Annual Reviews - Replication

Annu. Rev. Biochem. 2005. 74:283315 doi: 10.1146/annurev.biochem.73.011303.073859 Copyright c 2005 by Annual Reviews.
All rights reserved
CELLULAR DNA REPLICASES: Components

and Dynamics at the Replication Fork
Aaron Johnson2 and Mike ODonnell1,2
Annu. Rev. Biochem. 2005.74:283-315. Downloaded from www.annualreviews.org by Universidade Federal do Rio de Janeiro on 08/16/11. For personal use only.
Howard Hughes Medical Institute, 2 The Rockefeller University, New York City, New York 10021-6399; email: johnsoa@mail.rockefeller.edu, odonnel@mail.rockefeller.edu
Key Words DNA replication, DNA sliding clamps, DNA polymerase, processivity clamp loader, protein-DNA interactions Abstract Chromosomal DNA replicases are multicomponent machines that have evolved clever strategies to perform their function. Although the structure of DNA is elegant in its simplicity, the job of duplicating it is far from simple. At the heart of the replicase machinery is a heteropentameric AAA+ clamp-loading machine that couples ATP hydrolysis to load circular clamp proteins onto DNA. The clamps encircle DNA and hold polymerases to the template for processive action. Clamp-loader and sliding clamp structures have been solved in both prokaryotic and eukaryotic systems. The heteropentameric clamp loaders are circular oligomers, reecting the circular shape of their respective clamp substrates. Clamps and clamp loaders also function in other DNA metabolic processes, including repair, checkpoint mechanisms, and cell cycle progression. Twin polymerases and clamps coordinate their actions with a clamp loader and yet other proteins to form a replisome machine that advances the replication fork. CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE E. coli REPLICASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pol III Core . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Sliding Clamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Complex Clamp Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Holoenzyme Particle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Replisome Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein Trafcking on DNA Sliding Clamps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE EUKARYOTIC REPLICASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proliferating Cell Nuclear Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The RFC Clamp Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eukaryotic DNA Polymerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Eukaryotic Replisome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternate RFC Complexes and Other Roles for Clamp Loaders . . . . . . . . . . . . . . . . CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0066-4154/05/0707-0283$20.00 284 286 286 288 290 293 293 295 296 298 300 303 304 307 307
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INTRODUCTION
Chromosomal DNAs are exceedingly large molecules, and the vast repository of information they hold must be duplicated with precision [see (1) for overview]. The sheer size of these molecules may explain why all cells utilize a clamp loader to position a circular sliding clamp on DNA that tethers the DNA polymerases to their long substrates for highly processive synthesis (see Figure 1). The eukaryotic proliferating cell nuclear antigen (PCNA) and prokaryotic () clamp proteins have unrelated sequences, yet they have strikingly similar structures and thus share a common ancestor (2, 3). The clamps must be opened and closed around DNA, and this job is performed by a multiprotein clamp-loading ATPase [reviewed in (4)].
Figure 1 Components of cellular replicases. The table lists the three replicase components in the well-dened systems of Escherichia coli, eukaryotes, Archaea, and T4 phage. The scheme below is a generalized mechanism of replicase action. A multiprotein clamp-loader couples ATP binding and hydrolysis to loading of a ring-shaped processivity clamp that is then used by the replicative polymerase as a tether to the DNA template.
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The eukaryotic replication factor C (RFC) and the prokaryotic ( complex) clamploader subunits are arranged in a circle and are each members of the AAA+ protein family [briey reviewed in (4)]. The replicases of all cells function within a greater context, involving many other proteins. For example, two basic activities at a replication fork include a helicase to separate the duplex DNA and a primase that produces short RNA primers, which are required to initiate DNA synthesis. This larger machinery, frequently termed the replisome, is fairly well dened in prokaryotes (1, 58). In E. coli, and presumably other bacteria, the leading- and lagging-strand polymerases are connected to the clamp loader, which also binds a homohexameric helicase (DnaB in E. coli). The helicase acts on the lagging strand and activates the RNA primase (DnaG in E. coli). The replicase also makes a specic functional connection to single-stranded DNA-binding protein (SSB), which is present at the fork to protect single-stranded DNA (ssDNA) and melt hairpins. The eukaryotic replisome, in contrast, involves many more proteins and is less dened at the current time [reviewed in (911)]. The eukaryotic helicase, primase, and SSB are all heterooligomers. The helicase is thought to be the heterohexameric MCM complex (12), and the primase is the four-subunit DNA polymerase /primase that makes a hybrid RNA/DNA primer (13). The heterotrimeric replication protein A (RPA) functions as the SSB (14). Several additional eukaryotic proteins without prokaryotic counterparts are thought to act during replication initiation and fork progression as well (e.g., Cdc45, GINS complex, Dpb11, Sld2, and Sld3) (1517). The functions of most of these components are presently unknown. In addition, the leading- and lagging-strand polymerases are thought to be different enzymes, Pol (34 subunits) and Pol (4 subunits). It is still unclear which strands these polymerases act upon. Finally, many of these proteins are regulated by posttranslational modication, including the PCNA clamp which is ubiquitinated and sumoylated (1820). The clamp and clamp loaders of cellular replicases, prokaryote and eukaryote alike, also function in several other processes besides replication. For example, PCNA and bind DNA ligase as well as mismatch and excision repair proteins, although the exact role of these interactions is still not entirely clear (2123). A weak consensus sequence for proteins that bind PCNA reveals a broad array of additional proteins that bind this clamp and are generally involved in repair, chromatin structure, or cell cycle control (21). The clamps also function with other DNA polymerases (23, 24), probably helping to target them to sites where their action is required. Eukaryotes even utilize alternative RFC clamp loaders in which one subunit is replaced by a unique protein that presumably specializes the complex for the alternative function (2527, 234243). These alternative clamp loaders load PCNA clamps at specic target sites or, in one case, load an alternate PCNA-like clamp. Because of space limitations, this review focuses on prokaryotic and eukaryotic replicases and forgoes a description of archaebacterial, bacteriophage, and viral replicases. However, archaebacteria also utilize a similar clamp and clamp-loader
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strategy, as does the T4 bacteriophage (see the table in Figure 1) [reviewed in (7, 28), respectively]. We regret that space limitations prevent discussion of the phage T4 replisome in a similar manner; it is an enormously valuable system from which many basic early concepts of replisome action emerged, and it continues to provide fresh insights. A recent review in this series (7) expertly distills the T4 contributions to date and how they relate to the E. coli system. The replicative polymerases of most bacteriophage and viruses do not utilize a clamp and clamp loader, but these polymerases generally require one or two accessory factors that confer processivity [see T7 phage (7, 29, 30), vaccinia virus (31, 32), herpes virus (33, 34), and Pol (35)]. The authors have also refrained from discussing DNA replication initiation due to space limitation. Many useful reviews exist on this topic [see (1, 28, 36, 37)].
THE E. coli REPLICASE

The rst cellular replicase to be studied, E. coli DNA polymerase III holoenzyme, was isolated as an intact particle from E. coli extracts in the early 1980s, and its structure and function serve as a suitable paradigm for its eukaryotic counterpart (1). We, therefore, begin this review with the prokaryotic replicase machinery.
Pol III Core

