Title: A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase α within the eukaryotic replisome
Abstract: Article6 August 2009free access A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase α within the eukaryotic replisome Agnieszka Gambus Agnieszka Gambus Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UKPresent address: Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dow Street, Dundee DD1 5EH, UK Search for more papers by this author Frederick van Deursen Frederick van Deursen Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Dimitrios Polychronopoulos Dimitrios Polychronopoulos Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Athens, Greece Search for more papers by this author Magdalena Foltman Magdalena Foltman Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Richard C Jones Richard C Jones FDA-NCTR, Jefferson, AR, USAPresent address: NextGen Sciences, Inc, 4401 Varsity Drive, Suite E, Ann Arbor, MI 48108, USA Search for more papers by this author Ricky D Edmondson Ricky D Edmondson FDA-NCTR, Jefferson, AR, USAPresent address: Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, 4301 W Markham #776, Little Rock, AR 72205, USA Search for more papers by this author Arturo Calzada Arturo Calzada Centro Nacional de Biotecnología, Campus de Cantoblanco, Madrid, Spain Search for more papers by this author Karim Labib Corresponding Author Karim Labib Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Agnieszka Gambus Agnieszka Gambus Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UKPresent address: Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dow Street, Dundee DD1 5EH, UK Search for more papers by this author Frederick van Deursen Frederick van Deursen Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Dimitrios Polychronopoulos Dimitrios Polychronopoulos Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Athens, Greece Search for more papers by this author Magdalena Foltman Magdalena Foltman Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Richard C Jones Richard C Jones FDA-NCTR, Jefferson, AR, USAPresent address: NextGen Sciences, Inc, 4401 Varsity Drive, Suite E, Ann Arbor, MI 48108, USA Search for more papers by this author Ricky D Edmondson Ricky D Edmondson FDA-NCTR, Jefferson, AR, USAPresent address: Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, 4301 W Markham #776, Little Rock, AR 72205, USA Search for more papers by this author Arturo Calzada Arturo Calzada Centro Nacional de Biotecnología, Campus de Cantoblanco, Madrid, Spain Search for more papers by this author Karim Labib Corresponding Author Karim Labib Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Author Information Agnieszka Gambus1,‡, Frederick van Deursen1,‡, Dimitrios Polychronopoulos2, Magdalena Foltman1, Richard C Jones3, Ricky D Edmondson3, Arturo Calzada4 and Karim Labib 1 1Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK 2Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Athens, Greece 3FDA-NCTR, Jefferson, AR, USA 4Centro Nacional de Biotecnología, Campus de Cantoblanco, Madrid, Spain ‡These authors contributed equally to this work *Corresponding author. Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. Tel.: +44 161 446 8168; Fax: +44 161 446 3109; E-mail: [email protected] The EMBO Journal (2009)28:2992-3004https://doi.org/10.1038/emboj.2009.226 Present address: Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dow Street, Dundee DD1 5EH, UK PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The eukaryotic replisome is a crucial determinant of genome stability, but its structure is still poorly understood. We found previously that many regulatory proteins assemble around the MCM2-7 helicase at yeast replication forks to form the replisome progression complex (RPC), which might link MCM2-7 to other replisome components. Here, we show that the RPC associates with DNA polymerase α that primes each Okazaki fragment during lagging strand synthesis. Our data indicate that a complex of the GINS and Ctf4 components of the RPC is crucial to couple MCM2-7 to DNA polymerase α. Others have found recently that the Mrc1 subunit of RPCs binds DNA polymerase epsilon, which synthesises the leading strand at DNA replication forks. We show that cells lacking both Ctf4 and Mrc1 experience chronic activation of the DNA damage checkpoint during chromosome replication and do not complete the cell cycle. These findings indicate that coupling MCM2-7 to replicative polymerases is an important feature of the regulation of chromosome replication in eukaryotes, and highlight a key role for Ctf4 in this process. Introduction The replication of large chromosomes that are compacted into chromatin is a major challenge for the proliferation of eukaryotic