- Email: [email protected]
Intricacies in ATP-Dependent Clamp Loading
Structure, Vol. 9, 999–1004, November, 2001, 2001 Elsevier Science Ltd. All rights reserved.
Intricacies in ATP-Dependent Clamp Loading: Variations across Replication Systems Michael A. Trakselis and Stephen J. Benkovic Department of Chemistry 414 Wartik Laboratory Pennsylvania State University University Park, Pennsylvania 16802
Summary DNA replication requires the coordinated effort of many proteins to create a highly processive biomachine able to replicate entire genomes in a single process. The clamp proteins confer on replisomes this property of processivity but in turn require clamp loaders for their functional assembly onto DNA. A more detailed view of the mechanisms for holoenzyme assembly in replication systems has been obtained from the advent of novel solution experiments and the appearance of low- and high-resolution structures for the clamp loaders. Functional biomachines working to replicate DNA require the interaction of many protein parts to form a replisome in which many of these proteins have been structurally conserved throughout evolution to perform the same function. DNA polymerases constitute the core of the replisome and are able to synthesize complementary DNA in the 5⬘-to-3⬘ direction (reviewed in [1–3]), but alone they generally only produce strands of less than ten nucleotides before dissociation [4]. Processivity factors, or clamps, are proteins with toroidal structures that tether the polymerase onto DNA and thus effectively increase the amount of continuous replication on the leading strand to hundreds of thousands of bases [5]. DNA replication also occurs in the 5⬘-to-3⬘ direction on the lagging strand, but in a discontinuous fashion; such replication synthesizes Okazaki fragments of about 2000 base pairs in length and thus also requires a clamp protein. Crystal structures of the clamps from bacteriophage T4 (gp45) [6], Escherichia coli ( subunit) [7], eukaryotic proliferating cell nuclear antigen (PCNA) [8], and archaeal Pyrococcus furiosus (PCNA) [9] were all found to adopt a common ring-shaped structure with a central channel large enough to encircle duplex DNA (Figure 1). Even though the individual clamps exist in different oligomeric states, each has pseudo-6-fold symmetry with six domains in the active complex. The problem of opening such clamps to load them onto DNA is surmounted by cognate clamp-loader proteins. Recent low- and high-resolution structures of the clamp-loader proteins from E. coli, P. furiosus, and H. sapiens now provide important insights into how they function [10– 14]. Similarities in the structures of replication proteins across kingdoms suggest that their function in replisome assembly and activity are comparable, although Correspondence: [email protected]
PII S0969-2126(01)00676-1
Minireview
slight structural divergence would predict subtle mechanistic differences.
Mechanistic Comparison of Clamp Loading The clamp loader is a dynamic protein complex that undergoes and imposes a variety of structural changes while assembling the polymerase holoenzyme onto DNA. In E. coli, the clamp loader is minimally a 5 protein assembly termed the ␥ complex, which is composed of three ATP binding subunits, ␥1, ␥2, and ␥3, and two other subunits, ␦ and ␦⬘. In bacteriophage T4, the gene product, gp44/62; in eukaryotes, the replication factor C (RFC); and in archaea, an RFC-like complex act as clamp-loader proteins. Gp44 is a 4 subunit protein able to bind four equivalents of ATP; gp62 is monomeric. RFC is a heteropentameric complex composed of one large and four small subunits, each individually have nucleotide binding motifs and potential ATPase activity. All clamp loaders are ATPases similar in sequence to members of the AAA⫹ protein superfamily (ATPases associated with a variety of cellular activities) [15]. Detailed mechanistic studies of the clamp-loading process for bacteriophage T4 [16, 17] and E. coli [18–20] have outlined the kinetic steps involved in holoenzyme formation along with the role of ATP and have revealed similar sequential holoenzyme assembly mechanisms shaded by subtle differences that occur in the timing and stoichiometry of the ATP hydrolysis events that are affected by the clamp loader. In bacteriophage T4, two molecules of ATP bound to gp44 are reportedly hydrolyzed by gp44/62 upon interaction with gp45 to form an open clamp•clamp-loader complex, and an additional two are hydrolyzed after the interaction of the open clamp•clamp-loader complex with DNA that is associated with a rate-limiting step in holoenzyme assembly [21, 22]. A recent study, however, found that formation of the open clamp•clamp-loader complex does not require ATP hydrolysis and that only one molecule of ATP is hydrolyzed after the addition of DNA [23]. The reasons for this difference in timing and stoichiometry are not clear; however, unlike the E. coli and yeast clamp•clamp-loader complexes, ATP-␥-S is not functional in loading the T4 clamp