPA70kDa sub-
unit, and the role of the RPA32kDa and RPA14kDa
subunits is supplementary [124]. Indeed, RPA70kDa is
the major subunit of the complex having four ssDNA-
binding domains in the middle of the subunit. By

contrast, RPA32kDa and RPA14kDa each possess a
single DNA-binding domain, displaying only weak
binding affinity [2,125]. The contact surfaces in RPA
have been elucidated for several of its binding part-
ners. The results of these studies suggest that proteins
from distinct processing pathways may use a small
number of common sites to bind RPA and remodel
the mode of DNA binding [124].
The RPA32kDa subunit is phosphorylated during
progression of the cell cycle and in response to a wide
variety of DNA-damaging agents, such as ionizing
radiation, UV and camptothecin [120,126–128]. RPA
phosphorylation stimulated by DNA damage promotes
DNA binding and chromatin association of ATR
in vitro via ATR interacting protein [83,122,129]. RPA
is also required for recruitment and activation of the
Rad17 complexes during checkpoint signaling in vivo.
RPA may function in the sensing of DNA damage
[111]. In budding yeast, the middle subunit (32 kDa)
becomes phosphorylated in reactions that require the
Mec1 protein kinase, a central checkpoint regulator
and homolog of human ATR [71–74]. However, the
meiosis-specific protein kinase Ime2 is required for
normal meiotic progression [130]. A natural target of
Ime2 activity is also the middle subunit of RPA [130].
Ime2-dependent RPA phosphorylation first occurs
early in meiosis. The middle subunit is not supplemen-
tary, but is a signal acceptor for sensing various struc-
turally specific DNA sites. Furthermore, RPA32kDa is
reportedly related to viral DNA replication [124,131].

There is almost no information concerning the
molecular role of the RPA14kDa subunit. It is known
that RPA14kDa contains one weak DNA-binding
domain, which may slightly modify the mode of DNA
binding of RPA.
Consequently, it was generally believed that the
major roles of RPA had been elucidated. However, at
this stage, it was not known that RPA represented
more than one molecular species. Thus, most research-
ers did not consider the possibility of orthologs, para-
logs, analogs and heterologs of the RPA complex.
Multi-RPA systems
In contrast to the intensive studies of RPA in mam-
mals and yeasts, until 2001 little was known about this
protein in plants. Plants are affected by various envi-
ronmental stress factors. For example, DNA in plants
is continuously damaged by UV irradiation from sun-
light. UV is known to induce DNA damage [13],
although plants generally have a higher tolerance for
UV than animals. Field-grown crops such as wheat are
also known to suffer continuous UV-induced DNA
damage. Furthermore, the formation of reactive
oxygen species in cells due to UV irradiation, biotic
stresses and secondary metabolism, causes cellular
components, including DNA, to be oxidized and there-
K. Sakaguchi et al. The multi-replication protein A system
FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS 945
fore susceptible to oxidative modification. In addition,
the fidelity and integrity of DNA are constantly chal-
lenged by chemical substances in the environment, ion-

izing radiation and errors that occur during DNA
replication or proofreading. This accumulated damage
blocks a number of critical processes, such as tran-
scription and replication, and can eventually cause cell
death. Thus, UV damage can reduce the growth and
yield of plant crops. Indeed, there is no difference
between the abilities of animals and plants to remove
damaged DNA [13]. Plants have evolved several DNA-
repair pathways [13]. Whereas previous studies on
DNA repair have focused mostly on animals and yeast
cells, recent analyses of UV tolerance and DNA repair
have addressed the responses of plants to environmen-
tal factors and the mechanisms of stress resistance in
plants [13]. An additional basis for molecular analyses
has been provided by the completion of genome-
sequencing projects in model plants such as rice and
Arabidopsis. Completed genome sequences allow the
identification of entire gene groups related to DNA
repair in higher plants. In order to better understand
the mechanisms of DNA protection and plant DNA
repair systems, we attempted to isolate the gene encod-
ing plant RPA. Surprisingly, analysis of rice revealed a
new type of RPA complex gene [10–12].
In 1997, an ortholog of the RPA70kDa subunit (Os-
RPA1) was isolated from deepwater rice (Oryza sativa
L. cv. Pin Gaew 56), and its expression was induced
by gibberellin [132]. To use the OsRPA1 protein for
plant DNA replication studies, we aimed to clone the
cDNA and obtain the recombinant protein from rice
(O. sativa L. cv. Nipponbare). Although we failed to