cells. The presence of single strand DNA at replication forks makes them vulnerable to attack by nucleases, and defects in chromosome replication are a major source of genome instability. To combat these dangers and preserve the integrity of the chromosomes, eukaryotic cells have evolved mechanisms that regulate the progression of DNA replication forks, monitor defects in DNA synthesis, and deal with problems when they occur (DePamphilis, 2006; Branzei and Foiani, 2007; Tourriere and Pasero, 2007; Aguilera and Gomez-Gonzalez, 2008). Chromosome replication in eukaryotic cells is a highly complex process that is still understood much less well than in prokaryotes. One reason for the greater complexity in eukaryotes is that DNA synthesis is coupled to other processes at replication forks, such as the establishment of cohesion between sister chromatids and the duplication of complex epigenetic patterns of histone modifications throughout each chromosome. Our current understanding of eukaryotic DNA replication forks was inspired by earlier work with bacteria, in which chromosome replication is understood in much greater detail. In Escherichia coli, progression of DNA replication forks is dependent on the DnaB DNA helicase that unwinds the parental DNA duplex. The DnaB helicase is connected physically to a subset of the other replication factors to form a larger assembly called the replisome (Yao and O'Donnell, 2008). In addition to DnaB, the replisome includes the DNA polymerase that works on the leading and lagging strand templates, the primase that initiates each new DNA molecule, and the γ-complex that loads the β-clamp that serves as a processivity factor for the DNA polymerase. Interactions between the components of the replisome serve to ensure that unwinding by DnaB is coordinated with efficient DNA synthesis on the leading and lagging strand. This minimises the period during which single strand DNA is exposed at forks, as well as influencing the rate at which forks move (Pomerantz and O'Donnell, 2007). The replisome in eukaryotic cells has been characterised in much less detail, and indeed the identities of the replicative helicase and the leading and lagging strand polymerases were only determined relatively recently (Kunkel and Burgers, 2008; Stillman, 2008). Each new DNA molecule is initiated by the DNA polymerase α complex, which contains both primase and DNA polymerase subunits. The leading strand is then extended by DNA polymerase epsilon (Pursell et al, 2007), whereas each Okazaki fragment on the lagging strand is completed by DNA polymerase delta (Nick McElhinny et al, 2008). Progression of eukaryotic DNA replication forks is dependent on the activity of the Mcm2-7 DNA helicase, the activity of which is regulated very carefully in vivo so that each origin fires once and a single copy of the genome is produced during each cell cycle (Blow and Dutta, 2005; Sclafani and Holzen, 2007). It seems likely that MCM2-7 will be coupled physically to replisome components such as DNA polymerases and primase in an analogous manner to the situation in E. coli, to regulate fork progression and help preserve genome stability. For example, work with yeast shows that depletion of deoxynucleotides slows the progression of MCM2-7 in addition to slowing the movement of DNA polymerases (Aparicio et al, 1997; Katou et al, 2003). But the nature of such links between MCM2-7 and other replisome components is poorly understood. Previous work has shown that the MCM2-7 helicase associates with many other factors during the process of chromosome replication. These include Cdc45 and the four-protein GINS complex that are also important for fork progression (Tercero et al, 2000; Kanemaki et al, 2003; Kubota et al, 2003; Takayama et al, 2003; Pacek and Walter, 2004), and that form the ‘CMG’ complex (Cdc45-MCM-GINS) or ‘unwindosome’ together with MCM2-7 (Moyer et al, 2006; Pacek et al, 2006; Aparicio et al, 2009). Other factors associating with MCM2-7 in budding yeast include the checkpoint mediator Mrc1, the Tof1–Csm3 complex that is required for forks to pause at protein–DNA barriers, the histone chaperone FACT, the type I topoisomerase Top1, and Mcm10 and Ctf4 that are known to bind DNA polymerase α (Homesley et al, 2000; Katou et al, 2003; Ricke and Bielinsky, 2004; Nedelcheva et al, 2005; Gambus et al, 2006). By purifying GINS from budding yeast cell extracts, we found that all of the above proteins form a very large assembly called the replisome progression complex or RPC (Gambus et al, 2006). The RPC only exists during S-phase, occurs on chromatin, requires the prior loading of MCM2-7 at origins during G1-phase, and persists for as long as DNA replication forks are still present when nucleotide