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EMBO Rep. 2006 May; 7(5): 539–545.
Published online 2006 Feb 17. doi:  10.1038/sj.embor.7400649
PMCID: PMC1479547
Scientific Report

GINS, a central nexus in the archaeal DNA replication fork


In eukaryotes, the GINS complex is essential for DNA replication and has been implicated as having a role at the replication fork. This complex consists of four paralogous GINS subunits, Psf1, Psf2, Psf3 and Sld5. Here, we identify an archaeal GINS homologue as a direct interaction partner of the MCM helicase. The core archaeal GINS complex contains two subunits that are poorly conserved homologues of the eukaryotic GINS subunits, in complex with a protein containing a domain homologous to the DNA-binding domain of bacterial RecJ. Interaction studies show that archaeal GINS interacts directly with the heterodimeric core primase. Our data suggest that GINS is important in coordinating the architecture of the replication fork and provide a mechanism to couple progression of the MCM helicase on the leading strand with priming events on the lagging strand.

Keywords: replication, MCM, GINS, archaea, DNA


The archaeal DNA replication apparatus is fundamentally related to that of eukaryotes (Kelman & Kelman, 2003). In general, the archaeal machinery is a simplified version of that found in eukaryotes; for example, the presumptive replicative helicase, the MCM complex, is a heterohexamer in eukaryotes but a homo-multimer in archaea.

The eukaryotic GINS complex is composed of four subunits, Psf1, Psf2, Psf3 and Sld5, that are represented in all sequenced eukaryotic genomes (Kanemaki et al, 2003; Kubota et al, 2003; Takayama et al, 2003). Sequence comparisons show that these four subunits are distantly related to one another and, accordingly, seem to be ancient paralogues (Makarova et al, 2005). By contrast, most archaea encode a single GINS homologue.

GINS is essential for both initiation and elongation phases in eukaryotic DNA replication; however, its molecular basis of action is unclear at present. GINS binding to origins of replication requires MCM and is important for recruitment of the DNA polymerase (Kanemaki et al, 2003; Kubota et al, 2003; Takayama et al, 2003). There is increasing evidence that, although initially recruited to replication origins, GINS moves with the replication fork (Calzada et al, 2005). In the current work, we identify an archaeal GINS complex and show that it directly physically interacts with the archaeal MCM. Additional interaction studies implicate GINS as a central nexus in the replication fork, with the potential to act as a bridge between MCM and primase.

Results and Discussion

In the course of a two-hybrid screen to identify potential binding partners of the MCM protein of the hyperthermophilic archaeon Sulfolobus solfataricus, we identified open reading frame SSO0772 as a candidate interaction partner. As the initial construct identified did not contain the full-length SSO0772 open reading frame, we prepared plasmid constructs that would express full-length SSO0772 protein fused to GAL4 DNA-binding domain (DBD) or activation domain (AD) in yeast. Yeast cells were transformed with these constructs together with MCM-DBD- and MCM-AD-expressing plasmids. As can be seen in Fig 1A, the SSO0772 protein interacts with MCM in both DBD and AD fusion contexts. Next, we tested the ability of SSO0772 to interact with amino- and carboxy-terminally truncated MCM. The results shown in Fig 1B indicate that SSO0772 interacts with the N-terminal 266 residues of MCM.

Figure 1
An archaeal GINS homologue binds to MCM. (A) Yeast two-hybrid analysis showing interaction between SSO0772/Gins23 and MCM. Yeast cells were transformed with plasmids containing DNA-binding domain (DBD) or activation domain (AD) fused to MCM (M) or the ...

As shown previously, SSO0772 is one of the archaeal homologues of the eukaryotic GINS subunits (Makarova et al, 2005). Iterative database searches using the PSI-BLAST program with the predicted protein sequence of SSO0772 showed that it is most similar to Psf2 and Psf3 but also shows significant, though weaker, similarity to the other two GINS components, Sld5 and Psf1. Previous analyses have shown that the four eukaryotic GINS subunits are related to one another and, accordingly, must have evolved from a common ancestor by gene duplication events (Makarova et al, 2005). As SSO0772 is most similar to Psf2 and Psf3, we propose the name gins23 for this gene and will use this nomenclature in the following discussion.

