The multi-replication protein A RPA system – a newperspective Kengo Sakaguchi, Toyotaka Ishibashi*, Yukinobu Uchiyama and Kazuki Iwabata Department of Applied Biological Science, Tokyo U
Trang 1The 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 yeastindicated that they encoded a single copy of eachsubunit of the RPA complex [1,2] However, werecently found that higher plants have at least threedifferent species of complex (types A, B and C), eachdisplaying a different biological function [10–12] Orig-inally, we intended to investigate the plant repairsystem [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,
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 vivoand in vitro approaches, to be required for most aspects of eukaryoticDNA metabolism: replication, repair, telomere maintenance and homolo-gous recombination Here, we review recent data concerning the functionand biological importance of the multi-RPA complex There are distinctcomplexes of RPA found in the biological kingdoms, although for a longtime only one type of RPA complex was believed to be present in eukary-otes Each complex probably serves a different role In higher plants, threedistinct large and medium subunits are present, but only one species of thesmallest subunit Each of these protein subunits forms stable complexeswith their respective partners They are paralogs as complex Humans pos-sess two paralogs and one analog of RPA The multi-RPA system can beregarded as universal in eukaryotes Among eukaryotic kingdoms, para-logs, orthologs, analogs and heterologs of many DNA synthesis-relatedfactors, including RPA, are ubiquitous Convergent evolution seems to beubiquitous 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.
Trang 2serendipitously 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 activareplica-tion 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 conservedthroughout eukaryotes and that the function is funda-mental irrespective of DNA synthesis Many data wereobtained on the assumption that there is just one RPAcopy 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 inDNA repair and is required for nucleotide excision
A
B
Fig 1 (A) RPA in the DNA replication (B) The role of RPA in NER.
Trang 3repair (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 proteinsfrom distinct processing pathways may use a smallnumber of common sites to bind RPA and remodelthe mode of DNA binding [124]
The RPA32kDa subunit is phosphorylated duringprogression of the cell cycle and in response to a widevariety of DNA-damaging agents, such as ionizingradiation, UV and camptothecin [120,126–128] RPAphosphorylation stimulated by DNA damage promotesDNA binding and chromatin association of ATR
in vitro via ATR interacting protein [83,122,129] RPA
is also required for recruitment and activation of theRad17 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 theMec1 protein kinase, a central checkpoint regulatorand homolog of human ATR [71–74] However, themeiosis-specific protein kinase Ime2 is required fornormal meiotic progression [130] A natural target ofIme2 activity is also the middle subunit of RPA [130].Ime2-dependent RPA phosphorylation first occursearly in meiosis The middle subunit is not supplemen-tary, but is a signal acceptor for sensing various struc-turally specific DNA sites Furthermore, RPA32kDa isreportedly related to viral DNA replication [124,131].There is almost no information concerning themolecular role of the RPA14kDa subunit It is knownthat RPA14kDa contains one weak DNA-bindingdomain, which may slightly modify the mode of DNAbinding of RPA
Consequently, it was generally believed that themajor roles of RPA had been elucidated However, atthis stage, it was not known that RPA representedmore 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 mals and yeasts, until 2001 little was known about thisprotein in plants Plants are affected by various envi-ronmental stress factors For example, DNA in plants
mam-is continuously damaged by UV irradiation from light UV is known to induce DNA damage [13],although plants generally have a higher tolerance for
sun-UV than animals Field-grown crops such as wheat arealso known to suffer continuous UV-induced DNAdamage Furthermore, the formation of reactiveoxygen species in cells due to UV irradiation, bioticstresses and secondary metabolism, causes cellularcomponents, including DNA, to be oxidized and there-
Trang 4fore 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 Surprisencod-ingly, 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 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-DNAinsertion or RNA interference (RNAi), the line could
