A Single Subunit Directs the Assembly of the Escherichia coli DNA Sliding Clamp Loader

SUMMARY

Multi-protein clamp loader complexes are required to load sliding clamps onto DNA. In Escherichia coli the clamp loader contains three DnaX (τ/γ) proteins, δ, and δ′, which together form an asymmetric pentameric ring that also interacts with ψχ. Here we used mass spectrometry to examine the assembly and dynamics of the clamp loader complex. We find that γ exists exclusively as a stable homotetramer, while τ is in a monomer-dimer-trimer-tetramer equilibrium. δ′ plays a direct role in the assembly as a τ/γ oligomer breaker, thereby facilitating incorporation of lower oligomers. With δ′, both δ and ψχ stabilize the trimeric form of DnaX, thus completing the assembly. When τ and γ are present simultaneously, mimicking the situation in vivo, subunit exchange between τ and γ tetramers occurs rapidly to form heterocomplexes but is retarded when δδ′ is present. The implications for intracellular assembly of the DNA polymerase III holoenzyme are discussed.

INTRODUCTION

Chromosomal replication in Escherichia coli is carried out by the DNA polymerase III (Pol III) holoenzyme, a complex containing ten different subunits. Three subcomplexes, the replicase core (the α polymerase subunit, the ε proofreader, and the accessory factor θ), the sliding clamp (dimeric β), and the seven-subunit clamp loader complex (comprised of subunits τ₂γδδ′ψχ or τ₃δδ′ψχ) act together in the holoenzyme to deliver highly processive, accurate, and rapid DNA replication (Bloom, 2009; Johnson and O’Donnell, 2005; Kornberg and Baker, 1992; Schaeffer et al., 2005). The clamp loader complex is responsible for loading/unloading of the β₂ sliding clamp (Naktinis et al., 1995), which encircles the primed template DNA strand and maintains processivity by tethering the replicase core to the template (Jeruzalmi et al., 2001, 2002). The clamp loader comprises a central τ₂γδδ′ or τ₃δδ′ pentameric ring that provides the site of ATPase activity, attached to a ψχ heterodimer through interaction of ψ with the three τ or γ subunits (Dallmann and McHenry, 1995; Gulbis et al., 2004; Xiao et al., 1993a, 1993b). The χ subunit mediates a further interaction between the clamp loader and the single-stranded DNA-binding protein SSB (Yuzhakov et al., 1999).

Unusually, τ and γ are produced from the same gene, dnaX; γ is a truncated form of τ, produced by a programmed translational frameshift (Tsuchihashi and Kornberg, 1990) such that the two are identical in their first 431 residues (Domains I–III). All combinations of (τ/γ)₃ in the clamp loader complex have been shown to form a functioning clamp loader, i.e., τ₃δδ′ψχ, τ₂γδδ′ψχ, τγ₂δδ′ψχ, and γ₃δδ′ψχ (Blinkowa and Walker, 1990; Flower and McHenry, 1990; McHenry, 2003). Although the minimal unit functional in clamp loading is the γ complex γ₃δδ′ (O’Donnell and Kuriyan, 2006), coupled leading and lagging strand DNA replication requires the presence of at least two τ subunits that dimerize the replicase core through the unique C-terminal domains of τ. These domains, not present in γ, interact with both the α subunit of the core and the hexameric DnaB helicase (Gao and McHenry, 2001a, 2001b; Jergic et al., 2007; Lopez de Saro et al., 2003).

Clamp loaders from bacteria, archaea, and eukaryotes all contain a heteropentameric central ring of proteins in the AAA+ superfamily of ring-shaped ATPases (Cann and Ishino, 1999; Cullmann et al., 1995; Neuwald et al., 1999; O’Donnell et al., 1993). In eukaryotes, five different proteins build the central ring, whereas in bacteriophage T4, a monomer of the protein gp62 combines with a tetramer of gp44. Similar to T4, the archaeal clamp loader consists of two different subunits with stoichiometry that is still unclear. In E. coli, the composition is particularly intriguing since a previous study indicated that while DnaX (τ/γ) is in a monomer-tetramer equilibrium in solution, δδ′ somehow forces DnaX into a trimer in creating the central ring (Pritchard et al., 2000). The mechanism by which this occurs, however, has not been established.