DNA polymerase III (Pol III) was originally identied in a mutant strain of E. coli, polA (38). This strain lacked the comparatively strong DNA polymerase I activity, unmasking the replicative polymerase. The identity of this activity was likely the core polymerase subcomplex. The specic activity of Pol III core is similar to Pol I, but as will be described below, Pol III core functions with accessory proteins that convert it to an exceedingly efcient enzyme having the highest specic activity of any E. coli DNA polymerase (1). Pol III core is a 1:1:1 heterotrimer of the polymerase, 3 -5 proofreading exonuclease, and subunits (39, 40) (see Table 1). The subunit (encoded by dnaE) contains the DNA polymerase activity, incorporating 8 nucleotides/second (ntd/s), similar to Pol III core (20 ntd/s) (41). The (dnaQ) subunit is the proofreading 3 -5 exonuclease. It is interesting to note that without the processivity of holoenzyme is markedly reduced from >50 kb to about 1.5 kb (42), thereby ensuring that the proofreading subunit is present during genome duplication. In contrast, the small subunit (holE) has no known function besides a slight stimulation of (43) and the holE gene can be deleted with little consequence (44). Little structural information is available for Pol III core. The subunit is a member of the C family of DNA polymerases (45). Members of this family are present only in bacteria, and it remains the only major family for which no crystal structure has been solved. One may presume that it will contain the characteristic palm, thumb, and ngers domains and two-metal mechanism of catalysis present
CELLULAR REPLICASES TABLE 1 Escherichia coli replisome components and associated functionsa
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in all other polymerases (46). Deletion mutagenesis suggests that the active site is located in the N-terminal two thirds of , whereas contacts to other holoenzyme components are made through the C-terminal region (47, 48). Atomic resolution structures of portions of and are available (4951). The subunit is composed of two domains (52). The N-terminal domain (186 residues
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out of 243) contains the exonuclease active site and the binding site. The crystal structure of this active fragment has been solved (49), and it is structurally similar to other polymerase-associated exonucleases and the exonuclease domain of DNA polymerase I. The preferred DNA substrate for is a single-stranded 3 terminus, and a fully base-paired recessed 3 terminus is a poor substrate for alone (53, 54). However, tightly associates with the C terminus of and stimulates activity on a recessed 3 terminus, probably by bringing to the primer site. preferentially degrades a primer/template with a mismatched recessed 3 terminus. In addition, polymerization is inhibited by a mismatch, which provides more time to complete the excision. The structure of bears a resemblance to a DNA-interacting domain of eukaryotic DNA polymerase (50, 51). This structural similarity may underlie the slight stimulation of by and increased mutator phenotype of a holE-null mutant in an mutant background (44).
The Sliding Clamp

Pol III core by itself is slow, incorporating 20 ntd/s, and weakly processive, extending only 110 bases per binding event (41). In fact, no matter how much time or enzyme is available, the Pol III core cannot extend a unique primer full circle around an M13 ssDNA genome (55, 56). To become an efcient replicase, the core polymerase requires the clamp. Coupled to , core becomes exceedingly fast (750 ntd/s) and processive (>50 kb). Biochemical studies initially revealed that binds DNA topologically (57), implying it has a ring shape and encircles the duplex, whereupon it freely slides along it. This hypothesis was quickly proven by structure analysis (3) (Figure 2). Core polymerase directly associates with (5759), initially occupying about 22 base pairs (bp) of primer (60, 61). As core extends DNA, it pulls the clamp along behind it. The crystal structure of the dimer (3) shows that the two crescent-shaped protomers form a ring with a large central channel of 35 A in diameter that may easily accommodate double-stranded (ds) DNA modeled inside (Figure 2a,b). In fact, room exists for one or two layers of water between the DNA and , suggesting may ice skate along the duplex. The center of the ring is lined with 12 -helices, pairs of which are supported by an outer -sheet. This helix pair and sheet motif forms one globular domain, which is repeated six times around the ring, creating a sixfold pseudosymmetry. Each monomer consists of three domains and dimerizes head-to-tail with another to produce two structurally distinct faces. One face has several loops and protruding C termini. This is the face of the ring that interacts with other proteins as discussed below (62, 63). The clamp is a tight dimer, and its half-life on DNA is over 1 h at 37 C (64, 65). The subunit of complex can open by itself, as determined by its ability to rapidly remove the clamp from DNA (64, 66). It appears that only one binds 2 , and it does not dissociate the dimer into monomers, even though it
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Figure 2 Structure and dynamics of the sliding clamp. (a,b) Crystal structure of the dimer with modeled double-stranded DNA through the central channel (courtesy of D. Jeruzalmi and J. Kuriyan). Panel (b) highlights the two faces of the ring. The C-terminal face (right) is implicated in many of the interactions of with other proteins. (c) Superposition of one subunit from the dimer structure (purple) and the monomer from the 1 1 crystal structure (yellow), using domain II as a reference. (d ) Model of an open clamp made by arranging two monomer structures from 1 1 to create one dimer interface [described in (67)]. Panels (c) and (d ) adapted from (67), copyright 2001, with permission from Elsevier.
must destabilize one interface (62, 65, 66). This idea is consistent with the ability of complex to load that is cross-linked across one dimer interface (66). A cocrystal structure of bound to a monomeric mutant of has been solved (67). This structure shows two distinct points of contact between and . The contact points are located on the opposite ends of the same -helix, which is located in the
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N-terminal domain of . One end of the helix binds a hydrophobic pocket between domains II and III of . The other end appears to push on a loop at the dimer interface, leading to a distorted interface that can no longer close. It is proposed that gets a grip on via the hydrophobic pocket and pushes on the loop to crack the interface or to hold the interface open. Mutational analysis of these contact points has conrmed their roles in clamp-loader interaction and clamp opening (68). The - structure explains how the interface is cracked but not how the ring opens to accommodate DNA in the central channel. Comparison of the dimer and monomer (from the - structure) shows signicant rigid body motions between the domains, leading to a shallower crescent shape in the monomer (67) (see Figure 2c). This implies that the ring is under spring tension in the closed state until disrupts one interface, allowing tension between the domains to relax and producing a gap for DNA-strand passage (see Figure 2d). The strong interactions at the dimer interfaces likely maintain this tension.
The Complex Clamp Loader

