The internal workings of a DNA polymerase clamp‐loading machine

Jennifer Turner; Manju M. Hingorani; Zvi Kelman; Mike O'Donnell

doi:10.1093/emboj/18.3.771

The internal workings of a DNA polymerase clamp‐loading machine

Turner, Jennifer; Hingorani, Manju M.; Kelman, Zvi; O'Donnell, Mike 1999-02-01 00:00:00 The EMBO Journal Vol.18 No.3 pp.771–783, 1999 The internal workings of a DNA polymerase clamp-loading machine 1 2 These protein rings are assembled onto DNA by their Jennifer Turner , Manju M.Hingorani , 1,3 2,4,5 respective clamp loaders in an ATP-dependent reaction Zvi Kelman and Mike O’Donnell (Kelman and O’Donnell, 1995). Unlike sequence-speciﬁc 2 4 The Rockefeller University and The Howard Hughes Medical DNA-binding proteins, the circular clamps form a topo- Institute, 1230 York Avenue, Cornell University Medical College, logical link with DNA and slide along the duplex without 1300 York Avenue and Sloan-Kettering Institute, 1275 York Avenue, localizing to a speciﬁc region. Therefore, when the DNA New York, NY 10021, USA polymerase binds its clamp, it is endowed with high Corresponding author processivity, allowing it to move continuously along the e-mail: [email protected] template during chain extension. The E.coli replicase, DNA polymerase III holoenzyme, Replicative DNA polymerases are multiprotein consists of 10 different polypeptide chains (Kornberg and machines that are tethered to DNA during chain Baker, 1992; Kelman and O’Donnell, 1995). Within the extension by sliding clamp proteins. The clamps are holoenzyme are two core polymerases (αεθ) and a clamp designed to encircle DNA completely, and they are loader (γ complex), that are held together by τ, a connector manipulated rapidly onto DNA by the ATP-dependent protein (Onrust et al., 1995a). Both core polymerases activity of a clamp loader. We outline the detailed become highly processive when tethered to DNA by mechanism of γ complex, a ﬁve-protein clamp loader β sliding clamps (Stukenberg et al., 1991). As the holo- that is part of the Escherichia coli replicase, DNA enzyme moves along the replication fork, the processive polymerase III holoenzyme. The γ complex uses ATP polymerase extends DNA continuously on the leading to open the β clamp and assemble it onto DNA. strand. On the lagging strand, the other polymerase releases Surprisingly, ATP is not needed for γ complex to crack its sliding clamp upon completion of each Okazaki frag- open the β clamp. The function of ATP is to regulate ment, and is cycled back to the replication fork by targeting the activity of one subunit, δ, which opens the clamp to a new clamp assembled at an upstream RNA primer simply by binding to it. The δ subunit acts as a by the clamp loader (O’Donnell, 1987; Stukenberg modulator of the interaction between δ and β.On et al., 1994). binding ATP, the γ complex is activated such that the The E.coli clamp loader, γ complex, consists of ﬁve δ subunit permits δ to bind β and crack open the ring different subunits: γ, δ, δ, χ and ψ (Maki and Kornberg, at one interface. The clamp loader–open clamp protein 1988). Within the γ complex are two to four γ subunits complex is now ready for an encounter with primed and one each of δ, δ, χ and ψ (Maki and Kornberg, DNA to complete assembly of the clamp around DNA. 1988; Onrust et al., 1995b). The proteins are assembled Interaction with DNA stimulates ATP hydrolysis which such that both δ and ψ bind directly to γ, while δ and χ ejects the γ complex from DNA, leaving the ring to bind the δ and ψ subunits, respectively (Onrust et al., close around the duplex. 1995b). Previous studies have shown that a γδδ complex Keywords: ATPase/clamp loader/DNA polymerase/ is sufﬁcient to load β onto DNA (Onrust et al., 1991). processivity/sliding clamp The χ and ψ subunits are not essential for β loading onto DNA; χ binds single-stranded DNA binding protein (SSB) and facilitates displacement of the primase from RNA Introduction primers, an event that must occur prior to clamp loading on the lagging strand (Kelman et al., 1998; Yuzhakov Replicases are efﬁcient DNA-synthesizing enzymes that et al., 1999). The δ subunit is the major contact point duplicate long chromosomes with high speed and pro- between γ complex and β, and δ binds tightly to β in the cessivity. These biological machines are comprised of complete absence of the other clamp loader proteins three functional components in both prokaryotic and (Naktinis et al., 1995). When δ is part of the γ complex, eukaryotic organisms: (i) a DNA polymerase, (ii) a pro- however, it exhibits only a low afﬁnity for β in the absence cessivity factor or sliding clamp protein and (iii) a multi- of ATP. In the presence of ATP, the γ complex undergoes protein clamp loader. Replicases from Escherichia coli, a conformational change that now allows δ to bind β with Saccharomyces cerevisiae, humans and bacteriophage T4 an afﬁnity comparable with that of the free δ–β interaction have these three components. The DNA polymerases lack (Naktinis et al., 1995). γ is the only clamp loader subunit high processivity alone but upon association with their that binds and hydrolyzes ATP (Maki and Kornberg, 1988; respective sliding clamps, they can replicate several thou- Tsuchihashi and Kornberg, 1989; Onrust et al., 1991). sand bases continuously (reviewed in Kelman and Recent studies show that ATP binding powers a change O’Donnell, 1995). The sliding clamps are ring-shaped homo-oligomers that encircle duplex DNA (Stukenberg in γ subunit conformation (Hingorani and O’Donnell, et al., 1991; Kong et al., 1992; Burgers and Yoder, 1993; 1998), which may underlie the ATP-induced change in γ Krishna et al., 1994; Gulbis et al., 1996; Yao et al., 1996). complex conformation that leads to tight interaction with © European Molecular Biology Organization 771 J.Turner et al. β (Naktinis et al., 1995; Hingorani and O’Donnell, 1998). Thus, the γ subunit transduces the energy from ATP to expose the δ subunit for interaction with the β ring. This report describes the mechanics of the γ complex- catalyzed process of β assembly onto DNA. We show that the energy of ATP binding powers the γ complex machinery to bind β and open the circular clamp at one interface. Once γ complex binds β, its ATPase activity is supressed until it binds DNA. The correct DNA substrate stimulates ATP hydrolysis, which is coupled to the release of γ complex from β on DNA. On DNA, β reassumes its lowest free energy state and forms a closed ring, now with DNA passing through the center. The molecular details that underlie the clamp loader activity have been explored and as a result, speciﬁc functions have been assigned to the three integral com- ponents of the γ complex: γ is the ‘motor’, δ is the ‘ring opener’ and δ is the ‘modulator’. The γ subunits are the only components of γ complex to interact with ATP, and their function is to drive ATP-induced conformational changes of other subunits in the clamp loader. The δ subunit contains the intrinsic clamp-opening activity, as it can open the β ring in the complete absence of ATP and the other subunits. The δ subunit binds δ and probably modulates the ability of β to gain access to δ. The ATP- driven γ motor either moves or alters the δ modulator such that the δ clamp opener gains access to the β clamp and opens the ring. This action requires only ATP binding to the γ complex. The resulting clamp loader–open clamp composite then binds primed DNA with high afﬁnity. Upon interaction with primed DNA, two or three molecules of ATP are hydrolyzed, resulting in closure of the β ring around DNA and ejection of γ complex off the DNA and back into solution. Results The γ complex uses energy from ATP binding to open the β ring The β sliding clamp is composed of two crescent-shaped Fig. 1. γ complex opens the β clamp on binding ATP. The scheme protomers arranged in a tight head-to-tail dimer (Kong indicates the position of a cysteine residue (S) within the dimer et al., 1992). This circular protein clamp is transferred interface that is accessible for reaction only when the ring is opened. The structure of the [ P]maleimide reagent used to label cysteine is onto circular DNA by the ATP-dependent clamp-loading also shown. (A) β (3 μM) incubated with [ P]maleimide in the activity of the γ complex. How does γ complex assemble absence or presence of γ complex (3 μM), primed oligoDNA (3 μM) the β ring around DNA? How is ATP used in this reaction? 32 or ATP (2 mM), and analyzed by SDS–PAGE. P-labeled β is Is ATP binding sufﬁcient, or is hydrolysis necessary? indicated by an arrow, showing a 2- and 10-fold increase in β labeling in the presence of γ complex and γ complex ATP, respectively. What is the role of DNA in this process? To begin (B) γ complex-catalyzed ring opening assayed for varying times in the addressing these questions, we developed a novel ‘Cys- presence of ATP (left) or ATP-γ-S (right). (C) γ complex-catalyzed labeling’ assay to detect and characterize the ring-opening ATP hydrolysis compared with ATP-γ-S hydrolysis. γ complex step in clamp assembly. (0.1 μM), incubated with β (0.2 μM), hydrolyzes ATP with a k cat –3 1.7/min (d) and hydrolyzes ATP-γ-S with a k 410 /min (j). Leu273, a buried hydrophobic residue in the β dimer cat interface, was substituted with cysteine (β crystal structure; Kong et al., 1992). This buried cysteine residue should modiﬁcation was made in L273C-β to ensure that only not be accessible to a thiol-reactive reagent, such as a the interface cysteine residue would be labeled; the reactive maleimide, when the L273C-β ring is closed. When the Cys333 at the protein surface was changed to serine. The interface is opened, however, the buried cysteine should modiﬁed β is a dimer and is fully active in DNA replication become accessible to the surface and reactive to maleimide. assays with γ complex and core polymerase (data not To follow this reaction, we synthesized a radioactive shown). reagent by linking the N-terminus of a P-labeled peptide The results of the ring-opening assay are shown in with an N-hydroxysuccinimide (NHS) ester coupled to Figure 1A. The [ P]maleimide reagent was incubated maleimide (Figure 1). If the dimer interface opens, the with β in the absence or presence of γ complex, ATP and interface cysteine residue will react with the [ P]maleim- DNA, and then the reactions were analyzed on an SDS– ide reagent, resulting in a radiolabeled β clamp. One other polyacrylamide gel. The autoradiogram of the gel shows 772 Mechanics of clamp assembly on DNA very low reactivity of β alone (Figure 1A, lane 1). When non-reducing SDS–polyacrylamide gel to determine if β is mixed with γ complex, there is a 2-fold increase in disulﬁde cross-links had formed. Figure 2A shows that in β labeling (Figure 1A, lane 2). However, when both the absence of dithiothreitol (DTT), more than half of the S–S γ complex and ATP are present in the reaction, the β is β migrates as an 80 kDa band of cross-linked dimers; 10-fold more reactive with the [ P]maleimide (Figure the rest migrates as a 40 kDa band of monomers (lane 1). 1A, lane 4). The results indicate that in the presence of Native β migrates as a single band at 40 kDa (lane 2). S–S ATP, γ complex can open the β ring or at least change Incubation of β with DTT reduces the disulﬁde cross- S–S the ring conformation enough to expose the cysteine links such that at 2 and 20 mM DTT essentially all β residue buried in the dimer interface. When DNA is migrates as monomers on the SDS–polyacrylamide gel present in the reaction, β is labeled to the same extent as (lanes 3 and 4, respectively). S–S in the absence of DNA (compare lanes 4 and 5), indicating Next we analyzed the cross-linked β for assembly 32 S–S that γ complex can open the β ring even in the absence around a circular DNA substrate. The P-labeled β of DNA. was incubated with γ complex and DNA in the presence Next, the assay was used to examine whether ATP binding of ATP, and then assayed by gel ﬁltration over a Bio-Gel or hydrolysis is required to stimulate β ring opening. To A-15m column (this large pore resin includes proteins but separate the effects of ATP binding from ATP hydrolysis, excludes the large DNA substrate as well as any protein S–S we substituted ATP with its analog, ATP-γ-S (Figure 1B). bound to it). Figure 2B shows that about half of the β The turnover number for ATP hydrolysis catalyzed by is assembled on DNA in the absence of DTT (β on DNA, γ complex (in the presence of β) is 1.5–2/min at 37°C fractions 9–13; free β, fractions 18–27). Treatment of S–S (Figure 1C). Therefore, in a 10 min assay, each γ complex β with DTT prior to the clamp loading reaction results S–S hydrolyzes 15–20 molecules of ATP. The γ complex binds in assembly of nearly all the β on DNA (fractions 9– ATP ATP-γ-S with similar high afﬁnity as it does ATP (K 13), and is comparable with assembly of native β on DNA ATP-γ-S 2 μM and K 5 μM, respectively; Hingorani and (Figure 2B). Presumably in the absence of DTT the O’Donnell, 1998), but the turnover number for ATP-γ-S disulﬁde cross-links at the interface prevent some (or all) –3 S–S hydrolysis is very low (4–610 /min; see Figure 1C). of the β dimers from being assembled on DNA. S–S Since γ complex catalyzes hydrolysis of only one ATP-γ-S To determine if the β clamps assembled onto DNA every 2–3 h, ATP-γ-S can be considered a non-hydrolyzable in the absence of DTT contain any cross-links, the analog of ATP within the time frame of the ring-opening column fractions were analyzed on a non-reducing SDS– assay. polyacrylamide gel (Figure 2C). As expected, non-cross- S–S The data shown in Figure 1B demonstrate that ATP and linked β was assembled onto DNA (monomers in ATP-γ-S facilitate γ complex-catalyzed ring opening to fractions 9–13). However, a large fraction of the cross- S–S the same extent. Therefore, ATP binding appears sufﬁcient linked β was also assembled onto DNA (dimers in to drive γ complex-catalyzed opening of the β ring. Other fractions 9–13), while the remainder eluted as free protein S–S non-hydrolyzable ATP analogs such as AMP-PNP and (dimers in fractions 18–27). Presumably the β dimers AMP-PCP were also tested in the assay, but ring opening on DNA contain only one cross-link, and therefore can S–S was not detected (data not shown). However, this negative still be opened at one interface. The β dimers that were result may be explained by the fact that γ complex binds not assembled on DNA may contain two disulﬁde cross- these analogs with much lower afﬁnity than ATP (K links that preclude ring opening and assembly onto DNA. S–S ~1 mM versus 2 μM for ATP), and this binding energy To conﬁrm that cross-linked β on DNA (dimers in may be insufﬁcient to support ring opening (H.Xiao and fractions 9–13; Figure 2C) is cross-linked at only one M.O’Donnell, unpublished data). interface, the experiment in the absence of DTT was Next, the ring-opening step was examined in greater repeated and split into two halves. One half was analyzed detail to determine whether the clamp loader opens β at by gel ﬁltration as described above (Figure 2C), to conﬁrm S–S both interfaces (e.g. monomerizes the dimer to assemble that β was assembled on DNA. The other half was it around DNA), or if opening the β dimer at only one analyzed by gel ﬁltration in the presence of SDS (Figure S–S interface is sufﬁcient to allow entry of DNA into the ring. 2D). If the β on DNA contains one disulﬁde cross-link, then SDS denaturation will result in a dimer opened at The γ complex need open only one interface of the the non-cross-linked interface that will dissociate from S–S β dimer for clamp loading DNA during gel ﬁltration. If the β on DNA were to To determine whether γ complex needs to open one or contain two disulﬁde cross-links, the protein ring will both interfaces of the β dimer for assembly of the clamp remain covalently sealed even after SDS denaturation, and around