In the S. solfataricus genome (She et al, 2001), the gins23 gene is located immediately upstream of the gene for the MCM protein (Fig 1C). Indeed, the two open reading frames overlap by 11 bp. This organization is suggestive of co-transcription of the gins23 and mcm genes. We tested this hypothesis by performing reverse transcription–PCR (RT–PCR) using primers within the gins23 and mcm open reading frames. The data confirm that a transcript containing Gins23 and MCM coding sequences can be detected in RNA isolated from S. solfataricus cells, indicative of coexpression of the genes (Fig 1D).

Gins23 was expressed in Escherichia coli as a recombinant C-terminally hexahistidine-tagged protein. The protein was highly insoluble and could only be purified in the presence of 8 M urea. The insolubility did not seem to be a function of the presence of the His6 tag on the protein, as native-length, non-tagged, protein expressed in E. coli was similarly insoluble (data not shown). The purified His-tagged protein was used to generate rabbit polyclonal antisera. The antisera recognized a single protein of 21 kDa in extracts prepared from S. solfataricus cells (Fig 2A). Western blotting of whole-cell extracts prepared from logarithmically growing and stationary-phase S. solfataricus (Fig 2B) showed that, like MCM, the protein was still present in non-replicating cells, in contrast to the initiator protein Cdc6-1 (Robinson et al, 2004).

Figure 2
(A) Anti-Gins23 antisera recognize a single band in Sulfolobus whole-cell extracts. Western blots were performed on 10 μg of whole-cell extracts with pre-immune (Pre) or anti-Gins23 (Imm) antisera. (B) Western blotting of extracts prepared from ...

To test whether the MCM–Gins23 interaction detected in the yeast two-hybrid assay was representative of a physiologically relevant interaction in S. solfataricus cells, we carried out immunoprecipitation assays. The results indicate that Gins23 and MCM can be co-immunoprecipitated. Further, the insensitivity of the co-immunoprecipitation to the presence of ethidium bromide or DNaseI (data not shown) indicates that DNA is unlikely to be acting as a bridge between MCM and Gins23 (Fig 2C). We tested the ability of MCM and Gins23 to co-precipitate from extracts prepared from cells in early and mid-logarithmic growth and also from stationary phase. We find a very modest increase in co-immunoprecipitation of MCM with Gins23 from the extract prepared from mid-logarithmic cells, perhaps indicative of a higher proportion of cycling cells in this population (Fig 2D).

Whereas recombinant Gins23 was chronically insoluble in E. coli cells, native Gins23 seemed to be highly soluble in extracts from S. solfataricus cells prepared in salt concentrations ranging from 50 mM to 1 M NaCl (data not shown). We speculated therefore that the endogenous Gins23 might be associated with other proteins in S. solfataricus, thereby facilitating its solubility. To test this, we undertook purification of the endogenous Gins23. First, we fractionated crude cell extract by anion exchange chromatography and then used immunoaffinity chromatography to isolate endogenous Gins23-containing complex. As can be seen in Fig 3A, three proteins eluted specifically from the anti-Gins23 antisera column. These were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry as Gins23 and the products of the SSO0295 and SSO1049 open reading frames. The immediate genomic environment of SSO0295 does not contain any genes of known or predicted function in DNA replication. Intriguingly, however, as shown in Fig 3B, SSO1049 is encoded immediately downstream of the gene for the catalytic subunit of the heterodimeric DNA primase, in an operon-like organization that also contains the gene for the sliding clamp subunit PCNA2 (Dionne et al, 2003) in addition to ribosome- and translation-associated factors.

Figure 3
Isolation of a Gins23-containing complex from Sulfolobus solfataricus extracts. (A) Immunoaffinity purification of GINS. The sizes of markers (M) are shown on the left of the panel. Input material is shown (In). The eluted material from columns coupled ...