The E. coli clamp loader is a complex composed of ve different subunits in a dened stoichiometry: 3 1 1 1 1 (69, 70). The complex harnesses the energy of ATP binding and hydrolysis to topologically link to a primed DNA, then it ejects from DNA, leaving the closed clamp behind (57, 66, 71). It is convenient to dissect the clamp loader by the primary function of each subunit (4). The three subunits are the only subunits that bind ATP (72, 73) and have thus been termed the motor of the complex. The subunit is called the wrench because it is the main clamp-interacting subunit, and it can open the dimer interface by itself. The subunit modulates - contact (66). appears to be a rigid protein (74). Unlike and , the domains of have more intramolecular interactions and assume the same orientation in the structure and within 3 . This feature has earned the term stator, the stationary part of a machine upon which other parts move. In contrast, the three domains of assume different orientations in the trimer. The domains of also assume different orientations in - compared to 3 . The 3 1 1 complex is termed the minimal clamp loader, as it is sufcient to place on a DNA template (75). The and subunits are not essential for the clamp-loading mechanism (76), but links the clamp loader to SSB and primase (77, 78), which will be discussed below. serves as a connector to (76) and also strengthens the 3 complex (79). A broad outline of the clamp-loading mechanism has been determined from biochemical studies (4, 66). The nucleotide-free clamp loader has a low afnity for the clamp (62). The subunit, which binds tightly to , is likely sequestered by the other subunits without ATP present. Upon ATP binding, the complex undergoes a conformational change (62, 71) that allows tight binding to the clamp, whereupon opens one dimer interface of the clamp (65). The clamp-clamp loader complex has a strong afnity for DNA, particularly a primed template (71, 80). The DNA, presumably threaded through the clamp, stimulates ATPase activity in
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complex, and this allows the ring to close around DNA and the clamp loader to eject, thereby recycling it for repeated rounds of clamp loading (66, 81). Hydrolysis of ATP leaves the clamp loader in a low-afnity DNA-binding state, presumably until ADP dissociates and the complex can be recharged with triphosphate nucleotides (80, 8284). The crystal structure of the 3 complex provided deeper insight into clamploader action (70). This accomplishment was followed two years later by the crystal structure of the eukaryotic clamp loader bound to its clamp (85), which has lled in many details that will be discussed later. The ve subunits of E. coli 3 are arranged in a circular fashion (70) (Figure 3b). The three subunits are adjacent to one another, anked on one side by and the other by , which in turn interact with each other to complete the ring. Each subunit consists of three domains, and oligomerization is mediated mainly by the C-terminal domain III (Figure 3a,b). The N-terminal domains I and II of all ve subunits adopt the chain fold of the AAA+ (ATPases associated with a variety of cellular activities) family (Figure 3a) [reviewed in (86, 87)]. Although only binds ATP (72), has conserved elements of the AAA+ family (88). In contrast, the sequence has diverged, making the discovery of its AAA+ fold surprising. The ATP sites of complex are located at subunit interfaces and are supplemented with residues from the adjacent subunit (see Figure 3c). The interfacial location of ATP sites is typical of AAA+ oligomers and is presumed to promote communication among the subunits. contributes a key arginine to ATP Site 1 of the complex, as seen in the left panel of Figure 3c (red residue) with an ATP molecule modeled along the phosphate-binding loop (blue). This arginine is located in a conserved serine-arginine-cysteine (SRC) motif that is present in all known clamp loaders. Mutation of the Arg, and the homologous residue in , causes a severe defect in clamp-loading and ATPase activity and also disrupts interaction with DNA ( mutants) or ( mutants) (89, 90). Mutation of the P-loop of has more severe consequences that result in complete loss of activity (73). These observations highlight the coordination of the ATP cycle with the clamploading mechanism, suggesting regulation of substrate binding by conformational changes in distinct ATP sites during nucleotide binding and hydrolysis. The C termini (domains III) of the ve subunits of the minimal complex form a tight ring, or collar (91), but the N-terminal AAA+ domains (I and II) are more loosely associated, with a total lack of contact between and in these domains, creating a gap in the N-terminal portion of the ring (70) (Figure 3b). This gap may function to allow DNA to pass into positioned under the clamp-loader complex, as discussed in the RFC section of this review. Both ATP- and -clamp binding occur in these N-terminal domains (67, 92). The loose connections of these domains has led to the suggestion that some conformational freedom of the N termini is essential for function (93). The structure of 3 lacks ATP and thus is in the inactive state. Consistent with this, a dimer cannot be docked onto the complex (replacing with -) without signicant clashes, even with the substantial gap between and (70). Biophysical experiments in solution indicate that the distance between
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Figure 3 Crystal structure of the E. coli 3 1 1 complex. (a) Isolated subunits from the structure of the complex, domains IIII noted [adapted from (87), copyright 2002 Nature Publishing Group (http://www.nature.com)]. (b) Side view of 3 (left). The -interacting helix of is marked in yellow. View from the C termini of 3 (right) [adapted from (70), copyright 2001, with permission from Elsevier]. (c) The ATP sites of the ring-shaped complex are located at subunit interfaces, as illustrated in the cartoon (right) and taken from the structure of ATP Site 1 (left) with ATP (purple) modeled against the P-loop (blue). The Ser-Arg-Cys (SRC) motif of , conserved in all clamp loaders, points its arginine (red ) toward the phosphate of ATP [adapted with permission from (89)].
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and does not change dramatically during clamp association (93). Hence, some other conformational change may occur that does not necessitate increasing the gap between and . For example, may be pushed downward relative to , thus exposing the -binding element of without changing the distance between and . In addition, the subunit also binds (94). Taken together, these observations imply that an extensive surface of interaction is formed between the clamp and clamp loader. How the clamp loader binds the clamp and DNA is illuminated by the RFC-PCNA structure discussed later in this review.
The Holoenzyme Particle