production is inhibited (Gambus et al, 2006). It thus appears that the RPC represents the fraction of MCM2-7 helicase that is present at DNA replication forks and is therefore likely to be a key component of the replisome. The consequences of RPC formation are poorly understood but are likely to include activation of the MCM2-7 helicase by recruitment of other factors required for fork progression. The Cdc45 protein is required in addition to MCM2-7 for the unwinding of the parental DNA duplex at forks (Tercero et al, 2000; Pacek and Walter, 2004), and GINS is also important for fork progression and is required to preserve a stable interaction between MCM2-7 and Cdc45 (Kanemaki et al, 2003; Gambus et al, 2006). A second reason for RPC assembly could be to connect the MCM2-7 helicase to other components of the replisome such as DNA polymerases. Consistent with this view, recent work has shown that Mrc1 associates with DNA polymerase epsilon throughout the cell cycle and is a key determinant of the rate of progression of DNA replication forks (Szyjka et al, 2005; Tourriere et al, 2005; Hodgson et al, 2007; Lou et al, 2008). In addition, interaction between Mcm10 and Ctf4 has been shown to be important for recruitment of Pol α to chromatin during initiation of chromosome replication in extracts of Xenopus eggs (Zhu et al, 2007). But the subsequent contribution of Mcm10 and Ctf4 at forks to the stable link between DNA polymerase α and the MCM2-7 helicase was not addressed. Here, we provide direct evidence that the RPC is associated at forks with DNA polymerase α. Our data indicate that a complex of GINS and Ctf4 has an important function in coupling MCM2-7 to Pol α. Ctf4 and Mrc1 are crucial for the normal regulation of chromosome replication and their combined absence leads to chronic activation of the DNA damage checkpoint and cell cycle arrest. Results The RPC associates with DNA polymerase α As a first step towards characterising how the RPC might interact with other components of the replisome, we wanted to isolate the complex from budding yeast under very mild conditions, and then use mass spectrometry to identify any associated proteins in an unbiased manner. Originally, we purified RPCs from cell extracts that contained 300 or 700 mM potassium acetate, as the RPC is stable even at very high concentrations of salt (Gambus et al, 2006). It seemed likely, however, that the interaction of the RPC with other replisome components might not survive these rather harsh conditions. We repeated the purification, therefore, using a cell extract containing 50 mM potassium acetate, to try and preserve weaker ionic interactions between the RPC and other replication factors that might normally occur under physiological conditions. We found previously that GINS could still interact in the presence of 50 mM potassium acetate with all RPC components except Mcm10, which associates preferentially with the RPC in the presence of higher salt concentrations, perhaps through hydrophobic interactions (Gambus et al, 2006). We used budding yeast cells in which the Sld5 subunit of GINS was fused to the TAP tag, and in which the Mcm4 subunit of the MCM2-7 helicase ended with five copies of the FLAG epitope. We grew an 8L asynchronous culture of TAP-SLD5 MCM4-5FLAG cells, together with a culture of MCM4-5FLAG control cells in which the Sld5 subunit of GINS was untagged. Cell extracts were generated in the presence of 50 mM potassium acetate and chromosomal DNA was digested to completion with DNase I. We then incubated the extracts with IgG Sepharose to isolate GINS from the TAP-SLD5 strain but not from the control, before purifying MCM2-7 from the resultant material to yield RPCs. The purified samples from the two strains were separated in an SDS–PAGE gel, and each lane was cut subsequently into 40 slices. Mass spectrometry was then used to identify all the factors that were only present in the sample purified from the TAP-SLD5 MCM4-5FLAG strain. As expected, the purified sample contained all of the previously identified RPC components with the exception of Mcm10 (Figure 1; Supplementary Figure 1). Strikingly, however, the purified RPC material contained four additional proteins that were not detected in our previous experiments using cell extracts containing 300 or 700 mM potassium acetate. These factors comprised the four subunits of DNA polymerase α (Figure 1), indicating that a fraction of Pol α exists in a common complex with MCM2-7 and GINS at DNA replication forks. These findings imply that one role of the RPC is to connect the MCM helicase to DNA polymerase α. Figure 1.The RPC associates with DNA polymerase α. A cell extract of TAP-SLD5 MCM4-5FLAG (YAG236-2) was generated in the presence of 50 