onto DNA. In contrast, for E. coli, ATP binding is sufficient to open the  clamp [24], but the hydrolysis of 2–3 molecules of ATP is necessary for closing the clamp onto DNA and for its departure [20]. Recent studies of the yeast RFC clamp-loader protein detail the initial binding of two molecules of ATP and the successive binding of two additional ATP molecules after the interaction of RFC with PCNA and DNA [25, 26]. In both E. coli and yeast systems, ATP-␥-S stalls the loading process at the stage of an ATP-␥-S•clamp•clamp-loader•DNA complex, favoring the conclusion that hydrolysis of ATP after interaction with DNA causes the release of either yeast RFC or the ␥ complex from the PCNA•DNA or •DNA assembly, respectively. After the completion of DNA replication, clamp un-
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Figure 1. X-Ray Structures of the Clamp from Bacteriophage T4, E. coli, H. sapiens, and P. furiosus
loading in E. coli relies specifically on the ␦ subunit of the ␥ complex to unload  from DNA [27]. The E. coli system is thus unlike the bacteriophage T4 system, in which gp45 departs from DNA through subunit dissociation [28]. Although the mechanistic schemes for various clamp loadings share common characteristics that span the various replication systems, specific differences emerge. In particular, the kinetic data require supplementation with direct evidence for the state of the clamp proteins—open, closed, closed around DNA—for a more precise comparison. Important Clamp-Loader Structural Features The recent low- and high-resolution structures of the clamp loaders show notable similarities in structure, which is not too surprising because the clamp loaders act upon clamps, all of which have been shown to have conserved structures (Figure 1). In the crystal structure of the ␥ complex [11], all five subunits have essentially the same fold, with each subunit having three domains (Figure 2a; b). The subunits are arranged in a circular fashion, with the C-terminal domains forming a tight stabilizing structure that allows the N-terminal domains to form an asymmetric arrangement necessary for catalysis. The three ␥ subunits each have an ATP binding domain between domains 1 and 2 and a RecA-like fold at the N terminus. In this respect, they are similar to
many other AAA⫹ ATPases, but they also possess a C terminus (domain 3) that is unique to the clamp loader (Figure 2b). A sensor 1 region located in domain 1 and a sensor 2 region located in domain 2 are implicated in the transmission of conformational changes due to nucleotide binding. Once ATP is bound to the ␥ complex, the sensors cause conformational changes that allow interaction with the  clamp. The ␦ and ␦⬘ subunits have been named the “wrench” and “stator,” respectively. The structure of ␦ was found to be very similar to that of ␦⬘, the only subunit of the ␥ complex whose crystal structure had been solved previously [29]. Domains 1 and 2 of ␦ and ␦⬘ have common structural features with those domains in ␥. Differences from ␥ occur around the nucleotide binding site. In the ␦⬘ subunit, an N-terminal extension blocks the nucleotide binding site by forming a hydrophobic patch on the surface. ␦⬘ interactions with ␥1 are thought be important for ATP binding by holding open the subunit interface between ␦⬘ and ␥1 and thus allowing easier access for ATP. The lack of a flexible linker between domains 1 and 2 makes ␦⬘, or the stator, more rigid than the other subunits. The crystal structure of the small subunit of the clamp loader from P. furiosus has also been solved, and the structural features are similar to those of the ␥ complex described above [12]. The archaeal clamp loader (RFC
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ation of a high-resolution hexameric ring model of RFCS, which is consistent with the low-resolution solution structure revealed by electron microscopy [14]. Likewise, the human clamp loader (RFC complex) observed at low resolution by atomic force microscopy and transmission electron microscopy revealed a heteropentameric RFC aligned in a circular fashion with two subunits interacting with PCNA [13]. The conformation of the individual subunits is influenced by the presence of a hydrolysable form of ATP, which appears to drive an opening of the protein fold, a conformation that is maintained in the presence of PCNA. Although the quaternary structure of RFCS in the crystal structure differs from the E. coli ␥ complex, similar subunit architecture and packing are observed in both structures. Four of the six RFCS subunits bind ADP molecules, whereas the remaining two subunits are free of nucleotides. Additional similarities to the ␥ subunits from E. coli include the presence of: three domains for each subunit, nucleotide binding regions located in domain 1, and sensor 1 and 2 motifs responsible for nucleotide binding detection. The overall retention of key structural features across organisms is consistent with the common mechanistic features noted earlier.