On the basis of intracellular Pol III subunit concentrations, a portion of clamp loader is associated with the Pol III holoenzyme, but a majority of the clamp loader is free in solution (64, 95). The clamp loader associated with the holoenzyme contains a different form of the dnaX gene. The subunit is produced through a ribosomal frameshift in the dnaX gene, which causes almost immediate termination of translation to produce a 47.5-kDa protein (96, 97). The full-length product of dnaX is the subunit (71.1 kDa), which contains the sequence plus a unique 23.6 kDa C-terminal region ( c ) (Figure 4a). The 23.6 kDa c is comprised of two domains, IV and V, that bind DnaB and Pol III core (through ), respectively (98, 99). The c region is not required for clamp loading but is essential for cell viability (100), probably owing to its ability to organize the replisome as discussed below. The DnaX protein is present in three copies in the clamp-loader complex, and thus various species may assemble in stoichiometries of 3 , 2 1 , 1 2 , and 3 (69). Each C terminus will recruit one polymerase, and therefore the more present in the clamp loader, the more core polymerase molecules it may bind. At least two polymerases are required for concurrent synthesis of leading and lagging strands (Figure 4b). Because of this requirement, it is thought that the E. coli replicase contains two Pol III cores attached to a 2 1 clamp loader and that a specic order of assembly leads to this particle (69, 101, 102), which is termed Pol III (or Pol III star). The clamp associates with Pol III in an ATP-dependent manner to form the Pol III holoenzyme. The single-copy subunits of the clamp loader dene an inherent asymmetry, and thus by denition, the two cores attached to the two subunits are in somewhat different environments (8). The consequence of this asymmetric structure might be minimal because a proline-rich segment of separates the clamp-loader and polymerase-interacting domains, suggesting a exible connection. However, DnaB or other holoenzyme subunits may hold the cores in dened asymmetric positions (103). It has been proposed that the asymmetric structure imposes distinct properties onto the two polymerases, modeling their behavior to t the different needs of replicating the leading and lagging strands (8, 102, 104109).
Replisome Dynamics
The subunits of Pol III holoenzyme not only connect two core polymerases to the central clamp loader, but also connect the replicase to the DnaB helicase
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Figure 4 Organization and dynamics of the E. coli replisome. (a) The subunit of DNA polymerase III holoenzyme is comprised of the clamp-loader domains IIII ( sequence) and the replisome organization domains IV and V that bind the DnaB helicase and Pol III core, respectively. (b) Polymerase cycling at the replication fork. As the replisome advances, the clamp loader loads a clamp on an RNA primer (pink) synthesized by DnaG (upper right). When the lagging-strand polymerase replicates to a nick, it dissociates from DNA and (lower right) and cycles to the newly loaded clamp (lower left).
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(see Figure 4b). The homohexameric DnaB encircles the lagging strand, and when coupled to DNA synthesis through , its unwinding rate increases from 35 bp/s to near holoenzyme speed (98, 99, 103, 110112). As the replisome advances, the polymerase on the leading strand simply extends DNA in a continuous fashion. Presumed impediments to leading strand extension include sites of DNA damage and the consequent collapse of the fork. Repair and restart of synthesis is an elaborate process detailed in recent reviews (113, 114). Pol III holoenzyme has been implicated in mediating the restart of DNA replication after fork stalling (115). Lagging-strand replication is a discontinuous process of ts and starts that repeats in a cycle time of 13 s. An overview of the lagging-strand cycle is illustrated in Figure 4b. Each Okazaki fragment is initiated by primase, which synthesizes an RNA primer of about 1012 nucleotides (116, 117). Primase action requires interaction with DnaB, which involves a C-terminal region of primase (118, 119). Primase extends the RNA in the opposite direction of helicase unwinding and is presumed to separate from DnaB, which may account for its observed distributive action (120). Primase remains attached to the RNA primed site through its interaction with SSB (121123). Although primase eventually dissociates, release of primase is accelerated by the subunit of the clamp loader, which binds SSB in a competitive fashion, recruiting the clamp loader to the DNA template to compete with primase (124). The clamp loader then places onto the primer for the lagging-strand polymerase. As the lagging polymerase extends a fragment, a loop is generated because it is connected to the leading polymerase (via the clamp loader), yet extends DNA in the opposite direction (see top left diagram), as originally proposed (125) and recently conrmed (126) in the T4 system. The 13 kb Okazaki fragment will be completed within a few seconds (Figure 4b, top right diagram), and at this point, the core must rapidly release from DNA to start the next fragment (bottom right diagram). The highly processive Pol III requires a specic mechanism for this release step, which disengages core from , leaving the clamp behind on the nished fragment. The release step occurs only at a nick, thus ensuring completion of the fragment, and requires the subunit (127130). The lagging-strand core is now free to bind a new clamp placed on the next RNA primer by the clamp loader (bottom left diagram). Replication fork progression has been studied in E. coli, using rolling-circle DNA templates (103, 107, 116, 120, 131133). That clamps accumulate on the lagging strand and that the single clamp loader can rapidly load clamps on DNA repeatedly during fork progression have been demonstrated using this system (103). The accumulated clamps can be recycled by the unloading action of complex and also by the vefold excess of free subunit in the E. coli cell (64).
Protein Trafcking on DNA Sliding Clamps

Many different proteins function with sliding clamps. An understanding of the way proteins coordinate their trafc ow on clamps is now emerging. To illustrate
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how proteins may switch positions to utilize the clamp, we briey describe three important switches on that occur during the progression of the E. coli replication fork. The / clamp loader and Pol III core are known to bind the same face of in essentially the same spot, and therefore these two factors compete for the clamp (63, 134). Yet both the core and / complex must function with at the start of each DNA chain. This protein trafcking event is facilitated by the fact that ATP hydrolysis ejects the clamp loader from (Figure 5a) (71). In addition, the clamp loader remains in a reduced activity state for a short interval, presumably owing to slow ADP release (80, 83). The core polymerase is then free to bind the abandoned clamp and, in fact, binds tighter on DNA than off (63). Switching on is also thought to occur when the leading polymerase stalls. In this case it is thought that a bypass polymerase, either Pol IV or Pol V, takes possession of the clamp to extend DNA through the stall site. Recent structural analysis suggests that Pol IV can associate in two ways with (135, 136). Pol IV binds the edge of the ring and one hydrophobic pocket, but Pol IV is angled off the DNA (135). It is possible that in this binding mode Pol IV may bind to simultaneously with Pol III (see Figure 5b). Upon stalling of Pol III, Pol IV must break its interaction with the side of the ring and swing down to the DNA, presumably maintaining its hold on the hydrophobic pocket of . This action would displace Pol III from DNA and perhaps disrupts the Pol III- contact as well. Pol IV is distributive, even with , allowing Pol III to regain the clamp after the lesion is bypassed. Recent evidence with Pol III, Pol V, and suggests that a similar trade-off may occur (137). At the end of an Okazaki fragment, the normally highly processive Pol III rapidly dissociates from (127130), freeing the polymerase to extend the next Okazaki fragment. This trafcking event is mediated by the c portion of . The subunit binds via the extreme C-terminal residues (134). c also binds the C terminus and disrupts core- interaction. However, c binds ssDNA, and this prevents from binding the C-terminal residues of . Hence, so long as there is ssDNA template, c is turned off, and core functions with . But when all available ssDNA is converted to duplex, c turns on and separates core from (see Figure 5c).These switch processes explain how interactions with the clamp are modulated by ATP or DNA structure to promote the trafcking of different proteins on sliding clamps to ensure progression of replication.
THE EUKARYOTIC REPLICASE

Although the complexity of architecture, interaction, and regulation of DNA replication is far greater in eukaryotes than in bacteria, the core replicase components are structurally and functionally more similar than different. However, beyond this basic machinery lies a much larger network of proteins required for propagation of the replication fork and regulation of its advance as well as coordinating its
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Figure 5 Three examples of protein trafcking on sliding clamps. Interactions with the clamp from E. coli. (a) The complex clamp loader associates tightly with when bound to ATP. DNA triggers ATP hydrolysis, resulting in low afnity for and DNA. (b) When Pol III, the replicative polymerase, encounters a lesion in the DNA template, it stalls, unable to overcome its inherent delity to incorporate opposite a damaged base. Stalling allows an error-prone polymerase, such as Pol IV (red ) passively traveling on , an opportunity to trade places with Pol III on to replicate past the lesion. [Adapted with permission from (135).] (c) Pol III maintains a tight grip on via the polymerase C terminus. However, when it completely replicates its substrate DNA, the polymerase must release from to recycle to the next primed site. The subunit modulates this interaction, binding the polymerase C tail only when no more single-stranded template is present. This severs the connection between the polymerase and the clamp [adapted with permission from (134), copyright 2003, National Academy of Sciences, U.S.A.].
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activity with other DNA metabolic machineries. Many of these details are only now coming into focus.
Proliferating Cell Nuclear Antigen