DNA, we constructed a β dimer in which the thus remain topologically linked to DNA in the presence S–S monomers could be cross-linked at the dimer interface. of SDS. The results show that the cross-linked β Arg103 and Ile305, two residues close to each other but dissociates from DNA during gel ﬁltration in the presence on opposing sides of the dimer interface (C atoms ~6 Å of SDS, indicating that the rings were cross-linked at only apart; β cyrstal structure; Kong et al., 1992), were substi- one interface (dimers in fractions 18–27; Figure 2D). tuted with cysteine. Due to the head-to-tail arrangement Thus, the γ complex can load clamps that are sealed at of the β dimer, the two interfaces are identical. It was one interface, and does not need to monomerize the expected that oxidation would result in formation of β dimer to load it onto DNA. disulﬁde cross-links between the cysteine residues, and yield β dimers covalently linked at one or both interfaces. δ is the ring opener S–S 32 Initially, the modiﬁed β (β ) was P-labeled at an We performed the Cys-labeling assay described in Figure 1 N-terminal kinase recognition site and analyzed on a with individual subunits and subassemblies of the 773 J.Turner et al. Fig. 2. γ complex opens the β clamp at one interface. Cysteine residues were placed at the β dimer interface to design a clamp with disulﬁde cross- S–S S–S links at the interface (β ). (A) Native β and β analyzed on a non-reducing SDS–polyacrylamide gel with no DTT, 2 mM DTT and 20 mM DTT 32 32 S–S in the reaction. (B) Gel ﬁltration analysis of native [ P]β (d) and [ P]β clamps assembled onto DNA by γ complex in the absence of DTT (s) S–S and in the presence of DTT (u). The next scheme shows a mix of β rings with two, one or zero cross-links at the interface, assembled on DNA S–S and analyzed by gel ﬁltration followed by non-reducing SDS–PAGE. (C) Analysis of β from a clamp-loading reaction without DTT [as in (B)]. (D) The same reaction analyzed with SDS present during gel ﬁltration. Fraction numbers are indicated beneath the gels. The positions of non-cross- S–S S–S linked β (monomers) and cross-linked β (dimers) are indicated by arrows. γ complex, to determine the minimal subunit requirement simultaneous presence of all three subunits, γ, δ and δ for ring opening. The minimal subassembly that tested (Onrust et al., 1991). However, the Cys-labeling assay for positive for ring opening was the three-subunit γδδ ring opening does not rigorously exclude the possibility complex, which, like γ complex, needs ATP or ATP-γ-S that a smaller subassembly than γδδ, or even just one to crack open the β dimer interface (data not shown). subunit, can crack open the dimer interface transiently. Individual subunits or combinations of two subunits did For example, the assay requires time for the [ P]maleimide not give a detectable signal. This result implies that more to react with an exposed cysteine residue, and is likely to than two proteins are needed to detect ring opening in the proceed more efﬁciently the longer the β dimer interface Cys-labeling assay, that γδδ is sufﬁcient for ring opening is held open. To investigate if any one subunit can open and that the χ and ψ subunits are not absolutely required the β ring at least transiently, we designed a more direct for the mechanics of this process. This conclusion is assay to detect opening of the dimer interface. This assay consistent with earlier data showing that efﬁcient stimula- is based on the following rationale: once β is assembled tion of processive DNA polymerase activity requires the onto DNA, the β–DNA complex is very stable (t 1h 1/2 774 Mechanics of clamp assembly on DNA Fig. 3. A ring-unloading assay reveals that δ opens β.[ P]β clamps were assembled onto circular DNA and incubated with γ, δ or δ in the absence or presence of ATP and analyzed by agarose gel electrophoresis (scheme). (A) An autoradiogram of the agarose gel shows the [ P]β on DNA separated from the [ P]β unloaded off DNA. (B) The gel stained with ethidium bromide shows that DNA is not degraded during the reaction. at 37°C; Yao et al., 1996). However, if the dimer interface δ and β compete for access to δ were to open even for a short time, the β ring could slip Previous studies identiﬁed δ as the only subunit of γ free of DNA. The experiment described below examines complex with a detectable interaction with β (Naktinis the ring-opening activity of the γ, δ and δ subunits by et al., 1995). When δ is part of the γ complex, the observing the unloading of circular clamps from a circular interaction with β is not favorable in the absence of ATP DNA substrate. (Naktinis et al., 1995). In the presence of ATP, however, In this experiment, P-labeled β clamps were assembled γ complex undergoes a conformational change and binds onto circular DNA by γ complex, and the [ P]β on DNA β with similar afﬁnity as δ alone (Naktinis et al., 1995; was separated from free γ complex and free [ P]β by gel Hingorani and O’Donnell, 1998). These observations sug- ﬁltration. The [ P]β–DNA complex was incubated further gest that δ is partially buried in the γ complex and that with either γ, δ or δ, in the absence or presence of ATP. the ATP-induced conformational change in γ complex Following this, the reactions were analyzed on an agarose results in exposure of δ for interaction with β. Which 32 32 gel to separate [ P]β trapped on DNA from the [ P]β subunit of the γ complex is responsible for modulating released from DNA. The results in Figure 3A, show that the access of β to δ? Our previous studies have shown the δ subunit by itself can release [ P]β from DNA, and that δ binds stably to the δ subunit (Onrust and O’Donnell, this process does not require ATP. The lack of ATP 1993). Therefore, we examined whether δ might interfere dependence is consistent with earlier evidence that δ binds with the interaction between δ and β. β in the absence of ATP (Naktinis et al., 1995; note In the experiment shown in Figure 4, we examined the neither δ nor β binds ATP). It also veriﬁes that the [ P]β interactions between δ, δ and β by gel ﬁltration using a unloading (mediated by δ) observed in this assay is not a Superose 12 sizing column. Figure 4A, B and C shows result of a γ complex contaminant in the δ protein the elution proﬁles of β (25 μM dimer), δ (5 μM) and δ preparation, since γ complex requires ATP to release β (5 μM), respectively. Figure 4D shows that β (25 μM) from DNA (Naktinis et al., 1996). The gel was also mixed with the δ subunit (5 μM) forms a δβ complex, stained with ethidium bromide (Figure 3B), which showed and Figure 4E shows that a 1:1 mix of δ (5 μM) and δ that the circular DNA remained intact during incubation (5 μM) results in a stable δδ complex. Finally, Figure 4F with each protein, including δ. The data conﬁrm that β shows that when β (25 μM) is added to puriﬁed δδ release from DNA is a result of ring opening and not due complex (5 μM), the δ subunit interacts preferentially to the ring sliding off linearized DNA, as the circular with β (to form δβ), resulting in the displacement of δ. DNA substrate is not degraded during the assay. The δβ complex is favored over δδ even when the These results show that clamp opening is actually reaction contains a 3-fold excess of δ over β (data performed by only one subunit of the γ complex, and is not shown). the result of a simple protein–protein interaction. It should These protein–protein interaction studies indicate that be noted that even though δ opens β enough to allow it β competes with δ for interaction with δ, and provide to slip free from DNA, it cannot load β onto DNA. Thus, some insight into how the γ complex subunits may work the γ and δ subunits play a critical role in assembly of during clamp loading. If, within the γ complex, δ binds the open clamp around DNA. The following experiments δ and blocks access of β to δ, then the ATP-dependent examine how the δ and γ subunits function in clamp change in γ complex conformation may help remove the loading. δ block and favor δ–β interaction over the δ–δ interaction. 775 J.Turner et al. Fig. 5. ATP binding to γ complex facilitates interaction with DNA. Interactions of γ complex and β with DNA were assayed in the presence of ATP or ATP-γ-S. [ H]γ complex (500 fmol) β (500 fmol) or [ P]β γ complex were incubated with primed M13mp18 DNA (400 fmol) and 2 mM ATP or ATP-γ-S at 37°C, and analyzed by gel ﬁltration. The fraction numbers are indicated beneath the elution Fig. 4. β and δ compete for binding to δ. Protein–protein interactions proﬁles and the fractions corresponding to proteins ‘on DNA’ and were analyzed on a Superose 12 sizing column, and the column ‘free’ protein are indicated above the proﬁles. (A and B) The elution fractions were analyzed by SDS–PAGE. Fraction numbers are 32 3 proﬁles of [ P]β (d) and [ H]γ complex (r), respectively, in the indicated above the gels, and the positions of β, δ and δ are absence of nucleotide. (C and D) In the presence of ATP-γ-S, β (d) indicated. (A) The elution proﬁle of β alone (25 μM dimer), (B) δ and γ complex (r) are bound to DNA. (E and F) In the presence of (5 μM) and (C) δ (5 μM). Protein mixtures were incubated at 25°C hydrolyzable ATP, β is retained on DNA (d) and γ complex is for 15 min prior to analysis. (D) The elution proﬁle of a mixture of δ released (r). (5 μM) and β (25 μM), (E) the δδ complex (5 μM) and (F) the proﬁle of β (25 μM) mixed with puriﬁed δδ (5 μM). strated that energy from ATP binding to γ powers the Later in the reaction, when the γ complex binds DNA, events leading to ring opening. The next experiment the δ–β interaction may be severed to allow the β ring to examines interaction of γ complex and β with DNA, and close around DNA. Thus, δ may serve as a modulator the role of ATP binding and ATP hydrolysis in the clamp- during clamp assembly, ﬁrst by facilitating access of δ to loading reaction. Mixtures of [ H]β and γ complex, as β, allowing it to open the ring, and then by facilitating well as β and [ H]γ complex, were used to follow their the dissociation of δ from β, thereby allowing the ring to presence in complex with a primed DNA substrate. In the 3 3 close around DNA (see Discussion; see Figure 7). Detailed absence of ATP, no binding of [ H]β or [ H]γ complex to studies with modiﬁed δ protein constructs are underway DNA is detectable (Figure 5A and B, respectively). In the 3 3 to explore further its role as a modulator in the mechanics presence of ATP-γ-S, both [ H]β and [ H]γ complex co- of clamp loader activity. migrate with the DNA (Figure 5C and D, respectively). No interaction between the γ subunit and δ or β has β appears essential for stable binding of γ complex to been detected. Therefore, if β were to completely severe DNA, as no interaction of γ complex with DNA can be the δ–δ contact in γδδ, the δβ complex would be detected by gel ﬁltration in the absence of β, whether or expected to dissociate from γδ. Since this does not occur, not ATP or ATP-γ-S is present in the reaction (Hingorani the γ or δ subunits presumably maintain some contact and O’Donnell, 1998). In the presence of ATP, only [ H]β with δ and/or β in the γδδ–β complex. is observed on the DNA, whereas [ H]γ complex elutes as free protein (Figure 5E and F, respectively; see also ATP binding signals γ complex–β to bind DNA and Stukenberg et al., 1991). These data indicate that ATP ATP hydrolysis ejects γ complex from β on DNA binding to γ complex locks the clamp loader, clamp and Clamp loading is a multistep process involving more than DNA in a ternary complex that is stable to gel ﬁltration, simple opening of the β ring. For example, after δ binds while ATP hydrolysis leads to release of the γ complex, to β and opens the ring, β must be positioned at a primed leaving the β ring on DNA. DNA site and then closed to form a continuous protein The next experiment was designed to measure the ring around DNA. The experiment in Figure 1 demon- number of ATP molecules hydrolyzed by γ complex for 776 Mechanics of clamp assembly on DNA Table I. ATP hydrolyzed per clamp assembled on DNA Table II. ATPase activity of the clamp loader γ complex β on DNA ATP hydrolyzed ATP/β ratio γ complex substrate k (per min) cat (nM) (fmol) (fmol) None 12 25 446 850 1.9 β 1.7 50 748 1884 2.5 Primed M13 DNA 55 100 946 2928 3.1 ss M13 DNA 35 200 1405 3640 2.6 β primed M13 DNA 110 β ss M13 DNA 25 32 3 [ P]ATP hydrolysis and [ H]β assembly onto DNA were measured in Primed oligoDNA 72 the same experiment, by TLC and gel ﬁltration, respectively. ss oligoDNA 108 Quantitation of ADP formed and β on DNA yielded the molar ratio of β primed oligoDNA 350 ATP hydrolyzed per clamp assembled on DNA. β ss oligoDNA 120 γ complex (0.1 μM)-catalyzed ATP (1 mM) hydrolysis was measured at 37°C, in the absence or presence of β (0.2 μM) and DNA each β clamp assembled on DNA. The clamp-loading (0.1–0.5 μM). The k was determined by dividing the rate of cat reaction was performed with H-labeled β in the presence formation of ADP by γ complex concentration. of [ P]ATP. The reactions were analyzed by thin-layer chromatography (TLC) to determine the amount of ATP hydrolyzed, and by gel ﬁltration to quantitate the number to minimize futile cycles of ATP hydrolysis until γ complex of β clamps loaded on DNA. The experiment was repeated interacts with DNA. at four different concentrations of γ complex. In each DNA substrates stimulate the ATP hydrolysis activity case, ~2–3 ATP were hydrolyzed for each β clamp of γ complex (Onrust et al., 1991). In the absence of β, assembled onto DNA (Table I). single-stranded DNA and primed DNA increase the There is some ambiguity regarding the stoichiometry γ complex ATPase rate from 12/min (γ complex alone) to of γ subunits per γ complex. The free γ subunit can form 35 and 55/min, respectively (Table II). We have observed tetramers (Tsuchihashi and Kornberg, 1989; Dallmann and that γ complex dissociates from DNA after hydrolyzing McHenry, 1995; Onrust et al., 1995a); however, within γ ATP (Figure 5). Thus, DNA may hasten the turnover of complex, the estimates range from two to four γ subunits γ complex, such that on binding DNA, the clamp loader per complex (Maki and Kornberg, 1988; Dallmann and rapidly hydrolyzes ATP and releases DNA to return to its McHenry, 1995; Onrust et al., 1995a,b). A recent analysis original conformation. of the molecular mass of γ complex by multi-angle laser Finally, the ATPase activity of γ complex was measured light scattering indicates a molar ratio of 2.5 γ subunits in the presence of both the β clamp and DNA. Addition per γ complex (unpublished data). Furthermore, quantitat- of DNA to the reaction containing γ complex–β resulted ive ATP binding experiments yield a stoichiometry of two in a large increase in γ complex ATPase activity (Figure ATP molecules per γ complex (Hingorani and O’Donnell, 6A; Table II). Primed single-stranded DNA (ssDNA) is a 1998). These data suggest that even if there are more than better effector of ATP hydrolysis than ssDNA (k 110 cat two γ subunits per clamp loader, only two bind ATP, and 25/min, respectively, at 37°C). The above experiments which is consistent with hydrolysis of 2–3 ATP per were performed with 1 mM ATP in the reaction to ensure β clamp assembled on DNA as shown above. The results maximum ATPase activity (γ complex K for ATP ranges with the γ complex clamp loader are also consistent with from 10 to 30 μM with unprimed ssDNA and primed an earlier study indicating that two ATP are hydrolyzed ssDNA substrates). Furthermore, to ensure saturating con- during formation of a processive DNA polymerase III centrations of the DNA substrates, we repeated the meas- holoenzyme complex on primed DNA (Burgers and urements in the presence of an excess of a synthetic Kornberg, 1982). ssDNA 100mer or a synthetic primed template DNA (28mer primer/100mer template), which serves as a tem- Coupling of ATP hydrolysis to clamp assembly plate for β loading (Bloom et al., 1996). Again, the depends on the DNA structure γ complex hydrolyzes ATP at a higher rate in the presence The previous experiments demonstrated that ATP binding of primed oligoDNA (350/min) than in the presence of ss drives γ complex to open the β ring and to bind DNA, oligoDNA (120 min; Table II). whereas ATP hydrolysis ejects γ complex from β–DNA. In Figure 6B, we investigated whether ATP hydrolysis Next, we examined γ complex ATPase activity to determine stimulated by ssDNA leads to assembly of β clamps on the effects of clamp-loading substrates (β and DNA) on ssDNA. The results show that no loading of [ H]β clamps ATP hydrolysis. The ATPase rate was quantitated in the can be detected on unprimed, single-stranded M13DNA. absence and presence of β at 37°C (Table II). The data Thus, γ complex ATPase activity is not coupled to clamp show that the γ complex ATPase activity is markedly assembly on this substrate. The speciﬁcity of γ complex inhibited by β, dropping from a turnover rate of 12/min activity for clamp loading on primed DNA reﬂects the to 1.7/min, respectively (see also Figure 1C). The need for sliding clamps at primed sites on DNA that can γ complex still binds ATP with high afﬁnity in the presence be extended by the DNA polymerase. The uncoupled of β (K 2 μM; Hingorani and O’Donnell, 1998). ATPase activity at unprimed ssDNA may in fact play an Therefore, this result implies that binding of β to important role during lagging strand synthesis by helping γ complex stabilizes it in the ATP-bound state, leading to γ complex locate primed sites on DNA by scanning inhibition of ATP hydrolysis. Presumably, inhibition of long stretches of ssDNA via repeated DNA binding and ATPase activity is important in the clamp-loading pathway release events. 