larg-be viable [10–12] The surviving mutant was logically normal except for hypersensitivity towardssome mutagens, such as UV and methyl methanesulfo-nate (MMS) [10–12] Plants are naturally exposed to
morpho-UV for much longer than animals or yeast [133–135]and depend on sunlight for their development Becauseseed plants synthesize DNA under relatively high levels
of UV irradiation, the RPA system might be morecomplicated in plants than in animals
Therefore, we attempted to screen for rice RPA genes
in the genome (O sativa L cv Nipponbare) We foundthree different genes encoding the largest (RPA70kDa)and middle subunits (RPA32kDa), but only onegene encoding the smallest (RPA14kDa) [12] EachOsRPA70s 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] TheRPA70bsubunit is the ubiquitous RPA70 subunit found
in all eukaryotes [10] The various subunits do not domly associate with other subunits, but form a distinctcomplex Three different RPA complexes (A, B or Ctype) were composed of these subunits in vivo Types A,
ran-B and C were OsRPA70a–OsRPA32-2–OsRPA14,OsRPA70b–OsRPA32-1–OsRPA14 and OsRPA70c–OsRPA32-3–OsRPA14, respectively [11,12] Only thesmallest subunit is common to all the complexes.Because the system was also present in A thaliana[11,12], these properties may be universal in higherplants In conclusion, higher plants have a multi-RPAsystem [11,12]
The RPA complexes are spatially segregated inplants Type A is localized to the chloroplast, whereastypes B and C are found in the nuclear region [11] Inhuman and yeast cells, the middle subunit exists in thenucleus and cytoplasm, whereas the large subunit ispresent 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 freeheterodimer complexes such as OsRPA32-1–OsRPA14,OsRPA32-2–OsRPA14 and OsRPA32-3–OsRPA14[11,12]
In rice, co-regulation of OsRPA70b and OsRPA32-1during the cell cycle, and regulation of OsRPA32-1 inresponse to UV has been reported [43] RPA70kDahas 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
Trang 5the 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 OsRPA32kDasubunits were low [12] The B type complex wasthought 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-bindingactivity (Fig 2B) RPA70kDa also contains the DBD-
F domain, which has been shown to interact withmultiple proteins and to interact weakly with DNA(Fig 2B) The primary amino acid sequences ofDBD-A, DBD-B, DBD-C and DBD-F domains arevery similar [12] RPA32kDa has only a single ssDNA-binding domain (DBD-D) [12] Furthermore, all thedomains have high levels of sequence homology withtheir counterparts in human and yeast RPAs [12] TheDBD-E domain is in the RPA14kDa subunit, and isalso highly conserved [12]
In yeast, RPA1 (largest subunit) can only bind tothe RPA2⁄ 3 dimer (middle and smallest subunitdimer) The DBD-C and DBD-D regions of rice arequite similar to the DBD-C and DBD-D regions of
S cerevisiae [139], but OsRPA14 has only low larity to RPA3 This sequence divergence mayaccount for the differences in binding observedbetween the yeast and rice proteins Rice DBD-A andDBD-B domains are more conserved than DBD-Cand DBD-F, implying that the primary function ofOsRPA70a and OsRPA70b is to bind DNA, and thatthis function has been conserved during evolution,even though the secondary functions of these proteinsmay have diverged Based on this analysis the B typecomplex corresponds to the mammalian and yeastRPA
simi-In plant, human and yeast, the domains of DBD-Aand DBD-B are more homologous than those ofDBD-C and DBD-F, and the biochemical characteris-tics are common among OsRPA70a, OsRPA70b andOsRPA70c It is well established that the RPA70kDasubunit accumulates along stretches of ssDNA gener-ated by stalled replication forks and⁄ or DNA damage[1,82–84] In the RPA70kDa subunit, DBD-A andDBD-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 andDBD-D have a weak ssDNA–binding activity [12],whereas DBD-F interacts physically with the tumorsuppressor p53 and nucleosome remodeling complexFACT The interaction with DBD-F can also contrib-ute to an additional binding of structurally distortedDNA (i.e damaged DNA) By analogy, the primaryfunction of all the OsRPA70kDa subunits must be tofind special regions of DNA with which to bind Isthere a divergence in biochemical function among the
Trang 6various 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 establishwhether plants have paralogs or orthologs of hSSB Inparticular, we need to investigate the in vivo functions
of each of the A, B and C types of plant multi-RPAsystems
In vivo roles of the multi-RPA system
If the multi-RPA system is unique in plants, some ofthe 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 Asystem is thought to be plant specific, whereas types Band 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.