In this study, we use electrospray ionization (ESI) mass spectrometry (MS) to examine the assembly of the E. coli clamp loader in solution. MS of intact complexes is capable of identifying noncovalent protein interactions unambiguously under dynamic equilibrium conditions where multiple intermediates can be populated, revealing their stoichiometry and compositions in real time (reviewed in Hernández and Robinson, 2001; Loo, 1997; Sharon and Robinson, 2007; van den Heuvel and Heck, 2004). We set out to assemble the complete clamp loader complex from its individual components and to study the subunit exchange dynamics of τ and γ in solution. Our results reveal that γ₄ is more stable than its τ₄ counterpart and that the principal role of δ′ in the initiation of assembly is to break the tetramers into smaller oligomeric species. In the presence of δ′, δ and the ψχ dimer further direct the assembly of the clamp loader by stabilizing the trimeric form of (τ/γ)₃ to form the full clamp loader complex. Our results further show that incubation of tetrameric γ₄ with τ₄ results in rapid exchange of subunits to yield a statistical distribution of heterotetramers. The presence of δδ′, in contrast, prevents τ/γ exchange such that subunit interactions in the clamp loader must be preserved on the time scale of chromosomal replication.

RESULTS

τ and γ Are Predominantly Tetramers in Isolation

To identify the preferred oligomeric states of the component subunits that form the clamp loader (τ, γ, δ, δ′, and χ), mass spectra were recorded for all proteins in isolation. The proteins δ, δ′, and χ were found to exist primarily as monomers (see Table S1 available online). For τ, charge state series were observed corresponding to four distinct species (Figure 1A). The measured mass of 285,422 ± 44 Da for the major charge state series of τ is in close agreement with the calculated mass of the τ tetramer, 284,548 Da (Table S1). The mass increase observed experimentally is attributed to the incomplete removal of solvent and/or buffer ions under MS instrument conditions used here (McKay et al., 2006). Additional series of peaks observed in this spectrum are assigned to populations of monomers, dimers, and trimers of τ, indicating that all four oligomeric species are present in equilibrium. In contrast, only one charge state series was observed for γ, centered on a 32⁺ ion, indicating a homogeneous tetrameric state (Figure 1B). Only by using higher ionic strength buffer (>1 M NH₄OAc) were we able to observe all four oligomeric species of γ (Figure 1C). This shows that γ₄ is less stable at high ionic strength, dissociating to intermediate species similar to those formed by τ at lower ionic strength (0.1 M NH₄OAc). Overall, therefore, γ₄ appears to be more stable to dissociation than τ₄.

Figure 1. Mass Spectra of the τ and γ Subunits in Isolation.

(A) Mass spectrum of τ in 0.1 M NH₄OAc at pH 7.6 and 1 mM dithiothreitol. The major charge state series corresponds to a tetrameric form of τ. Series of τ in monomeric, dimeric, and trimeric forms are also observed as the minor charge state series.

(B) Mass spectrum of γ in 0.1 M NH₄OAc at pH 6.9. Well resolved charge states indicate that γ forms a well defined tetramer.

(C) Mass spectrum of all four oligomeric species of γ in 1 M NH₄OAc at pH 6.9. See also Table S1.

δ′ Is an Oligomer Breaker Creating Smaller Oligomers of τ and γ

To investigate how the tetrameric states of τ/γ adopt their established trimeric forms in the clamp loader, we used MS to measure changes in their oligomeric states as either δ′ or δ was added to solutions containing the tetramers. The mass spectrum of τ and δ′, at 1:2 mole ratio in solution, showed three distinct charge state series corresponding to τ₃δ′, τ₂δ′, and τδ′ (Figure 2A; Table S1). Furthermore, comparing this result to τ alone, we observe a dramatic increase in the population of smaller oligomers of τ in the presence of δ′ (Figure 2A compared with Figure 1A). This indicates that δ′ alone is capable of trapping the smaller oligmeric forms of τ. For solutions of γ with δ′, again smaller oligomers appeared; the subcomplexes γ₂δ′ and γ₃δ′ were observed (Figure S1A). For neither τ nor γ were τ₄δ′ or γ₄δ′ subcomplexes detected. Together these data indicate that the δ′ subunit plays the role of a τ₄/γ₄ “oligomer breaker” by selectively interacting with and trapping trimers and smaller oligomers in solution.