777 J.Turner et al. Fig. 6. ATP hydrolysis is coupled to clamp assembly at a primed site on DNA. (A) Primed M13mp18 ssDNA stimulates the ATPase activity of γ complex (in the presence of β) from 1.7/min (Table I) to 110/min (m), while ssDNA stimulates the ATPase rate to 25/min (j). (B)A gel ﬁltration analysis of γ complex-catalyzed assembly of [ H]β on primed DNA (m) compared with assembly on ssDNA (j). The fraction numbers are indicated beneath the elution proﬁle, and the fractions corresponding to ‘β on DNA’ and ‘free β’ are indicated above the proﬁle. Discussion Fig. 7. A model of subunit dynamics during γ complex-catalyzed Key elements of the clamp-loading mechanism assembly of β onto DNA. (A) γ is the ‘C’-shaped motor subunit of the clamp loader that harnesses the energy of ATP for assembly of β onto The γ complex catalyzes assembly of the β clamp onto a DNA. On binding ATP, a conformational change in γ affects δ, circular DNA molecule to form a protein–DNA catenane. another ‘C’-shaped protein, which leads to exposure of the δ ring In a process fueled by ATP, the clamp loader opens the opener. (B) δ binds β and opens the ring at one interface. (C) The clamp at the dimer interface, binds DNA, and then releases ATPase activity of this protein complex is suppressed until it binds DNA. (D) An encounter with primed DNA stimulates ATP hydrolysis, the clamp so that it forms a closed ring around DNA. The which is coupled to release of the β clamp and DNA. The open β δ subunit is the key component of γ complex that opens dimer snaps shut to form a ring around DNA. the β ring. The interaction between δ and β yields sufﬁcient energy to destabilize the β dimer interface. Therefore, ring-opening does not require ATP binding or hydrolysis, Dissociation of γ complex from β is necessary for its but is a result of a simple protein–protein interaction. catalytic activity and, more importantly, it is a prerequisite Nevertheless, the δ ‘ring opener’ cannot facilitate assembly for processive DNA synthesis, as the clamp loader and of the open β ring onto DNA by itself. This process polymerase compete for the same site on β (Naktinis requires the combined activities of γ, δ and δ, indicating et al., 1996). Once γ complex releases β, the polymerase that clamp loading is a more complicated process than can bind the clamp and initiate DNA synthesis (Naktinis simply opening the ring. Thus, γ complex must perform et al., 1996). In DNA polymerase III holoenzyme, the several functions to complete the process of clamp assem- γ complex and both leading and lagging strand polymerases bly on DNA. For example, the open β ring and DNA are held together by a τ dimer (Onrust et al., 1995a). The must be positioned correctly for topological linkage, the γ complex catalyzes rapid assembly of sliding clamps ring must be closed and, ﬁnally, γ complex must dissociate onto DNA for use by both polymerases (Yuzhakov et al., from the ring on DNA. 1996). This is particularly signiﬁcant during lagging strand 778 Mechanics of clamp assembly on DNA synthesis. On the lagging strand, the polymerase releases primed DNA with high afﬁnity to form a γ complex– its β clamp upon ﬁnishing an Okazaki fragment; it then ATP–open β–DNA composite (Figure 7C). The DNA rapidly targets to a new β that has been assembled at a stimulates γ complex ATPase activity which leads to fresh upstream primed site by the γ complex. The deposition of the β clamp onto DNA, and ejection of γ complex also recycles the used β clamps that have been γ complex from β on DNA (Figure 7D). Two to three abandoned on completed Okazaki fragments (Naktinis molecules of ATP are hydrolyzed per clamp-loading event. et al., 1996). Thus, γ complex and polymerase work in Accordingly, the model in Figure 7 indicates that both concert for clamp loading and primer extension, resulting γ subunits hydrolyze ATP and that after hydrolysis (or in rapid and efﬁcient duplication of E.coli genomic DNA. upon ADP P dissociation), they return to their original ‘C’ shape. Presumably, at this point, the δ–β interaction A model for γ complex-catalyzed clamp loading is disrupted, possibly facilitated by the rebinding of δ to The γ complex can be described as a ‘switch’ protein that δ, thereby facilitating release of γ complex from β and alternates between the ‘on’ state (ATP bound) and the closure of the ring around DNA (Figure 7D). ‘off’ state (either ADP or no nucleotide bound). It is, The energy from ATP hydrolysis may be used simply however, more than a simple switch protein because in to orchestrate dissociation of the γ complex from the each state, and in the transition between these states, the clamp and DNA. Alternatively, ATP hydrolysis may fuel clamp loader machinery performs multistep functions that a more active process such as closing of the clamp around together result in clamp loading. A model summarizing DNA. At this time, we favor the former, ‘clamp release’ the current information is presented in Figure 7 and the mechanism, keeping in mind that ATP binding powers structure–function details are described below. The model assembly of all three components (γ complex, β and DNA) shows only two γ subunits, although estimates of the in close proximity, and that the lowest free energy state stoichiometry of γ in γ complex range from two to four of the free clamp is a closed ring. Therefore, when β is (Maki and Kornberg, 1988; Dallmann and McHenry, 1995; released, it should be capable of closing around DNA Onrust et al., 1995a,b). without further input of energy from ATP hydrolysis. Pre- The crystal structure of the δ subunit has been solved steady-state studies of clamp loading and γ complex recently (Guenther et al., 1997). The protein is composed ATPase activity are underway to deﬁne more precisely of three domains organized in the form of the letter ‘C’. the role of ATP hydrolysis in clamp loading. The top and bottom domains are barely connected through a small domain that may function as a hinge. The sequence Clamp assembly in vivo identity between δ and γ predicts that γ also has a ‘C’ DNA polymerase III holoenzyme contains two copies shape (Guenther et al., 1997). The structure of γ, modeled each of γ and τ (Onrust et al., 1995a). The γ subunit is after δ, predicts that the ATP-binding region lies very approximately the N-terminal two-thirds of the τ subunit, close to the hinge region between the top and bottom and it is created by a frameshift during protein expression domains of the γ ‘C’. This central position suggests that from the dnaX gene (Tsuchihashi and Kornberg, 1989; ATP binding to γ may perturb the ‘C’ shape, perhaps Blinkova and Walker, 1990; Flower and McHenry, 1990). resulting in movement of the top and bottom domains, as The frameshift allows addition of one unique amino acid speculated in Figure 7A. Recent reports of an ATP- to γ, followed by a stop codon. In vitro, both γ and induced conformational change in γ, and in γ complex, τ subunits can be assembled into functional clamp loaders are consistent with perturbation of the γ ‘C’ shape (Naktinis with the δ, δ, χ and ψ subunits (Onrust et al., 1995b). et al., 1995; Hingorani and O’Donnell, 1998). In addition, τ has the unique ability to bind the core The ATP-induced changes in γ suggest that it functions polymerase (McHenry, 1982; Studwell-Vaughan and as a ‘motor’, using the energy of ATP binding to power O’Donnell, 1991) as well as the DnaB helicase with high the conformational change in γ complex that takes δ from afﬁnity (Kim et al., 1996; Yuzhakov et al., 1996). These a buried position into one that is exposed and allows properties require the extra 213 amino acids in τ, which interaction with the β ring (Naktinis et al., 1996). The are absent in γ. Speciﬁcally a τ dimer, not γ, dimerizes present study indicates that δ may be the subunit that the two core DNA polymerases in the holoenzyme and prevents δ from binding β in the absence of ATP. Hence, contacts the DnaB helicase during DNA replication. ATP binding to γ may re-position δ and/or affect the ring Which protein, γ or τ, functions as the motor subunit opener δ, such that it prefers binding to β rather than δ. for the clamp loader within the holoenzyme, or do both γ In Figure 7A, this is depicted as a change in the δ subunit and τ form the clamp loader (i.e. a γτ complex)? This ‘C’ shape in response to the ATP-induced change in the question has been addressed in a previous study of the γ γ subunit ‘C’ shape (i.e. the δ conformation is guided by and τ proteins. ATP-binding site mutants of γ and τ were γ). The resulting arrangement leaves γ complex in the ‘on’ constructed which, when constituted into either γ complex state in which δ can bind the β clamp and open the ring or τ complex with δ, δ, χ and ψ, were unable to hydrolyze at one interface (Figure 7B). The resulting γ complex– ATP and were inactive in DNA replication assays with ATP–open β ring composite is stabilized by the decrease core polymerase and β (Xiao et al., 1995). The mutant in ATPase activity of the γ complex (Table II). If ATP proteins were also assembled into DNA polymerase III were hydrolyzed, the clamp loader would release the holoenzymes. The holoenzyme containing mutant γ clamp and ‘turn over’. Thus the inhibition of ATPase wild-type τ was inactive in DNA replication. The holo- activity probably maintains the integrity of the γ complex– ATP–open β ring composite for an encounter with a DNA enzyme containing mutant τ wild-type γ was as active substrate. as the wild-type DNA polymerase III holoenzyme. These The γ complex–ATP–open β ring composite binds results demonstrate that the ATP site of γ is needed for 779 J.Turner et al. holoenzyme function, and thus γ is the clamp loading DNA polymerase δ requires contact with the same face motor in the holoenzyme. of PCNA as RF-C. Hence RF-C must release PCNA for In normal cells, the holoenzyme contains both γ and τ the polymerase to gain access to the ring. Studies of (Maki et al., 1988). However, if the frameshift signal in human RF-C have shown that a three-subunit subassembly dnaX is mutated such that γ is not produced, the cells (36, 37 and 40 kDa subunits) as well as the 40 kDa remain viable (Blinkova et al., 1993). Presumably the subunit alone is capable of removing PCNA clamps from holoenzyme in these γ-less cells contains four τ subunits, DNA in the absence of ATP (Cai et al., 1997). These where two τ subunits dimerize the polymerases and the observations indicate that the RF-C clamp loaders may other two substitute for γ as the clamp loader motor. utilize a similar modular design and mechanism for However, even though the τ subunit can substitute for γ, assembly of rings around DNA as the E.coli clamp loader. its ATPase activity and clamp-loading activity is not The γ complex subunits also provide ancillary functions; essential when the γ subunit is present in the holoenzyme, for example the χ subunit binds SSB and is necessary for as demonstrated by the ATP site mutant study (Xiao displacement of the primase from RNA primers (Kelman et al., 1995). et al., 1998; Yuzhakov et al., 1999). It is tempting to The τ complex has the unique ability to utilize ATP- speculate that eukaryotic clamp loader subunits may also γ-S to provide the core polymerase with β-dependent provide additional functions at the site of DNA synthesis, processivity for DNA synthesis, although the level of other than clamp assembly on DNA. synthesis is much lower than in the presence of ATP; the The bacteriophage T4 clamp loader contains two differ- γ complex does not function in this capacity (Dallmann ent subunits (gp44 and gp62), and a circular sliding clamp et al., 1995). This phenomenon is interesting since neither (gp45), which is a loosely associated trimer that lacks the γ complex nor τ complex hydrolyze ATP-γ-S signiﬁcantly, stability of the β and PCNA rings (Yao et al., 1996). and ATP hydrolysis is required by both clamp loaders for Recent studies on the role of ATP in gp45 assembly on the completion of clamp assembly onto DNA (Hingorani DNA suggest that ATP hydrolysis is needed to open the and O’Donnell, 1998; unpublished data). The results of this gp45 ring (Latham et al., 1996; Sexton et al., 1996; report offer a new insight into the molecular mechanism by Pietroni et al., 1997). In contrast, in E.coli, ATP binding which the τ complex plus core, or the holoenzyme, may is sufﬁcient for ring opening, and ATP hydrolysis is used use ATP-γ-S for processive DNA replication. In as much to eject γ complex from the β ring on DNA. The unstable as τ and γ are closely related, and ATP-γ-S induces γ nature of the trimeric gp45 ring compared with the complex to bind β and open the ring, it may be presumed β dimer may account for this signiﬁcantly different role that ATP-γ-S has the same effect on τ complex. Since τ, for ATP hydrolysis in the T4 system compared with E.coli. unlike γ, can bind the core polymerase, it may form a For example, gp44/62-catalyzed opening of the gp45 bridge between the polymerase and the clamp, resulting trimer may enhance dissociation of the protomers and in an enzyme assembly that can function processively. preclude reclosure of the clamp around DNA. Therefore, The inability of γ to bind core would prevent formation it is possible that the T4 system has evolved to allow of such an assembly, and, therefore, γ complex cannot assembly of the clamp loader and closed clamp on DNA facilitate β-dependent DNA synthesis by the core in the before ATP hydrolysis, which powers both ring opening presence of ATP-γ-S. Thus, even though the γ complex and loading of the ring onto DNA at the same time. appears to be the primary clamp loader in DNA polymerase III holoenzyme, under artiﬁcial conditions, such as in the Clamp loaders in cell cycle regulation presence of ATP-γ-S (Dallmann et al., 1995) or when γ Recently, a number of clamp loader or clamp loader-like is absent from the holoenzyme (Blinkova et al., 1993), τ proteins have been implicated in cell cycle checkpoint is eminently capable of supporting clamp assembly, in pathways (reviewed in Elledge, 