mM potassium acetate and the RPC was purified as described in Materials and methods. The final material was separated in a 4–12% SDS–PAGE gradient gel and the lane was then cut into 40 slices. The graphs illustrate the total number of peptides that were identified by mass spectrometry in each slice of the gel, for a representative selection of the identified proteins. More details are shown in Supplementary Figure 1. The numbers in brackets indicate the combined Mascot score for each protein, corresponding to the sum of the unique peptide scores. Download figure Download PowerPoint Ctf4 is required to couple DNA polymerase α to the RPC To begin to explore the significance of the association of the RPC with DNA polymerase α, we wanted to determine which RPC subunits are responsible for the interaction. Work with yeast has shown that Mcm10, Ctf4 and FACT are all able to associate individually with Pol α (Miles and Formosa, 1992b; Wittmeyer and Formosa, 1997; Ricke and Bielinsky, 2004), and the orthologues of Mcm10 and Ctf4 in human cells and Xenopus laevis have also been found to interact with DNA polymerase α (Chattopadhyay and Bielinsky, 2007; Zhu et al, 2007). However, the contribution of these factors to the stable association at forks of DNA polymerase α with MCM2-7 helicase has not been addressed earlier. It is interesting to note that Pol α was found to co-purify with the RPC in our experiments under conditions in which Mcm10 is displaced from the RPC. This indicates that other factors contribute to the interaction between MCM2-7 and Pol α at DNA replication forks. We found earlier that both Mcm10 and FACT co-purify with the MCM2-7 helicase during G1-phase and S-phase, whereas Ctf4 only interacts with MCM2-7 during S-phase as part of the RPC (Gambus et al, 2006). To determine when MCM2-7 associates with DNA polymerase α, we synchronised cells in G1-phase by treatment with mating pheromone, and then washed cells into fresh medium lacking mating pheromone so that they could enter S-phase (Figure 2A). At each time point, we generated cell extracts and digested the chromosomal DNA, before isolating the Mcm4 protein by immunoprecipitation and analysing the associated proteins by immunoblotting. In contrast to Mcm10 and FACT (Gambus et al, 2006), DNA polymerase α did not associate with the MCM2-7 helicase during G1-phase. Instead, Pol α co-purified with MCM2-7 during S-phase with similar kinetics to Ctf4 and other RPC components (Figure 2B). The interaction is likely to be independent of DNA, as the chromosomal DNA was digested to undetectable levels during the course of the experiment (Figure 2C), and the interaction was not affected by addition of ethidium bromide to the cell extract (Figure 2D). We note that Pol α is also recruited to chromatin later than Mcm10 during chromosome replication in extracts of Xenopus eggs, with similar timing to And-1/Ctf4 (Zhu et al, 2007). Taken together, these findings suggested that Ctf4 might be important to maintain the link between MCM2-7 and DNA polymerase α at replication forks. Figure 2.MCM2-7 associates with DNA polymerase α only during S-phase when RPCs are present. (A) An asynchronous culture of MCM4-5FLAG POL1-6HA PRI1-9MYC (YFJD62) was arrested in G1-phase at 24°C with mating pheromone before release into S-phase for the indicated times. DNA content was measured by flow cytometry. (B) Cell extracts were generated from the same experiment and treated with benzonase to digest chromosomal DNA as described in Materials and methods, before centrifugation and isolation of Mcm4-5FLAG by immunoprecipitation. The indicated proteins were analysed by immunoblotting. (C) Digestion of chromosomal DNA was monitored in an analogous experiment to that described in (A). Most DNA was digested during the initial 30′ treatment with Benzonase before centrifugation (compare lanes 1 and 2). The remainder was removed by centrifugation at 16 000 g for 30′ (lane 3). Even without centrifugation, all chromosomal DNA was degraded to undetectable levels during the time taken for the complete immunoprecipitation procedure (lane 4). (D) The experiment in (A) was repeated and samples taken 20′ after release from G1-phase. Benzonase and ethidium bromide were added to the initial extract as indicated, before immunoprecipitation of Mcm4. Download figure Download PowerPoint To test this idea more directly, we purified RPCs from extracts of ctf4Δ cells in the presence of 50 mM potassium acetate and then analysed the resulting material as above. As shown in Figure 3A and Supplementary Figure 2, Pol α did not co-purify with RPCs in the absence of Ctf4. In addition, however, the Spt16 and Pob3 subunits of the FACT complex were largely but not completely displaced from