clone the OsRPA1 cDNA, we unexpectedly obtained
cDNA of the RPA70kDa subunit alternative. The new
alternative gene differed greatly from OsRPA1, having
closer homology with its counterpart in Arabdop-
sis thaliana reported in the database [10]. We found
that A. thaliana also has a homolog of OsRPA1, sug-
gesting that two different RPA types are universally
present in seed plants [10]. Rice has two different types
of RPA70kDa subunit, renamed OsRPA70a (newly
found) and OsRPA70b (OsRPA1), respectively [10].
We discovered their homologs in A. thaliana, and
described the substantial properties of the T-DNA
insertion lines [11]. Transcripts of OsRPA70a are
expressed in proliferating tissues, such as root tips and
young leaves that contain meristem, but also more
weakly in the mature leaves, whereas OsRPA70b is
expressed mostly in proliferating tissues [10].
The existence of these genes gives rise to an intrigu-
ing evolutionary question. Why do mammals and yeast
have only one copy of the gene for the RPA70kDa
subunit in their genome? Furthermore, is only the larg-
est subunit of the RPA complex duplicated in plant,
and what are the roles of the two RPA types? Interest-
ingly, when the RPA70a subunit lacked the T-DNA
insertion or RNA interference (RNAi), the line could
be viable [10–12]. The surviving mutant was morpho-
logically normal except for hypersensitivity towards
some mutagens, such as UV and methyl methanesulfo-
nate (MMS) [10–12]. Plants are naturally exposed to
UV for much longer than animals or yeast [133–135]

and depend on sunlight for their development. Because
seed plants synthesize DNA under relatively high levels
of UV irradiation, the RPA system might be more
complicated in plants than in animals.
Therefore, we attempted to screen for rice RPA genes
in the genome (O. sativa L. cv. Nipponbare). We found
three different genes encoding the largest (RPA70kDa)
and middle subunits (RPA32kDa), but only one
gene encoding the smallest (RPA14kDa) [12]. Each
OsRPA70s and OsRPA32s gene was not a pseudogene
or redundant gene. We designated the subunits from rice
as OsRPA70a, OsRPA70b, OsRPA70c, OsRPA32-1,
OsRPA32-2, OsRPA32-3 and OsRPA14 [12]. The
RPA70bsubunit is the ubiquitous RPA70 subunit found
in all eukaryotes [10]. The various subunits do not ran-
domly associate with other subunits, but form a distinct
complex. Three different RPA complexes (A, B or C
type) were composed of these subunits in vivo. Types A,
B and C were OsRPA70a–OsRPA32-2–OsRPA14,
OsRPA70b–OsRPA32-1–OsRPA14 and OsRPA70c–
OsRPA32-3–OsRPA14, respectively [11,12]. Only the
smallest subunit is common to all the complexes.
Because the system was also present in A. thaliana
[11,12], these properties may be universal in higher
plants. In conclusion, higher plants have a multi-RPA
system [11,12].
The RPA complexes are spatially segregated in
plants. Type A is localized to the chloroplast, whereas
types B and C are found in the nuclear region [11]. In
human and yeast cells, the middle subunit exists in the

nucleus and cytoplasm, whereas the large subunit is
present only in the nucleus [11]. The RPA32kDa sub-
units probably exist as each protein alone (OsRPA32-
1, OsRPA32-2, OsRPA32-3 or OsRPA14) or as free
heterodimer complexes such as OsRPA32-1–OsRPA14,
OsRPA32-2–OsRPA14 and OsRPA32-3–OsRPA14
[11,12].
In rice, co-regulation of OsRPA70b and OsRPA32-1
during the cell cycle, and regulation of OsRPA32-1 in
response to UV has been reported [43]. RPA70kDa
has been reported to be unstable when not in a com-
plex. Because expression of OsRPA70a was observed
at both the mRNA and protein levels, we suggest that
The multi-replication protein A system K. Sakaguchi et al.
946 FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS
the rice genome contains another protein, distinct from
OsRPA32-2 that might form a stable complex with
OsRPA70a. As described earlier, the RPA32kDa sub-
unit is phosphorylated in response to cell-cycle phase
transitions and a wide variety of DNA-damaging
agents, suggesting that RPA activities are regulated by
the extent of phosphorylation [120,126–128]. Rice had
three different RPA32kDa subunits. This infers the
existence of independent phosphorylation systems that
control each type of RPA complex. Does the phos-
phorylation occur on the same RPA complex?
Are such phenomena limited in the RPA system?
Drosophila has two paralogs of proliferating cell
nuclear antigen (PCNA) and a ‘heterolog’ (Rad9–
Rad1–Hus1) [65,136,137]. Moreover, the fungus

Coprinus cinereus generates two different PCNAs by
alternative splicing, although there is only a single
copy of the gene in the genome [138]. Even the plural-
izing recipe of PCNA is also phylogenetically diversi-
fied. The roles of PCNA are probably diversified, and
a division of labor occurs [65]. Like RadA and hSSB,
we also found another FEN-1-like analog, SEND-1
and GEN [25,63,66]. All are transcribed and translated
and therefore do not represent pseudogenes. Knock-
down of one of their genes in the same category seems
to lead to lethality, although there is little published
data on this subject. The diversification must be closely
related to the point at which biochemical control sys-
tems divide [65]. Similar considerations probably apply
to the multi-RPA system.
Phylogenetic aspects of multi-RPA
systems
Sophisticated studies are required to verify whether
a specific subunit (OsRPA32-1, OsRPA32-2 or
OsRPA32-3) is responsible for phosphorylational
control. Furthermore, which RPA complex corresponds
to the RPA found in mammals and yeast? Are no other
RPA types present in animals and yeasts? Whether
mammals and yeasts evolved a multi-RPA system,
which was subsequently lost over evolutionary time is so
far unclear. We have investigated the plant multi-RPA
system in terms of phylogenetics.
Two large RPA subunits, RPA70 and RPA32, and a
small subunit, RPA14, are relatively well conserved
among eukaryotes (Fig. 2A). The deduced amino