1996; Mossi and Hubscher, addition to dimerizing the polymerases and maintaining 1998). Classical checkpoint proteins sense particular contact with the helicase during DNA replication aspects of a cell’s environment or condition and ‘decide’ whether to restrict or promote cell cycle progression from A common mechanism for clamp loading? one phase to the next. For example, some checkpoint Clamp loaders of three other well studied systems include proteins monitor the integrity of DNA prior to and after the gp44/62 complex from bacteriophage T4, yRF-C from replication. If DNA is damaged, the cell cycle can be S.cerevisiae and hRF-C from humans. These clamp loaders arrested in G phase, slowed in S phase and arrested in use ATP to assemble their respective circular clamps, T4 G phase, to allow repair of the DNA prior to, during and gp45, yeast proliferating cell nuclear antigen (PCNA) and after DNA replication, respectively. Similarly, if DNA human PCNA, onto DNA. synthesis is incorrect or incomplete, DNA replication Like γ complex, both the yeast and human clamp loaders checkpoints can inhibit transition of the cell cycle (e.g. are ﬁve-protein machines (O’Donnell et al., 1993). Earlier from G to mitosis) until the speciﬁc problems are ﬁxed. studies have shown that ATP-γ-S leads to formation of an Three of the ﬁve subunits of the S.cerevisiae clamp RF-C–PCNA–DNA composite, but this is not competent loader, RF-C1, RF-C2 and RF-C5, are now thought for extension by DNA polymerase δ in the absence of participate in cell cycle checkpoint pathways involving hydrolyzable ATP (Lee and Hurwitz, 1990; Burgers, 1991). DNA metabolism (Cullmann et al., 1995; Sugimoto et al., A possible interpretation of this result is that RF-C 1996; Noskov et al., 1998). Although the underlying functions similarly to γ complex, in that ATP binding is mechanism by which these proteins regulate the cell cycle sufﬁcient for ring opening and interaction with DNA, but is not yet understood, it is possible that the regulatory ATP hydrolysis is needed to eject RF-C, allowing the pathways involve loading of PCNA onto DNA. For PCNA ring to close around DNA. As in the E.coli system, example, a clamp loader is crucial at an early stage in DNA 780 Mechanics of clamp assembly on DNA treatment results in ~30 primers per DNA circle (assuming an average replication for initiation of processive DNA synthesis; primer length of 25 nucleotides). Synthetic ssDNA 100mer and synthetic therefore, a shutdown of its clamp-loading activity can primed template DNA were prepared as described (Hingorani and prevent entry into S phase. The large subunit of human O’Donnell, 1998). RF-C (p140) has several putative phosphorylation sites Buffer A is 20 mM Tris–HCl pH 7.5, 0.1 mM EDTA and 10% glycerol. Superose 12 gel ﬁltration buffer is 20 mM Tris–HCl pH 7.5, for protein kinase A, protein kinase C and tyrosine kinases, 0.1 mM EDTA, 10% glycerol, 5 mM DTT and 100 mM NaCl. Reaction and this has led to speculation that the PCNA loading buffer is 20 mM Tris–HCl pH 7.5, 0.1 mM EDTA, 4% glycerol and activity of RF-C may be regulated by phosphorylation 8 mM MgCl . Bio-Gel A-15m gel ﬁltration buffer is reaction buffer events in checkpoint pathways (Mossi and Hubscher, plus 100 mM NaCl. Loading buffer is 50 mM Tris–HCl (pH 6.8), 1998). Alternatively, clamp loaders may serve as signal 100 mM DTT, 2% SDS, 0.1% bromophenol blue and 10% glycerol. sensors in checkpoint pathways through recognition of speciﬁc DNA structures produced during DNA replication Proteins The γ complex was constituted from pure γ, δ, δ, χ and ψ, followed by and/or repair processes. For example, recent evidence isolation from the free subunits on a MonoQ column (Pharmacia) as suggests that RF-C5 may interact with Spk1 (Rad53/ described (Onrust et al., 1995b). Puriﬁcation of γ (Studwell and Mec2/Sad1), an essential protein kinase for the transition O’Donnell, 1990), δ and δ (Dong et al., 1993), χ and ψ (Xiao et al., of S phase to mitosis (Sugimoto et al., 1996). 1993) was performed as described earlier. The δ and β proteins were Structural homologs of clamp loader subunits are also H-labeled by reductive methylation as described (Kelman et al., 1995a). The catalytic subunit of cAMP-dependent protein kinase produced in known to participate in cell cycle regulation. RAD24 in E.coli was the gift of Dr S.Taylor (University of California, San S.cerevisiae, which is homologous to E.coli γ and δ Diego, CA). proteins as well as all the RF-C subunits, is required for The modiﬁed versions of β were constructed by PerImmune, Inc. DNA damage checkpoint pathways, and its homolog in using a DNA oligonucleotide site-directed method. The mutant dnaN genes were placed into vectors encoding an N-terminal hexa-histidine Schizosaccharomyces pombe, RAD17, is required for both tag and a kinase recognition sequence (pHK vector; Kelman et al., DNA replication and damage checkpoints (Elledge, 1996). 1995b). The speciﬁc β modiﬁcations are described in detail later. Below Perhaps RAD24 and RAD17 interact with RF-C and is a summary of the puriﬁcation protocol. Modiﬁed β was overexpressed regulate its clamp-loading activity, or they may be subunits in E.coli BL21(DE3) pLysS cells by inducing cultures at OD 0.6 of other protein loaders that play a role in checkpoint with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were harvested, and heat lysed by the method of Wickner (1976). The pathways. A recent study has shown that Cdc6p, an cell lysate was precipitated sequentially with 35 and 70% ammonium S.cerevisiae protein essential for entry of cells into S phase, sulfate and the pellet was resuspended in 20 mM Tris–HCl (pH 7.9) also shares homology with prokaryotic and eukaryotic 500 mM NaCl and 5 mM imidazole. The protein was loaded onto a clamp loader subunits (Perkins and Difﬂey, 1998). It has 5 ml chelating Sepharose resin (Pharmacia) charged with NiSO , and bound proteins were eluted with a linear gradient of imidazole (60– also been suggested that Cdc6p may be involved in 500 mM) in the above buffer. Fractions were mixed immediately with assembly of Mcm proteins onto chromatin, and thereby EDTA (50 mM ﬁnal) to chelate any nickel in the eluate that tends to serves a function analogous to that of a clamp loader in precipitate protein. β was identiﬁed by SDS–PAGE, and peak fractions assembling proteins onto DNA (Perkins and Difﬂey, 1998). were pooled and dialyzed against buffer A. The pool was loaded onto an 8 ml MonoQ column equilibrated in buffer A, and the bound protein Although quite speculative in nature, these ﬁndings imply was eluted with a linear NaCl gradient (0–500 mM) in buffer A. Peak that the use of clamp loaders and their underlying mechan- fractions were pooled and frozen at –70°C. Each of the modiﬁed β isms may generalize to other processes beyond loading subunits had replication activity levels comparable with wild-type β. sliding clamps onto DNA. Studies of E.coli DNA replication have been a guide to Ring-opening assay understanding similar mechanisms in complex eukaryotes, The β clamp was modiﬁed by changing Leu273 to cysteine and Cys333 including humans. This report provides a detailed mechan- to serine. The resulting Cys273 residue is buried in the dimer interface (L273C), and the highly reactive surface Cys333 is removed. The ism for the function of the γ complex clamp loader, a remaining three cysteine residues in β were found unreactive to thiol- multiprotein enzyme that assembles sliding clamps onto labeling reagents and were left unchanged. The reagent used to probe DNA for processive and rapid DNA replication. The the interface cysteine was prepared by labeling 300 nmol of peptide structural and functional homology between γ complex, (NH -LRRASVP-COOH; Chiron mimotopes) with 0.03 U of cAMP- RF-C and other important cell cycle regulatory proteins dependent protein kinase catalytic subunit and [γ- P]ATP 200 μCi (3000 Ci/mmol) in 150 μl of reaction buffer (30 min at 37°C). Then, suggests that insights gleaned from the molecular mechan- ATP was added to a ﬁnal concentration of 10 mM and the mixture was ism of the γ complex will generalize to eukaryotic clamp incubated for an additional 30 min. The mixture was incubated with loaders, and perhaps to yet other critical processes in 5 mg of sulfosuccinimidylc 4-[N-maleimidomethyl] cyclohexane-1- DNA metabolism. carboxylate (Sulfo-SMCC; Pierce) at 25°C for 1 h, and the P-labeled maleimide was puriﬁed on a 24 ml Superdex-Peptide column (Pharmacia) equilibrated in 0.1 M sodium phosphate (pH 7.2), 0.15 M NaCl, using the Pharmacia SMART system. Aftera7ml void volume, 500 μl Materials and methods fractions were collected. The peak (fractions 17–18) containing [ P]ma- Materials leimide was identiﬁed by the co-incidence of the radioactive peak Radioactive nucleotides were purchased from Dupont-NEN. M13mp18 and the peak of absorbance at 305nm (λ for N-ethylmaleimide). max ssDNA was prepared by phenol extraction of puriﬁed M13mp18 phage [ P]maleimide concentration was determined at 305 nm, using a molar that had been banded twice in CsCl gradients as described (Turner and extension coefﬁcient of 620/M/cm for maleimide, and was typically at O’Donnell, 1995). Nicked circular DNA was prepared by incubating 400 μM. 200 μg of doubly CsCl-banded supercoiled Bluescript plasmid (pBS) The ring-opening assay was performed in 15 μl of reaction buffer with 100 U of mung bean nuclease (NEB) for 15 min, followed by containing 3 μM β,3 μM γ complex, 2 mM ATP and 3 μM primed phenol extraction and ethanol precipitation. Mung bean nuclease is a oligoDNA. Proteins were mixed on ice and the reaction was initiated single strand-speciﬁc nuclease that nicks supercoiled DNA once, and by with 5 μlof [ P]maleimide and a shift to 37°C. The reactions were relaxing the plasmid precludes further nicking. The DNA was ~90% quenched after 12 min with 5 μl of loading buffer 5 μlof1M nicked. Multiprimed DNA was prepared by incubating 0.6 μM DnaG DTT. Samples were analyzed on a 10% SDS–polyacrylamide gel that primase and 0.6 μM DnaB helicase with 1 mM NTPs for 30 min at subsequently was dried and analyzed on a PhosphorImager. The γ com- 37°C, followed by phenol extraction and ethanol precipitation. This plex subunits exhibit some background labeling by [ P]maleimide; 781 J.Turner et al. however, they are easily resolved from β during SDS–PAGE and do not assembled onto DNA. ATP hydrolysis was quantitated by TLC on PEI interfere with the detection of β ring opening. cellulose F sheets (EM Science), using 0.6 M potassium phosphate In experiments using ATP-γ-S, 5 μM each of γ complex and β were buffer, pH 3.4. The TLC sheet was dried and the molar amount of ADP incubated in 30 μl of reaction buffer with either 200 μM ATP or ATP- P formed was determined by analysis on a PhosphorImager. γ-S. Reactions were initiated by adding 10 μlof [ P]maleimide, and ATPase assays (or ATP-γ-Sase assays) measuring γ complex turnover 4 μl aliquots were removed and quenched at 0, 0.5, 1, 2, 5 and 10 min, rate were performed at 37°C with 0.1 μM γ complex, with or without and analyzed as described above. 0.2 μM β and with 1 mM ATP [α- P]ATP (or 0.5 mM ATP-γ-S [γ- S]ATP-γ-S). At various times, 5 μl aliquots of the reaction were Cross-linked β loading assay quenched with equal volumes of 0.5 M EDTA and analyzed as described For this experiment, Arg103 and Ile305 in β were changed to cysteine above. To compare the effect of primed and ssDNA on γ complex– S–S S–S to create a disulﬁde-linked β (β ). Native β and β were radiolabeled β-catalyzed ATP hydrolysis, similar reactions were performed, except with the catalytic subunit of cAMP-dependent protein kinase and with 0.1 μM M13 ssDNA coated with 17 μM SSB or with 0.1 μM [γ- P]ATP as described (Kelman et al., 1995a), except that DTT was primed M13 DNA coated with SSB in the reaction (or 0.5 μM ss- S–S omitted in order to maintain the disulﬁde cross-links in β . The oligoDNA and primed oligoDNA). The molar amounts of products γ complex used in this assay was constituted in the absence of DTT. formed were plotted versus time of reaction, and the slope of each The assembly reaction was performed in 50 μl of reaction buffer time course was divided by γ complex concentration to yield the containing 500 fmol of pBS DNA, 19 pmol of β, 5.4 pmol of γ complex turnover number. and 500 μM ATP, in the absence or presence of 20 mM DTT. The reaction was allowed to proceed for 10 min at 37°C and then loaded onto a 5 ml Bio-Gel A-15m gel ﬁltration column (Bio-Rad), equilibrated Acknowledgements in A-15m buffer in the presence or absence of 1% SDS. Fractions (200 μl) were collected and 40 μl aliquots were quantitated by liquid We are grateful to Dr Susan Taylor for the catalytic subunit of the scintillation counting or analyzed on a 12% SDS–polyacrylamide gel cAMP-dependent protein kinase. This work was supported by NIH grant under non-reducing conditions (no DTT). The gels were dried and GM38839. M.M.H. was supported by a grant from the Charles H.Revson exposed to ﬁlm or analyzed on a PhosphorImager (Molecular Dynamics) Foundation. Clamp-unloading assay β (1.6 μM), P-labeled at a C-terminal kinase site (Naktinis et al., References 1996), was incubated with 3.3 μM γ complex and 5 pmol of pBS DNA in 75 μl of reaction buffer containing 500 μM ATP for 10 min at 37°C. Blinkova,A.L. and Walker,J.L. (1990) Programmed ribosomal The reaction was applied to a 5 ml A-15m gel ﬁltration column and frameshifting generates the Escherichia coli DNA polymerase III γ eluted with A-15m buffer. Peak fractions, containing β on DNA, were subunit from within the τ subunit reading frame. Nucleic Acids Res., pooled, and 25 μl aliquots (containing 0.2 μM β) were incubated with 18, 1725–1729. 1 μM γ, δ or δ with or without 500 μM ATP. After 10 min, each sample Blinkova,A.L., Hervas,C., Stukenberg,P.T., Onrust,R., O’Donnell,M. and was mixed with glycerol (15% ﬁnal) and analyzed on a 1% agarose gel Walker,J.R. (1993) The Escherichia coli DNA polymerase III (89 mM Tris–borate buffer with 10 ng/ml ethidium bromide). After holoenzyme contains both products of the dnaX gene, τ and γ, but electrophoresis for 2 h at 100 V, the agarose gel was photographed under only τ is essential. J. Bacteriol., 175, 6018–6027. UV light and exposed to ﬁlm. Bloom,L.B., Turner,J.T., Kelman,Z., Beechem,J.M., O’Donnell,M. and Goodman,M. (1996) Dynamics of loading the β sliding clamp of Analysis of β, δ and δ interactions DNA polymerase III onto DNA. J. Biol. Chem., 271, 30699–30708. A δ complex was formed by mixing 2 mg of δ and 3mgof δ in buffer Burgers,P.M.J. (1991) Saccharomyces cerevisiae replication factor C. II. A followed by puriﬁcation on a 1 ml MonoQ column (with a linear Formation and activity of complexes with the proliferating cell nuclear gradient of 50–500 mM NaCl in buffer A). Then 5 μM of the pure δδ antigen and with DNA polymerases δ and ε. J. Biol. Chem., 266, complex was mixed with 25 μM β (dimer) in 200 μl of buffer A for 22698–22706. 15 min at 25°C, and analyzed by gel ﬁltration on a Superose 12 column Burgers,P.M.J. and Kornberg,A. (1982) ATP activation of DNA equilibrated with buffer A containing 100 mM NaCl. After a void polymerase III holoenzyme from Escherichia coli. II. Initiation volume of 7 ml, 180 μl fractions were collected, and 20 μl aliquots complex: stoichiometry and reactivity. J. Biol. Chem., 257, 11474– were analyzed by SDS–PAGE and visualized by Coomassie staining of 11478. the gels. The control gel ﬁltration proﬁles of β alone, δ alone, δ alone, Burgers,P.M.J. and Yoder,B.L. 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(1995a) Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader and sliding clamps in one holoenzyme http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals http://www.deepdyve.com/lp/springer-journals/the-internal-workings-of-a-dna-polymerase-clamp-loading-machine-GFpN0FQCSN