Trang 7to 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
AtRPA70aalso displayed lethality However, RNAi of
AtRPA70bwas 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 H2O2
After 1 week, growth of the wild-type plants was
inhibited by UV-B, MMS or H2O2 Compared with
wild-type plants, the growth of atrpa70b mutant and
AtRPA70bRNAi line seedlings was more inhibited by
UV-B, and was completely stopped by MMS [11,12].Mutants showed little increase in sensitivity to H2O2.Like the yeast rpa70 mutants, the atrpa70b mutantand AtRPA70b RNAi line are more sensitive thanwild-type to UV and MMS, suggesting that At-RPA70b is involved in the repair system for DNAdamaged by these mutagens [11,12]
The lethality of both the T-DNA insertion mutantand the RNAi line of AtRPA70a indicate that theAtRPA70a–AtRPA32-2–AtRPA14 complex plays anessential role, such as DNA replication, in the chlorop-lasts of living cells (Fig 3) By contrast, the mutantand RNAi line of AtRPA70b were viable but showedhigh 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 replicationand transcription (Fig 3) Subcellular localizationanalysis suggested that the type A RPA complex isrequired for chloroplast DNA metabolism, whereastypes B and C function in nuclear DNA metabolism[12]
Recently, RPA70 and RPA32 subunits from plantshave been reported to play a role in viral and transpo-son DNA syntheses [131,144] It will be intriguing toinvestigate how the RPA complex functions in thesemechanisms Higher plants may have evolved thetype A for the chloroplast to offer protection againsthigh levels of UV irradiation Indeed, as mentionedearlier, plants are exposed to UV radiation for muchlonger than animals or yeast Higher plants depend onexposure 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 inanimals and yeast
The human homologs of RPA32, HsRPA2 andHsRPA4 [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 inS-phase cells and hSSB1 deficiency did not influenceS-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 andanti-cancer drugs can induce DNA DSBs, which arehighly cytotoxic lesions Cells deficient in hSSB1 exhib-ited increased radiosensitivity, defective checkpointactivation and enhanced genomic instability coupledwith a diminished capacity for DNA repair Thus,
Trang 8hSSB1 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 formconnect-ing 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 thelethality (i.e the type C), but type 2 RPA can surviveunless the DNA is damaged (i.e type B) Therefore,subunit variety and function of the various subunits ofRPA 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 action, and recruited the incision proteinsERCC1⁄ XPF and XPG to the damaged site [89] TheRPA 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 thesefunctions in eukaryote kingdoms
inter-The reported biological functions of mammalian andyeast RPA are mostly involved in meiosis The middlesubunit has an important role in regulating synaptine-mal complex (SC) formation and meiotic recombina-tion at meiotic prophase, mainly at zygotene andpachytene [71–74,114,115,130] The protein factors,such as DNA polymerases and recombinases, aremajor 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 tion and in several types of repair, DNA polymerase isassisted by RPA In eukaryotes, recent investigationshave revealed at least 14 types of DNA polymerase
replica-Fig 3 Hypothetic model of the cellular function of A-, B- and C-type RPA com- plexes.
Trang 9(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 (lateleptotene to early zygotene), with RPA, the homology-search recombinase complex is involved in homologouschromosome synapsis, preventing the formation ofsuperfluous reciprocal recombinant events (Fig 4)[114,115] Both Rad51 and Lim15⁄ Dmc1 were identi-fied as being involved in this process, although thespecific function of each protein is not yet known [44].Are the DNA polymerase and recombinasefunctions mediated by one species of RPA complex?Interestingly, dephosphorylation of transformed nod-ule-associated histone H2AX chromatin occurs at thistime This suggests annealing of single strands orrepair of DSBs By a similar mechanism, if the middlesubunit of RPA is also dephosphorylated, RPA wouldlose the function of maintaining the noncross-overcondition 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 therepair synthesis of DNA occurs at pachytene (cross-over DNA synthesis) [172,173] The two sequentialDNA synthesis reactions play a role in the progression
of meiosis It is possible that a complex of RPA andpol 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.
Trang 10ssDNA 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 DNAsequence ‘Ortholog’ is a gene in different species thatevolved from a common ancestral gene ‘Paralog’ is agene related by duplication within a genome Orthologsretain the same function in the course of evolution,whereas paralogs evolve new functions ‘Analog’ is a genethat has common activity but not a common origin
‘Heterolog’ is a gene that differs in both origin and ity Heterolog does not classify homolog, ortholog, par-alog or analog It may be also said that heterolog is used
activ-as a synonym of ‘just different protein (gene)’, bactiv-asically.For example, PCNA is not one copy [65,138,159];two PCNA paralogs and one PCNA-like heterotrimer(Rad9–Rad1–Hus1) (‘analog’ or ‘heterolog’) werefound in Drosophila [65,136,137] Rad9–Rad1–Hus1 isfound universally in eukaryotes Plant SYCP1 andyeast Zip1 mediate the same role in meiosis, despitedisplaying no significant homology (‘analog’ or ‘het-erologs’) [174,175] Similarly, human mus81–Eme1 isfunctionally the same as Escherichia coli RuvC (‘ana-log’ or ‘heterologs’) [176–178] In plants, two recA-likeprotein paralogs (Rad51 and Lim15⁄ Dmc1) as well as
a prokaryotic recA homolog (RadA) were found logs’) [42] Furthermore, this is not the plastid compo-nent [42] As described earlier, in addition to the twosubtypes of RPA (HsRPA2 and HsRPA4) two humanSSB 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 forDNA repair, are greatly diversified in eukaryotes[76,182,183] DNA polymerase b (pol b) for shortpatch base excision repair are found only in verte-brates [45]; plant short patch base excision repair usespol f instead [33,39,45] However, as yet, a recBCDhomolog has not been found in the eukaryotic recom-bination process Prokaryotic homologs such as RadAand hSSB are often found in eukaryotes (‘analog’ or
(‘ana-‘heterolog’), although there are the eukaryotic tional alternatives [42,70] All the protostomic animalslack any X family DNA polymerases essential fordevelopment of the nervous and immune system [45]
func-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