PCNA derives its name from the early nding that the protein is abundant in proliferating cells (138). PCNA was shown to be directly involved in DNA replication through its ability to stimulate DNA polymerase in replicating long stretches of primed DNA (139141). This characteristic suggested it may be analogous to the E. coli subunit, although neither were known to be sliding clamps at the time. Subsequent work showed the importance of PCNA for simian virus 40 (SV40) DNA replication and its enhanced activity in the presence of RFC and ATP (142). As a monomeric unit, PCNA is about two-thirds the size of , and accordingly, each protomer contains two structurally similar domains instead of the three domains found in (2). The chain folds of the two PCNA domains are the same as those found in the domains of . PCNA and form very similar ring-shaped structures, except PCNA must trimerize to form a six-domain ring (Figure 6a). Like , the PCNA ring is quite stably attached to DNA (t1/2 = 24 min) (143). The PCNA protomers are also arranged head-to-tail to create two distinct faces of the ring, mirroring . As in the E. coli system, the eukaryotic clamp loader and polymerase compete for binding the same face of the PCNA ring, the face from which the C termini project (144, 145). A variety of proteins involved in DNA repair and cell cycle control interact with PCNA, but this topic is covered elsewhere [reviewed in (21, 23)]. Relevant to the current review, a weak PCNA-binding consensus sequence has emerged: Q-x-x-h-X-X-a-a, where x = any residue; h = L,I,M; and a = F,Y (21, 23). The crystal structure of human PCNA in complex with a peptide derived from the cell cycle regulator p21WAF1/Cip1 was the rst to demonstrate that these clamp-binding
Figure 6 Structures of the eukaryotic clamp and clamp loader from Saccharomyces cerevisiae. (a) View of the C-terminal face of PCNA. The ring-shaped PCNA is a headto-tail trimer of a two-domain monomer. The sixfold pseudosymmetry of the clamp is evident in PCNA as well. (b) The structure of replication factor C (RFC) bound to PCNA reveals the structural similarity between RFC and complex. RFC binds to the C-terminal face of PCNA. (ce) The RFC subunits are arranged in a helix that tracks the minor groove of B-form DNA modeled through the PCNA ring. (d ) In this cartoon, the 5 terminus of a recessed primer template is positioned to exit the central channel of the clamp and clamp loader through the gap between RFC1 and RFC5. (e) N-terminal regions of the ve RFC subunits and the PCNA ring from the RFC-PCNA structure. Two conserved helices in each RFC subunit (yellow) are in position to interact with DNA (orange/green) that passes through the central channel of PCNA (gray) with the 5 terminus (green spheres) exiting between RFC1 and RFC5. [Adapted with permission from (85), copyright 2004 Nature Publishing Group (http://www.nature.com).]
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proteins interact with PCNA at a hydrophobic pocket located between the two domains of a protomer (146). The nding generalizes to the position at which DNA polymerase binds the T4 gp45 clamp (147149). Also, the subunit of Pol III core and subunit of complex bind E. coli at a spot between domains II and III (24, 67, 150).
The RFC Clamp Loader

The eukaryotic clamp loader was rst isolated as a required component of the in vitro SV40 genome replication system (151) [reviewed in (152)]. The activity was originally named replication factor C (RFC) (51) or activator-1 (153). RFC is a DNA-dependent ATPase that functions with PCNA to confer processivity on DNA polymerase (142) [reviewed in (154, 155)]. The similarity between RFC and E. coli complex was apparent early on. RFC consists of ve different proteins that are homologous to each other and to and of E. coli complex (156, 157). Common sequence motifs among these clamp-loader AAA+ proteins have been termed RFC boxes. In overview, RFC acts similarly to complex. In an ATP-dependent reaction, RFC loads PCNA onto a recessed 3 primer/template junction and then dissociates, allowing PCNA to function with Pol (158, 159). The RFC subunits in S. cerevisiae are referred to as RFC15 (157) (see Table 2 for human RFC nomenclature). RFC25 share a similar molecular weight and three-domain architecture characteristic of the E. coli subunit (85, 160). RFC1 also contains these three domains, along with sizable N- and C-terminal extensions (161). The N-terminal region (residues 1275 in S. cerevisiae) has clear homology to DNA ligases, although there is no evidence of ligase activity. The removal of the RFC1 N terminus results in sensitivity to DNA-damaging agents (162), but this region is not necessary for in vitro clamp loading (163) or cell viability (162). The C-terminal domain (residues 660861 in S. cerevisiae) has not yet been characterized genetically or biochemically. The ve-subunit composition of RFC bears a close similarity to the minimal E. coli 3 complex (85). On the basis of subunit interactions and sequence similarity to complex subunits, the subunit arrangement of the RFC pentamer was proposed (4, 164) and has since been proven by structure analysis (85). RFC1 shares characteristics of the subunit in its conserved clamp-interacting residues and position in the pentamer (163, 165). RFC24 are considered -like in their ability to form a trimeric ATPase subassembly (166). RFC5 is in the position of , and like it contains an SRC motif but lacks a consensus phosphate-binding loop (P-loop). Like the 3 complex, the ATP sites of RFC are located at subunit interfaces. However, unlike 3 , RFC contains four competent ATPase sites because RFC1 also binds ATP where does not. In fact, RFC5 also binds a nucleotide in the crystal structure. During the RFC ATPase cycle in S. cerevisiae, the complex initially binds two ATP, then a third upon PCNA binding, and a fourth when it locates the
CELLULAR REPLICASES TABLE 2 Replisome component RFCa Eukaryotic replisome components Saccharomyces cerevisiae (kDa) RFC (277.7)a RFC1 (94.9) RFC2 (39.7) RFC3 (38.2) RFC4 (36.1) RFC5 (39.9) PCNA (28.9) Pol (220.2)a Pol3 (124.6) Pol31 (55.3) Pol32 (40.3) Pol a Pol (378.7)a Pol2 (255.7) Dpb2 (78.3) Dpb3 (22.7) Dpb4 (22.0) Pol a Pol (355.6)a Pol1 (166.8) Pol12 (78.8) Pri2 (62.3) Pri1 (47.7) MCM (605.6)a Mcm2 (98.8) Mcm3 (107.5) Mcm4 (105.0) Mcm5 (86.4) Mcm6 (113.0) Mcm7 (94.9) RPAa RPA (114)a RPA70 (70.3) RPA30 (29.9) RPA14 (13.8) Function and remarks [Schizosaccharomyces pombe name (S.p.)] Pentameric clamp loadera Binds ATP; phosphorylated Binds ATP Binds ATP Binds ATP Binds ATP or ADP 87 kDa homotrimeric processivity sliding clampa
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Human (kDa) RFC (314.9)a p140 (128.2) p37 (39.2) p36 (40.6) p40 (39.7) p38 (38.5) PCNA (28.7) Pol (238.7)a p125 (123.6) p50 (51.3) p66 (51.4) p12 (12.4) Pol (350.3)a p261 (261.5) p59 (59.5) p17 (17.0) p12 (12.3) Pol (340.6)a p180 (165.9) p68 (66.0) p55 (58.8) p48 (49.9) MCM (535)a Mcm2 (91.5) Mcm3 (91.0) Mcm4 (96.6) Mcm5 (82.3) Mcm6 (92.3) Mcm7 (81.3) RPA (100.5)a RPA70 (70.3) RPA30 (29) RPA14 (13.5)
PCNA Pol a
Replicative DNA polymerasea DNA polymerase, 3 -5 exonuclease, binds PCNA; subunit A (S.p. Pol3) Structural subunit; subunit B (S.p. Cdc1) Binds PCNA; subunit C (S.p. Cdc27); binds Pol large subunit Structural, stimulates processivity; subunit D (S.p. Cdm1) Replicative DNA polymerasea DNA polymerase, 3 -5 exonuclease (S.p. Pol2/cdc20) Binds polymerase subunit (S.p. Dpb2) Binds Dpb4 Present in ISW2/yCHRAC chromatinremodeling complex (S.p. Dpb4) DNA polymerase/primasea DNA polymerase Structural subunit Interacts tightly with p48 RNA primase catalytic subunit Putative 3 -5 replicative helicasea Phosphorylated by Dbf4-dependent kinase Ubiquitinated, acetylated Helicase with MCM6,7; phosphorylated by CDK; aka Cdc54 Aka Cdc46; Bob1 is a mutant form Helicase with MCM4,7 Helicase with MCM4,6; ubiquitinated Single-stranded DNA-binding proteina Binds DNA, stimulates Pol Binds RPA70 and 14, phosphorylated Binds RPA30
MCMa
Information concerns a protein complex.
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primer/template DNA, which triggers ATP hydrolysis, causing RFC to eject and leave the closed PCNA ring on DNA (167). This mechanism excludes the route of RFC rst encountering DNA and subsequently recruiting PCNA. ATP site mutational studies have demonstrated that, in general, disrupting any one of the four consensus ATP sites has a signicant effect on the activity of RFC (168170), although ATP binding may sufce to rescue this defect in at least one ATP site (in RFC4 for S. cerevisiae). These single P-loop mutants still bind PCNA readily but are decient in DNA binding (170). A crystal structure of an RFC-PCNA complex has been solved (85). To promote stability of this nucleotide-dependent interaction by preventing hydrolysis, ATP S was used, and RFC was produced with R to Q mutations in the SRC motifs. The RFC-ATP S-PCNA crystal structure has provided a detailed view of how a clamp loader interacts with its cognate clamp (see Figure 6b). In addition, the structure has revealed how DNA binds to a clamp loader. The main PCNA contacts occur through RFC1 and RFC3, which bind to the hydrophobic pocket between the domains of two different PCNA protomers. RFC1 interacts extensively with PCNA, whereas RFC3 seems to be only partially engaged with PCNA and the ring is closed. In the RFC-ATP S-PCNA structure, RFC2 and RFC5 do not bind PCNA at all. This suboptimal interaction of RFC with PCNA may explain why the clamp remains closed and only slightly perturbed from its unbound structure. Alternatively, the closed PCNA may be due to crystal packing forces, instability of an open-ring complex, or the arginine to glutamine mutation in the four SRC motifs. Although this mutation should ensure that no ATP S becomes hydrolyzed, it may have prevented or perturbed some necessary RFC subunit-PCNA interaction needed for clamp opening. The way RFC binds DNA is suggested by the helical arrangement of RFC subunits in the structure, with the helical axis passing through the central channel of the closed PCNA (see Figure 6c,e). The helix begins at RFC1, the subunit that is fully engaged with the hydrophobic pocket on one PCNA protomer. Adjacent to RFC1 is RFC4, displaced by the helical operator of 61 rotation and 5.5 A translation. Overall, the helical operations that relate all ve subunits have a pitch of 5.6 A per 60 rotation, ending with RFC5, which lies 25 A above PCNA and is signicantly separated from the ATPase domain of RFC1. This right-handed helix and pitch mimics that of duplex B-form DNA. Furthermore, there is sufcient space in the center of RFC to model a DNA duplex, which passes right through the center of PCNA. Each RFC subunit has two -helices that are oriented so their positive dipole tracks the minor groove phosphate backbone of DNA modeled into the structure (Figure 6e). Several basic residues on these helices are conserved in E. coli and eukaryotic clamp loaders, consistent with the idea that DNA binds within this central area of RFC. Modeling a 3 -recessed, primed template from another crystal structure into the center of the ring shows that the specic recognition of a primer/template by RFC might occur by a simple clash of the primed-template junction with the domain III cap of the RFC subunits. A stiff duplex DNA could not proceed through the RFC structure because it would hit the cap and could
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not bend to exit out the side of RFC. The exible ssDNA of a primed junction may bend to exit through the gap between RFC1 and 5, perhaps assisted by a positive patch on the RFC1 surface (see Figure 6e). This hypothesis of specic primer/template recognition is in agreement with recent biophysical evidence of RFC/DNA association (171, 172).
Eukaryotic DNA Polymerases