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V. Naktinis, J. Turner, M. O’Donnell (1996)
A Molecular Switch in a Replication Machine Defined by an Internal Competition for Protein Rings
Cell, 84
P. Burgers, B. Yoder (1993)
ATP-independent loading of the proliferating cell nuclear antigen requires DNA ends.
The Journal of biological chemistry, 268 27
H Xiao, N Naktinis, M O'Donnell (1995)
Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader and sliding clamps in one holoenzyme particle. IV. ATP‐binding site mutants identify the clamp loader
J Biol Chem, 270
Z. Kelman, A. Yuzhakov, Jelena Andjelkovic, M. O’Donnell (1998)
Devoted to the lagging strand—the χ subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly
The EMBO Journal, 17

Publisher: Springer Journals
Copyright: Copyright © European Molecular Biology Organization 1999
ISSN: 0261-4189
eISSN: 1460-2075
DOI: 10.1093/emboj/18.3.771
Publisher site: See Article on Publisher Site

Abstract

The EMBO Journal Vol.18 No.3 pp.771–783, 1999 The internal workings of a DNA polymerase clamp-loading machine 1 2 These protein rings are assembled onto DNA by their Jennifer Turner , Manju M.Hingorani , 1,3 2,4,5 respective clamp loaders in an ATP-dependent reaction Zvi Kelman and Mike O’Donnell (Kelman and O’Donnell, 1995). Unlike sequence-speciﬁc 2 4 The Rockefeller University and The Howard Hughes Medical DNA-binding proteins, the circular clamps form a topo- Institute, 1230 York Avenue, Cornell University Medical College, logical link with DNA and slide along the duplex without 1300 York Avenue and Sloan-Kettering Institute, 1275 York Avenue, localizing to a speciﬁc region. Therefore, when the DNA New York, NY 10021, USA polymerase binds its clamp, it is endowed with high Corresponding author processivity, allowing it to move continuously along the e-mail: [email protected] template during chain extension. The E.coli replicase, DNA polymerase III holoenzyme, Replicative DNA polymerases are multiprotein consists of 10 different polypeptide chains (Kornberg and machines that are tethered to DNA during chain Baker, 1992; Kelman and O’Donnell, 1995). Within the extension by sliding clamp proteins. The clamps are holoenzyme are two core polymerases (αεθ) and a clamp designed to encircle DNA completely, and they are loader (γ complex), that are held together by τ, a connector manipulated rapidly onto DNA by the ATP-dependent protein (Onrust et al., 1995a). Both core polymerases activity of a clamp loader. We outline the detailed become highly processive when tethered to DNA by mechanism of γ complex, a ﬁve-protein clamp loader β sliding clamps (Stukenberg et al., 1991). As the holo- that is part of the Escherichia coli replicase, DNA enzyme moves along the replication fork, the processive polymerase III holoenzyme. The γ complex uses ATP polymerase extends DNA continuously on the leading to open the β clamp and assemble it onto DNA. strand. On the lagging strand, the other polymerase releases Surprisingly, ATP is not needed for γ complex to crack its sliding clamp upon completion of each Okazaki frag- open the β clamp. The function of ATP is to regulate ment, and is cycled back to the replication fork by targeting the activity of one subunit, δ, which opens the clamp to a new clamp assembled at an upstream RNA primer simply by binding to it. The δ subunit acts as a by the clamp loader (O’Donnell, 1987; Stukenberg modulator of the interaction between δ and β.On et al., 1994). binding ATP, the γ complex is activated such that the The E.coli clamp loader, γ complex, consists of ﬁve δ subunit permits δ to bind β and crack open the ring different subunits: γ, δ, δ, χ and ψ (Maki and Kornberg, at one interface. The clamp loader–open clamp protein 1988). Within the γ complex are two to four γ subunits complex is now ready for an encounter with primed and one each of δ, δ, χ and ψ (Maki and Kornberg, DNA to complete assembly of the clamp around DNA. 1988; Onrust et al., 1995b). The proteins are assembled Interaction with DNA stimulates ATP hydrolysis which such that both δ and ψ bind directly to γ, while δ and χ ejects the γ complex from DNA, leaving the ring to bind the δ and ψ subunits, respectively (Onrust et al., close around the duplex. 1995b). Previous studies have shown that a γδδ complex Keywords: ATPase/clamp loader/DNA polymerase/ is sufﬁcient to load β onto DNA (Onrust et al., 1991). processivity/sliding clamp The χ and ψ subunits are not essential for β loading onto DNA; χ binds single-stranded DNA binding protein (SSB) and facilitates displacement of the primase from RNA Introduction primers, an event that must occur prior to clamp loading on the lagging strand (Kelman et al., 1998; Yuzhakov Replicases are efﬁcient DNA-synthesizing enzymes that et al., 1999). The δ subunit is the major contact point duplicate long chromosomes with high speed and pro- between γ complex and β, and δ binds tightly to β in the cessivity. These biological machines are comprised of complete absence of the other clamp loader proteins three functional components in both prokaryotic and (Naktinis et al., 1995). When δ is part of the γ complex, eukaryotic organisms: (i) a DNA polymerase, (ii) a pro- however, it exhibits only a low afﬁnity for β in the absence cessivity factor or sliding clamp protein and (iii) a multi- of ATP. In the presence of ATP, the γ complex undergoes protein clamp loader. Replicases from Escherichia coli, a conformational change that now allows δ to bind β with Saccharomyces cerevisiae, humans and bacteriophage T4 an afﬁnity comparable with that of the free δ–β interaction have these three components. The DNA polymerases lack (Naktinis et al., 1995). γ is the only clamp loader subunit high processivity alone but upon association with their that binds and hydrolyzes ATP (Maki and Kornberg, 1988; respective sliding clamps, they can replicate several thou- Tsuchihashi and Kornberg, 1989; Onrust et al., 1991). sand bases continuously (reviewed in Kelman and Recent studies show that ATP binding powers a change O’Donnell, 1995). The sliding clamps are ring-shaped homo-oligomers that encircle duplex DNA (Stukenberg in γ subunit conformation (Hingorani and O’Donnell, et al., 1991; Kong et al., 1992; Burgers and Yoder, 1993; 1998), which may underlie the ATP-induced change in γ Krishna et al., 1994; Gulbis et al., 1996; Yao et al., 1996). complex conformation that leads to tight interaction with © European Molecular Biology Organization 771 J.Turner et al. β (Naktinis et al., 1995; Hingorani and O’Donnell, 1998). Thus, the γ subunit transduces the energy from ATP to expose the δ subunit for interaction with the β ring. This report describes the mechanics of the γ complex- catalyzed process of β assembly onto DNA. We show that the energy of ATP binding powers the γ complex machinery to bind β and open the circular clamp at one interface. Once γ complex binds β, its ATPase activity is supressed until it binds DNA. The correct DNA substrate stimulates ATP hydrolysis, which is coupled to the release of γ complex from β on DNA. On DNA, β reassumes its lowest free energy state and forms a closed ring, now with DNA passing through the center. The molecular details that underlie the clamp loader activity have been explored and as a result, speciﬁc functions have been assigned to the three integral com- ponents of the γ complex: γ is the ‘motor’, δ is the ‘ring opener’ and δ is the ‘modulator’. The γ subunits are the only components of γ complex to interact with ATP, and their function is to drive ATP-induced conformational changes of other subunits in the clamp loader. The δ subunit contains the intrinsic clamp-opening activity, as it can open the β ring in the complete absence of ATP and the other subunits. The δ subunit binds δ and probably modulates the ability of β to gain access to δ. The ATP- driven γ motor either moves or alters the δ modulator such that the δ clamp opener gains access to the β clamp and opens the ring. This action requires only ATP binding to the γ complex. The resulting clamp loader–open clamp composite then binds primed DNA with high afﬁnity. Upon interaction with primed DNA, two or three molecules of ATP are hydrolyzed, resulting in closure of the β ring around DNA and ejection of γ complex off the DNA and back into solution. Results The γ complex uses energy from ATP binding to open the β ring The β sliding clamp is composed of two crescent-shaped Fig. 1. γ complex opens the β clamp on binding ATP. The scheme protomers arranged in a tight head-to-tail dimer (Kong indicates the position of a cysteine residue (S) within the dimer et al., 1992). This circular protein clamp is transferred interface that is accessible for reaction only when the ring is opened. The structure of the [ P]maleimide reagent used to label cysteine is onto circular DNA by the ATP-dependent clamp-loading also shown. (A) β (3 μM) incubated with [ P]maleimide in the activity of the γ complex. How does γ complex assemble absence or presence of γ complex (3 μM), primed oligoDNA (3 μM) the β ring around DNA? How is ATP used in this reaction? 32 or ATP (2 mM), and analyzed by SDS–PAGE. P-labeled β is Is ATP binding sufﬁcient, or is hydrolysis necessary? indicated by an arrow, showing a 2- and 10-fold increase in β labeling in the presence of γ complex and γ complex ATP, respectively. What is the role of DNA in this process? To begin (B) γ complex-catalyzed ring opening assayed for varying times in the addressing these questions, we developed a novel ‘Cys- presence of ATP (left) or ATP-γ-S (right). (C) γ complex-catalyzed labeling’ assay to detect and characterize the ring-opening ATP hydrolysis compared with ATP-γ-S hydrolysis. γ complex step in clamp assembly. (0.1 μM), incubated with β (0.2 μM), hydrolyzes ATP with a k cat –3 1.7/min (d) and hydrolyzes ATP-γ-S with a k 410 /min (j). Leu273, a buried hydrophobic residue in the β dimer cat interface, was substituted with cysteine (β crystal structure; Kong et al., 1992). This buried cysteine residue should modiﬁcation was made in L273C-β to ensure that only not be accessible to a thiol-reactive reagent, such as a the interface cysteine residue would be labeled; the reactive maleimide, when the L273C-β ring is closed. When the Cys333 at the protein surface was changed to serine. The interface is opened, however, the buried cysteine should modiﬁed β is a dimer and is fully active in DNA replication become accessible to the surface and reactive to maleimide. assays with γ complex and core polymerase (data not To follow this reaction, we synthesized a radioactive shown). reagent by linking the N-terminus of a P-labeled peptide The results of the ring-opening assay are shown in with an N-hydroxysuccinimide (NHS) ester coupled to Figure 1A. The [ P]maleimide reagent was incubated maleimide (Figure 1). If the dimer interface opens, the with β in the absence or presence of γ complex, ATP and interface cysteine residue will react with the [ P]maleim- DNA, and then the reactions were analyzed on an SDS– ide reagent, resulting in a radiolabeled β clamp. One other polyacrylamide gel. The autoradiogram of the gel shows 772 Mechanics of clamp assembly on DNA very low reactivity of β alone (Figure 1A, lane 1). When non-reducing SDS–polyacrylamide gel to determine if β is mixed with γ complex, there is a 2-fold increase in disulﬁde cross-links had formed. Figure 2A shows that in β labeling (Figure 1A, lane 2). However, when both the absence of dithiothreitol (DTT), more than half of the S–S γ complex and ATP are present in the reaction, the β is β migrates as an 80 kDa band of cross-linked dimers; 10-fold more reactive with the [ P]maleimide (Figure the rest migrates as a 40 kDa band of monomers (lane 1). 1A, lane 4). The results indicate that in the presence of Native β migrates as a single band at 40 kDa (lane 2). S–S ATP, γ complex can open the β ring or at least change Incubation of β with DTT reduces the disulﬁde cross- S–S the ring conformation enough to expose the cysteine links such that at 2 and 20 mM DTT essentially all β residue buried in the dimer interface. When DNA is migrates as monomers on the SDS–polyacrylamide gel present in the reaction, β is labeled to the same extent as (lanes 3 and 4, respectively). S–S in the absence of DNA (compare lanes 4 and 5), indicating Next we analyzed the cross-linked β for assembly 32 S–S that γ complex can open the β ring even in the absence around a circular DNA substrate. The P-labeled β of DNA. was incubated with γ complex and DNA in the presence Next, the assay was used to examine whether ATP binding of ATP, and then assayed by gel ﬁltration over a Bio-Gel or hydrolysis is required to stimulate β ring opening. To A-15m column (this large pore resin includes proteins but separate the effects of ATP binding from ATP hydrolysis, excludes the large DNA substrate as well as any protein S–S we substituted ATP with its analog, ATP-γ-S (Figure 1B). bound to it). Figure 2B shows that about half of the β The turnover number for ATP hydrolysis catalyzed by is assembled on DNA in the absence of DTT (β on DNA, γ complex (in the presence of β) is 1.5–2/min at 37°C fractions 9–13; free β, fractions 18–27). Treatment of S–S (Figure 1C). Therefore, in a 10 min assay, each γ complex β with DTT prior to the clamp loading reaction results S–S hydrolyzes 15–20 molecules of ATP. The γ complex binds in assembly of nearly all the β on DNA (fractions 9– ATP ATP-γ-S with similar high afﬁnity as it does ATP (K 13), and is comparable with assembly of native β on DNA ATP-γ-S 2 μM and K 5 μM, respectively; Hingorani and (Figure 2B). Presumably in the absence of DTT the O’Donnell, 1998), but the turnover number for ATP-γ-S disulﬁde cross-links at the interface prevent some (or all) –3 S–S hydrolysis is very low (4–610 /min; see Figure 1C). of the β dimers from being assembled on DNA. S–S Since γ complex catalyzes hydrolysis of only one ATP-γ-S To determine if the β clamps assembled onto DNA every 2–3 h, ATP-γ-S can be considered a non-hydrolyzable in the absence of DTT contain any cross-links, the analog of ATP within the time frame of the ring-opening column fractions were analyzed on a non-reducing SDS– assay. polyacrylamide gel (Figure 2C). As expected, non-cross- S–S The data shown in Figure 1B demonstrate that ATP and linked β was assembled onto DNA (monomers in ATP-γ-S facilitate γ complex-catalyzed ring opening to fractions 9–13). However, a large fraction of the cross- S–S the same extent. Therefore, ATP binding appears sufﬁcient linked β was also assembled onto DNA (dimers in to drive γ complex-catalyzed opening of the β ring. Other fractions 9–13), while the remainder eluted as free protein S–S non-hydrolyzable ATP analogs such as AMP-PNP and (dimers in fractions 18–27). Presumably the β dimers AMP-PCP were also tested in the assay, but ring opening on DNA contain only one cross-link, and therefore can S–S was not detected (data not shown). However, this negative still be opened at one interface. The β dimers that were result may be explained by the fact that γ complex binds not assembled on DNA may contain two disulﬁde cross- these analogs with much lower afﬁnity than ATP (K links that preclude ring opening and assembly onto DNA. S–S ~1 mM versus 2 μM for ATP), and this binding energy To conﬁrm that cross-linked β on DNA (dimers in may be insufﬁcient to support ring opening (H.Xiao and fractions 9–13; Figure 2C) is cross-linked at only one M.O’Donnell, unpublished data). interface, the experiment in the absence of DTT was Next, the ring-opening step was examined in greater repeated and split into two halves. One half was analyzed detail to determine whether the clamp loader opens β at by gel ﬁltration as described above (Figure 2C), to conﬁrm S–S both interfaces (e.g. monomerizes the dimer to assemble that β was assembled on DNA. The other half was it around DNA), or if opening the β dimer at only one analyzed by gel ﬁltration in the presence of SDS (Figure S–S interface is sufﬁcient to allow entry of DNA into the ring. 2D). If the β on DNA contains one disulﬁde cross-link, then SDS denaturation will result in a dimer