Figure 2. X-ray Structures of the ␥ Complex, ␥ Subunit, and ␦ Complexed with  from E. coli (a) Top or C-terminal view of the X-ray structure of the ␥ complex from E. coli. (b) Close-up of the ␥1 subunit. (c) X-ray structure of the ␦ subunit complex with the  monomer from E. coli. The structure highlights the interacting region with a surface representation.
complex) comprises one large subunit (RFCL) and four small subunits (RFCS), both of which share significant sequence identity and biochemical characteristics with their eukaryotic homologs [30]. The dimer-of-trimers structure identified by crystallography allowed the cre-
Structural Features of Clamp Loading A fascinating aspect of the high-resolution crystal structures is the complex between the ␦ subunit of the ␥ complex and the  clamp (Figure 2c; [10]). This complex provides the first detailed indication of how ␥ interacts with . A mutant monomeric  clamp was crystallized with either the full-length ␦ or a C-terminally truncated ␦1–140. Only the N-terminal domain 1 of ␦ was found to interact with , through a protruding helical hydrophobic region, which is inserted into a hydrophobic cleft on the surface of  near the subunit interface. A conformational change in the ␥ complex facilitates this contact and allows translation of the hydrophobic region in ␦, or the wrench, to the cleft in . Superimposition of the ␦- complex upon the previously solved  dimer highlights curvature changes in  that are consistent with a relief of strain within the clamp structure upon opening. Currently, the high-resolution structure and model of the clamp•clamp loader only identifies the interaction between ␦ and . More recently, all of the subunits of the ␥ complex have been shown to interact with , although with different affinities [31]. Additional details on the clamp loading process have been obtained with the T4 bacteriophage system from the vantage of the clamp protein by the use of fluorescence resonance energy transfer triangulation measurements [17]. Presuming one can generalize structures across the various systems, a representation of the clamp loader has been modeled that presumes that gp44/62 would act through the binding of gp62 with an intrasubunit region in gp45 (Figure 3). Nonspecific crosslinking experiments have revealed the capture of both gp44 and gp62 by gp45, and in the presence of ATP, major structural rearrangements occurred that resulted in new crosslinks from gp45 to gp44 and gp62 [32–34]. The further opening of gp45 (this clamp is partially open in solution [35]) in the presence of the gp44/
Figure 3. Mechanisms of Holoenzyme Assembly in E. coli and Bacteriophage T4 The E. coli mechanism includes: (1) initial complexes of ␥ and , (2) ATP binding to ␥ and subsequent interaction with , (3) loading  onto DNA, (4) hydrolysis of ATP to close , (5) ␥ dissociation, and (6) holoenzyme formation. The T4 mechanism includes: (1) gp44/62, (2) ATP binding to gp44/62, (3) interaction with gp45 and hydrolysis of ATP to open gp45, (4) closing of gp45 out of plane onto DNA through ATP hydrolysis, (5) chaperone property of gp44/62, and (6) holoenzyme formation.