purified RPCs in the absence of Ctf4 (Figure 3A; Supplementary Figure 2). It was possible, therefore, that FACT also helps to maintain the link between MCM2-7 and Pol α, but our preliminary findings indicate that this is not the case (Magdalena Foltman and Karim Labib, unpublished data). Moreover, the stability of Pol α, Mcm10 and FACT in yeast cell extracts is independent of Ctf4 (Figure 3B). As Ctf4 is known to bind directly to Pol α (Miles and Formosa, 1992b), and both factors associate with MCM2-7 with similar kinetics (Figure 2; Gambus et al, 2006), these data indicate that Ctf4 has a direct role in preserving the link between PoI α and the RPC. Figure 3.Ctf4 is required for stable association of the RPC with DNA polymerase α. (A) RPC material was purified from ctf4Δ TAP-SLD5 MCM4-5FLAG (YAG374-2) and analysed as described above for Figure 1. The graphs illustrate a representative selection of the identified proteins, and the full data set is shown in Supplementary Figure 2. (B) The levels of Pol1, Mcm10, Spt16 and Pob3 were monitored by immunoblotting in extracts of control and ctf4Δ cells. Download figure Download PowerPoint Ctf4 binds directly to the GINS component of the RPC To understand how Ctf4 contributes to the link between Pol α and the MCM2-7 helicase within the RPC, it was necessary to determine how Ctf4 itself associates with the RPC. The interaction of most components of the RPC with either GINS or MCM2-7 only occurs during S-phase and is dependent on prior loading of the MCM2-7 helicase at origins of DNA replication. These interactions might require post-translational modifications that only occur during the initiation reaction at origins of DNA replication, or could involve multiple RPC components that are only brought together at origins when the whole complex is assembled. In contrast, Mcm10 and FACT co-purify with MCM2-7 (but not GINS) even from cells in G1-phase and these interactions are thought to be direct. Ctf4 co-purifies with MCM2-7 only during S-phase (Figure 2; Gambus et al, 2006) but is unique amongst RPC subunits as a fraction of Ctf4 can associate with GINS throughout the cell cycle (Figure 4A). Consistent with this fact, Ctf4 still associates with GINS when RPC formation is blocked by inhibiting the prior loading of the MCM2-7 helicase at origins during G1-phase, in contrast to other RPC components such as MCM2-7 or Cdc45 (Figure 4B). These findings show that at least some fraction of GINS and Ctf4 form part of a common complex even away from the RPC. Moreover, we showed earlier that GINS is not only required to recruit Ctf4 to origins and to the MCM2-7 helicase, but is also needed subsequently to maintain the interaction of MCM2-7 with Ctf4 within the RPC (Gambus et al, 2006). Taken together, these data suggest that the association of Ctf4 with RPCs might involve direct interaction of Ctf4 with GINS. Figure 4.A fraction of Ctf4 can interact with GINS throughout the cell cycle and not just in RPCs. (A) Cultures of PSF2-TAP (YAG187-1) were grown at 24°C either asynchronously (Asyn.), or arrested in G1-phase with mating pheromone (G1), or released into S-phase from G1 in the presence of 0.2 M hydroxyurea for 60′ (S), or arrested in G2/M-phase with nocodazole (G2/M). Psf2-TAP was isolated by immunoprecipitation and the indicated proteins analysed by immunoblotting. (B) A cdc6Δ∷GAL-CDC6 TAP-SLD5 strain (YAG258-1) was grown at 24°C in rich medium containing galactose (YPGal), in parallel with a TAP-SLD5 control strain (YAG236-2), and cells were arrested in G2-M-phase with nocodazole. Expression of GAL-CDC6 was then repressed in fresh medium containing glucose (YPD) as well as nocodazole, before cells were released into fresh YPD medium to allow synchronization in the following G1-phase with mating pheromone. At this stage, the MCM helicase was assembled at origins into prereplicative complexes in the control strain (+ pre-RCs), but not in the GAL-CDC6 strain (− pre-RCs). Finally, cells were released into S-phase for 60′ in the presence of 0.2 M HU. TAP-Sld5 was isolated by immunoprecipitation and the indicated proteins analysed by immunoblotting. Download figure Download PowerPoint To test whether GINS and Ctf4 do indeed bind each other directly, we determined whether these factors are able to interact in an extract of E. coli cells, in the absence of other eukaryotic proteins. We generated an E. coli strain that expressed the four GINS proteins as part of a common operon, and a second E. coli strain that expressed Ctf4. We then mixed the cells and generated a single cell extract containing GINS, Ctf4 and all the native E. coli proteins. We purified GINS from the extract by virtue of a GST tag on the Psf3 subunit of GINS, and found that GINS