Figure 2. Mass Spectra of τ in the Presence of Various Subunits.

(A) In the presence of δ′, τ forms three subcomplexes, τ₃δ′, τ₂δ′, and τδ′ but not τ₄δ′.

(B) None of the oligomeric species of τ associates with δ.

(C) The ψχ complex binds to τ, forming τ₄ψχ and τ₃ψχ.

(D) δ does not associate with τ in the presence of ψχ. See also Figure S1 for the γ subunit interaction and Table S1.

In contrast, the analogous reaction with δ in place of δ′ showed no change in the distributions of τ/γ oligomers and no new interactions between the two components (Figure 2B; Figure S1B). Thus, even though τ₃ was present in solution, δ did not bind to it to form the τ₃δ subcomplex. This suggests two possibilities. One is that the specific conformation of τ/γ produced by prior binding of δ′ is critical for association of δ. The other is that δ requires δ′ to be present for binding τ/γ more tightly through cooperative interactions with τ/γ. In either case, only δ′ is capable of initiating the assembly of the clamp loader by breaking tetrameric (τ/γ)₄ oligomers and making possible the binding of δ. As expected, when both δ and δ′ are added to τ/γ, one predominant series of peaks was observed at high m/z and assigned to the complete (τ/γ)₃δδ′ pentamer (Figure 3A; Figure S2A compared with Figure 2A and Figure S1A, where multiple assemblies are formed in the presence of δ′ alone). This implies that after δ′ breaks the tetramer of τ or γ, binding of δ stabilizes the trimeric state to drive the correct stoichiometry of τ/γ in the clamp loader complex.

Figure 3. Assembly Pathway of the Clamp Loader and Mass Spectra of Complexes Formed Along the Pathway.

First, δ′ initiates the assembly by dissociating tetramers of τ into trimers, the functional unit of the clamp loader. Both δ or ψχ interact with the initial τ₃δ′ subcomplex, resulting in either τ₃δδ′ or τ₃δ′ψχ subcomplexes. Further addition of ψχ or δ leads to the final clamp loader, τ₃δδ′ψχ. Mass spectra are recorded for τ₃δδ′ (A), τ₃δ′ψχ (B), and τ₃δδ′ψχ (C and D), when ψχ was added to (A) and δ to (B). For γ, analogous results were observed in Figure S2, suggesting identical assembly pathways. See also Table S1. Note that although all four species of oligomers of τ are present in solution, only tetramers of τ are shown initially.

Interaction between the Pentameric Ring and ψχ

Assembly of the complete clamp loader complex requires binding of the ψχ heterodimer to the central (τ/γ)₃δδ′ ring to form (τ/γ)₃δδ′ψχ. We predicted that ψχ would stabilize the trimer of τ/γ, since the recent crystal structure of γ₃δδ′ bound to the N-terminal interacting peptide of ψ shows that the peptide interacts with all three γ subunits at the collar domain but neither δ nor δ′ (Simonetta et al., 2009). To investigate this, first we examined the interaction of ψχ with other clamp loader subunits (γ, τ, δ, and δ′) individually. We did not see any interaction between ψχ and δ or δ′ as would be expected from the crystal structure (Simonetta et al., 2009) and other biochemical studies (Gao and McHenry, 2001c; Xiao et al., 1993b). In the mass spectrum of ψχ with τ (Figure 2C), two distinct series of peaks were observed, corresponding to homogenous populations of τ₄ψχ and τ₃ψχ (Table S1). Since neither τ₂ψχ nor τψχ was observed, it can be inferred that ψχ binds more stably to τ₄/τ₃ and directs assembly to (or traps) higher oligomeric species of τ. Similarly, the ψχ complex associates with γ₄, resulting in γ₄ψχ and γ₃ψχ complexes (Figure S1C and Table S1). The effect of ψχ in stabilizing higher oligmeric species is in contrast to δ′, which, as we have seen, specifically disrupts τ and γ tetramers. Interestingly, for both τ and γ, τ₄ψχ and γ₄ψχ are major species, not τ₃ψχ and γ₃ψχ. This indicates that ψχ preferentially associates with tetramers of τ/γ rather than trimers. This may be simply because ψ binds to only three subunits of γ in a tetramer, consequently there could be two different overlapping binding sites for ψ.