Many DNA polymerases are now known to function in the eukaryotic cell. These polymerases all share a common catalytic mechanism [reviewed in (11)], but most serve a specic function outside of basic genome duplication. DNA polymerases , , and are the established replicative polymerases and are thought to function together at the replication fork to copy genomic DNA in a semi-discontinuous manner. Pol , , and are all members of the B-family of DNA polymerases (45).
DNA POLYMERASE
DNA polymerase (Pol ) was initially thought to be the main replicase before Pol was discovered. Pol is unique in its ability to initiate DNA synthesis by rst synthesizing its own 12-nucleotide RNA primer and then extending it with about 20 bases of DNA (173, 174) [reviewed in (13)]. Pol is now thought to be the eukaryotic primase, making a hybrid RNA/DNA primer, followed by a polymerase switch allowing the replicase to take over elongation (175). The switch is mediated by RFC, which displaces Pol from the primer via competition for RPA (176, 177). Pol consists of four subunits (13). The DNA polymerase activity is found in the largest subunit (p180), and primase activity is located in the smallest subunit (p48). The exact functions of the middle two subunits are not clear, but all four subunits are present in Pol isolated from yeast, human, Xenopus, and Drosophila.
DNA POLYMERASE
DNA polymerase (Pol ) in ssion yeast, human, and other eukaryotic organisms is composed of four essential subunits (178, 179). Interestingly, in S. cerevisiae, Pol has only three subunits, and no apparent homologue exists for the fourth subunit (180). Furthermore, the third subunit can be deleted from budding yeast, although cell growth is compromised (181). A unied subunit nomenclature for Pol has recently been proposed (182). On the basis of Schizosaccharomyces pombe Pol subunits and their homologues in other eukaryotes, the subunits have been renamed AD for the Pol3(A), Cdc1(B), Cdc27(C), and Cdm1(D) polypeptides. Much debate has centered on the possibility of the Pol complex self-associating into a dimer. The most recent evidence in multiple systems indicates that Pol complex contains only one copy of each subunit across a wide range of concentrations and has an elongated shape that resulted in the earlier confusion over whether it was a dimeric polymerase particle (183, 184). Pol subcomplexes can also be isolated as a core A/B dimer (180, 185). A zinc-nger module in subunit A interacts with subunit B, which acts as a bridge to subunits C and D (when present) (183, 186, 187). The polymerase and
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3 -5 exonuclease activities of Pol are present in the large A subunit. The Pol polymerase extends DNA with high delity, but the exonuclease seems surprisingly ineffective in vitro in S. pombe (188) and may be most important when Pol is clamped onto DNA by PCNA (189, 190). Pol associates with PCNA via interactions with at least two of its subunits. The clamp is positioned behind the polymerase (191), preventing polymerase dissociation as it extends a primer (192). The strongest interaction is between PCNA and subunit C (193). Subunit C contains the consensus PCNA interaction motif, and studies in yeast show that this PCNA-interacting subunit stimulates Pol (183, 194). Surprisingly, stimulation is not dependent upon the PCNA-interacting motif but mainly on the domain involved in connection to the A/B complex. The A/B heterodimer alone can also associate with PCNA, presumably through subunit A (180). The stimulation of subunit C on Pol activity might be due to protein complex stabilization rather than catalytic enhancement. Even the small D subunit from the human four-subunit Pol complex signicantly stimulated the A/B/C subcomplex in assays with RFC and PCNA (179). Studies showing Pol is essential in yeast placed it at the replication fork (195). Indeed, chromatin immunoprecipitation (ChIP) assays in yeast indicate that Pol is located at origins prior to S phase (196) and moves away from origins upon releasing an S phase block (197), consistent with a role in chromosome replication. However, there are some conicting genetic studies on whether the intrinsic DNA polymerase activity is required for replication or whether the protein may instead serve another role, either as a DNA sensor or checkpoint protein (198200), or perhaps it holds together other proteins that are essential in the replisomal particle (201). Although further work will be required to fully understand the exact role of Pol , it is widely believed to be directly involved in DNA synthesis at the replication fork. A recent report on Pol concludes that it is a heterotetramer with a stoichiometry of 1:1:1:1, presumably in all eukaryotes (202). The DNA polymerase and 3 -5 exonuclease of Pol reside in the largest subunit and appear to have a higher delity than Pol /PCNA (203). The third-largest subunit, Dpb4, is also a member of a complex that appears to be involved in chromatin remodeling (204) (A. Tackett and B. Chait, personal communication). Pol activity does not absolutely require PCNA, but PCNA stimulation increases as the ionic strength is raised (205207). The PCNA interaction motif on the large subunit of Pol is not essential for cell viability, but mutational analysis of this sequence suggests a role in DNA repair (208).
DNA POLYMERASE (POL )
The Eukaryotic Replisome