co-purified with Ctf4, in contrast to the 4000 native E. coli proteins (Figure 5A, E1). Ctf4 was then isolated from this material through a six-histidine tag at its amino terminus, and was again seen to co-purify specifically with the four GINS proteins (Figure 5A, E2). This shows that GINS and Ctf4 are able to interact directly to form a specific and stable complex. Figure 5.GINS and Ctf4 interact directly to form a stable complex. (A) Recombinant GINS was purified from an E. coli cell extract that also contained recombinant Ctf4 as described in Materials and methods, by virtue of a GST tag on the Psf3 subunit of GINS (E1). The purified material was separated in an SDS–PAGE gel that was stained with Coomassie blue. The band marked by an asterisk (*) was identified by mass spectrometry and corresponds to free GST that might have been derived as a degradation product from GST-Psf3. Ctf4 was then isolated from the purified GINS sample using a six-histidine tag at its amino terminus, and the figure shows the flow through of this purification step (FT), the washes (W1–W3), and the final eluate that represents purified GINS–Ctf4 complex (E2). The identity of the various bands was confirmed by mass spectrometry. (B) A similar experiment was performed using an E. coli extract containing recombinant Sld5–Psf2 subcomplex of GINS (with the GST tag on Sld5), together with 6His-Ctf4. (C) Summary of the interaction of truncated forms of recombinant Ctf4 with GINS in an E. coli extract, in an assay analogous to that described above. (D) An extract of E. coli was generated that contained recombinant Ctf4-ΔNT with 6His at its amino terminus, and recombinant GINS with the Streptag III epitope at the amino terminus of Psf3 as well as a truncated form of Psf1 (Psf1-ΔCT, amino acids 1–164) to improve visualisation of the four GINS proteins in the subsequent gel. StreptagIII-Psf3 was isolated from the extract and 6His-Ctf4ΔNT was then purified from the resultant material. The purified complex of GINS and Ctf4ΔNT was applied to a gel filtration column, and migrated with a retention volume of 50 ml (distinct from the void volume of 46 ml). The peak fractions were combined and concentrated before separation in an SDS–PAGE gel that was then stained with Coomassie blue. (E) Comparison of the migration through gel filtration columns of purified recombinant versions of GINS-Ctf4ΔNT, GINS and Ctf4ΔNT. Download figure Download PowerPoint To investigate which subunits of GINS might be required to interact with Ctf4, we generated recombinant subcomplexes comprising Sld5–Psf1–Psf2 and Sld5–Psf2 and found that both could still interact directly with Ctf4 in extracts of E. coli cells (Figure 5B, and F van Deursen unpublished data). This indicates that Ctf4 interacts with either or both of the Sld5 and Psf2 subunits of the GINS complex. Unfortunately, Sld5 and Psf2 are not soluble on their own, and so a more complete understanding of the interaction between Ctf4 and Sld5–Psf2 awaits future structural studies of these factors. Direct binding of Ctf4 to GINS and the amino terminus of Pol1 does not require the WD40 domain of Ctf4 We then investigated which domains of the Ctf4 protein are required for its interaction with GINS. The tertiary structure of Ctf4 has not been determined, but BLAST searches indicate that the protein begins with a WD40 domain, which is thought to mediate protein–protein interactions (Figure 5C). Orthologues of Ctf4 have a similar predicted structure although Ctf4 in higher eukaryotes has an additional HMG-like domain at the carboxy terminus. We found that the WD40 domain is dispensable for the interaction of Ctf4 with GINS, which instead is mediated by the remainder of the protein (Figure 5C; Supplementary Figure 3). To illustrate this fact, we generated an extract of E. coli cells containing GINS and Ctf4-ΔNT, purified the GINS–Ctf4ΔNT complex in an analogous manner to that described above, and ran the purified material through a gel filtration column. This generated a single peak of protein that comprised the recombinant GINS–Ctf4ΔNT complex (Figure 5D), and that migrated more quickly than either GINS or Ctf4-ΔNT (Figure 5E). Note that the Ctf4 protein (and Ctf4-ΔNT) behaves as a multimer, and this is true of the endogenous protein in yeast extracts as well the purified recombinant protein (A Gambus and K Labib, unpublished data). One of the original studies to identify Ctf4 showed that it could be isolated from a yeast extract using an affinity column bearing the Pol1 catalytic subunit of DNA polymerase α (Miles and Formosa, 1992b), although the nature of the interaction between Ctf4 and DNA polymerase α is not well understood. A later study combined Ctf4 in a two-hybrid assay with the first