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62 clamp loader is observed only with ATP and not ATP␥-S, implying that hydrolysis of ATP is necessary for steps 2 and 3 in the T4 system. Ring closure (steps 3 and 4) is also an ATP hydrolysis-associated event that leads to a structure in which the clamp subunits are initially out-of-plane. Pre-steady-state kinetics have revealed that productive binding of the polymerase requires a chaperone activity of the clamp-loader protein, which transiently binds the same face of the clamp as the polymerase protein (steps 4 and 5) [36, 37]. Docking of the polymerase with the clamp proceeds with insertion of the former’s C terminus into the subunit interface of the clamp protein and the establishment of additional contacts between them (steps 5 and 6) [38]. The structure of this final complex is derived from X-ray crystallography of a C-terminal peptide bound to gp45 [39] augmented by site-specific crosslinking experiments and biochemical affinity experiments [38]. In parallel, Figure 3 illustrates steps identified kinetically for the E. coli clamp-loading process; it emphasizes the overall features that are retained. Obviously, additional details elaborating the panoply of contacts between the various proteins remain to be determined. Conclusions Common structural features and their structural rearrangement have been observed in prokaryotic, eukaryotic, and archaeal clamp-loading complexes. Like their target clamp proteins, the clamp loaders progress from a closed state to an open state upon interaction with the clamp and then retrace these states upon departure from the clamps. The binding and hydrolysis of ATP powers different steps in the loading sequence, dependent on the origin of the complex, and the recent structures of the clamp loaders provide insight into how this may occur structurally. One is struck with the architectural and functional beauty of the resulting holoenzymes; what better than a ring-imbedded polymerase to slide along a string of DNA to provide for processive replication?
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Acknowledgments We thank Dr. Faoud Ishmael for help with the preparation of the figures and Dr. Morikawa and his lab for providing the Protein Data Bank coordinates for the trimeric PCNA from P. furiosus.
21.
22. References 1. Baker, T.A., and Bell, S.P. (1998). Polymerases and the replisome: machines within machines. Cell 92, 295–305. 2. Keck, J.L., and Berger, J.M. (2000). DNA replication at high resolution. Chem. Biol. 7, R63–R71. 3. Benkovic, S.J., Valentine, A.M., and Salinas, F. (2001). Replisome mediated DNA replication. Annu. Rev. Biochem. 70, 181–208. 4. Mace, D.C., and Alberts, B.M. (1984). T4 DNA polymerase. Rates and processivity on single-stranded DNA templates. J. Mol. Biol. 177, 295–311. 5. Kuriyan, J., and O’Donnell, M. (1993). Sliding clamps of DNA polymerases. J. Mol. Biol. 234, 915–925. 6. Moarefi, I., Jeruzalmi, D., Turner, J., O’Donnell, M., and Kuriyan, J. (2000). Crystal structure of the DNA polymerase processivity factor of T4 bacteriophage. J. Mol. Biol. 296, 1215–1223. 7. Kong, X.P., Onrust, R., O’Donnell, M., and Kuriyan, J. (1992). Three-dimensional structure of the beta subunit of E. coli DNA
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27. Leu, F.P., Hingorani, M.M., Turner, J., and O’Donnell, M. (2000). The delta subunit of DNA polymerase III holoenzyme serves as a sliding clamp unloader in Escherichia coli. J. Biol. Chem. 275, 34609–34618. 28. Soumillion, P., Sexton, D.J., and Benkovic, S.J. (1998). Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly. Biochemistry 37, 1819–1827. 29. Guenther, B., Onrust, R., Sali, A., O’Donnell, M., and Kuriyan, J. (1997). Crystal structure of the delta⬘ subunit of the clamploader complex of E. coli DNA polymerase III. Cell 91, 335–345. 30. Cann, I.K., and Ishino, Y. (1999). Archaeal DNA replication: identifying the pieces to solve a puzzle. Genetics 152, 1249–1267. 31. Leu, F.P., and O’Donnell, M. (2001). Interplay of a clamp loader subunits in opening the {beta} sliding clamp of E. coli DNA polymerase III holoenzyme. J. Biol. Chem., in press. 32. Latham, G.J., Bacheller, D.J., Pietroni, P., and von Hippel, P.H. (1997). Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. II. The gp44/62 clamp loader interacts with a single defined face of the sliding clamp ring. J. Biol. Chem. 272, 31677– 31684. 