opened at The γ complex need open only one interface of the the non-cross-linked interface that will dissociate from S–S β dimer for clamp loading DNA during gel ﬁltration. If the β on DNA were to To determine whether γ complex needs to open one or contain two disulﬁde cross-links, the protein ring will both interfaces of the β dimer for assembly of the clamp remain covalently sealed even after SDS denaturation, and around DNA, we constructed a β dimer in which the thus remain topologically linked to DNA in the presence S–S monomers could be cross-linked at the dimer interface. of SDS. The results show that the cross-linked β Arg103 and Ile305, two residues close to each other but dissociates from DNA during gel ﬁltration in the presence on opposing sides of the dimer interface (C atoms ~6 Å of SDS, indicating that the rings were cross-linked at only apart; β cyrstal structure; Kong et al., 1992), were substi- one interface (dimers in fractions 18–27; Figure 2D). tuted with cysteine. Due to the head-to-tail arrangement Thus, the γ complex can load clamps that are sealed at of the β dimer, the two interfaces are identical. It was one interface, and does not need to monomerize the expected that oxidation would result in formation of β dimer to load it onto DNA. disulﬁde cross-links between the cysteine residues, and yield β dimers covalently linked at one or both interfaces. δ is the ring opener S–S 32 Initially, the modiﬁed β (β ) was P-labeled at an We performed the Cys-labeling assay described in Figure 1 N-terminal kinase recognition site and analyzed on a with individual subunits and subassemblies of the 773 J.Turner et al. Fig. 2. γ complex opens the β clamp at one interface. Cysteine residues were placed at the β dimer interface to design a clamp with disulﬁde cross- S–S S–S links at the interface (β ). (A) Native β and β analyzed on a non-reducing SDS–polyacrylamide gel with no DTT, 2 mM DTT and 20 mM DTT 32 32 S–S in the reaction. (B) Gel ﬁltration analysis of native [ P]β (d) and [ P]β clamps assembled onto DNA by γ complex in the absence of DTT (s) S–S and in the presence of DTT (u). The next scheme shows a mix of β rings with two, one or zero cross-links at the interface, assembled on DNA S–S and analyzed by gel ﬁltration followed by non-reducing SDS–PAGE. (C) Analysis of β from a clamp-loading reaction without DTT [as in (B)]. (D) The same reaction analyzed with SDS present during gel ﬁltration. Fraction numbers are indicated beneath the gels. The positions of non-cross- S–S S–S linked β (monomers) and cross-linked β (dimers) are indicated by arrows. γ complex, to determine the minimal subunit requirement simultaneous presence of all three subunits, γ, δ and δ for ring opening. The minimal subassembly that tested (Onrust et al., 1991). However, the Cys-labeling assay for positive for ring opening was the three-subunit γδδ ring opening does not rigorously exclude the possibility complex, which, like γ complex, needs ATP or ATP-γ-S that a smaller subassembly than γδδ, or even just one to crack open the β dimer interface (data not shown). subunit, can crack open the dimer interface transiently. Individual subunits or combinations of two subunits did For example, the assay requires time for the [ P]maleimide not give a detectable signal. This result implies that more to react with an exposed cysteine residue, and is likely to than two proteins are needed to detect ring opening in the proceed more efﬁciently the longer the β dimer interface Cys-labeling assay, that γδδ is sufﬁcient for ring opening is held open. To investigate if any one subunit can open and that the χ and ψ subunits are not absolutely required the β ring at least transiently, we designed a more direct for the mechanics of this process. This conclusion is assay to detect opening of the dimer interface. This assay consistent with earlier data showing that efﬁcient stimula- is based on the following rationale: once β is assembled tion of processive DNA polymerase activity requires the onto DNA, the β–DNA complex is very stable (t 1h 1/2 774 Mechanics of clamp assembly on DNA Fig. 3. A ring-unloading assay reveals that δ opens β.[ P]β clamps were assembled onto circular DNA and incubated with γ, δ or δ in the absence or presence of ATP and analyzed by agarose gel electrophoresis (scheme). (A) An autoradiogram of the agarose gel shows the [ P]β on DNA separated from the [ P]β unloaded off DNA. (B) The gel stained with ethidium bromide shows that DNA is not degraded during the reaction. at 37°C; Yao et al., 1996). However, if the dimer interface δ and β compete for access to δ were to open even for a short time, the β ring could slip Previous studies identiﬁed δ as the only subunit of γ free of DNA. The experiment described below examines complex with a detectable interaction with β (Naktinis the ring-opening activity of the γ, δ and δ subunits by et al., 1995). When δ is part of the γ complex, the observing the unloading of circular clamps from a circular interaction with β is not favorable in the absence of ATP DNA substrate. (Naktinis et al., 1995). In the presence of ATP, however, In this experiment, P-labeled β clamps were assembled γ complex undergoes a conformational change and binds onto circular DNA by γ complex, and the [ P]β on DNA β with similar afﬁnity as δ alone (Naktinis et al., 1995; was separated from free γ complex and free [ P]β by gel Hingorani and O’Donnell, 1998). These observations sug- ﬁltration. The [ P]β–DNA complex was incubated further gest that δ is partially buried in the γ complex and that with either γ, δ or δ, in the absence or presence of ATP. the ATP-induced conformational change in γ complex Following this, the reactions were analyzed on an agarose results in exposure of δ for interaction with β. Which 32 32 gel to separate [ P]β trapped on DNA from the [ P]β subunit of the γ complex is responsible for modulating released from DNA. The results in Figure 3A, show that the access of β to δ? Our previous studies have shown the δ subunit by itself can release [ P]β from DNA, and that δ binds stably to the δ subunit (Onrust and O’Donnell, this process does not require ATP. The lack of ATP 1993). Therefore, we examined whether δ might interfere dependence is consistent with earlier evidence that δ binds with the interaction between δ and β. β in the absence of ATP (Naktinis et al., 1995; note In the experiment shown in Figure 4, we examined the neither δ nor β binds ATP). It also veriﬁes that the [ P]β interactions between δ, δ and β by gel ﬁltration using a unloading (mediated by δ) observed in this assay is not a Superose 12 sizing column. Figure 4A, B and C shows result of a γ complex contaminant in the δ protein the elution proﬁles of β (25 μM dimer), δ (5 μM) and δ preparation, since γ complex requires ATP to release β (5 μM), respectively. Figure 4D shows that β (25 μM) from DNA (Naktinis et al., 1996). The gel was also mixed with the δ subunit (5 μM) forms a δβ complex, stained with ethidium bromide (Figure 3B), which showed and Figure 4E shows that a 1:1 mix of δ (5 μM) and δ that the circular DNA remained intact during incubation (5 μM) results in a stable δδ complex. Finally, Figure 4F with each protein, including δ. The data conﬁrm that β shows that when β (25 μM) is added to puriﬁed δδ release from DNA is a result of ring opening and not due complex (5 μM), the δ subunit interacts preferentially to the ring sliding off linearized DNA, as the circular with β (to form δβ), resulting in the displacement of δ. DNA substrate is not degraded during the assay. The δβ complex is favored over δδ even when the These results show that clamp opening is actually reaction contains a 3-fold excess of δ over β (data performed by only one subunit of the γ complex, and is not shown). the result of a simple protein–protein interaction. It should These protein–protein interaction studies indicate that be noted that even though δ opens β enough to allow it β competes with δ for interaction with δ, and provide to slip free from DNA, it cannot load β onto DNA. Thus, some insight into how the γ complex subunits may work the γ and δ subunits play a critical role in assembly of during clamp loading. If, within the γ complex, δ binds the open clamp around DNA. The following experiments δ and blocks access of β to δ, then the ATP-dependent examine how the δ and γ subunits function in clamp change in γ complex conformation may help remove the loading. δ block and favor δ–β interaction over the δ–δ interaction. 775 J.Turner et al. Fig. 5. ATP binding to γ complex facilitates interaction with DNA. Interactions of γ complex and β with DNA were assayed in the presence of ATP or ATP-γ-S. [ H]γ complex (500 fmol) β (500 fmol) or [ P]β γ complex were incubated with primed M13mp18 DNA (400 fmol) and 2 mM ATP or ATP-γ-S at 37°C, and analyzed by gel ﬁltration. The fraction numbers are indicated beneath the elution Fig. 4. β and δ compete for binding to δ. Protein–protein interactions proﬁles and the fractions corresponding to proteins ‘on DNA’ and were analyzed on a Superose 12 sizing column, and the column ‘free’ protein are indicated above the proﬁles. (A and B) The elution fractions were analyzed by SDS–PAGE. Fraction numbers are 32 3 proﬁles of [ P]β (d) and [ H]γ complex (r), respectively, in the indicated above the gels, and the positions of β, δ and δ are absence of nucleotide. (C and D) In the presence of ATP-γ-S, β (d) indicated. (A) The elution proﬁle of β alone (25 μM dimer), (B) δ and γ complex (r) are bound to DNA. (E and F) In the presence of (5 μM) and (C) δ (5 μM). Protein mixtures were incubated at 25°C hydrolyzable ATP, β is retained on DNA (d) and γ complex is for 15 min prior to analysis. (D) The elution proﬁle of a mixture of δ released (r). (5 μM) and β (25 μM), (E) the δδ complex (5 μM) and (F) the proﬁle of β (25 μM) mixed with puriﬁed δδ (5 μM). strated that energy from ATP binding to γ powers the Later in the reaction, when the γ complex binds DNA, events leading to ring opening. The next experiment the δ–β interaction may be severed to allow the β ring to examines interaction of γ complex and β with DNA, and close around DNA. Thus, δ may serve as a modulator the role of ATP binding and ATP hydrolysis in the clamp- during clamp assembly, ﬁrst by facilitating access of δ to loading reaction. Mixtures of [ H]β and γ complex, as β, allowing it to open the ring, and then by facilitating well as β and [ H]γ complex, were used to follow their the dissociation of δ from β, thereby allowing the ring to presence in complex with a primed DNA substrate. In the 3 3 close around DNA (see Discussion; see Figure 7). Detailed absence of ATP, no binding of [ H]β or [ H]γ complex to studies with modiﬁed δ protein constructs are underway DNA is detectable (Figure 5A and B, respectively). In the 3 3 to explore further its role as a modulator in the mechanics presence of ATP-γ-S, both [ H]β and [ H]γ complex co- of clamp loader activity. migrate with the DNA (Figure 5C and D, respectively). No interaction between the γ subunit and δ or β has β appears essential for stable binding of γ complex to been detected. Therefore, if β were to completely severe DNA, as no interaction of γ complex with DNA can be the δ–δ contact in γδδ, the δβ complex would be detected by gel ﬁltration in the absence of β, whether or expected to dissociate from γδ. Since this does not occur, not ATP or ATP-γ-S is present in the reaction (Hingorani the γ or δ subunits presumably maintain some contact and O’Donnell, 1998). In the presence of ATP, only [ H]β with δ and/or β in the γδδ–β complex. is observed on the DNA, whereas [ H]γ complex elutes as free protein (Figure 5E and F, respectively; see also ATP binding signals γ complex–β to bind DNA and Stukenberg et al., 1991). These data indicate that ATP ATP hydrolysis ejects γ complex from β on DNA binding to γ complex locks the clamp loader, clamp and Clamp loading is a multistep process involving more than DNA in a ternary complex that is stable to gel ﬁltration, simple opening of the β ring. For example, after δ binds while ATP hydrolysis leads to release of the γ complex, to β and opens the ring, β must be positioned at a primed leaving the β ring on DNA. DNA site and then closed to form a continuous protein The next experiment was designed to measure the ring around DNA. The experiment in Figure 1 demon- number of ATP molecules hydrolyzed by γ complex for 776 Mechanics of clamp assembly on DNA Table I. ATP hydrolyzed per clamp assembled on DNA Table II. ATPase activity of the clamp loader γ complex β on DNA ATP hydrolyzed ATP/β ratio γ complex substrate k (per min) cat (nM) (fmol) (fmol) None 12 25 446 850 1.9 β 1.7 50 748 1884 2.5 Primed M13 DNA 55 100 946 2928 3.1 ss M13 DNA 35 200 1405 3640 2.6 β primed M13 DNA 110 β ss M13 DNA 25 32 3 [ P]ATP hydrolysis and [ H]β assembly onto DNA were measured in Primed oligoDNA 72 the same experiment, by TLC and gel ﬁltration, respectively. ss oligoDNA 108 Quantitation of ADP formed and β on DNA yielded the molar ratio of β primed oligoDNA 350 ATP hydrolyzed per clamp assembled on DNA. β ss oligoDNA 120 γ complex (0.1 μM)-catalyzed ATP (1 mM) hydrolysis was measured at 37°C, in the absence or presence of β (0.2 μM) and DNA each β clamp assembled on DNA. The clamp-loading (0.1–0.5 μM). The k was determined by dividing the rate of cat reaction was performed with H-labeled β in the presence formation of ADP by γ complex concentration. of [ P]ATP. The reactions were analyzed by thin-layer chromatography (TLC) to determine the amount of ATP hydrolyzed, and by gel ﬁltration to quantitate the number to minimize futile cycles of ATP hydrolysis until γ complex of β clamps loaded on DNA. The experiment was repeated interacts with DNA. at four different concentrations of γ complex. In each DNA substrates stimulate the ATP hydrolysis activity case, ~2–3 ATP were hydrolyzed for each β clamp of γ complex (Onrust et al., 1991). In the absence of β, assembled onto DNA (Table I). single-stranded DNA and primed DNA increase the There is some ambiguity regarding the stoichiometry γ complex ATPase rate from 12/min (γ complex alone) to of γ subunits per γ complex. The free γ subunit can form 35 and 55/min, respectively (Table II). We have observed tetramers (Tsuchihashi and Kornberg, 1989; Dallmann and that γ complex dissociates from DNA after hydrolyzing McHenry, 1995; Onrust et al., 1995a); however, within γ ATP (Figure 5). Thus, DNA may hasten the turnover of complex, the estimates range from two to four γ subunits γ complex, such that on binding DNA, the clamp loader per complex (Maki and Kornberg, 1988; Dallmann and rapidly hydrolyzes ATP and releases DNA to return to its McHenry, 1995; Onrust et al., 1995a,b). A recent analysis original conformation. of the molecular mass of γ complex by multi-angle laser Finally, the ATPase activity of γ complex was measured light scattering indicates a molar ratio of 2.5 γ subunits in the presence of both the β clamp and DNA. Addition per γ complex (unpublished data). Furthermore, quantitat- of DNA to the reaction containing γ complex–β resulted ive ATP binding experiments yield a stoichiometry of two in a large increase in γ complex ATPase activity (Figure ATP molecules per γ complex (Hingorani and O’Donnell, 6A; Table II). Primed single-stranded DNA (ssDNA) is a 1998). These data suggest that even if there are more than better effector of ATP hydrolysis than ssDNA (k 110 cat two γ subunits per clamp loader, only two bind ATP, and 25/min, respectively, at 37°C). The above experiments which is consistent with hydrolysis of 2–3 ATP per were performed with 1 mM ATP in the reaction to ensure β clamp assembled on DNA as shown above. The results maximum ATPase activity (γ complex K for ATP ranges with the γ complex clamp loader are also consistent with from 10 to 30 μM with unprimed ssDNA and primed an earlier study indicating that two ATP are hydrolyzed ssDNA substrates). Furthermore, to ensure saturating con- during formation of a processive DNA polymerase III centrations of the DNA substrates, we repeated the meas- holoenzyme complex on primed DNA (Burgers and urements in the presence of an excess of a synthetic Kornberg, 1982). ssDNA 100mer or a synthetic primed template DNA (28mer primer/100mer template), which serves as a tem- Coupling of ATP hydrolysis to clamp assembly plate for β loading (Bloom et al., 1996). Again, the depends on the DNA structure γ complex hydrolyzes ATP at a higher rate in the presence The previous experiments demonstrated that ATP binding of primed oligoDNA (350/min) than in the presence of ss drives γ complex to open the β ring and to bind DNA, oligoDNA (120 min; Table II). whereas ATP hydrolysis ejects γ complex from β–DNA. In Figure 6B, we investigated whether ATP hydrolysis Next, we examined γ complex ATPase activity to determine stimulated by ssDNA leads to assembly of β clamps on the effects of clamp-loading substrates (β and DNA) on ssDNA. The results show that no loading of [ H]β clamps ATP hydrolysis. The ATPase rate was quantitated in the can be detected on unprimed, single-stranded M13DNA. absence and presence of β at 37°C (Table II). The data Thus, γ complex ATPase activity is not coupled to clamp show that the γ complex ATPase activity is markedly assembly on this substrate. The speciﬁcity of γ complex inhibited by β, dropping from a turnover rate of 12/min activity for clamp loading on primed DNA reﬂects the to 1.7/min, respectively (see also Figure 1C). The need for sliding clamps at primed sites on DNA that can γ complex still binds ATP with high afﬁnity in the presence be extended by the DNA polymerase. The uncoupled of β (K 2 μM; Hingorani and O’Donnell, 1998). ATPase activity at unprimed ssDNA may in fact play an Therefore, this result implies that binding of β to important role during lagging strand synthesis by helping γ complex stabilizes it in the ATP-bound state, leading to γ complex locate primed sites on DNA by scanning inhibition of ATP hydrolysis. Presumably, inhibition of long stretches of ssDNA via repeated DNA binding and ATPase activity is important in the clamp-loading pathway release events. 