Communication between subunits within an archaeal clamp-loader complex
2006; Springer Nature; Volume: 25; Issue: 10 Linguagem: Inglês
10.1038/sj.emboj.7601093
ISSN1460-2075
AutoresAnja Seybert, Martin R. Singleton, Nicola Cook, David R. Hall, Dale B. Wigley,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle20 April 2006free access Communication between subunits within an archaeal clamp-loader complex Anja Seybert Anja Seybert Search for more papers by this author Martin R Singleton Martin R Singleton Search for more papers by this author Nicola Cook Nicola Cook Search for more papers by this author David R Hall David R Hall Search for more papers by this author Dale B Wigley Corresponding Author Dale B Wigley Clare Hall Laboratories, Cancer Research UK, London Research Institute, South Mimms Potters Bar, Herts, UK Search for more papers by this author Anja Seybert Anja Seybert Search for more papers by this author Martin R Singleton Martin R Singleton Search for more papers by this author Nicola Cook Nicola Cook Search for more papers by this author David R Hall David R Hall Search for more papers by this author Dale B Wigley Corresponding Author Dale B Wigley Clare Hall Laboratories, Cancer Research UK, London Research Institute, South Mimms Potters Bar, Herts, UK Search for more papers by this author Author Information Anja Seybert‡, Martin R Singleton‡, Nicola Cook, David R Hall and Dale B Wigley 1 1Clare Hall Laboratories, Cancer Research UK, London Research Institute, South Mimms Potters Bar, Herts, UK ‡These authors contributed equally to this work *Corresponding author. Clare Hall Laboratories, Cancer Research UK, London Research Institute, Blanche Lane, South Mimms Potters Bar, Herts EN6 3LD, UK. Tel.: +44 207 269 3930; Fax: +44 207 269 3803; E-mail: [email protected] The EMBO Journal (2006)25:2209-2218https://doi.org/10.1038/sj.emboj.7601093 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have investigated the communication between subunits in replication factor C (RFC) from Archaeoglobus fulgidus. Mutation of the proposed arginine finger in the small subunits results in a complex that can still bind ATP but has impaired clamp-loading activity, a process that normally only requires binding of nucleotide. The small subunit alone forms a hexameric ring that is six-fold symmetric in the absence of ATP. However, this symmetry is broken when the nucleotide is bound to the complex. A conformational change associated with nucleotide binding may relate to the opening of PCNA rings by RFC during the loading reaction. The structures also reveal the importance of the N-terminal helix of each subunit at the ATP-binding site. Analysis of mutant protein complexes containing subunits lacking this N-terminal helix reveals key distinct regulatory roles during clamp loading that are different for the large and small subunits in the RFC complex. Introduction Replicative polymerases from eukaryotes to prokaryotes obtain processivity using ring-shaped DNA sliding clamps that are loaded onto DNA by clamp-loader proteins in ATP-dependent reactions. Although the amino-acid sequences and the protein complex compositions differ between the various systems, both the overall structure of the clamp/clamp-loader proteins and the molecular mechanisms of the clamp-loading process appear to be conserved (Jeruzalmi et al, 2002). The Escherichia coli γ complex, which has an in vivo composition of γ3δδ′χψ (Pritchard et al, 2000), is the most thoroughly analysed clamp loader. A heteropentamer consisting only of γ3δδ′ has been shown to be able to load the dimeric β sliding clamp onto DNA (Onrust and O'Donnell, 1993). Crystal structures of the γ3δδ′ complex and of the δ subunit in a complex with a mutant monomeric form of β have been published (Jeruzalmi et al, 2001a, 2001b). Together with earlier biochemical data (Naktinis et al, 1995), these crystal structures suggested that the δ subunit contacts the β clamp and traps β in a conformation where one of the two clamp interfaces is open. ATP binding alone, but not hydrolysis, is essential for the E. coli clamp loader to open the sliding clamp and to load it onto a primer/template junction of DNA (Turner et al, 1999). Biochemical studies have shown that this is also true for replication factor C (RFC), the eukaryotic and archaeal clamp loader (Gomes and Burgers, 2001; Seybert and Wigley, 2004). Unlike the E. coli clamp loader, eukaryotic RFC consists of five different subunits (RFC1–RFC5) (Cullmann et al, 1995). However, like the minimal E. coli clamp loader (γ3δδ′), the composition of RFC is heteropentameric (Lee et al, 1991). The crystal structure of a mutant yeast RFC complexed with PCNA revealed details of the interactions within the complex (Bowman et al, 