PSI-BLAST searches indicated that the protein encoded by SSO0295 was related to the DBD of the bacterial RecJ family of single-stranded DNA exonucleases (Yamagata et al, 2002); however, SSO0295 lacked a counterpart to the C-terminal, catalytic domain of RecJ. Hereinafter, we refer to this protein as RecJdbh, after RecJ DNA-binding domain homologue (data not shown). Extensive PSI-BLAST searches with the sequence of SSO1049 showed that it was a highly diverged member of the GINS family of proteins (Fig 3C), more closely related to Psf1 and Sld5 than to Psf2 and Psf3. Accordingly, we shall refer to this gene as gins15.

To test that the immunoaffinity approach used to purify the complex described above had not led to artefactual co-purification of the three proteins, we undertook a partial purification of the putative Gins23-containing complex using conventional chromatography (Fig 3D). As can been seen in Fig 3E, the two GINS family members and RecJdbh co-purified in eight steps, supporting the notion that they exist as a stable complex in cells.

We next performed further interaction studies to investigate the architecture of the Gins23–Gins15–RecJdbh complex and to determine whether further interactions could be detected with other DNA replication-associated proteins. A two-hybrid matrix indicated that Gins15 could interact with itself, with Gins23 and with RecJdbh. As described above, Gins23 interacted with MCM and also with both large and small subunits of the archaeal primase (Fig 4A). No interactions could be detected between any of Gins23, Gins15 or RecJdbh and Cdc6-1, Cdc6-2, Cdc6-3, SSB, UDG1, DNA polB1, Fen1, the two RFC subunits or any of the three Sulfolobus PCNA subunits (data not shown).

Figure 4
Architecture of GINS and a model for the replication fork. (A) Summary of two-hybrid analyses. ‘++' indicates growth on media lacking histidine and adenine and ‘+' indicates growth on media lacking histidine. ( ...

To test further the interactions with primase and MCM, we coexpressed Gins23 and Gins15 in E. coli. This permitted purification of a soluble complex containing both proteins in an equimolar ratio (Fig 4B). Analytical gel filtration gave an apparent molecular mass of 67 kDa. As a dimer of Gins23 and Gins15 would have a mass of 37 kDa, it seems that the core archaeal GINS complex is tetrameric, containing a dimer of dimers. The tetrameric structure of the archaeal GINS complex is in good agreement with the proposed quaternary structure of the eukaryal GINS complex (Kubota et al, 2003; Takayama et al, 2003).

The purified archaeal GINS complex was next used in ‘pull-down' assays with His6-tagged primase (tagged on the PriL subunit; Lao-Sirieix & Bell, 2004) and His6-tagged MCM. As can be seen in the upper panels of Fig 4C,D, GINS was retained on columns containing MCM or primase, but not by the matrix alone, or by matrix containing PCNA2 (not shown). Finally, we tested whether addition of the S. solfataricus GINS complex to MCM helicase assays or primase assays could have any influence on the activity of primase or MCM. However, no significant effects could be detected (data not shown).

Our results describe the first characterization of an archaeal GINS complex. As shown in Fig 4E, our data indicate that the complex is a tetramer containing two copies of Gins15 and Gins23. As Gins15 can self-interact and also bind to Gins23, we propose that a Gins15 homodimer lies at the heart of the complex, with each Gins15 protomer binding to one Gins23 subunit. These data are in agreement with the proposed architecture of the eukaryal GINS complex (Kanemaki et al, 2003; Kubota et al, 2003; Takayama et al, 2003).