777 J.Turner et al. Fig. 6. ATP hydrolysis is coupled to clamp assembly at a primed site on DNA. (A) Primed M13mp18 ssDNA stimulates the ATPase activity of γ complex (in the presence of β) from 1.7/min (Table I) to 110/min (m), while ssDNA stimulates the ATPase rate to 25/min (j). (B)A gel ﬁltration analysis of γ complex-catalyzed assembly of [ H]β on primed DNA (m) compared with assembly on ssDNA (j). The fraction numbers are indicated beneath the elution proﬁle, and the fractions corresponding to ‘β on DNA’ and ‘free β’ are indicated above the proﬁle. Discussion Fig. 7. A model of subunit dynamics during γ complex-catalyzed Key elements of the clamp-loading mechanism assembly of β onto DNA. (A) γ is the ‘C’-shaped motor subunit of the clamp loader that harnesses the energy of ATP for assembly of β onto The γ complex catalyzes assembly of the β clamp onto a DNA. On binding ATP, a conformational change in γ affects δ, circular DNA molecule to form a protein–DNA catenane. another ‘C’-shaped protein, which leads to exposure of the δ ring In a process fueled by ATP, the clamp loader opens the opener. (B) δ binds β and opens the ring at one interface. (C) The clamp at the dimer interface, binds DNA, and then releases ATPase activity of this protein complex is suppressed until it binds DNA. (D) An encounter with primed DNA stimulates ATP hydrolysis, the clamp so that it forms a closed ring around DNA. The which is coupled to release of the β clamp and DNA. The open β δ subunit is the key component of γ complex that opens dimer snaps shut to form a ring around DNA. the β ring. The interaction between δ and β yields sufﬁcient energy to destabilize the β dimer interface. Therefore, ring-opening does not require ATP binding or hydrolysis, Dissociation of γ complex from β is necessary for its but is a result of a simple protein–protein interaction. catalytic activity and, more importantly, it is a prerequisite Nevertheless, the δ ‘ring opener’ cannot facilitate assembly for processive DNA synthesis, as the clamp loader and of the open β ring onto DNA by itself. This process polymerase compete for the same site on β (Naktinis requires the combined activities of γ, δ and δ, indicating et al., 1996). Once γ complex releases β, the polymerase that clamp loading is a more complicated process than can bind the clamp and initiate DNA synthesis (Naktinis simply opening the ring. Thus, γ complex must perform et al., 1996). In DNA polymerase III holoenzyme, the several functions to complete the process of clamp assem- γ complex and both leading and lagging strand polymerases bly on DNA. For example, the open β ring and DNA are held together by a τ dimer (Onrust et al., 1995a). The must be positioned correctly for topological linkage, the γ complex catalyzes rapid assembly of sliding clamps ring must be closed and, ﬁnally, γ complex must dissociate onto DNA for use by both polymerases (Yuzhakov et al., from the ring on DNA. 1996). This is particularly signiﬁcant during lagging strand 778 Mechanics of clamp assembly on DNA synthesis. On the lagging strand, the polymerase releases primed DNA with high afﬁnity to form a γ complex– its β clamp upon ﬁnishing an Okazaki fragment; it then ATP–open β–DNA composite (Figure 7C). The DNA rapidly targets to a new β that has been assembled at a stimulates γ complex ATPase activity which leads to fresh upstream primed site by the γ complex. The deposition of the β clamp onto DNA, and ejection of γ complex also recycles the used β clamps that have been γ complex from β on DNA (Figure 7D). Two to three abandoned on completed Okazaki fragments (Naktinis molecules of ATP are hydrolyzed per clamp-loading event. et al., 1996). Thus, γ complex and polymerase work in Accordingly, the model in Figure 7 indicates that both concert for clamp loading and primer extension, resulting γ subunits hydrolyze ATP and that after hydrolysis (or in rapid and efﬁcient duplication of E.coli genomic DNA. upon ADP P dissociation), they return to their original ‘C’ shape. Presumably, at this point, the δ–β interaction A model for γ complex-catalyzed clamp loading is disrupted, possibly facilitated by the rebinding of δ to The γ complex can be described as a ‘switch’ protein that δ, thereby facilitating release of γ complex from β and alternates between the ‘on’ state (ATP bound) and the closure of the ring around DNA (Figure 7D). ‘off’ state (either ADP or no nucleotide bound). It is, The energy from ATP hydrolysis may be used simply however, more than a simple switch protein because in to orchestrate dissociation of the γ complex from the each state, and in the transition between these states, the clamp and DNA. Alternatively, ATP hydrolysis may fuel clamp loader machinery performs multistep functions that a more active process such as closing of the clamp around together result in clamp loading. A model summarizing DNA. At this time, we favor the former, ‘clamp release’ the current information is presented in Figure 7 and the mechanism, keeping in mind that ATP binding powers structure–function details are described below. The model assembly of all three components (γ complex, β and DNA) shows only two γ subunits, although estimates of the in close proximity, and that the lowest free energy state stoichiometry of γ in γ complex range from two to four of the free clamp is a closed ring. Therefore, when β is (Maki and Kornberg, 1988; Dallmann and McHenry, 1995; released, it should be capable of closing around DNA Onrust et al., 1995a,b). without further input of energy from ATP hydrolysis. Pre- The crystal structure of the δ subunit has been solved steady-state studies of clamp loading and γ complex recently (Guenther et al., 1997). The protein is composed ATPase activity are underway to deﬁne more precisely of three domains organized in the form of the letter ‘C’. the role of ATP hydrolysis in clamp loading. The top and bottom domains are barely connected through a small domain that may function as a hinge. The sequence Clamp assembly in vivo identity between δ and γ predicts that γ also has a ‘C’ DNA polymerase III holoenzyme contains two copies shape (Guenther et al., 1997). The structure of γ, modeled each of γ and τ (Onrust et al., 1995a). The γ subunit is after δ, predicts that the ATP-binding region lies very approximately the N-terminal two-thirds of the τ subunit, close to the hinge region between the top and bottom and it is created by a frameshift during protein expression domains of the γ ‘C’. This central position suggests that from the dnaX gene (Tsuchihashi and Kornberg, 1989; ATP binding to γ may perturb the ‘C’ shape, perhaps Blinkova and Walker, 1990; Flower and McHenry, 1990). resulting in movement of the top and bottom domains, as The frameshift allows addition of one unique amino acid speculated in Figure 7A. Recent reports of an ATP- to γ, followed by a stop codon. In vitro, both γ and induced conformational change in γ, and in γ complex, τ subunits can be assembled into functional clamp loaders are consistent with perturbation of the γ ‘C’ shape (Naktinis with the δ, δ, χ and ψ subunits (Onrust et al., 1995b). et al., 1995; Hingorani and O’Donnell, 1998). In addition, τ has the unique ability to bind the core The ATP-induced changes in γ suggest that it functions polymerase (McHenry, 1982; Studwell-Vaughan and as a ‘motor’, using the energy of ATP binding to power O’Donnell, 1991) as well as the DnaB helicase with high the conformational change in γ complex that takes δ from afﬁnity (Kim et al., 1996; Yuzhakov et al., 1996). These a buried position into one that is exposed and allows properties require the extra 213 amino acids in τ, which interaction with the β ring (Naktinis et al., 1996). The are absent in γ. Speciﬁcally a τ dimer, not γ, dimerizes present study indicates that δ may be the subunit that the two core DNA polymerases in the holoenzyme and prevents δ from binding β in the absence of ATP. Hence, contacts the DnaB helicase during DNA replication. ATP binding to γ may re-position δ and/or affect the ring Which protein, γ or τ, functions as the motor subunit opener δ, such that it prefers binding to β rather than δ. for the clamp loader within the holoenzyme, or do both γ In Figure 7A, this is depicted as a change in the δ subunit and τ form the clamp loader (i.e. a γτ complex)? This ‘C’ shape in response to the ATP-induced change in the question has been addressed in a previous study of the γ γ subunit ‘C’ shape (i.e. the δ conformation is guided by and τ proteins. ATP-binding site mutants of γ and τ were γ). The resulting arrangement leaves γ complex in the ‘on’ constructed which, when constituted into either γ complex state in which δ can bind the β clamp and open the ring or τ complex with δ, δ, χ and ψ, were unable to hydrolyze at one interface (Figure 7B). The resulting γ complex– ATP and were inactive in DNA replication assays with ATP–open β ring composite is stabilized by the decrease core polymerase and β (Xiao et al., 1995). The mutant in ATPase activity of the γ complex (Table II). If ATP proteins were also assembled into DNA polymerase III were hydrolyzed, the clamp loader would release the holoenzymes. The holoenzyme containing mutant γ clamp and ‘turn over’. Thus the inhibition of ATPase wild-type τ was inactive in DNA replication. The holo- activity probably maintains the integrity of the γ complex– ATP–open β ring composite for an encounter with a DNA enzyme containing mutant τ wild-type γ was as active substrate. as the wild-type DNA polymerase III holoenzyme. These The γ complex–ATP–open β ring composite binds results demonstrate that the ATP site of γ is needed for 779 J.Turner et al. holoenzyme function, and thus γ is the clamp loading DNA polymerase δ requires contact with the same face motor in the holoenzyme. of PCNA as RF-C. Hence RF-C must release PCNA for In normal cells, the holoenzyme contains both γ and τ the polymerase to gain access to the ring. Studies of (Maki et al., 1988). However, if the frameshift signal in human RF-C have shown that a three-subunit subassembly dnaX is mutated such that γ is not produced, the cells (36, 37 and 40 kDa subunits) as well as the 40 kDa remain viable (Blinkova et al., 1993). Presumably the subunit alone is capable of removing PCNA clamps from holoenzyme in these γ-less cells contains four τ subunits, DNA in the absence of ATP (Cai et al., 1997). These where two τ subunits dimerize the polymerases and the observations indicate that the RF-C clamp loaders may other two substitute for γ as the clamp loader motor. utilize a similar modular design and mechanism for However, even though the τ subunit can substitute for γ, assembly of rings around DNA as the E.coli clamp loader. its ATPase activity and clamp-loading activity is not The γ complex subunits also provide ancillary functions; essential when the γ subunit is present in the holoenzyme, for example the χ subunit binds SSB and is necessary for as demonstrated by the ATP site mutant study (Xiao displacement of the primase from RNA primers (Kelman et al., 1995). et al., 1998; Yuzhakov et al., 1999). It is tempting to The τ complex has the unique ability to utilize ATP- speculate that eukaryotic clamp loader subunits may also γ-S to provide the core polymerase with β-dependent provide additional functions at the site of DNA synthesis, processivity for DNA synthesis, although the level of other than clamp assembly on DNA. synthesis is much lower than in the presence of ATP; the The bacteriophage T4 clamp loader contains two differ- γ complex does not function in this capacity (Dallmann ent subunits (gp44 and gp62), and a circular sliding clamp et al., 1995). This phenomenon is interesting since neither (gp45), which is a loosely associated trimer that lacks the γ complex nor τ complex hydrolyze ATP-γ-S signiﬁcantly, stability of the β and PCNA rings (Yao et al., 1996). and ATP hydrolysis is required by both clamp loaders for Recent studies on the role of ATP in gp45 assembly on the completion of clamp assembly onto DNA (Hingorani DNA suggest that ATP hydrolysis is needed to open the and O’Donnell, 1998; unpublished data). The results of this gp45 ring (Latham et al., 1996; Sexton et al., 1996; report offer a new insight into the molecular mechanism by Pietroni et al., 1997). In contrast, in E.coli, ATP binding which the τ complex plus core, or the holoenzyme, may is sufﬁcient for ring opening, and ATP hydrolysis is used use ATP-γ-S for processive DNA replication. In as much to eject γ complex from the β ring on DNA. The unstable as τ and γ are closely related, and ATP-γ-S induces γ nature of the trimeric gp45 ring compared with the complex to bind β and open the ring, it may be presumed β dimer may account for this signiﬁcantly different role that ATP-γ-S has the same effect on τ complex. Since τ, for ATP hydrolysis in the T4 system compared with E.coli. unlike γ, can bind the core polymerase, it may form a For example, gp44/62-catalyzed opening of the gp45 bridge between the polymerase and the clamp, resulting trimer may enhance dissociation of the protomers and in an enzyme assembly that can function processively. preclude reclosure of the clamp around DNA. Therefore, The inability of γ to bind core would prevent formation it is possible that the T4 system has evolved to allow of such an assembly, and, therefore, γ complex cannot assembly of the clamp loader and closed clamp on DNA facilitate β-dependent DNA synthesis by the core in the before ATP hydrolysis, which powers both ring opening presence of ATP-γ-S. Thus, even though the γ complex and loading of the ring onto DNA at the same time. appears to be the primary clamp loader in DNA polymerase III holoenzyme, under artiﬁcial conditions, such as in the Clamp loaders in cell cycle regulation presence of ATP-γ-S (Dallmann et al., 1995) or when γ Recently, a number of clamp loader or clamp loader-like is absent from the holoenzyme (Blinkova et al., 1993), τ proteins have been implicated in cell cycle checkpoint is eminently capable of supporting clamp assembly, in pathways (reviewed in Elledge, 1996; Mossi and Hubscher, addition to