33. Pietroni, P., Young, M.C., Latham, G.J., and von Hippel, P.H. (1997). Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. I. Conformational changes within the gp44/62-gp45ATP complex during clamp loading. J. Biol. Chem. 272, 31666– 31676. 34. Alley, S.C., Ishmael, F.T., Jones, A.D., and Benkovic, S.J. (2000). Mapping protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme using a novel trifunctional photocrosslinking and affinity reagent. J. Am. Chem. Soc. 122, 6126– 6127. 35. Alley, S.C., Shier, V.K., Abel-Santos, E., Sexton, D.J., Soumillion, P., and Benkovic, S.J. (1999). Sliding clamp of the bacteriophage T4 polymerase has open and closed subunit interfaces in solution. Biochemistry 38, 7696–7709. 36. Kaboord, B.F., and Benkovic, S.J. (1996). Dual role of the 44/ 62 protein as a matchmaker protein and DNA polymerase chaperone during assembly of the bacteriophage T4 holoenzyme complex. Biochemistry 35, 1084–1092. 37. Latham, G.J., Bacheller, D.J., Pietroni, P., and von Hippel, P.H. (1997). Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. III. The gp43 DNA polymerase binds to the same face of the sliding clamp as the clamp loader. J. Biol. Chem. 272, 31685–31692. 38. Alley, S.C., Trakselis, M.A., Mayer, M.U., Ishmael, F.T., Jones, A.D., and Benkovic, S.J. (2001). Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme. J. Biol. Chem. 276, 39340–39349. 39. Shamoo, Y., and Steitz, T.A. (1999). Building a replisome from interacting pieces: sliding clamp complexed to a peptide from DNA polymerase and a polymerase editing complex. Cell 99, 155–166.
Intricacies in ATP-Dependent Clamp Loading: Variations across Replication Systems Michael A. Trakselis and Stephen J. Benkovic Department of Chemistry 414 Wartik Laboratory Pennsylvania State University University Park, Pennsylvania 16802
Summary DNA replication requires the coordinated effort of many proteins to create a highly processive biomachine able to replicate entire genomes in a single process. The clamp proteins confer on replisomes this property of processivity but in turn require clamp loaders for their functional assembly onto DNA. A more detailed view of the mechanisms for holoenzyme assembly in replication systems has been obtained from the advent of novel solution experiments and the appearance of low- and high-resolution structures for the clamp loaders. Functional biomachines working to replicate DNA require the interaction of many protein parts to form a replisome in which many of these proteins have been structurally conserved throughout evolution to perform the same function. DNA polymerases constitute the core of the replisome and are able to synthesize complementary DNA in the 5⬘-to-3⬘ direction (reviewed in [1–3]), but alone they generally only produce strands of less than ten nucleotides before dissociation [4]. Processivity factors, or clamps, are proteins with toroidal structures that tether the polymerase onto DNA and thus effectively increase the amount of continuous replication on the leading strand to hundreds of thousands of bases [5]. DNA replication also occurs in the 5⬘-to-3⬘ direction on the lagging strand, but in a discontinuous fashion; such replication synthesizes Okazaki fragments of about 2000 base pairs in length and thus also requires a clamp protein. Crystal structures of the clamps from bacteriophage T4 (gp45) [6], Escherichia coli ( subunit) [7], eukaryotic proliferating cell nuclear antigen (PCNA) [8], and archaeal Pyrococcus furiosus (PCNA) [9] were all found to adopt a common ring-shaped structure with a central channel large enough to encircle duplex DNA (Figure 1). Even though the individual clamps exist in different oligomeric states, each has pseudo-6-fold symmetry with six domains in the active complex. The problem of opening such clamps to load them onto DNA is surmounted by cognate clamp-loader proteins. Recent low- and high-resolution structures of the clamp-loader proteins from E. coli, P. furiosus, and H. sapiens now provide important insights into how they function [10– 14]. Similarities in the structures of replication proteins across kingdoms suggest that their function in replisome assembly and activity are comparable, although Correspondence: [email protected]
PII S0969-2126(01)00676-1
Minireview
slight structural divergence would predict subtle mechanistic differences.