The interaction of Gins23 with primase and MCM suggests that GINS could act as a molecular bridge between MCM and the primase. We have tested for, but failed to detect, direct interactions between the archaeal primase and MCM (Fig 4A, and C,D, lower panels). This contrasts with the situation in bacteria, in which the primase DnaG forms a functional complex with the helicase DnaB (Soultanas, 2005). However, there is an important difference between the bacterial helicase and the MCM complex. Bacterial helicases have 5′ to 3′ polarity and so, in the classical model of the replication fork, track along the lagging strand. The physical association of bacterial primase and helicase therefore serves to couple initiation of Okazaki fragment synthesis on the lagging strand with replication fork progression. In contrast, MCM has 3′ to 5′ polarity and thus would track along the leading strand. By interacting with the trailing N-terminal domains of the MCM helicase and serving as a bridge to the primase, GINS might facilitate coupling of MCM progression on the leading strand with the action of primase on the lagging strand template (Fig 4E). Given the conservation of GINS and MCM and the requirement for GINS in both initiation and elongation phases of replication, we propose that this architecture is conserved in eukarya (Kanemaki et al, 2003; Kubota et al, 2003; Takayama et al, 2003). Indeed, recent work from Karim Labib and colleagues has identified an MCM- and GINS-containing ‘replisome progression complex' in yeast (K. Labib, personal communication). Finally, the interaction of a homologue of the DBD of RecJ with the archaeal GINS complex is intriguing. In E coli, RecJ is important in the processing of stalled replication forks (Courcelle et al, 2003). It is tempting to speculate that, in Sulfolobus, RecJdbh, by virtue of its interaction with the GINS complex, might have a role in detection and signalling of stalled replication fork structures. Thus, archaeal GINS might have dual roles in facilitating monitoring and maintenance of replication fork progression.


Yeast two-hybrid screen. This was performed using a library of S. solfataricus genomic DNA as described previously (Dionne & Bell, 2005). The bait construct contained the full-length open reading frame of S. solfataricus MCM in the pGBKT7 vector (Clontech, Mountain View, CA, USA).

Immunoprecipitation. Frozen cell pellets of exponentially growing S. solfataricus were resuspended in 20 ml of 1 × TBS (150 mM NaCl, 10 mM Tris–HCl (pH 8.0)) per gram of pellet. Triton X-100 was added to 0.1% to lyse the cells. After a 5 min incubation on ice, lysates were clarified (35,000g, 20 min, 4°C) twice and passed through a 0.45 μm syringe filter. Filtered lysates (100 μl) were incubated with 2 μl of the specified antisera and 24 μg/ml ethidium bromide, if applicable, at 4°C. After 90–120 min, 50 μl of protein A beads equilibrated in TBST (1 × TBS, 0.1% (v/v) Tween 20) and 20 μg/ml ethidium bromide, if applicable, were added, and the incubation was repeated. After 90–120 min, the supernatants were removed and saved for analysis. Beads and associated material were washed on ice four times in 1 ml TBST over a period of 30 min, resuspended in 50 μl 1 × LB (1.05% (w/v) SDS, 62.5 mM Tris–HCl (pH 6.8), 10% (v/v) glycerol, 0.35 M β-mercaptoethanol, 0.0125% (w/v) bromophenol blue), boiled (5 min), resolved by SDS–polyacrylamide gel electrophoresis and immunoblotted.

Immunopurification of native GINS complex. A 5 g portion of S. solfataricus P2 biomass was resuspended in 25 ml of 10 mM Tris (pH 8.0), following which Triton X-100 was added to 0.1%, incubated for 5 min at 4°C and then sonicated. The extract was clarified by centrifugation (15 min, 4°C, 35,000g). The extract was dialysed overnight against 10 mM Tris (pH 8.0), re-clarified by centrifugation and applied to an 18 ml Q-Sepharose column, then washed and eluted with a linear gradient to 10 mM Tris (pH 8.0) and 1 M NaCl. Then, 10 ml fractions were collected and Gins23-positive fractions identified by western blotting. The three peak fractions were pooled, dialysed against 10 mM Tris (pH 8.0), 150 mM NaCl and 0.1% Tween 20, and 15 ml was applied to either specific or control immunoaffinity columns. Immunoaffinity columns were prepared using 1 ml HiTrap rProtein A columns to which 2 ml of either pre-immune or anti-Gins23 antisera was bound and crosslinked using dimethylpimelimidate. Following application to the columns, the columns were washed with 10 mM Tris (pH 8.0) and 700 mM NaCl and eluted with glycine (pH 2.5)+0.5% SDS. Then, 1 ml fractions were collected and concentrated by trichloroacetic acid precipitation before resolution by SDS–polyacrylamide gel electrophoresis. The GINS complex eluted after 4 column volumes of glycine (pH 2.5)+0.5% SDS had been applied. Protein species were identified by MALDI-TOF mass spectrometry in the MRC Laboratory of Molecular Biology.