acid sequence among OsRPA70a, OsRPA70b and
OsRPA70c showed low identity levels ( 50%) between
them [12]. Similarly, the deduced amino acid sequence
among OsRPA32-1, OsRPA32-2 and OsRPA32-3 was
compared; each type also displayed low identity levels
[12]. In the system, the sequence homologies among the
OsRPA70kDa subunits and among the OsRPA32kDa
subunits were low [12]. The B type complex was
thought to be ubiquitous in eukaryotes [12].
RPA70kDa has two RPA ssDNA-binding domains,
DBD-A and DBD-B for binding ssDNA, and a third,
DBD-C, which displays only weak ssDNA-binding
activity (Fig. 2B). RPA70kDa also contains the DBD-
F domain, which has been shown to interact with
multiple proteins and to interact weakly with DNA
(Fig. 2B). The primary amino acid sequences of
DBD-A, DBD-B, DBD-C and DBD-F domains are
very similar [12]. RPA32kDa has only a single ssDNA-
binding domain (DBD-D) [12]. Furthermore, all the
domains have high levels of sequence homology with
their counterparts in human and yeast RPAs [12]. The
DBD-E domain is in the RPA14kDa subunit, and is
also highly conserved [12].
In yeast, RPA1 (largest subunit) can only bind to
the RPA2 ⁄ 3 dimer (middle and smallest subunit
dimer). The DBD-C and DBD-D regions of rice are
quite similar to the DBD-C and DBD-D regions of
S. cerevisiae [139], but OsRPA14 has only low simi-
larity to RPA3. This sequence divergence may
account for the differences in binding observed

between the yeast and rice proteins. Rice DBD-A and
DBD-B domains are more conserved than DBD-C
and DBD-F, implying that the primary function of
OsRPA70a and OsRPA70b is to bind DNA, and that
this function has been conserved during evolution,
even though the secondary functions of these proteins
may have diverged. Based on this analysis the B type
complex corresponds to the mammalian and yeast
RPA.
In plant, human and yeast, the domains of DBD-A
and DBD-B are more homologous than those of
DBD-C and DBD-F, and the biochemical characteris-
tics are common among OsRPA70a, OsRPA70b and
OsRPA70c. It is well established that the RPA70kDa
subunit accumulates along stretches of ssDNA gener-
ated by stalled replication forks and ⁄ or DNA damage
[1,82–84]. In the RPA70kDa subunit, DBD-A and
DBD-B possess the strongest ssDNA-binding activity.
Indeed, DBD-A and DBD-B were the first to be iden-
tified as DNA-binding domains [12]. DBD-C and
DBD-D have a weak ssDNA–binding activity [12],
whereas DBD-F interacts physically with the tumor
suppressor p53 and nucleosome remodeling complex
FACT. The interaction with DBD-F can also contrib-
ute to an additional binding of structurally distorted
DNA (i.e. damaged DNA). By analogy, the primary
function of all the OsRPA70kDa subunits must be to
find special regions of DNA with which to bind. Is
there a divergence in biochemical function among the
K. Sakaguchi et al. The multi-replication protein A system

FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS 947
various domains? What is the specialization of hSSBs
(analogs of RPA), which appeared by convergent
evolution [70]?
Furthermore, why are the middle subunits diversified
phylogenetically? As discussed earlier, the major role
of the middle subunits is not to bind to DNA,
although they may be involved in the controlling signal
via phosphorylation. Indeed, in humans, HsRPA2
interacts with uracil–DNA glycosylase and XPA, but
HsRPA4 does not [67–69]. Moreover, the small sub-
unit is presumably responsible for linking the other
subunits (large and middle). The driving force behind
the diversification of the small subunit is an interesting
question that needs to be addressed.
The phylogenetic data suggest that the multi-RPA
(or the SSB–RPA mixed) systems are universal in
eukaryotes. However, it is important to establish
whether plants have paralogs or orthologs of hSSB. In
particular, we need to investigate the in vivo functions
of each of the A, B and C types of plant multi-RPA
systems.
In vivo roles of the multi-RPA system
If the multi-RPA system is unique in plants, some of
the in vivo roles may also be specific for plants.
OsRPA70a (type A complex) is localized in the chloro-
plast, but OsRPA70b (type B) and OsRPA70c (type C)
are found in the nuclear compartment [12]. The type A
system is thought to be plant specific, whereas types B
and C could be universal. Fortunately, the homologs

of OsRPA70a, OsRPA70b and OsRPA70c were found
A
B
Fig. 2. (A) Pairwise comparison of each
OsRPA subunit with human (HsRPA),
Schizosaccharomyces pombe (SpRF-A)
and Drosophila melanogaster (DmRPA). (B)
Domain structures of OsRPAs. Each color
box indicates each DBD domain shown as
the lower half of the figure. DBD domain
are classified into A, B, C, D, E and F.
The multi-replication protein A system K. Sakaguchi et al.
948 FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS
to be present in A. thaliana (AtRPA70a, AtRPA70b
and AtRPA70c) [11,12].
Interestingly, the AtRPA70a deletion mutant
(SALK017580) was lethal, but the AtRPA70b deletion
mutant (SALK088429) was viable and hypersensitive
to UV and MMS [12]. Therefore, type A may be
essential for DNA replication and transcription (and
also DNA repair) in the chloroplast. Type B may have
at least some role in nuclear DNA repair [12]. Intrigu-
ingly, the AtRPA70c deletion mutant does not appear
to be viable. Type C shows nuclear localization, and
the AtRPA70c deletion mutant may be lethal, suggest-
ing that type C is essential for DNA replication and
transcription (and possibly DNA repair) in the nucleus
[12].
To investigate the function of the various proteins,
RNAi of AtRPA70a and AtRPA70b were performed