Mechanistic Comparison of Clamp Loading The clamp loader is a dynamic protein complex that undergoes and imposes a variety of structural changes while assembling the polymerase holoenzyme onto DNA. In E. coli, the clamp loader is minimally a 5 protein assembly termed the ␥ complex, which is composed of three ATP binding subunits, ␥1, ␥2, and ␥3, and two other subunits, ␦ and ␦⬘. In bacteriophage T4, the gene product, gp44/62; in eukaryotes, the replication factor C (RFC); and in archaea, an RFC-like complex act as clamp-loader proteins. Gp44 is a 4 subunit protein able to bind four equivalents of ATP; gp62 is monomeric. RFC is a heteropentameric complex composed of one large and four small subunits, each individually have nucleotide binding motifs and potential ATPase activity. All clamp loaders are ATPases similar in sequence to members of the AAA⫹ protein superfamily (ATPases associated with a variety of cellular activities) [15]. Detailed mechanistic studies of the clamp-loading process for bacteriophage T4 [16, 17] and E. coli [18–20] have outlined the kinetic steps involved in holoenzyme formation along with the role of ATP and have revealed similar sequential holoenzyme assembly mechanisms shaded by subtle differences that occur in the timing and stoichiometry of the ATP hydrolysis events that are affected by the clamp loader. In bacteriophage T4, two molecules of ATP bound to gp44 are reportedly hydrolyzed by gp44/62 upon interaction with gp45 to form an open clamp•clamp-loader complex, and an additional two are hydrolyzed after the interaction of the open clamp•clamp-loader complex with DNA that is associated with a rate-limiting step in holoenzyme assembly [21, 22]. A recent study, however, found that formation of the open clamp•clamp-loader complex does not require ATP hydrolysis and that only one molecule of ATP is hydrolyzed after the addition of DNA [23]. The reasons for this difference in timing and stoichiometry are not clear; however, unlike the E. coli and yeast clamp•clamp-loader complexes, ATP-␥-S is not functional in loading the T4 clamp onto DNA. In contrast, for E. coli, ATP binding is sufficient to open the  clamp [24], but the hydrolysis of 2–3 molecules of ATP is necessary for closing the clamp onto DNA and for its departure [20]. Recent studies of the yeast RFC clamp-loader protein detail the initial binding of two molecules of ATP and the successive binding of two additional ATP molecules after the interaction of RFC with PCNA and DNA [25, 26]. In both E. coli and yeast systems, ATP-␥-S stalls the loading process at the stage of an ATP-␥-S•clamp•clamp-loader•DNA complex, favoring the conclusion that hydrolysis of ATP after interaction with DNA causes the release of either yeast RFC or the ␥ complex from the PCNA•DNA or •DNA assembly, respectively. After the completion of DNA replication, clamp un-
Structure 1000
Figure 1. X-Ray Structures of the Clamp from Bacteriophage T4, E. coli, H. sapiens, and P. furiosus
loading in E. coli relies specifically on the ␦ subunit of the ␥ complex to unload  from DNA [27]. The E. coli system is thus unlike the bacteriophage T4 system, in which gp45 departs from DNA through subunit dissociation [28]. Although the mechanistic schemes for various clamp loadings share common characteristics that span the various replication systems, specific differences emerge. In particular, the kinetic data require supplementation with direct evidence for the state of the clamp proteins—open, closed, closed around DNA—for a more precise comparison. Important Clamp-Loader Structural Features The recent low- and high-resolution structures of the clamp loaders show notable similarities in structure, which is not too