The essential nature of both Pol and Pol and the fact that they both function with PCNA as monomeric polymerase particles have led to renewed proposals that they function together to replicate the leading and lagging strands. Xenopus
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extracts have shown that depletion of either polymerase results in a dramatic drop in replication (209, 210). Depletion of Pol gave rise to many short nascent strands that may have been improperly elongated Okazaki fragments, whereas Pol depletion merely slowed down the replication machinery. Viability of Pol -deleted strains of yeast suggests that Pol is not essential for cell viability (198, 201, 211). However, an inactive point mutant of Pol resulted in cell death (212). One possible explanation for this apparent contradiction is that another polymerase, presumably Pol , can carry out DNA replication in the complete absence of Pol but that the Pol point mutant acted as a dominant negative to stop the fork. Intensive studies of in vitro replication from the SV40 origin have provided great insight into eukaryotic fork function, but the SV40 T antigen lls many roles ordinarily performed by host proteins, including helicase function (213). Study of polymerases in this system show that Pol and Pol , and even Pol alone under some conditions, are sufcient for replication. However, the small size of the genome and use of T antigen may minimize the requirements for replication [reviewed in (152)]. Reconstitution of a eukaryotic replisome on a rolling-circle template may greatly facilitate our understanding of the roles of Pol and Pol and which strand(s) they operate on. The 10-fold-smaller size of eukaryotic lagging-strand fragments (200 bp) compared to E. coli is counterbalanced by the 10-fold-slower rate of fork movement. Stoichiometric use of PCNA clamps during lagging-strand replication has not been demonstrated but is presumed to occur as it does with the E. coli clamp. That PCNA clamps are left behind on lagging-strand fragments is implied by the fact that PCNA interacts with and, in some cases, stimulates the factors necessary for Okazaki fragment maturation (214216). There is also an interesting observation that PCNA-DNA complexes may persist through mitosis, marking chromosomes for epigenetic inheritance (217, 218). The eukaryotic replisome factors that contain helicase, primase, and SSB are each composed of multi-protein assemblies in eukaryotes [reviewed in (9)]. This complexity is in contrast to the single-subunit factors in E. coli. For example, even the SSB in eukaryotes, termed RPA, is composed of three subunits in a 1:1:1 heterotrimer [reviewed in (14)]. It is widely believed that the eukaryotic helicase is the heterohexameric MCM2-7 complex [reviewed in (12)], although this conclusion is not yet rm. Like E. coli DnaB, the hexameric MCM complex is ring shaped, but each MCM subunit is a different polypeptide. Subunit arrangements for the MCM2-7 complex have been proposed (219, 220). Helicase activity has been observed only for the MCM4/6/7 subcomplex (221223), yet all six MCM genes are essential in a variety of systems (224) [also, see references within (12)]. These ndings have led to the suggestion that one or more of the MCM2,3,5 subunits act as regulators (219, 222, 225). Consistent with this view, MCM2 inhibits the MCM4/6/7 helicase. The MCM complex is also a target of phosphorylation and ubiquitination and is thought to require activation for helicase action after assembly on DNA. MCM subunits are AAA+ proteins and thus are thought to have an evolutionary origin distinct from DnaB, which is constructed from the RecA module.
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Figure 7 Hypothetical arrangement of proteins at the eukaryotic replication fork. The hexameric MCM complex encircles the leading strand. In this cartoon, Pol is placed on the leading strand and Pol on the lagging, with RFC bridging the two polymerases and helicase. Pol /primase action places it on the lagging strand along with RPA bound to the looping single-stranded DNA. Other factors involved in replication and known to bind certain proteins at the replication fork include Cdc45, Sld2, Sld3, Dpb11, and the heterotetrameric GINS complex.
Furthermore, these helicases translocate on DNA with opposite polarities (221 223), thereby placing the MCMs on the leading strand (see Figure 7). However, like DnaB, the MCMs have been shown to be capable of encircling two DNA strands (223, 226), and evidence that they may form a double hexamer exists in both Archaea (227, 228) and S. pombe (229) systems. One line of evidence for this comes from an archaeal single-gene MCM that produces a circular double hexamer with helicase activity (227). MCM helicase activity is rather weak, somewhat reminiscent of the relatively weak helicase activity of E. coli DnaB. As described earlier, DnaB becomes highly active when coupled to Pol III holoenzyme, and this coupling occurs through the subunit of the clamp loader (111). It seems likely that a similar arrangement may
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exist in eukaryotes. In the scheme of Figure 7, RFC is proposed to act as a scaffold similar to the / complex in E. coli, bridging the two polymerases and coupling them to the helicase. The proposed connections are simply inferred because only scant evidence exists for an RFC-Pol interaction (176, 177), and none as yet exists for RFC binding either Pol or the MCM complex. However, evidence exists for the presence of many other protein actors required for DNA synthesis and presumed to act at the eukaryotic replication fork. Although the individual functions of these factors are largely unknown, protein interaction studies indicate a network as illustrated schematically in Figure 7. Cdc45 has been known for some time to be required for replication, and interaction between Cdc45 and MCMs has been documented (230, 231). Newer actors include Sld3, which binds Cdc45 (17), and the heterotetramer GINS complex, which appears to have a ring shape (15, 232). The GINS complex also appears to bind Pol , and similar to Sld3, assembles at origins just prior to DNA synthesis (15). Dpb11 is thought to bind both Pol and Pol (233), and it also forms a complex with Sld2 (16). Biochemical study of these various factors, alone and in combinations, will be required to understand their individual roles in chromosome replication and to determine whether they all function together at each replication fork.
Alternate RFC Complexes and Other Roles for Clamp Loaders