Given that the only oligomer breaker among the canonical clamp loader subunits is δ′, we investigated the order of binding of δ and ψχ to τ₃δ′/τ₂δ′/τδ′. When δ was added to solutions containing both τ and δ′, the central ring of the clamp loader, τ₃δδ′, formed rapidly, without incubation (Figure 3A). No detectable intermediate states were detected (e.g., τ₂δδ′), equivalent to when δ and δ′ were added together to τ. As might be expected from the γ₃δδ′-ψ peptide structure (Simonetta et al., 2009), addition of ψχ to τ₃δ′/τ₂δ′/τδ′ produced the τ₃δ′ψχ complex as the major species (Figure 3B). Our insight into the assembly grows further by noting that little τ₂δ′ψχ was detected, implying that in the presence of δ′, ψχ forms the most stable complex with the τ trimer. The role of ψχ in stabilizing the canonical clamp loader assembly is more clearly observed when the complex is constructed using γ rather than τ, since no γ₂δ′ψχ was observed in solution (Figure S2B). In addition, similar results were observed when δ′ was added to the (τ/γ)₄ψχ and (τ/γ)₃ψχ (Figure 3B; Figure S2B). Therefore, ψχ stabilizes the trimer of τ/γ in the presence of δ′, guiding the assembly of the functional stoichiometry of τ/γ in the clamp loader. This role is analogous to that of δ in the formation of the heteropentamer.

It is possible, however, that alternative assembly routes exist. To investigate this we examined whether ψχ can support δ in binding of τ/γ. Mass spectra recorded after addition of δ to solutions of (τ/γ)₄ψχ and (τ/γ)₃ψχ showed no evidence of (τ/γ)₃δψχ formation (Figure 2D; Figure S1D). Unlike δ′, therefore, the ψχ complex does not assist δ in binding. Thus, we have shown conclusively that only δ′ can initiate the assembly of the full clamp loader and subsequently enable binding of ψχ and/or δ (Figure 3).

Subunit Exchange Dynamics of τ and γ

So far we have studied the clamp loader assembly with either τ or γ, but since all combinations of (τ/γ)₃ form functional clamp loaders, it is important to consider how the incorporation of τ versus γ is established. First, we examined subunit exchange between τ and γ in the absence of other clamp loader proteins. Under the same buffer conditions and concentrations, solutions of τ and γ were combined and mass spectra were recorded at fixed time points to monitor the exchange reaction. All five possible tetrameric species of τ and γ (τ₄, τ₃γ, τ₂γ₂, τγ₃, and γ₄) were formed immediately in a statistical distribution (Figure 4A) that showed no further changes with time (Figure S3). This indicates that subunit exchange within homotetramers of τ and γ occurs rapidly and reaches equilibrium within seconds in vitro.

Figure 4. Subunit Exchange Dynamics between τ and γ.

(A) τ and γ undergo rapid subunit exchange at 0°C and form all five possible tetrameric species, τ₄, τ₃γ, τ₂γ₂, τγ₃, and γ₄, with the dead time of the experiment <15 s. See also Figure S3.

(B) Mass spectra of τ₃δδ′ in the presence of γ₄. At τ = 0, free δδ′ interacts with γ₄ directly and forms the γ complex (bottom spectrum). At τ = 1 hr, low intensity peaks are observed (not labeled, middle spectrum), which increase in intensity after 24 hr and are assigned to all four possible complexes, γ₃δδ′, γτ₂δδ′, γ₂τδδ′, and τ₃δδ′ (top spectrum). See also Table S1.

