Architectures of archaeal GINS complexes, essential DNA replication initiation factors.

Abstract

In the early stage of eukaryotic DNA replication, the template DNA is unwound by the MCM helicase, which is activated by forming a complex with the Cdc45 and GINS proteins. The eukaryotic GINS forms a heterotetramer, comprising four types of subunits. On the other hand, the archaeal GINS appears to be either a tetramer formed by two types of subunits in a 2:2 ratio (α2β2) or a homotetramer of a single subunit (α4). Due to the low sequence similarity between the archaeal and eukaryotic GINS subunits, the atomic structures of the archaeal GINS complexes are attracting interest for comparisons of their subunit architectures and organization.
We determined the crystal structure of the α2β2 GINS tetramer from Thermococcus kodakaraensis (TkoGINS), comprising Gins51 and Gins23, and compared it with the reported human GINS structures. The backbone structure of each subunit and the tetrameric assembly are similar to those of human GINS. However, the location of the C-terminal small domain of Gins51 is remarkably different between the archaeal and human GINS structures. In addition, TkoGINS exhibits different subunit contacts from those in human GINS, as a consequence of the different relative locations and orientations between the domains. Based on the GINS crystal structures, we built a homology model of the putative homotetrameric GINS from Thermoplasma acidophilum (TacGINS). Importantly, we propose that a long insertion loop allows the differential positioning of the C-terminal domains and, as a consequence, exclusively leads to the formation of an asymmetric homotetramer rather than a symmetrical one.
The DNA metabolizing proteins from archaea are similar to those from eukaryotes, and the archaeal multi-subunit complexes are occasionally simplified versions of the eukaryotic ones. The overall similarity in the architectures between the archaeal and eukaryotic GINS complexes suggests that the GINS function, directed through interactions with other protein components, is basically conserved. On the other hand, the different subunit contacts, including the locations and contributions of the C-terminal domains to the tetramer formation, imply the possibility that the archaeal and eukaryotic GINS complexes contribute to DNA unwinding reactions by significantly different mechanisms in terms of the atomic details.

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RESEARCH ARTICLEOpen Access
Architectures of archaeal GINS complexes,
essential DNA replication initiation factors
Takuji Oyama1,5, Sonoko Ishino2, Seiji Fujino2, Hiromi Ogino2, Tsuyoshi Shirai3,5, Kouta Mayanagi4,5, Mihoko Saito3,
Naoko Nagasawa1, Yoshizumi Ishino2,5and Kosuke Morikawa1*
Abstract
Background: In the early stage of eukaryotic DNA replication, the template DNA is unwound by the MCM
helicase, which is activated by forming a complex with the Cdc45 and GINS proteins. The eukaryotic GINS forms a
heterotetramer, comprising four types of subunits. On the other hand, the archaeal GINS appears to be either a
tetramer formed by two types of subunits in a 2:2 ratio (a2b2) or a homotetramer of a single subunit (a4). Due to
the low sequence similarity between the archaeal and eukaryotic GINS subunits, the atomic structures of the
archaeal GINS complexes are attracting interest for comparisons of their subunit architectures and organization.
Results: We determined the crystal structure of the a2b2GINS tetramer from Thermococcus kodakaraensis
(TkoGINS), comprising Gins51 and Gins23, and compared it with the reported human GINS structures. The
backbone structure of each subunit and the tetrameric assembly are similar to those of human GINS. However, the
location of the C-terminal small domain of Gins51 is remarkably different between the archaeal and human GINS
structures. In addition, TkoGINS exhibits different subunit contacts from those in human GINS, as a consequence of
the different relative locations and orientations between the domains. Based on the GINS crystal structures, we
built a homology model of the putative homotetrameric GINS from Thermoplasma acidophilum (TacGINS).
Importantly, we propose that a long insertion loop allows the differential positioning of the C-terminal domains
and, as a consequence, exclusively leads to the formation of an asymmetric homotetramer rather than a
symmetrical one.
Conclusions: The DNA metabolizing proteins from archaea are similar to those from eukaryotes, and the archaeal
multi-subunit complexes are occasionally simplified versions of the eukaryotic ones. The overall similarity in the
architectures between the archaeal and eukaryotic GINS complexes suggests that the GINS function, directed
through interactions with other protein components, is basically conserved. On the other hand, the different
subunit contacts, including the locations and contributions of the C-terminal domains to the tetramer formation,
imply the possibility that the archaeal and eukaryotic GINS complexes contribute to DNA unwinding reactions by
significantly different mechanisms in terms of the atomic details.