2004). Despite being crystallised with a very poorly hydrolysable ATP analogue (ATPγS), the structure determined was of a catalytically inactive mutant protein in which the four ‘arginine fingers’ of the small subunits were replaced by glutamine. Furthermore, even though the structure contains bound nucleotides and it has been established that clamp loading by RFC requires ATP binding but not hydrolysis (Gomes et al, 2001; Seybert and Wigley, 2004), the complex is clearly not in a conformation that could load PCNA onto DNA because the PCNA ring is topologically closed. With sequence identities of 30–40%, the clamp loaders of Archaea seem to be a closely related, yet simplified, version of their eukaryotic counterparts. Thus, in contrast to eukaryotic clamp loaders in which each of the five different RFC subunits have been found to be individually essential (Cullmann et al, 1995), only two RFC-like subunits are found in each of the completely sequenced archaeal genomes (Cann and Ishino, 1999). The conserved RFC boxes II–VIII can be found in both the large and the small subunits that constitute archaeal clamp loaders (Cann and Ishino, 1999). Archaeal clamp loaders from Sulfolobus solfataricus, Methanobacterium thermoautotrophicum, Pyrococcus furiosus and Archaeoglobus fulgidus have been isolated and shown to stimulate the processivity of their replicative polymerases in PCNA-dependent reactions (Kelman and Hurwitz, 2000; Pisani et al 2000; Cann et al, 2001; Seybert and Wigley, 2004). The two subunits of archaeal RFC form a complex with 1:4 (large to small) stoichiometry (Pisani et al, 2000; Cann et al, 2001; Seybert et al, 2002). Electron microscopy (EM) studies of the wild-type archaeal RFC/PCNA complex from P. furiosus (Shiomi et al, 2000; Miyata et al, 2004, 2005) have revealed important insights into the assembly of the complex, with results that differed from the crystal structure of the mutant yeast RFC/PCNA (Bowman et al, 2004). Although the earlier low-resolution (23 Å) images were misinterpreted, once higher resolution (12 Å) images were available it became clear exactly how the complex was assembled. Unlike the yeast structure, the PCNA ring is opened in the EM images. This appears to be a consequence of the PCNA interacting with multiple subunits of RFC that are arranged in a spiral, as seen in all clamp-loader structures. This results in a conformation of the PCNA that is reminiscent of a ‘lock washer’. However, the crack in the PCNA ring is not wide enough (only 5 Å) to allow passage of duplex DNA, but could allow single-stranded DNA to enter the ring, although the EM analysis also showed that duplex DNA was bound at the centre of the RFC/PCNA complex. As a result, the authors suggest that either PCNA is loaded onto the single-strand DNA ahead of the template and the RFC/PCNA complex, then slides back onto the primer template junction or, alternatively, that there is a conformational change that might open up the ring further to allow entry of duplex DNA directly, the nature of which is presently unknown. However, just such a conformational change was observed upon binding of ATP using EM, atomic force microscopy and biochemical methods (Shiomi et al, 2000), although the limited resolution of these studies precluded details of the nature of this conformational change. Using mutant archaeal clamp loaders deficient in either ATP binding or hydrolysis in different subunits, it was revealed that the different subunits use ATP binding and hydrolysis in distinct ways at different steps in the loading process (Seybert and Wigley, 2004). Binding of nucleotide by the large subunit and three of the four small subunits is sufficient for clamp loading. However, ATP hydrolysis by the small subunits is required for release of PCNA to allow the subsequent formation of the complex between PCNA and the polymerase. By contrast, ATP hydrolysis by the large subunit is required for catalytic clamp loading. In the present study, we have investigated communication between subunits in the RFC holoenzyme from A. fulgidus. Mutation of the arginine finger in the small subunits results in a complex that is able to bind ATP but has impaired ATPase activity. However, quite unexpectedly, the mutant complex is also deficient in clamp loading, an activity that does not require ATP hydrolysis. Crystal structures of the small subunit alone reveal that it forms a hexameric ring that, in the absence of bound nucleotide, is six-fold symmetric. However, this symmetry is broken when nucleotide is bound to the complex. The large conformational change observed may relate to the opening of PCNA rings that is required for them to be loaded onto DNA substrates. The structures also suggest a key role for the N-terminal helix of each