To see whether the rapid exchange dynamics observed for τ and γ also occur in the presence of δδ′, we mixed solutions containing equimolar τ₃δδ′ and γ₄ for specific time intervals. Soon after mixing, we observed peaks corresponding to τ₃δδ′, γ₄, and, interestingly, γ₃δδ′ (Figure 4B, bottom). Given that τ₃δδ′ is in equilibrium with many subspecies such as τ₃ and δδ′ (Figure 3A), it is likely that the appearance of γ₃δδ′ is not due to γ₃ replacing τ₃ in τ₃δδ′, but to the availability of free δδ′ that could convert γ₄ into the γ₃δδ′ complex as described above. After incubation for 1 hr, small peaks indicate minimal exchange of τ/γ subunits (Figure 4B, middle). Nevertheless, all four forms of (τ/γ)₃δδ′ were readily identified after 24 hr (Figure 4B, top). These experiments demonstrate that in the presence of δδ′, subunit exchange between τ and γ takes place on a much longer time scale relative to the situation where τ and γ alone are incubated. Since this longer time scale is beyond the time required for chromosome replication (~40 min), we surmise that δδ′ prevents significant subunit exchange in vivo.

DISCUSSION

Unique Roles for δ and δ′ in the Clamp Loader Assembly

In this study we used MS to follow an assembly pathway for the seven-subunit E. coli DNA sliding clamp loader, an important example of an AAA+ motor class of multi-protein complex. Although this clamp loader in E. coli contains trimeric τ/γ proteins, previous studies using ultracentrifugation have suggested that τ/γ in isolation exists in both monomeric and tetrameric forms (Dallmann and McHenry, 1995). As a consequence, it was not clear how such an oligomeric population is converted into trimers in the presence of δδ′ to form the functional clamp loader. Here we confirm that both τ and γ adopt a predominantly tetrameric state but, in contrast to the earlier work, dimers and trimers were observed in addition to monomers. Furthermore, we found that γ₄ is more stable than τ₄ since, unlike τ, the tetramers of γ do not dissociate under similar experimental conditions.

By determining interactions among components of the clamp loader, we identified the crucial role of δ′ in breaking the τ/γ tetramer, to displace the equilibrium among τ/γ oligomers toward the smaller oligomeric species. It is not clear how δ′ displaces τ/γ within the τ/γ oligomers; however, we predict that the rigidity within δ′ offers constant binding sites for τ/γ, which subsequently secures a more stable conformation. It is also possible that in the presence of δ′, the fourth τ/γ may be prevented sterically from forming the circular architecture. In contrast, we found that the δ subunit alone could not interact with or disrupt any of the τ/γ oligomers, in accord with previous studies (Onrust et al., 1995a). In particular, although even in the absence of δ′ some τ₃ exists in equilibrium, we observed that δ did not interact even with τ₃. Therefore, the data led us to speculate that δ′ assists δ in binding to (τ/γ)₃ either by facilitating changes in the conformation within (τ/γ)₃, revealing interaction surfaces on τ/γ, or simply by placing δ next to γ as the result of binding δ′. Thus, we have identified two important roles of δ′ in the assembly of the clamp loader: first as a breaker of the τ/γ oligomer and second as a promoter for binding δ and consequently completing the pentameric central ring. Moreover, since we observed no smaller oligomeric species of τ/γ in the presence of both δ and δ′, it appears that δ not only completes the central ring but that it also stabilizes the trimeric τ/γ in the presence of δ′.

A similar stabilizing effect of ψχ on (τ/γ)₃ was observed, further ensuring the correct stoichiometry of τ/γ in the heteropentameric clamp loader complex. However, we found that this locking of τ/γ into a trimeric form by ψχ is only established in the presence of δ′. Reconciling our results with the γ₃δδ′-ψ peptide structure (Simonetta et al., 2009), we conclude that ψχ does not break the τ/γ tetramer; it may nonetheless stabilize a trimer within the tetramer. It may be that ψχ stabilizes the τ/γ trimer, which in turn assists in entry of δ′, providing an alternative assembly pathway of the clamp loader complex. Assembly dynamics of the clamp loader complex may also differ in the presence of nucleotides, although the clamp loader assembles independently in vitro without nucleotides. Nonetheless, it is clear that δ′ plays a crucial role as an oligomer breaker in directing the correct assembly of the clamp loader.