dimerizing the polymerases and maintaining 1998). Classical checkpoint proteins sense particular contact with the helicase during DNA replication aspects of a cell’s environment or condition and ‘decide’ whether to restrict or promote cell cycle progression from A common mechanism for clamp loading? one phase to the next. For example, some checkpoint Clamp loaders of three other well studied systems include proteins monitor the integrity of DNA prior to and after the gp44/62 complex from bacteriophage T4, yRF-C from replication. If DNA is damaged, the cell cycle can be S.cerevisiae and hRF-C from humans. These clamp loaders arrested in G phase, slowed in S phase and arrested in use ATP to assemble their respective circular clamps, T4 G phase, to allow repair of the DNA prior to, during and gp45, yeast proliferating cell nuclear antigen (PCNA) and after DNA replication, respectively. Similarly, if DNA human PCNA, onto DNA. synthesis is incorrect or incomplete, DNA replication Like γ complex, both the yeast and human clamp loaders checkpoints can inhibit transition of the cell cycle (e.g. are ﬁve-protein machines (O’Donnell et al., 1993). Earlier from G to mitosis) until the speciﬁc problems are ﬁxed. studies have shown that ATP-γ-S leads to formation of an Three of the ﬁve subunits of the S.cerevisiae clamp RF-C–PCNA–DNA composite, but this is not competent loader, RF-C1, RF-C2 and RF-C5, are now thought for extension by DNA polymerase δ in the absence of participate in cell cycle checkpoint pathways involving hydrolyzable ATP (Lee and Hurwitz, 1990; Burgers, 1991). DNA metabolism (Cullmann et al., 1995; Sugimoto et al., A possible interpretation of this result is that RF-C 1996; Noskov et al., 1998). Although the underlying functions similarly to γ complex, in that ATP binding is mechanism by which these proteins regulate the cell cycle sufﬁcient for ring opening and interaction with DNA, but is not yet understood, it is possible that the regulatory ATP hydrolysis is needed to eject RF-C, allowing the pathways involve loading of PCNA onto DNA. For PCNA ring to close around DNA. As in the E.coli system, example, a clamp loader is crucial at an early stage in DNA 780 Mechanics of clamp assembly on DNA treatment results in ~30 primers per DNA circle (assuming an average replication for initiation of processive DNA synthesis; primer length of 25 nucleotides). Synthetic ssDNA 100mer and synthetic therefore, a shutdown of its clamp-loading activity can primed template DNA were prepared as described (Hingorani and prevent entry into S phase. The large subunit of human O’Donnell, 1998). RF-C (p140) has several putative phosphorylation sites Buffer A is 20 mM Tris–HCl pH 7.5, 0.1 mM EDTA and 10% glycerol. Superose 12 gel ﬁltration buffer is 20 mM Tris–HCl pH 7.5, for protein kinase A, protein kinase C and tyrosine kinases, 0.1 mM EDTA, 10% glycerol, 5 mM DTT and 100 mM NaCl. Reaction and this has led to speculation that the PCNA loading buffer is 20 mM Tris–HCl pH 7.5, 0.1 mM EDTA, 4% glycerol and activity of RF-C may be regulated by phosphorylation 8 mM MgCl . Bio-Gel A-15m gel ﬁltration buffer is reaction buffer events in checkpoint pathways (Mossi and Hubscher, plus 100 mM NaCl. Loading buffer is 50 mM Tris–HCl (pH 6.8), 1998). Alternatively, clamp loaders may serve as signal 100 mM DTT, 2% SDS, 0.1% bromophenol blue and 10% glycerol. sensors in checkpoint pathways through recognition of speciﬁc DNA structures produced during DNA replication Proteins The γ complex was constituted from pure γ, δ, δ, χ and ψ, followed by and/or repair processes. For example, recent evidence isolation from the free subunits on a MonoQ column (Pharmacia) as suggests that RF-C5 may interact with Spk1 (Rad53/ described (Onrust et al., 1995b). Puriﬁcation of γ (Studwell and Mec2/Sad1), an essential protein kinase for the transition O’Donnell, 1990), δ and δ (Dong et al., 1993), χ and ψ (Xiao et al., of S phase to mitosis (Sugimoto et al., 1996). 1993) was performed as described earlier. The δ and β proteins were Structural homologs of clamp loader subunits are also H-labeled by reductive methylation as described (Kelman et al., 1995a). The catalytic subunit of cAMP-dependent protein kinase produced in known to participate in cell cycle regulation. RAD24 in E.coli was the gift of Dr S.Taylor (University of California, San S.cerevisiae, which is homologous to E.coli γ and δ Diego, CA). proteins as well as all the RF-C subunits, is required for The modiﬁed versions of β were constructed by PerImmune, Inc. DNA damage checkpoint pathways, and its homolog in using a DNA oligonucleotide site-directed method. The mutant dnaN genes were placed into vectors encoding an N-terminal hexa-histidine Schizosaccharomyces pombe, RAD17, is required for both tag and a kinase recognition sequence (pHK vector; Kelman et al., DNA replication and damage checkpoints (Elledge, 1996). 1995b). The speciﬁc β modiﬁcations are described in detail later. Below Perhaps RAD24 and RAD17 interact with RF-C and is a summary of the puriﬁcation protocol. Modiﬁed β was overexpressed regulate its clamp-loading activity, or they may be subunits in E.coli BL21(DE3) pLysS cells by inducing cultures at OD 0.6 of other protein loaders that play a role in checkpoint with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were harvested, and heat lysed by the method of Wickner (1976). The pathways. A recent study has shown that Cdc6p, an cell lysate was precipitated sequentially with 35 and 70% ammonium S.cerevisiae protein essential for entry of cells into S phase, sulfate and the pellet was resuspended in 20 mM Tris–HCl (pH 7.9) also shares homology with prokaryotic and eukaryotic 500 mM NaCl and 5 mM imidazole. The protein was loaded onto a clamp loader subunits (Perkins and Difﬂey, 1998). It has 5 ml chelating Sepharose resin (Pharmacia) charged with NiSO , and bound proteins were eluted with a linear gradient of imidazole (60– also been suggested that Cdc6p may be involved in 500 mM) in the above buffer. Fractions were mixed immediately with assembly of Mcm proteins onto chromatin, and thereby EDTA (50 mM ﬁnal) to chelate any nickel in the eluate that tends to serves a function analogous to that of a clamp loader in precipitate protein. β was identiﬁed by SDS–PAGE, and peak fractions assembling proteins onto DNA (Perkins and Difﬂey, 1998). were pooled and dialyzed against buffer A. The pool was loaded onto an 8 ml MonoQ column equilibrated in buffer A, and the bound protein Although quite speculative in nature, these ﬁndings imply was eluted with a linear NaCl gradient (0–500 mM) in buffer A. Peak that the use of clamp loaders and their underlying mechan- fractions were pooled and frozen at –70°C. Each of the modiﬁed β isms may generalize to other processes beyond loading subunits had replication activity levels comparable with wild-type β. sliding clamps onto DNA. Studies of E.coli DNA replication have been a guide to Ring-opening assay understanding similar mechanisms in complex eukaryotes, The β clamp was modiﬁed by changing Leu273 to cysteine and Cys333 including humans. This report provides a detailed mechan- to serine. The resulting Cys273 residue is buried in the dimer interface (L273C), and the highly reactive surface Cys333 is removed. The ism for the function of the γ complex clamp loader, a remaining three cysteine residues in β were found unreactive to thiol- multiprotein enzyme that assembles sliding clamps onto labeling reagents and were left unchanged. The reagent used to probe DNA for processive and rapid DNA replication. The the interface cysteine was prepared by labeling 300 nmol of peptide structural and functional homology between γ complex, (NH -LRRASVP-COOH; Chiron mimotopes) with 0.03 U of cAMP- RF-C and other important cell cycle regulatory proteins dependent protein kinase catalytic subunit and [γ- P]ATP 200 μCi (3000 Ci/mmol) in 150 μl of reaction buffer (30 min at 37°C). Then, suggests that insights gleaned from the molecular mechan- ATP was added to a ﬁnal concentration of 10 mM and the mixture was ism of the γ complex will generalize to eukaryotic clamp incubated for an additional 30 min. The mixture was incubated with loaders, and perhaps to yet other critical processes in 5 mg of sulfosuccinimidylc 4-[N-maleimidomethyl] cyclohexane-1- DNA metabolism. carboxylate (Sulfo-SMCC; Pierce) at 25°C for 1 h, and the P-labeled maleimide was puriﬁed on a 24 ml Superdex-Peptide column (Pharmacia) equilibrated in 0.1 M sodium phosphate (pH 7.2), 0.15 M NaCl, using the Pharmacia SMART system. Aftera7ml void volume, 500 μl Materials and methods fractions were collected. The peak (fractions 17–18) containing [ P]ma- Materials leimide was identiﬁed by the co-incidence of the radioactive peak Radioactive nucleotides were purchased from Dupont-NEN. M13mp18 and the peak of absorbance at 305nm (λ for N-ethylmaleimide). max ssDNA was prepared by phenol extraction of puriﬁed M13mp18 phage [ P]maleimide concentration was determined at 305 nm, using a molar that had been banded twice in CsCl gradients as described (Turner and extension coefﬁcient of 620/M/cm for maleimide, and was typically at O’Donnell, 1995). Nicked circular DNA was prepared by incubating 400 μM. 200 μg of doubly CsCl-banded supercoiled Bluescript plasmid (pBS) The ring-opening assay was performed in 15 μl of reaction buffer with 100 U of mung bean nuclease (NEB) for 15 min, followed by containing 3 μM β,3 μM γ complex, 2 mM ATP and 3 μM primed phenol extraction and ethanol precipitation. Mung bean nuclease is a oligoDNA. Proteins were mixed on ice and the reaction was initiated single strand-speciﬁc nuclease that nicks supercoiled DNA once, and by with 5 μlof [ P]maleimide and a shift to 37°C. The reactions were relaxing the plasmid precludes further nicking. The DNA was ~90% quenched after 12 min with 5 μl of loading buffer 5 μlof1M nicked. Multiprimed DNA was prepared by incubating 0.6 μM DnaG DTT. Samples were analyzed on a 10% SDS–polyacrylamide gel that primase and 0.6 μM DnaB helicase with 1 mM NTPs for 30 min at subsequently was dried and analyzed on a PhosphorImager. The γ com- 37°C, followed by phenol extraction and ethanol precipitation. This plex subunits exhibit some background labeling by [ P]maleimide; 781 J.Turner et al. however, they are easily resolved from β during SDS–PAGE and do not assembled onto DNA. ATP hydrolysis was quantitated by TLC on PEI interfere with the detection of β ring opening. cellulose F sheets (EM Science), using 0.6 M potassium phosphate In experiments using ATP-γ-S, 5 μM each of γ complex and β were buffer, pH 3.4. The TLC sheet was dried and the molar amount of ADP incubated in 30 μl of reaction buffer with either 200 μM ATP or ATP- P formed was determined by analysis on a PhosphorImager. γ-S. Reactions were initiated by adding 10 μlof [ P]maleimide, and ATPase assays (or ATP-γ-Sase assays) measuring γ complex turnover 4 μl aliquots were removed and quenched at 0, 0.5, 1, 2, 5 and 10 min, rate were performed at 37°C with 0.1 μM γ complex, with or without and analyzed as described above. 0.2 μM β and with 1 mM ATP [α- P]ATP (or 0.5 mM ATP-γ-S [γ- S]ATP-γ-S). At various times, 5 μl aliquots of the reaction were Cross-linked β loading assay quenched with equal volumes of 0.5 M EDTA and analyzed as described For this experiment, Arg103 and Ile305 in β were changed to cysteine above. To compare the effect of primed and ssDNA on γ complex– S–S S–S to create a disulﬁde-linked β (β ). Native β and β were radiolabeled β-catalyzed ATP hydrolysis, similar reactions were performed, except with the catalytic subunit of cAMP-dependent protein kinase and with 0.1 μM M13 ssDNA coated with 17 μM SSB or with 0.1 μM [γ- P]ATP as described (Kelman et al., 1995a), except that DTT was primed M13 DNA coated with SSB in the reaction (or 0.5 μM ss- S–S omitted in order to maintain the disulﬁde cross-links in β . The oligoDNA and primed oligoDNA). The molar amounts of products γ complex used in this assay was constituted in the absence of DTT. formed were plotted versus time of reaction, and the slope of each The assembly reaction was performed in 50 μl of reaction buffer time course was divided by γ complex concentration to yield the containing 500 fmol of pBS DNA, 19 pmol of β, 5.4 pmol of γ complex turnover number. and 500 μM ATP, in the absence or presence of 20 mM DTT. The reaction was allowed to proceed for 10 min at 37°C and then loaded onto a 5 ml Bio-Gel A-15m gel ﬁltration column (Bio-Rad), equilibrated Acknowledgements in A-15m buffer in the presence or absence of 1% SDS. Fractions (200 μl) were collected and 40 μl aliquots were quantitated by liquid We are grateful to Dr Susan Taylor for the catalytic subunit of the scintillation counting or analyzed on a 12% SDS–polyacrylamide gel cAMP-dependent protein kinase. This work was supported by NIH grant under non-reducing conditions (no DTT). The gels were dried and GM38839. M.M.H. was supported by a grant from the Charles H.Revson exposed to ﬁlm or analyzed on a PhosphorImager (Molecular Dynamics) Foundation. Clamp-unloading assay β (1.6 μM), P-labeled at a C-terminal kinase site (Naktinis et al., References 1996), was incubated with 3.3 μM γ complex and 5 pmol of pBS DNA in 75 μl of reaction buffer containing 500 μM ATP for 10 min at 37°C. Blinkova,A.L. and Walker,J.L. (1990) Programmed ribosomal The reaction was applied to a 5 ml A-15m gel ﬁltration column and frameshifting generates the Escherichia coli DNA polymerase III γ eluted with A-15m buffer. Peak fractions, containing β on DNA, were subunit from within the τ subunit reading frame. Nucleic Acids Res., pooled, and 25 μl aliquots (containing 0.2 μM β) were incubated with 18, 1725–1729. 1 μM γ, δ or δ with or without 500 μM ATP. After 10 min, each sample Blinkova,A.L., Hervas,C., Stukenberg,P.T., Onrust,R., O’Donnell,M. and was mixed with glycerol (15% ﬁnal) and analyzed on a 1% agarose gel Walker,J.R. (1993) The Escherichia coli DNA polymerase III (89 mM Tris–borate buffer with 10 ng/ml ethidium bromide). After holoenzyme contains both products of the dnaX gene, τ and γ, but electrophoresis for 2 h at 100 V, the agarose gel was photographed under only τ is essential. J. Bacteriol., 175, 6018–6027. UV light and exposed to ﬁlm. Bloom,L.B., Turner,J.T., Kelman,Z., Beechem,J.M., O’Donnell,M. and Goodman,M. (1996) Dynamics of loading the β sliding clamp of Analysis of β, δ and δ interactions DNA polymerase III onto DNA. J. Biol. Chem., 271, 30699–30708. A δ complex was formed by mixing 2 mg of δ and 3mgof δ in buffer Burgers,P.M.J. (1991) Saccharomyces cerevisiae replication factor C. II. A followed by puriﬁcation on a 1 ml MonoQ column (with a linear Formation and activity of complexes with the proliferating cell nuclear gradient of 50–500 mM NaCl in buffer A). Then 5 μM of the pure δδ antigen and with DNA polymerases δ and ε. J. Biol. Chem., 266, complex was mixed with 25 μM β (dimer) in 200 μl of buffer A for 22698–22706. 15 min at 25°C, and analyzed by gel ﬁltration on a Superose 12 column Burgers,P.M.J. and Kornberg,A. (1982) ATP activation of DNA equilibrated with buffer A containing 100 mM NaCl. After a void polymerase III holoenzyme from Escherichia coli. II. Initiation volume of 7 ml, 180 μl fractions were collected, and 20 μl aliquots complex: stoichiometry and reactivity. J. Biol. Chem., 257, 11474– were analyzed by SDS–PAGE and visualized by Coomassie staining of 11478. the gels. The control gel ﬁltration proﬁles of β alone, δ alone, δ alone, Burgers,P.M.J. and Yoder,B.L. 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The EMBO Journal – Springer Journals

Published: Feb 1, 1999

Keywords: ATPase; clamp loader; DNA polymerase; processivity; sliding clamp

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The internal workings of a DNA polymerase clamp‐loading machine

The internal workings of a DNA polymerase clamp‐loading machine

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The internal workings of a DNA polymerase clamp‐loading machine

The internal workings of a DNA polymerase clamp‐loading machine

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