[140–143]. The RNAi-mediated knockdown of
AtRPA70a also displayed lethality. However, RNAi of
AtRPA70b was viable and did not differ in phenotype
from wild-type. RT-PCR analysis was also carried out
using total RNA extract from seedlings of atrpa70b
mutant and the AtRPA70b RNAi line. No atRPA70b
transcript could be detected. Furthermore, western blot
analysis of total proteins from seedlings of wild-type
and atrpa70b mutant indicated very little AtRPA70b
[12].
These results indicated that AtRPA70a (probably,
the AtRPA70a–AtRPA32-2–AtRPA14 complex) has
an essential role, probably in DNA replication in the
chloroplast, whereas AtRPA70b (the AtRPA70b–At-
RPA32-1–AtRPA14 complex) is not essential under
normal growth conditions. However, it is known that
yeast rpa70 mutants are very sensitive to mutagens
such as UV and MMS [11,12]. To determine whether
AtRPA70b is similarly involved in mutagen tolerance,
the mutagen sensitivity of atrpa70b mutant and the
AtRPA70b RNAi line was tested. When 1-week-old
seedlings were exposed to various UV-B doses and
then grown for an additional week in the absence of
UV-B, there were no remarkable morphological differ-
ences between wild-type, atrpa70b mutant and
AtRPA70b RNAi line seedlings, although leaf yellow-
ing was somewhat increased in the mutant and RNAi
seedlings [11,12]. Compared with wild-type, the
amounts of chlorophyll (a + b) were decreased in
atrpa70b and the AtRPA70b RNAi lines [11,12]. One-

week-old seedlings were also grown on MS medium
containing various concentrations of MMS or H
2
O
2
.
After 1 week, growth of the wild-type plants was
inhibited by UV-B, MMS or H
2
O
2
. Compared with
wild-type plants, the growth of atrpa70b mutant and
AtRPA70b RNAi line seedlings was more inhibited by
UV-B, and was completely stopped by MMS [11,12].
Mutants showed little increase in sensitivity to H
2
O
2
.
Like the yeast rpa70 mutants, the atrpa70b mutant
and AtRPA70b RNAi line are more sensitive than
wild-type to UV and MMS, suggesting that At-
RPA70b is involved in the repair system for DNA
damaged by these mutagens [11,12].
The lethality of both the T-DNA insertion mutant
and the RNAi line of AtRPA70a indicate that the
AtRPA70a–AtRPA32-2–AtRPA14 complex plays an
essential role, such as DNA replication, in the chlorop-
lasts of living cells (Fig. 3). By contrast, the mutant

and RNAi line of AtRPA70b were viable but showed
high sensitivity to UV and MMS, suggesting involve-
ment of the AtRPA70b–AtRPA32-1–AtRPA14 com-
plex in the repair of damaged DNA (Fig. 3). However,
AtRPA70c deletion was thought to be lethal, suggest-
ing that the AtRPA70c–AtRPA32-3–AtRPA14 com-
plex may function mainly in nuclear DNA replication
and transcription (Fig. 3). Subcellular localization
analysis suggested that the type A RPA complex is
required for chloroplast DNA metabolism, whereas
types B and C function in nuclear DNA metabolism
[12].
Recently, RPA70 and RPA32 subunits from plants
have been reported to play a role in viral and transpo-
son DNA syntheses [131,144]. It will be intriguing to
investigate how the RPA complex functions in these
mechanisms. Higher plants may have evolved the
type A for the chloroplast to offer protection against
high levels of UV irradiation. Indeed, as mentioned
earlier, plants are exposed to UV radiation for much
longer than animals or yeast. Higher plants depend on
exposure to sunlight, including UV, for their develop-
ment because their energy is derived from photosyn-
thesis. Thus, the repair system in subcellular organelles
is presumably much more efficient in plants than in
animals and yeast.
The human homologs of RPA32, HsRPA2 and
HsRPA4 [67] may correspond to OsRPA32-1 (type B)
and OsRPA32-3 (type C) of plants, respectively,
although only the middle subunit is diversified. Inter-