Final Stoichiometry of τ versus γ in the Clamp Loader Complex

In vivo, DNA replication proceeds on both the leading and lagging template strands of DNA within a single DNA replisome. Each strand requires a replicative polymerase (the αεθ core), tethered to the clamp loader complex through α via the C terminus of a τ subunit within the assembly. Therefore, for “coupled” replication to take place, at least two τ subunits per clamp loader are required [i.e., (αεθ-τ)₂γδδ′ψχ]. However, whether there are two or three τ subunits within a functioning replisome in vivo is yet to be determined.

Prior work on assembly of the holoenzyme and clamp loader complexes from the component subunits in vitro (Onrust et al., 1995a; Pritchard and McHenry, 2001) is consistent with the proposition that exchange of the τ and γ subunits can only occur efficiently prior to their assembly with δ and δ′ into the active clamp loader complexes. Nevertheless, the picture has been complicated by belief that (a) simply mixing all ten subunits produces only the τ₃ holoenzyme (McInerney et al., 2007; Pritchard et al., 2000; Pritchard and McHenry, 2001), (b) that Pol III* (holoenzyme lacking β) isolated directly from E. coli contains both the τ₃ holoenzyme and the γ₃ clamp loader complex, lacking the heterotrimeric τ₁γ₂ and τ₂γ₁ species (McInerney et al., 2007), and (c) that Pol III’, a species with the composition (αεθ-τ)₂, is an intermediate in the holoenzyme assembly (Pritchard and McHenry, 2001). It has been shown that in vitro assembly of τ₁γ₂ and τ₂γ₁ clamp loader complexes requires lengthy preincubation of separately purified τ and γ to allow exchange before addition of the other subunits (Onrust et al., 1995a, 1995b) and that τ/γ exchange is prevented when the other clamp loader subunits are present (Onrust et al., 1995b; Pritchard and McHenry, 2001). Nevertheless, overexpression of τ and γ from wild-type dnaX in a single operon with genes encoding the other four subunits produced γ₃, τ₁γ₂, and τ₂γ₁ clamp loader complexes, indicating fast exchange of newly translated τ and γ in vivo, at least when at elevated concentrations (Pritchard et al., 2000).

These previous studies used chromatographic methods to study clamp loader assembly. In this study, we exploited the ability of ESI MS to rapidly determine compositions of heterogeneous complexes to show that all combinations of mixed tetramers of τ and γ were assembled immediately upon incubation of the two proteins under our buffer conditions (Figure 4A). This is expected given that the two proteins exist in solution as equilibrium mixtures of oligomers. That the distribution of mixed tetramers appears to be statistical (Figure 4B) indicates that there is no preferential association between τ subunits; i.e., all interactions that determine assembly among subunits are restricted to the N-terminal region common to the two proteins. Because subunit exchange occurs rapidly, it is reasonable to think that all combinations of τ and γ would assemble into mixed tetramers following translation in vivo, and thus it seems very likely that E. coli makes all Pol III*/clamp loader assemblies, including γ₃δδ′ψχ, (αεθ-τ)γ₂δδ′ψχ, (αεθ-τ)₂γδδ′ψχ, and (αεθ-τ)₃δδ′ψχ, although only the last two complexes are capable of coupled leading and lagging strand replication (McI-nerney et al., 2007). Interestingly, γ is not essential for cell viability (Blinkova et al., 1993), which implies that (αεθ-τ)₃δδ′ψχ not only accomplishes coupled DNA synthesis but must also be capable of clamp loading both in replisome assembly and elsewhere. It has been suggested that γ₃δδ′ψχ may function in clamp loading for other DNA maintenance functions (Bloom, 2009). We have also confirmed that δδ′ prevents the subunit exchange between τ and γ. Thus, the compositions of individual clamp loader/holoenzyme assemblies are likely to be preserved as they carry out their specific functions, (αεθ-τ)₃δδ′ψχ and (αεθ-τ)₂γδδ′ψχ in replication and γ₃δδ′ψχ in clamp loading (or unloading) for other activities. While there is no clear role for (αεθ-τ)γ₂δδ′ψχ, which would be incapable of lagging strand synthesis at a fork, its comparative lack of necessary protein-protein and DNA interactions would probably place it at a competitive disadvantage in functional replisome assembly.