surprising because the clamp loaders act upon clamps, all of which have been shown to have conserved structures (Figure 1). In the crystal structure of the ␥ complex [11], all five subunits have essentially the same fold, with each subunit having three domains (Figure 2a; b). The subunits are arranged in a circular fashion, with the C-terminal domains forming a tight stabilizing structure that allows the N-terminal domains to form an asymmetric arrangement necessary for catalysis. The three ␥ subunits each have an ATP binding domain between domains 1 and 2 and a RecA-like fold at the N terminus. In this respect, they are similar to
many other AAA⫹ ATPases, but they also possess a C terminus (domain 3) that is unique to the clamp loader (Figure 2b). A sensor 1 region located in domain 1 and a sensor 2 region located in domain 2 are implicated in the transmission of conformational changes due to nucleotide binding. Once ATP is bound to the ␥ complex, the sensors cause conformational changes that allow interaction with the  clamp. The ␦ and ␦⬘ subunits have been named the “wrench” and “stator,” respectively. The structure of ␦ was found to be very similar to that of ␦⬘, the only subunit of the ␥ complex whose crystal structure had been solved previously [29]. Domains 1 and 2 of ␦ and ␦⬘ have common structural features with those domains in ␥. Differences from ␥ occur around the nucleotide binding site. In the ␦⬘ subunit, an N-terminal extension blocks the nucleotide binding site by forming a hydrophobic patch on the surface. ␦⬘ interactions with ␥1 are thought be important for ATP binding by holding open the subunit interface between ␦⬘ and ␥1 and thus allowing easier access for ATP. The lack of a flexible linker between domains 1 and 2 makes ␦⬘, or the stator, more rigid than the other subunits. The crystal structure of the small subunit of the clamp loader from P. furiosus has also been solved, and the structural features are similar to those of the ␥ complex described above [12]. The archaeal clamp loader (RFC
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ation of a high-resolution hexameric ring model of RFCS, which is consistent with the low-resolution solution structure revealed by electron microscopy [14]. Likewise, the human clamp loader (RFC complex) observed at low resolution by atomic force microscopy and transmission electron microscopy revealed a heteropentameric RFC aligned in a circular fashion with two subunits interacting with PCNA [13]. The conformation of the individual subunits is influenced by the presence of a hydrolysable form of ATP, which appears to drive an opening of the protein fold, a conformation that is maintained in the presence of PCNA. Although the quaternary structure of RFCS in the crystal structure differs from the E. coli ␥ complex, similar subunit architecture and packing are observed in both structures. Four of the six RFCS subunits bind ADP molecules, whereas the remaining two subunits are free of nucleotides. Additional similarities to the ␥ subunits from E. coli include the presence of: three domains for each subunit, nucleotide binding regions located in domain 1, and sensor 1 and 2 motifs responsible for nucleotide binding detection. The overall retention of key structural features across organisms is consistent with the common mechanistic features noted earlier.
Figure 2. X-ray Structures of the ␥ Complex, ␥ Subunit, and ␦ Complexed with  from E. coli (a) Top or C-terminal view of the X-ray structure of the ␥ complex from E. coli. (b) Close-up of the ␥1 subunit. (c) X-ray structure of the ␦ subunit complex with the  monomer from E. coli. The structure highlights the interacting region with a surface representation.