estingly, hSSB1 did not localize to replication foci in
S-phase cells and hSSB1 deficiency did not influence
S-phase progression [70]. Depletion of hSSB1 abro-
gated the cellular response to DSBs, including activa-
tion of ATM and phosphorylation of ATM targets,
after ionizing radiation [70]. Ionizing radiation and
anti-cancer drugs can induce DNA DSBs, which are
highly cytotoxic lesions. Cells deficient in hSSB1 exhib-
ited increased radiosensitivity, defective checkpoint
activation and enhanced genomic instability coupled
with a diminished capacity for DNA repair. Thus,
K. Sakaguchi et al. The multi-replication protein A system
FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS 949
hSSB1 must influence diverse endpoints in the cellular
DNA damage response. In this way, hSSB1 resembles
the type B system.
Why are they not always found? The multi-RPA
types may resemble each other biochemically because
most of the subunits (large and ⁄ or middle) display a
significant degree of similarity. In many eukaryotes, the
multi-RPA system may diversify by exchanging some
subunits. For example, some of the non-homolog(s) of
hSSB1 are derived from convergent evolution. Further-
more, ubiquitous RPA (type B) is dispensable and can
easily be analyzed using the knockdown mutant,
whereas the type C or HsRPA complex (or hSSB2) is
lethal. However, very few researchers have studied these
mutants. Interestingly, the same phenomena was found
in Drosophila PCNAs, where the major PCNA is a
homolog of the ubiquitous PCNA in eukaryotes but is

dispensable [65]. Subsequently we analyzed the proper-
ties of these proteins in more detail. The role of the
miner subunit is not well understood because the
knockdown mutant is, as yet, unavailable [65].
A new perspective for RPA complexes
If multi-system RPAs are found to be universal each
of the corresponding functions should be reconsidered.
Nuclear RPAs may be divided into two categories: (a)
replication ⁄ transcription (plant C type), and (b)
repair ⁄ recombination (plant B type). The large subunit
may function as an agent for ssDNA stretching [1,2],
whereas the middle subunit may act as a signal trans-
duction acceptor. The small subunit may be a connect-
ing factor for forming the heterotrimeric complex.
Indeed, the small subunit mostly exists as a hetero-
dimer with the middle subunit, whereas the largest sub-
unit can be stabilized by binding to the dimer [10–12].
Genetic knockdown of the type 1 RPA increases the
lethality (i.e. the type C), but type 2 RPA can survive
unless the DNA is damaged (i.e. type B). Therefore,
subunit variety and function of the various subunits of
RPA must be reconsidered in view of these new find-
ings. For example, human RPA interacted with XPA
at sites of DNA damage, stimulated XPA–DNA inter-
action, and recruited the incision proteins
ERCC1 ⁄ XPF and XPG to the damaged site [89]. The
RPA must be a complex with HsRPA2, which corre-
sponds to type B. In NER and long-patch base exci-
sion repair, type B may be responsible for these
functions in eukaryote kingdoms.

The reported biological functions of mammalian and
yeast RPA are mostly involved in meiosis. The middle
subunit has an important role in regulating synaptine-
mal complex (SC) formation and meiotic recombina-
tion at meiotic prophase, mainly at zygotene and
pachytene [71–74,114,115,130]. The protein factors,
such as DNA polymerases and recombinases, are
major proteins involved in meiotic prophase events.
Nevertheless, RPA is known biochemically to interact
in vitro with DNA polymerases and recombinases
[6–8,13,31,40–42,44,72,85–88,138,145–169].
In fulfilling its biosynthetic roles in nuclear replica-
tion and in several types of repair, DNA polymerase is
assisted by RPA. In eukaryotes, recent investigations
have revealed at least 14 types of DNA polymerase
Fig. 3. Hypothetic model of the cellular
function of A-, B- and C-type RPA com-
plexes.
The multi-replication protein A system K. Sakaguchi et al.
950 FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS
(pol a, b, c, d, e, f, g, h, i, j, k, l, m and p) [45,170].
In a sense, all are analogs of each other. RPA is
reported to interact with at least pol a, d, e, k and j
[3,5–8,76,80,81,85–88]. RPA contributes to the high
fidelity of the polymerases during DNA synthesis. Of
the polymerase species, pol a, d and e replicate DNA
during S phase, but pol a is replication specific [80].
All the other polymerases are involved in DNA repair
and recombination [81]. We reported that in meiosis
two categories of DNA polymerases (a) pol a complex

and (b) pol k and l were expressed [165,168]. The
former is for replication at zygotene (or SC formation)
and the latter is for repair and recombination at late
zygotene to pachytene (Fig. 4) [155,165,168,171–173].
Using a D-loop recombination intermediate substrate,
we observed that either pol k or pol l can promote
the primer extension of an invading strand present in a
D-loop structure [168]. Both could fully extend the
primer in the D-loop substrate, suggesting that the
D-loop extension is an activity that is intrinsic to
the polymerases [168].
Two orthologs of the recombinases, Rad51 and
Lim15 ⁄ Dmc1, are present in meiosis [44,114,115,152–
154,161,162,167]. These recombinases occur at late
leptotene to early zygotene (Fig. 4). The interaction of
RPA and Rad51 is well established. Another meiotic
role of RPA was also found. At meiotic prophase (late
leptotene to early zygotene), with RPA, the homology-
search recombinase complex is involved in homologous
chromosome synapsis, preventing the formation of
superfluous reciprocal recombinant events (Fig. 4)
[114,115]. Both Rad51 and Lim15 ⁄ Dmc1 were identi-
fied as being involved in this process, although the
specific function of each protein is not yet known [44].
Are the DNA polymerase and recombinase
functions mediated by one species of RPA complex?
Interestingly, dephosphorylation of transformed nod-
ule-associated histone H2AX chromatin occurs at this
time. This suggests annealing of single strands or
repair of DSBs. By a similar mechanism, if the middle