Chromatographic purification of native GINS complex. During purification, the Gins23 complex was followed by western blotting of fractions. A measure of 100 ml of extract was prepared from 20 g of biomass as described above. Following clarification, polyethyleneimine was added slowly to a final concentration of 0.1% and the solution was incubated with mixing for 30 min at 4°C. Precipitated material was collected by centrifugation as before and resuspended in 50 ml of 10 mM Tris (pH 8.0) and 1 M NH4SO4 and dialysed overnight against 5 l of the same buffer. The resolubilized material was clarified as above and applied to a 20 ml Butyl Sepharose column at 2 ml/min, washed and eluted with 200 ml gradient to 10 mM Tris (pH 8.0). Gins23-containing fractions were dialysed overnight into 10 mM Tris (pH 8.0) and 50 mM NaCl and applied to a Mono Q HR10/10 column. The column was developed with a gradient to 10 mM Tris (pH 8.0) and 300 mM NaCl, positive fractions pooled and concentrated to 8 ml in Vivaspin 5k concentrator before application to Superdex 200 column in 10 mM Tris (pH 8.0) and 150 mM NaCl at 2 ml/min. Positive fractions were pooled, dialysed against 10 mM sodium phosphate (pH 6.6), applied to hydroxyapatite column and eluted with a gradient to 500 mM sodium phosphate (pH 6.6). Positive fractions were pooled and dialysed against 100 volumes of 10 mM Tris (pH 6.0) and 50 mM NaCl, and passed through a 5 ml HiTrap heparin column (the Gins23 complex eluted in the flowthrough). This material was then applied to a 1 ml Mono Q HR5/5 and eluted with a linear gradient to 10 mM Tris (pH 6.0) and 250 mM NaCl.

Reverse transcription–PCR. An S. solfataricus culture was grown to an OD600 of 0.27. RNA was extracted using the RNeasy Mini Kit (Qiagen, Crawley, UK) and treated with 7.5 U RNase-free DNaseI (Roche, Basel, Switzerland) for 1 h at 37°C in a 50 μl reaction containing 0.25 × NEBuffer 3 (New England Biolabs, Ipswich, MA, USA), and cleaned up with the RNeasy Mini Kit (Qiagen). A 10 μg portion of RNA was used with the OneStep RT–PCR Kit (Qiagen) under the following PCR conditions: 45 s denaturation, 45 s annealing (60°C), 29 cycles. Sequences of the gins23-and mcm-specific primers are available on request.

Purification of recombinant proteins. The Gins23 and Gins15 open reading frames were cloned into the pET-Duet vector (Novagen, EMD Biosciences, San Diego, CA, USA). This was transformed into BL21 AI (Stratagene, La Jolla, CA, USA) and protein expression induced according to the manufacturer's instructions. Cell lysate was prepared in 10 mM Tris (pH 8.0) and 300 mM NaCl and clarified by centrifugation. The soluble material was heated to 80°C for 30 min and re-centrifuged. The supernatant was dialysed against 10 mM Tris (pH 8.0) and 1 M NH4SO4 and purified over Butyl Sepharose, Mono Q and Superdex 200 columns as described above, before concentration in a Vivaspin concentrator. His-tagged and untagged MCM and primase were purified as described previously (Lao-Sirieix & Bell, 2004; McGeoch et al, 2005).

Supplementary information is available at EMBO reports online (http://www.emboreports.org).

Supplementary Material

Supplementary Information


We thank K. Labib for communicating results before publication and for invaluable discussions. We are indebted to S.Y. Peak-Chew of the MRC laboratory of Molecular Biology for mass spectrometry. Work in S.D.B.'s laboratory is funded by the Medical Research Council and the Nuffield Foundation.


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