Background
DNA replication is an essential event for all living
organisms, and thus the basic mechanism is conserved
from bacteria to eukaryotes. Genomic DNA replication
must be executed accurately and only once during the S
phase of the cell cycle. Rapid and accurate DNA replica-
tion requires the assembly of a large number of proteins,
termed the replisome, which directs major reactions,
such as origin recognition, template DNA unwinding,
and primer extension. Accumulating evidence has iden-
tified the essential proteins for DNA replication, which
have helped to provide a better understanding of the
complex puzzle of DNA replication [1]. For example, in
eukaryotes, a heterohexamer composed of the Mcm2-
Mcm7 subunits works as the MCM helicase to unwind
the template DNA, but this helicase activity is very low
in vitro [2]. While Schwacha’s group demonstrated the
significant helicase activity of yeast Mcm2-7 alone [3,4],
extra protein factors, which enhance the MCM helicase
* Correspondence: morikako@protein.osaka-u.ac.jp
1Laboratory of Protein Organic Chemistry, Institute for Protein Research,
Osaka University, Open Laboratories of Advanced Bioscience and
Biotechnology (OLABB), 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan
Full list of author information is available at the end of the article
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Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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activity, have been found, and in particular, Cdc45,
MCM, and GINS form a complex called the unwindo-
some or the CMG complex, which plays an essential
role in the template DNA unwinding reaction, and thus
this complex is considered to be the functional replica-
tive helicase [5-8]. GINS, named from the Japanese go-
ichi-ni-san, meaning 5-1-2-3, is a heterotetramer in
eukaryotes, comprising the Sld5, Psf1, Psf2, and Psf3
subunits, and is essential for both initiation and elonga-
tion in DNA replication [9-11]. GINS is recruited to the
replication origin with Dpb11 and Sld3 for the initiation
and may functionally interact with DNA polymerase ε
[9]. A functional interaction between human GINS and
DNA polymerase a/primase complex is also reported
[12], indicating its important role to move from initia-
tion to elongation of the DNA replication. GINS actually
forms a transient complex, designated as the preloading
complex (pre-LC), with DNA polymerase ε, Sld2, and
Dpb11, in a cyclin-dependent kinase (CDK)-dependent
manner in budding yeast [13].
In 2007, three research groups reported the crystal
structures of human GINS. Together, they showed a
rigid and stable tetrameric core structure, with multiple
flexible surface regions that are important for functional
interactions with other DNA replication proteins
[14-16]. Each subunit of human GINS consists of an a-
helical main domain and a b-stranded small domain,
and assembles into the heterotetramer, which exhibits a
unique trapezoidal or ellipsoidal shape. Interestingly, the
four subunits share a similar fold, in spite of the low
amino acid sequence identity of at most 15%. Further-
more, they are classified into two groups. One group,
including human Sld5 and Psf1, possesses the a-helical
(A) domain at the N-terminus and the b-stranded one
(B) at the C-terminus (this arrangement is called AB-
type), and the other group, comprising human Psf2 and
Psf3, is the permuted version (BA-type; Figure 1A)
[17-19].
It is well known that the DNA metabolizing proteins
from archaea are similar to those from eukaryotes, in
both structure and function [20]. Furthermore, the
archaeal DNA replication system appears to be a simpli-
fied version of the eukaryotic one. Therefore, structural
studies of the archaeal system could be beneficial to
understand the more complex DNA replication mechan-
ism of eukaryotes. Bioinformatics analyses using geno-
mic information from archaea and eukaryotes suggested
that the archaeal GINS complexes are simplified ver-
sions of the eukaryotic complexes. The archaeal com-
plexes are probably either a2b2 type tetramers
composed of the two types of subunits or a4type
homotetramers of a single subunit, suggesting that the
four different eukaryotic GINS subunits had diverged
from a common ancestor [17]. The functional tetrameric
assembly of the archaeal GINS has been proved by bio-
chemical analyses. An archaeal GINS homologue was
first identified in Sulfolobus solfataricus, as a binding
partner of MCM [18]. The GINS complex from Pyrococ-
cus furiosus increases the cognate MCM helicase activ-
ity, in a similar manner to eukaryotic GINS, in spite of
the lack of a eukaryotic Cdc45 homolog [21]. Due to the
low sequence similarity between the archaeal and eukar-
yotic GINS subunits, information about the three-
dimensional structure of archaeal GINS complexes is
crucial to obtain clearer insights into the functional sub-
unit assembly.