subunit of RPA is also dephosphorylated, RPA would
lose the function of maintaining the noncross-over
condition. We must also consider the role of the multi-
RPA system during the meiotic prophase events.
It is known that a small amount of DNA replicates
at zygotene (pairing DNA synthesis) and that the
repair synthesis of DNA occurs at pachytene (cross-
over DNA synthesis) [172,173]. The two sequential
DNA synthesis reactions play a role in the progression
of meiosis. It is possible that a complex of RPA and
pol a differs from the recombination-dependent RPA.
Because DNA polymerase searches for the RPA–
Fig. 4. Hypothetic model of meiotic cell cycle and its relation to RPA.
K. Sakaguchi et al. The multi-replication protein A system
FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS 951
ssDNA complex structure on the DNA, RPA
complexed with pol a are probably functionally inde-
pendent from RPA complexed with other repair
polymerases. Pol k and l were thought to be involved
in the ‘crossover DNA synthesis’ for DNA recombina-
tion. Because the pol k(or the pol l)-deficient mutant
is viable, RPA may be like the type B or HsRPA2
type. However, ‘pairing DNA replication’ appears to
be specific for SC formation. At that stage, the DNA
polymerase a-catalytic subunit and primase are pre-
sumably also present [165]. This replication could be
the basis for SC extension and formation of the transi-
tion nodules [44]. Indeed, this process probably
requires RPA, such as the type C form (Fig. 4).
During prophase, DNA polymerases as well as

paralogs and orthologs of PCNA, recombinases
and perhaps RPA are required (Fig. 4) [42,44,45,
151,152,155,157,159,160,165,168,171]. Electron micros-
copy data [115,117] suggest that meiotic functions
in vivo are shared by each of the paralogs and ortho-
logs, and maybe also the analogs and heterologs.
Indeed, control of the biological process could be more
finely tuned by sharing function amongst paralogs,
orthologs, analogs and heterologs.
Background for the screening of
multiple protein systems involved in
DNA metabolism
We have studied many protein factors in DNA replica-
tion ⁄ repair and their relation to the meiotic system in
higher plants (O. sativa and A. thaliana) [13–
43,45,156,171], a fungus (C. cinereus) [44,138,145–155,
157–169] and an arthropod (Drosophila melanogaster)
[44–66]. Each of the materials represents the biological
kingdom of plant, fungus and animal, respectively.
Our research aimed to comprehensively understand
these DNA synthesis-related events in phylogenetically
diverse species. In addition to RPA, we elucidated
many of the related factors, such as Rad51,
Lim15 ⁄ Dmc1, RadA, PCNA, DDB, XRCC1, Rad2
family nucleases and special nucleases, DNA polyme-
rases, ORC1, RFC, RecQ, DNA ligases, CAF-1,
mtTFA, Rrp1, Mer3, Snm1, Rad6, SUMOylation fac-
tors (Aos1, Uba2, Ubc9, SUMO), leucine aminopepti-
dase and 26S proteasome-related factors (Jab1, Sgt1,
DnaJ) (Table 1). During the course of our experi-

ments, we frequently observed that protein factors
involved in the same DNA metabolic processes are not
always homologs in eukaryotic cells. Although the
paralogs and orthologs are ubiquitous, evolutionally
different factors were often found to be involved in
the same biosystems, which are referred to as ‘analogs’
and ‘heterologs’. Indeed, convergent evolution might
be ubiquitous in eukaryotic DNA metabolic processes.
According to definition, ‘homolog’ is a gene related to
a second gene by descent from a common ancestral DNA
sequence. ‘Ortholog’ is a gene in different species that
evolved from a common ancestral gene. ‘Paralog’ is a
gene related by duplication within a genome. Orthologs
retain the same function in the course of evolution,
whereas paralogs evolve new functions. ‘Analog’ is a gene
that has common activity but not a common origin.
‘Heterolog’ is a gene that differs in both origin and activ-
ity. Heterolog does not classify homolog, ortholog, par-
alog or analog. It may be also said that heterolog is used
as a synonym of ‘just different protein (gene)’, basically.
For example, PCNA is not one copy [65,138,159];
two PCNA paralogs and one PCNA-like heterotrimer
(Rad9–Rad1–Hus1) (‘analog’ or ‘heterolog’) were
found in Drosophila [65,136,137]. Rad9–Rad1–Hus1 is
found universally in eukaryotes. Plant SYCP1 and
yeast Zip1 mediate the same role in meiosis, despite
displaying no significant homology (‘analog’ or ‘het-
erologs’) [174,175]. Similarly, human mus81–Eme1 is
functionally the same as Escherichia coli RuvC (‘ana-
log’ or ‘heterologs’) [176–178]. In plants, two recA-like