complex) comprises one large subunit (RFCL) and four small subunits (RFCS), both of which share significant sequence identity and biochemical characteristics with their eukaryotic homologs [30]. The dimer-of-trimers structure identified by crystallography allowed the cre-
Structural Features of Clamp Loading A fascinating aspect of the high-resolution crystal structures is the complex between the ␦ subunit of the ␥ complex and the  clamp (Figure 2c; [10]). This complex provides the first detailed indication of how ␥ interacts with . A mutant monomeric  clamp was crystallized with either the full-length ␦ or a C-terminally truncated ␦1–140. Only the N-terminal domain 1 of ␦ was found to interact with , through a protruding helical hydrophobic region, which is inserted into a hydrophobic cleft on the surface of  near the subunit interface. A conformational change in the ␥ complex facilitates this contact and allows translation of the hydrophobic region in ␦, or the wrench, to the cleft in . Superimposition of the ␦- complex upon the previously solved  dimer highlights curvature changes in  that are consistent with a relief of strain within the clamp structure upon opening. Currently, the high-resolution structure and model of the clamp•clamp loader only identifies the interaction between ␦ and . More recently, all of the subunits of the ␥ complex have been shown to interact with , although with different affinities [31]. Additional details on the clamp loading process have been obtained with the T4 bacteriophage system from the vantage of the clamp protein by the use of fluorescence resonance energy transfer triangulation measurements [17]. Presuming one can generalize structures across the various systems, a representation of the clamp loader has been modeled that presumes that gp44/62 would act through the binding of gp62 with an intrasubunit region in gp45 (Figure 3). Nonspecific crosslinking experiments have revealed the capture of both gp44 and gp62 by gp45, and in the presence of ATP, major structural rearrangements occurred that resulted in new crosslinks from gp45 to gp44 and gp62 [32–34]. The further opening of gp45 (this clamp is partially open in solution [35]) in the presence of the gp44/
Figure 3. Mechanisms of Holoenzyme Assembly in E. coli and Bacteriophage T4 The E. coli mechanism includes: (1) initial complexes of ␥ and , (2) ATP binding to ␥ and subsequent interaction with , (3) loading  onto DNA, (4) hydrolysis of ATP to close , (5) ␥ dissociation, and (6) holoenzyme formation. The T4 mechanism includes: (1) gp44/62, (2) ATP binding to gp44/62, (3) interaction with gp45 and hydrolysis of ATP to open gp45, (4) closing of gp45 out of plane onto DNA through ATP hydrolysis, (5) chaperone property of gp44/62, and (6) holoenzyme formation.
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62 clamp loader is observed only with ATP and not ATP␥-S, implying that hydrolysis of ATP is necessary for steps 2 and 3 in the T4 system. Ring closure (steps 3 and 4) is also an ATP hydrolysis-associated event that leads to a structure in which the clamp subunits are initially out-of-plane. Pre-steady-state kinetics have revealed that productive binding of the polymerase requires a chaperone activity of the clamp-loader protein, which transiently binds the same face of the clamp as the polymerase protein (steps 4 and 5) [36, 37]. Docking of the polymerase with the clamp proceeds with insertion of the former’s C terminus into the subunit interface of the clamp protein and the establishment of additional contacts between them (steps 5 and 6) [38]. The structure of this final complex is derived from X-ray crystallography of a C-terminal peptide bound to gp45 [39] augmented by site-specific crosslinking experiments and biochemical affinity experiments [38]. In parallel, Figure 3 illustrates steps identified kinetically for the E. coli clamp-loading process; it emphasizes the overall features that are retained. Obviously, additional details elaborating the panoply of contacts between the various proteins remain to be determined. Conclusions Common structural features and their structural rearrangement have been observed in prokaryotic, eukaryotic, and archaeal clamp-loading complexes. Like their target clamp proteins, the clamp loaders progress from a closed state to an open state upon interaction with the clamp and then retrace these states upon departure from the clamps. The binding and hydrolysis of ATP powers different steps in the loading sequence, dependent on the origin of the complex, and the recent structures of the clamp loaders provide insight into how this may occur structurally. One is struck with the architectural and functional beauty of the resulting holoenzymes; what better than a ring-imbedded polymerase to slide along a string of DNA to provide for processive replication?
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Acknowledgments We thank Dr. Faoud Ishmael for help with the preparation of the figures and Dr. Morikawa and his lab for providing the Protein Data Bank coordinates for the trimeric PCNA from P. furiosus.
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