In this paper, we report the crystal structure of the
a2b2GINS tetramer from Thermococcus kodakaraensis
(TkoGINS), which consists of the Gins51 and Gins23
subunits. This first crystal structure of an archaeal GINS
was compared with that of human GINS. The overall
fold of each subunit and the tetrameric assembly of Tko-
GINS are essentially similar to those of human GINS.
However, the locations of the C-terminal small domains
are strikingly different between the two GINS, thus
resulting in the formation of different subunit contacts
in the complexes. Based on the two GINS crystal struc-
tures, we built a homology model of the putative homo-
tetrameric GINS from Thermoplasma acidophilum
(TacGINS), and found that TacGINS could form the
tetrameric subunit structure, in a similar manner to the
a2b2TkoGINS tetramer and the human GINS heterote-
tramer. These results suggest that the basic function of
GINS in DNA replication is conserved across the
domains of life.
Results and Discussion
Structure of TkoGINS
The crystal structure of TkoGINS was determined by
the multiple isomorphous replacement method with
anomalous dispersion (MIRAS), and the atomic model
was refined at 2.65 Å (Table 1). The crystal contains
one pair of the Gins51 and Gins23 subunits in the
asymmetric unit, and the protein forms the a2b2tetra-
mer generated by the crystallographic two-fold symme-
try operation (Figure 1B).
Gins51 is composed of the larger a-helical domain at
the N-terminus, and the smaller b-stranded domain at
the C-terminus. In the Gins51 subunit, the long linker
region between the two domains is disordered in the
crystal, and 16 residues could not be assigned in the
electron density map. Therefore, although we built an
atomic model with the C-terminal domain contacting
the nearest N-terminal one, the positions of the C-term-
inal domains may be swapped between the two Gins51
subunits to those in the tetrameric structure. Gins23
also possesses similar structural domains as in Gins51,
but the order of the two domains in the sequence is
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permutated. Each domain of Gins23 can be separately
superimposed on that of Gins51. The Gins23 N-terminal
domain superimposed on the Gins51 C-terminal one
with a root mean square deviation (RMSD) of 0.81 Å,
using the corresponding 38 Ca atoms, and the Gins23
C-terminal domain superimposed on the Gins51 N-
terminal one with an RMSD of 1.36 Å, using 89 Ca
atoms.
Architectural comparison between the T. kodakaraensis
and human GINS complexes
The Tko GINS tetrameric structure is similar, in terms
of shape and size, to the human GINS complex, com-
prising the four different subunits, Sld5, Psf1, Psf2 and
Psf3 [14-16]. While both crystal structures contain a
narrow central hole, an electron microscopic analysis of
human GINS revealed a horseshoe-shaped structure
Figure 1 Crystal structure of Thermococcus kodakaraensis GINS. (A) Domain organization of GINS from human, Thermococcus kodakaraensis,
and Thermoplasma acidophilum. Cylinders represent A-domains mainly composed of a-helices and arrows show B-domains rich in b-strands.
This coloring is used throughout the manuscript. The B domain of human Psf1 is missing in the crystal structures. (B) Ribbon representation of
the structure. The Gins51 subunits are colored green, and the Gins23 subunits are cyan. Missing parts are shown with dotted lines. The
crystallographic two-fold axis is indicated. The figure was prepared with PYMOL http://www.pymol.org.
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with a large central hole [22]. It is tempting to speculate
that GINS undergoes a conformational change between
the open and closed forms, and the crystal structure
exhibits a closed state. As expected, the spatial location
of the TkoGins51 subunit in the tetramer corresponds
to those in human Sld5 and Psf1, and that of TkoGins23
corresponds to those of Psf2 and Psf3 (Figure 2A). Tko-
Gins51 from the upper layer superimposed onto human
Sld5 and Psf1 with RMSDs of 1.7 Å and 2.3 Å, using 87
and 85 Ca atoms in the A domains, respectively (Figure
2B, upper line). In the case of the lower layer subunits,
the two Gins23 subunit structures are entirely similar to
their human counterparts, including the relative loca-
tions between the two domains. Indeed, TkoGins23
superimposed onto Psf2 with an RMSD of 2.1 Å using
93 Ca atoms, and onto Psf3 with an RMSD of 1.6 Å
using 82 Ca atoms. These structural similarities
highlight the evolutionary conservation between the
archaeal and eukaryotic GINS complexes (Figure 2B,
lower column). Notably, Swiatek and MacNeill reported
the structural similarity between the B domains of the
human GINS proteins and the C-terminal domain
(CTD) of the primase small subunit (PriS)-CTD) from
Sulfolobus solfataricus [23]. The B domains of the Tko-
GINS subunits are also similar to PriS-CTD [see Addi-
tional file 1], supporting an interesting hypothesis that
archaeal PriS acquired its CTD by straightforward
sequence duplication.