protein paralogs (Rad51 and Lim15 ⁄ Dmc1) as well as
a prokaryotic recA homolog (RadA) were found (‘ana-
logs’) [42]. Furthermore, this is not the plastid compo-
nent [42]. As described earlier, in addition to the two
subtypes of RPA (HsRPA2 and HsRPA4) two human
SSB homologs are also present (‘analogs’) [70]. More-
over, in human, five Rad51 paralogs (Rad51B,
Rad51C, Rad51D, Xrcc2 and Xrcc3) have been found
[179–181]. Two FEN-1 paralogs (FEN-1a and FEN-
1b) and one analog (SEND-1) were found in plants
[25,26], and another FEN-1 analog occurs in Drosoph-
ila (GEN) [63,66]. DNA polymerases, especially for
DNA repair, are greatly diversified in eukaryotes
[76,182,183]. DNA polymerase b (pol b) for short
patch base excision repair are found only in verte-
brates [45]; plant short patch base excision repair uses
pol f instead [33,39,45]. However, as yet, a recBCD
homolog has not been found in the eukaryotic recom-
bination process. Prokaryotic homologs such as RadA
and hSSB are often found in eukaryotes (‘analog’ or
‘heterolog’), although there are the eukaryotic func-
tional alternatives [42,70]. All the protostomic animals
lack any X family DNA polymerases essential for
development of the nervous and immune system [45].
In Drosophila, AP endonuclease 1 homolog (Rrp1)
binds to pol f [64]. Plant XRCC1 lacks the polymer-
ase-binding domain [33,39]. Therefore, factor variation
(orthologs, paralogs, ‘analogs’ and ‘heterologs’) seems
to be ubiquitous in eukaryotic DNA metabolism.
The multi-replication protein A system K. Sakaguchi et al.

952 FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS
Table 1. The main role of DNA synthesis-related factor.
Protein Function Reference
RPA Required for DNA recombination, repair and replication.
The activity of RPA is mediated by ssDNA binding and protein
interactions
[1,10–12,30,43,124]
Rad51 May participate in a common DNA damage-response pathway
associated with the activation of homologous recombination and
double-strand break repair. Binds to ssDNA and dsDNA and exhibits
DNA-dependent ATPase activity. Unwinds duplex DNA and forms
helical nucleoprotein filaments
[152,154,184–186]
Lim15 ⁄ Dmc1 May participate in meiotic recombination, specifically in homologous
strand assimilation, which is required for the resolution of meiotic
double-strand breaks.
[99,152–154,159–163,
167,187]
RadA This family consists exclusively of archaeal RadA protein, a homolog of
bacterial RecA, eukaryotic Rad51 and archaeal RadB. This protein is
involved in DNA repair and recombination
[42,188]
PCNA PCNA, or cyclin, is a non-histone acidic nuclear protein that plays a key
role in the control of eukaryotic DNA replication. It acts as a co-factor
for DNA polymerase delta, which is responsible for leading strand
DNA replication
[21,30,31,34,65,138,
159,189,190]
DDB Required for DNA repair. Binds to DDB2 to form the UV-damaged
DNA-binding protein complex (the UV–DDB complex). The UV–DDB

complex may recognize UV-induced DNA damage and recruit proteins
of the NER pathway to initiate DNA repair
[27,30,36,52,54,58,
61,62,191]
XRCC1 DNA-repair protein Xrcc1 functions in the repair of ssDNA breaks in
mammalian cells and forms a repair complex with beta-Pol, ligase III
and PARP
[39,192,193]
Rad2 Single-stranded DNA endonuclease involved in excision repair of DNA
damaged with UV light, bulky adducts, or cross-linking agents.
Essential for the incision step of excision-repair
[25,63,66,194,195]
DNA polymerase DNA polymerase is an enzyme that catalyzes the polymerization of
deoxyribonucleotides into a DNA strand. Based on sequence
homology, DNA polymerases can be further subdivided into seven
different families: A, B, C, D, X, Y and RT
[16,22,24,33,38,40,45,46,
51,53,55,56,59,60,64,
76,145,151,155,157,158,
165,168,170,171,182,183]
ORC1 Component of the origin recognition complex that binds
origins of replication. It has a role in both chromosomal
replication and mating type transcriptional silencing.
Binds to the ARS consensus sequence
of origins of replication in an ATP-dependent manner
[19,41,196]
RFC A complex of five polypeptides in eukaryotes, and two in prokaryotes,
that loads the DNA polymerase processivity factor PCNA onto DNA,
thereby permitting processive DNA synthesis catalyzed by DNA
polymerase

[20,28,197]
RecQ The ATP-dependent DNA helicase RecQ is involved in genome
maintenance. All homologues tested to date unwind paired DNA,
translocating in a 3¢ to 5¢ direction and several have a preference for
forked or 4-way DNA structures (e.g. Holliday junctions) or for
G-quartet DNA
[37,198]
DNA ligase DNA ligase (polydeoxyribonucleotide synthase) is the enzyme that
joins two DNA fragments by catalyzing the formation of an
internucleotide ester bond between phosphate and deoxyribose.
It is active during DNA replication, DNA repair and DNA
recombination
[150,164,166,199]
CAF-1 Complex that is thought to mediate chromatin assembly in DNA
replication and DNA repair. Assembles histone octamers onto
replicating DNA in vitro. CAF-1 performs the first step of the
nucleosome assembly process, bringing newly synthesized histones
H3 and H4 to replicating DNA
[160,200,201]
K. Sakaguchi et al. The multi-replication protein A system
FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS 953
Table 1. Continued.
Protein Function Reference
mtTFA Involved in mitochondrial transcription regulation. Required for accurate
and efficient promoter recognition by the mitochondrial RNA
polymerase. Activates transcription by binding immediately upstream
of transcriptional start sites. Is able to unwind and bend DNA
[57,202,203]
Rrp1 Could promote homologous recombination at sites of DNA damage.
Has apurinic endonuclease and double-stranded DNA 3¢-exonuclease