subunit, a feature that is conserved across clamp-loading proteins and is known as RFC box II (Cullmann et al, 1995). Analysis of mutant protein complexes containing subunits lacking this N-terminal helix reveal different regulatory roles in each subunit, both of which are key to coupling ATP hydrolysis with clamp loading. Results Arginine finger mutant Many oligomeric NTPases have the NTP-binding site located between protein subunits, with residues from both subunits contributing to the NTPase activity. Commonly, there is an arginine residue extending from a neighbouring subunit into the NTP-binding site that interacts with the γ-phosphate, thereby promoting catalysis, often referred to as an ‘arginine finger’ (Scheffzek et al, 1997). RFC small subunits contain a conserved sequence motif (SRC) in which the central arginine residue is implicated as an arginine finger (Bowman et al, 2004) By contrast, the RFC large subunit lacks this motif. In order to test the effect of replacing this residue in the small subunits, we mutated Arg-152 of the small subunits to alanine and reconstituted RFC complexes containing a wild-type large subunit and mutant small subunits (denoted LaWT/SmSAC). Our first experiments were to determine the stoichiometry of nucleotide binding by the LaWT/SmSAC complex using a spin-column assay described previously (Seybert and Wigley, 2004) (Table I). These data revealed that the mutant complex, in the presence of PCNA, was able to bind four nucleotide molecules in common with the wild type, albeit with slightly reduced nucleotide affinity. We have shown previously that these four nucleotides are bound to the large subunit and three of the four small subunits (Seybert and Wigley, 2004). Although competent to bind nucleotide, the LaWT/SmSAC complex had an ATPase activity that was severely compromised (Table I), consistent with the yeast arginine finger mutant RFC complex (Bowman et al, 2004). However, we went on to investigate whether the archaeal mutant complex was able to catalyse clamp loading and primer extension, neither of which was analysed for the mutant yeast enzyme. Interestingly, the archaeal mutant complex was completely deficient in loading of PCNA onto circular DNA substrates (Figure 1) and, hence, was also unable to support primer extension (data not shown). The inability to load clamps is particularly interesting since we, and others, have shown previously that ATP binding without hydrolysis is sufficient for clamp loading (Turner et al, 1999; Gomes and Burgers, 2001; Seybert and Wigley, 2004). In particular, an RFC complex with wild-type large subunits and mutant small subunits that are able to bind, but not hydrolyse ATP, and which retain the arginine finger (LaWT/SmBmut), are fully competent in stoichiometric loading of PCNA (Figure 1A). Although deficient in PCNA release after loading, the LaWT/SmBmut complex is able to support primer extension under conditions where the polymerase level is not rate limiting (Seybert and Wigley, 2004). By contrast, the defect in the primer extension assay for the Lawt/SmSAC cannot be overcome even when the polymerase concentration is raised 10-fold (Figure 1B). Since both the the LaWT/SmSAC and LaWT/SmBmut mutant complexes are able to bind nucleotide, yet the LaWT/SmSAC alone is unable to load PCNA, this suggests that there is a step after ATP binding, but preceding hydrolysis, that is required for clamp loading and that this step requires an intact arginine finger. We sought information on the nature of this step from structural studies of the RFC small subunits, since these appear to be the key to understanding the link between the arginine finger at the ATP-binding site and loading of PCNA. Figure 1.(A) PCNA-loading assays were performed using a spin-column assay. Reactions contained 8 pmol 32P-PCNA, 4 pmol RFC complexes and 4 pmol nicked pUC19 DNA. Samples 1, 3 and 4 contained 5 mM ATP, sample 2 contained 5 mM ATPγS. The spin columns were pre-equilibrated in 200 mM NaCl. The eluates of the reactions were separated by SDS–PAGE in a 15% gel and quantified on a phosphorimager. Samples 1 and 2 contained wild-type RFC, sample 3 contained LaWT/SmBmut RFC complexes with small subunits that contained a mutation in the Walker B motif, which we have shown previously to be able to bind ATP, but has severely impaired ATPase activity (Seybert and Wigley, 2004), and sample 4 contained complexes in which the small subunits had the arginine finger mutation (LaWT/SmSAC). (B) DNA primer extension assay showing a defect of the Lawt/SmSAC complex in stimulating DNA synthesis catalysed by PolB1. Although this defect appears to be partially compensated by increasing the level of PolB1 polymerase in the assay, the presence of the mutant complex