Despite the similarities in the internal architecture of
each subunit and the overall morphology of the tetra-
meric subunit assembly, notable differences are observed
between the archaeal and human GINS complexes.
Firstly, the locations of the C-terminal B domains are
strikingly different between the archaeal Gins51 and the
Table 1 X-ray diffraction data collection and refinement
Data Collection Summary
Derivative
Ta6Br14
1.2544
50.0-3.16
(3.27-3.16)
127012
9676 (901)
97.6 (95.1)
23.8 (11.4)
13.1 (13.5)
5.6 (23.0)
Native
1.0000
50.0-2.65
(2.74-2.65)
227882
16278 (1598)
99.4 (100.0)
17.6 (9.9)
14.0 (14.0)
4.3 (38.8)
SeMet
0.9795
50.0-2.80
(2.90-2.80)
181671
13780(1256)
99.1 (92.8)
14.5 (7.1)
13.2 (9.7)
7.5 (33.9)
K2PtCl4
1.0717
50.0-3.19
(3.30-3.19)
112023
9034 (921)
94.5 (99.9)
14.2 (7.4)
12.4 (9.3)
6.7 (38.6)
Wavelength
Resolution
(Å)
(Å)
(Highest shell)
Measured reflections
Unique reflections
Completeness
I/s(I)
Redundancy
Rmerge
MIRAS Phasing Statistics
(%)
(%)
Riso(F) (%)
Number of Sites
Resolution
Phasing Power (Centric/Acentric)
Figure of merit (Centric/Acen.)
12.4
2
50.0-4.0
0.56/0.57
21.1
6
50.0-4.0
0.64/0.54
17.1
1
50.0-4.0
0.63/0.62
(Å)
0.32/0.36
Refinement
Resolution
Rwork/Rfreea
Number of atoms
(Å)
(%)
50.0-2.65
25.7/29.7
Protein
Water
2623
33
Average B-factor(Å2)
Protein
Water
65.7
56.9
r.m.s.d.
Bond Lengths
Angles
PDB code
(Å)
(°)
0.01
1.364
3ANW
aRmerge= (Σ|II< II> |)/ΣI|II|, where < II> is the mean IIover symmetry-equivalent reflections.
bRwork=Σ|FO- FC|/Σ|FO| for all data excluding data used to calculate Rfree.
cRfreewas calculated using 5% of the total reflections, which were chosen randomly and omitted from the refinement.
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human Sld5. In the superimposed structures, the Tko-
Gins51 B domain is located about 30 Å away from that
of human Sld5 (Figure 2B). In the human complex, the
B domain of Sld5 contributes to the stable tetramer for-
mation, and the observed location in the crystal struc-
ture is in fact important for its function [15].
On the other hand, the corresponding domain of
human Psf1 is highly mobile. Kamada et al. and Choi et
al. crystallized the GINS proteins lacking the Psf1 B
domain [14,15]. Although Chang et al. solved the crystal
structure of the full-length GINS, they could not
observe the electron density for the Psf1 B-domain in
the crystal [16]. Thus, to determine whether the Gins51
B domain is required for the stable tetramer formation,
we examined the oligomeric state of a TkoGINS trunca-
tion mutant, lacking the B domain of Gins51, by gel fil-
tration. As shown in Figure 3A, this truncation mutant
forms a stable tetramer, establishing that the C-terminal
B domain of Gins51 is not essential for stable tetramer
formation. Notably, the isolated Gins51 subunit forms a
dimer irrespective of the presence or absence of the C-
terminal B domain, and the isolated Gins23 also eluted
Figure 2 Comparison of the archaeal and human GINS complexes. (A) Comparison of the tetrameric structures between the Thermococcus
kodakaraensis and human GINS complexes. (B) Comparison of the corresponding subunits.
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