In summary we have addressed two major problems: first, how the central pentameric ring of the clamp loader in E. coli is formed from six proteins (a tetramer of τ/γ, δ, and δ′) and, second, how the cell avoids creating a replisome with fewer than two copies of τ. The first problem is solved in a surprising way, by δ′ first breaking the tetramers of τ/γ and then (and only then) enabling δ to bind and lock in the central pentameric ring. This solution to the first problem also turns out to be related to the second. Our subunit exchange studies between τ and γ have indicated that all combinations of (τ/γ)₃δδ′ are likely formed in vivo; yet, once any are formed, the presence of δ and δ′ prevents the exchange of τ and γ subunits within the time scale of DNA synthesis. Once the replisome is formed with either τ₂γδδ′ or τ₃δδ′, the stability of these central pentameric rings therefore prevents them from exchanging to a form with fewer than two copies of τ. Thus, the second problem is apparently solved, not by the abolition of τγ₂δδ′ and γ₃δδ′, but by their exclusion from the assembly of the functional replisome.

EXPERIMENTAL PROCEDURES

Proteins and Sample Preparation for MS

Each subunit, τ, γ, δ, δ′, χ, ψχ, and a, was overexpressed in E. coli and purified as described (Ozawa et al., 2008; Tanner et al., 2008; Wijffels et al., 2004; Xiao et al., 1993a). For mass spectra of individual proteins, τ (10.2 mg/ml), γ (3.2 mg/ml), δ (3.8 mg/ml), δ′ (4.0 mg/ml), χ (2.4 mg/ml), ψχ (4.8 mg/ml), and α (9.2 mg/ml) were buffer exchanged into appropriate NH₄OAc buffers using Vivaspin 500 with 10–50 kDa molecular weight cutoff, depending on the size of the protein (Sartorius). Various concentrations and pHs of NH₄OAc buffers were used for each protein: 0.1 M NH₄OAc at pH 7.6 containing 1 mM dithiothreitol for τ, 0.1 M NH₄OAc at pH 6.9 for γ, 1 M NH₄OAc at pH 6.9 for δ, 0.5 M NH₄OAc at pH 6.9 for δ′, and 0.1 M NH₄OAc at pH 7.6 for ψχ and χ. Concentrations for τ, γ, δ, δ′, χ, ψχ, and α were measured spectrophotometrically at 280 nm, using ε₂₈₀ = 46380, 20940, 46830, 60440, 29400, 53680, and 96880 M⁻¹ cm⁻¹, respectively. The final concentrations of the proteins were in the range 5 to 10 μM. For subunit exchange experiments, τ₄ or τ₃δδ′ and γ₄ at 5 μM, subsequent to buffer exchange in 0.1 M NH₄OAc at pH 7.6 and 1 mM dithiothreitol, were mixed at 0°C. MS were recorded every 10 min for 1 hr and after 24 hr.

Mass Spectrometry

Nanoflow ESI MS and tandem MS experiments were conducted on a high mass Q-TOF type instrument (Sobott et al., 2002) adapted for a QSTAR XL platform (Chernushevich and Thomson, 2004). Typically, 2 μl aliquots of solution were electrosprayed from gold-coated borosilicate capillaries prepared in-house as described (Hernández and Robinson, 2007). MS experiments were conducted at a capillary voltage up to 1.2 kV, declustering potential 100–150 V, focusing potential 200–250V, declustering potential two 15–55 V, collision energy up to 180 V, and MCP 2350 V. All spectra were calibrated externally using a solution of cesium iodide (100 mg/ml). Spectra are shown here with minimal smoothing and without background subtraction.