actually supports less primer extension than in the absence of RFC in the control lane. By contrast, the Lawt/SmBmut mutant complex that is able to bind but not hydrolyse ATP at the small subunits shows a stimulatory effect under similar conditions (Seybert and Wigley, 2004). The bars below the gels indicate how much PolB1, PCNA or RFC was present in each lane. Download figure Download PowerPoint Table 1. Nucleotide-binding and ATPase assays RFC/PCNA complex No. of ATPγS bound per RFC complex KD ATPγS (μM) kcat (s−1) Km ATP (μM) LaWTSmWT 4.1±0.3 0.5 1.7 25 LaWTSmSAC 3.6±0.2 4.0 1000 >50 LargeΔN/Smallwt 3.4 26 1.0 Largewt/SmallΔN 6.8a >>200 69 a Since the assays were carried out at 2 mM ATP, which is saturating for the wild type and complexes, given the high KM for these other mutant complexes, the actual rate will be higher; so these rates should be taken as an apparent kcat at 2 mM ATP, rather than a true kcat. Given the enhanced ATP turnover of the mutant complexes, we decided to investigate how this had affected clamp loading. The PCNA-loading assay that we employed utilises a circular plasmid DNA containing a single nick site that allows single-turnover loading of PCNA onto the substrate (Seybert and Wigley, 2004). Once loaded, the PCNA is topologically linked to the DNA. However, if the plasmid is linearised, any PCNA that has been loaded onto the DNA, but released from the RFC complex, is able to diffuse along the DNA and then dissociate from the free ends. In light of the enhanced turnover of ATP, we anticipated that the N-terminal deletion mutants would be proficient in loading PCNA. However, the results were surprising. The two mutant complexes containing truncated small subunits were in fact severely compromised in loading PCNA (Figure 7A). We therefore deduce that ATP hydrolysis has become uncoupled from clamp loading in these mutants, indicating a role for the N-terminal helix of the small subunits in linking these processes. By contrast, one mutant complex (LaΔNSmWT) showed almost three-fold enhancement of loading of PCNA in this assay. It has been shown previously (Gomes and Burgers, 2001; Seybert and Wigley, 2004) that while loading of PCNA onto DNA merely requires ATP binding, the release of the clamp requires hydrolysis by the small subunits. Consequently, if the circular DNA substrate is linearised, we observe release of PCNA from the DNA. However, if ATPγS is used in place of ATP to prevent hydrolysis, the PCNA remains bound to the RFC and hence complexed with the DNA. Interestingly, there is a complete loss of PCNA from the LaΔNSmwt complex once the DNA is linearised, irrespective of whether ATP or ATPγS is present, indicating that the link between ATP hydrolysis by the small subunits and PCNA release is now broken in this mutant. Figure 7.(A) Deletion of the N-terminal helix in the RFC small subunits inhibits the formation of stable PCNA–DNA complexes, whereas deletion of the N-terminal helix in the RFC large subunit stabilizes PCNA–DNA complexes. Loading assays were performed using a spin-column assay. Reactions contained 8 pmol 32P-PCNA, 4 pmol nicked pUC19 DNA (reactions 1–5), or 4 pmol nicked, linearised pUC19 DNA (reactions 6–13). Reactions 1, 3–5, 6, 8, 10 and 12 contained 5 mM ATP. Reactions 2, 7, 9, 11 and 13 contained 5 mM ATPγS. The mixtures also contained 4 pmol wild-type (samples 1, 2, 6 and 7) or mutant (samples 3–5 and 8–13) RFC complexes. The spin columns were pre-equilibrated in 200 mM NaCl. The eluates of the reactions were separated by SDS–PAGE in a 15% gel and quantified on a phosphorimager. (B) Comparison of the ability of the N-terminal helix truncated proteins with wild-type and Walker B mutant RFC complexes to stimulate DNA synthesis catalysed by PolB1. Reaction mixtures (20 μl) were supplemented with 200 mM NaCl and singly primed, closed circular M13mp18 DNA (25 fmol). After incubation for 30 min at 65°C, the DNA was precipitated and analysed by alkaline gel electrophoresis. Autoradiograms of dried gels are shown. Download figure Download PowerPoint Finally, we looked to see how these mutants had been affected in the primer extension assay. This assay looks at the complete process of clamp loading; so defects at any step in the reaction cycle result in reduced efficiency of product formation. As expected, the two mutant complexes that were deficient in clamp loading (LaWTSmΔN and LaΔNSmΔN) were also completely deficient in primer extension (Figure 7B). Under conditions where catalytic loading by RFC is rate limiting (Seybert and Wigley, 2004), primer extension was also significantly reduced with the LaΔNSmWT complex. However, by raising the concentration of RFC/PCNA so that this is no longer rate li
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