activities and carries out single-stranded DNA renaturation in a
Mg
2+
-dependent manner. Activity is more efficient in purine-rich
regions of dsDNA than in pyrimidine-rich regions
[64,204–206]
Mer3 DNA-dependent ATPase. Required in the control of double strand
breaks transition and crossover during meiosis. Unwinds DNA in the
3¢ to 5¢ direction. Prefers ssDNA
[207–211]
Snm1 May be required for DNA interstrand cross-link repair. Also required for
checkpoint mediated cell cycle arrest in early prophase in response to
mitotic spindle poisons
[35,212,213]
Rad6 Ubiquitin-conjugating enzyme (E2), involved in postreplication repair
(with Rad18p), sporulation, telomere silencing, and ubiquitin-mediated
N-end rule protein degradation (with Ubr1p)
[32,214,215]
SUMOylation
(Aos1, Uba2, Ubc9, SUMO)
SUMO (small ubiquitin-like modifier) or SUMO proteins are a family of
small proteins that are covalently attached to and detached from
other proteins in cells to modify their function. SUMOylation is a
post-translational modification involved in various cellular processes,
such as nuclear–cytosolic transport, transcriptional regulation,
apoptosis, protein stability, response to stress and progression
through the cell cycle. In an ATP-dependent reaction, SUMO is
activated by a single specific E1-activating enzyme, the heterodimer
between Aos1 and Uba2 (or SAE1 ⁄ SAE2) resulting in an E1–SUMO
thioester linkage. SUMO is then transferred to the E2-conjugating

enzyme Ubc9, again forming a thioester. The catalytic cleft of Ubc9
directly interacts with many substrates via their SUMO consensus
motif. Target modification therefore often depends on a third class of
enzymes, the E3 ligases which enhance SUMO transfer from the E2
to the substrate
[44,163,216–219]
Leucine amino
peptidase
Presumably involved in the processing and regular turnover of
intracellular proteins. Catalyzes the removal of unsubstituted
N-terminal amino acids from various peptides
[146,220,221]
26S proteasome
related factor (Jab1)
Probable protease subunit of the COP9 signalosome complex (CSN), a
complex involved in various cellular and developmental processes.
The CSN complex is an essential regulator of the ubiquitin (Ubl)
conjugation pathway by mediating the deneddylation of the cullin
subunits of the SCF-type E3 ligase complexes, leading to decrease
the Ubl ligase activity of SCF-type complexes such as SCF, CSA or DDB2
[29,222]
26S proteasome
related factor (Sgt1)
Sgt1 is a novel subunit of the SCF ubiquitin ligase complex. Involved
in ubiquitination and subsequent proteosomal degradation of target
proteins. Required for both entry into S phase and kinetochore
function
[32,223]
26S proteasome
related factor (DnaJ)

Participates actively in the response to hyperosmotic and heat shock
by preventing the aggregation of stress-denatured proteins and by
disaggregating proteins, also in an autonomous, dnaK-independent
fashion. Unfolded proteins bind initially to dnaJ; upon interaction with
the dnaJ-bound protein, dnaK hydrolyzes its bound ATP, resulting in
the formation of a stable complex. GrpE releases ADP from dnaK;
ATP binding to dnaK triggers the release of the substrate protein,
thus completing the reaction cycle. Several rounds of ATP-dependent
interactions between dnaJ, dnaK and grpE are required for fully
efficient folding
[34,224,225]
The multi-replication protein A system K. Sakaguchi et al.
954 FEBS Journal 276 (2009) 943–963 ª 2009 The Authors Journal compilation ª 2009 FEBS
In conclusion, DNA synthetic systems are basically the
same from lower to higher eukaryotes, although related
factors are not necessarily homologs. In relation to
biological events, most, or all, of the repair factors are
involved in SC formation and meiotic recombination.
The presence of orthologs, paralogs, analogs and hetero-
logs should always be considered in the process. In meio-
sis, many of the factors require a protein that firstly
recognizes DNA structure to generate a highly organized
DNA–protein complex (i.e., replisome, repairsome or
recombinosome). Proteins such as RPA, PCNA, DDB or
FEN-1 may be responsible for defining the factor that
first recognizes the DNA structure. In our experiments,
many of the structure-recognition proteins tended to
diversify, suggesting that each of the factors can partici-
pate in generating the DNA–protein complexes.
In summary, we have used recent findings to specu-

late on the biological function of RPA. Figure 4 shows
the hypothetical model of the meiotic cell cycle at
prophase.
Acknowledgements
We thank Drs Yoichi Takakusagi, Seisuke Kimura,
Yoshihiro Kanai, Tatsushi Ruike and Taichi Yamam-
oto of our Department, for helpful discussions.
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