Sliding clamps are used in a variety of DNA metabolic processes (21, 23), and one may presume that their respective clamp loader is also involved in most of these processes. In addition, the subunit composition of the eukaryotic clamp loader is altered to perform novel functions in DNA metabolism. This alteration involves the use of an alternate clamp-loader subunit, in place of RFC1. In ssion yeast and human, the Rad17 subunit (Rad24 in S. cerevisiae) replaces p140 (RFC1) in complex with RFC25 (25, 234). The Rad17-RFC complex is involved in the DNA damage checkpoint response, along with a novel PCNA-like sliding clamp formed from the trimer Rad9/Rad1/Hus1 (Ddc1/Rad17/Mec3 in S. cerevisiae) [reviewed in (235)]. This clamp loader loads the 911 clamp onto primed DNA in an RPAstimulated reaction (236239) and does so with opposite polarity to RFC (238). The function of the 911 clamp is not clear, but it presumably recruits other factors when loaded on DNA. There may be a 3 -5 exonuclease activity in Rad9 or Hus1, thus providing a biochemical activity for the ring itself (240). RFC1 can also be replaced by Ctf18/Chl12 (26, 241) or Elg1 (27, 242, 243), which are involved in cohesion and genome stability, respectively, although the specic roles of these complexes are not understood.
CONCLUDING REMARKS
Each passing year brings signicant advances in our understanding of replication fork mechanisms. However, for each question answered, 10 more crop up. Even in the relatively well-dened and intensively studied prokaryotic system
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there remain numerous questions. We still do not know how the proteins are truly arranged at the replication fork. How are the two polymerases oriented, do they face the same direction? Does the helicase encircle one or more strands? Does it act as a double hexamer? How is the helicase positioned on the replicase? How is the length of Okazaki fragments measured and regulated? What happens when the replisome encounters a lesion on the leading or lagging strand? Does the helicase continue to unwind DNA, and if so, how far does it go before it stops? To advance past a lesion, E. coli mounts the SOS response and gets past the lesion by recombinative repair processes, collectively termed replication restart [reviewed in (113, 114)]. How does the replication machinery coordinate its action with those of repair and recombination? How does the replisome deal with transcribing RNA polymerase and other proteins tightly bound to DNA? The sliding clamps in both prokaryotes and eukaryotes interact with many different DNA polymerases and repair proteins. For example, and PCNA both interact with mismatch repair and excision repair proteins. What is the role of the clamps in these processes? The clamps also bind a variety of specialized DNA polymerases such as those of the Y-family, which have low delity but can bypass certain lesions. How do these polymerases trade places with the replicase at the right time and place to bypass lesions? How is the use of the clamp by these low-delity enzymes restricted and handed back to the high-delity replicase after a lesion is bypassed? PCNA appears to be modied by ubiquitination and/or sumoylation to assist this process. How do these modications control the trafcking of different polymerases on PCNA? Eukaryotes use a variety of alternate clamp loaders in which RFC1 is replaced by another protein. These RFC1 substitutes appear to be involved in the DNA damage response, chromatin cohesion, and genome stability. How do these alternate clamp loaders function and do they interface with the normal replication machinery? Clearly much work remains to be done to address these important issues. Knowledge of the eukaryotic replisome composition is rapidly becoming a complex challenge. How many more factors are there? How do they connect and what are their individual functions? Do all these proteins function within the same replisome or are there various types of eukaryotic replisomes, specialized for different regions of the chromosome or for different times in S phase? How is replisome assembly and function regulated at the start of S phase? How does this tie in with growth factors that work at the cell surface, and the complex kinase and cell cycle control networks that reach to the nucleus? How do DNA damage checkpoint and other checkpoint mechanisms exert their inuence during ongoing S-phase events? The authors hope this review highlighted several important advances in our knowledge of replicase structure and function. However, these concluding remarks underscore how much is yet to be learned and perhaps provide some sense of how far we have yet to go.
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ACKNOWLEDGMENTS The authors are grateful to the following individuals for helpful comments and discussions: Greg Bowman, Brian Chait, Megan Davey, David Jeruzalmi, John Kuriyan, and Alan Tackett. We are also grateful to Nina Yao for providing help with artwork. This work was supported by a grant from the National Institutes of Health (GM38839) and by the Howard Hughes Medical Institute. The Annual Review of Biochemistry is online at http://biochem.annualreviews.org
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Annual Review of Biochemistry Volume 74, 2005
CONTENTS
FROM PROTEIN SYNTHESIS TO GENETIC INSERTION,
Paul Zamecnik
1 29 53 83 115 129 179 199
THE BIOCHEMISTRY OF PARKINSONS DISEASE,

Mark R. Cookson
APPLICATIONS OF DNA MICROARRAYS IN BIOLOGY,

Roland B. Stoughton
ZONA PELLUCIDA DOMAIN PROTEINS, Luca Jovine, Costel C. Darie,

Eveline S. Litscher, and Paul M. Wassarman
PROLINE HYDROXYLATION AND GENE EXPRESSION,

William G. Kaelin Jr.
STRUCTURAL INSIGHTS INTO TRANSLATIONAL FIDELITY,

James M. Ogle and V. Ramakrishnan
ORIGINS OF THE GENETIC CODE: THE ESCAPED TRIPLET THEORY,

Michael Yarus, J. Gregory Caporaso, and Rob Knight AN ABUNDANCE OF RNA REGULATORS, Gisela Storz, Shoshy Altuvia, and Karen M. Wassarman
MEMBRANE-ASSOCIATED GUANYLATE KINASES REGULATE ADHESION AND PLASTICITY AT CELL JUNCTIONS, Lars Funke, Srikanth Dakoji,
and David S. Bredt 219
STRUCTURE, FUNCTION, AND FORMATION OF BIOLOGICAL IRON-SULFUR CLUSTERS, Deborah C. Johnson, Dennis R. Dean,
Archer D. Smith, and Michael K. Johnson 247 283
CELLULAR DNA REPLICASES: COMPONENTS AND DYNAMICS AT THE REPLICATION FORK, Aaron Johnson and Mike ODonnell EUKARYOTIC TRANSLESION SYNTHESIS DNA POLYMERASES: SPECIFICITY OF STRUCTURE AND FUNCTION, Satya Prakash,
Robert E. Johnson, and Louise Prakash
317
NOD-LRR PROTEINS: ROLE IN HOST-MICROBIAL INTERACTIONS AND INFLAMMATORY DISEASE, Naohiro Inohara, Mathias Chamaillard,
Christine McDonald, and Gabriel Nu ez n 355
vi
CONTENTS
REGULATION OF PROTEIN FUNCTION BY GLYCOSAMINOGLYCANSAS EXEMPLIFIED BY CHEMOKINES, T.M. Handel, Z. Johnson, S.E. Crown,
E.K. Lau, M. Sweeney, and A.E. Proudfoot 385 411
STRUCTURE AND FUNCTION OF FATTY ACID AMIDE HYDROLASE,

Michele K. McKinney and Benjamin F. Cravatt
NONTEMPLATE-DEPENDENT POLYMERIZATION PROCESSES: POLYHYDROXYALKANOATE SYNTHASES AS A PARADIGM,

JoAnne Stubbe, Jiamin Tian, Aimin He, Anthony J. Sinskey, Adam G. Lawrence, and Pinghua Liu
433 481
EUKARYOTIC CYTOSINE METHYLTRANSFERASES, Mary Grace Goll

and Timothy H. Bestor
MONITORING ENERGY BALANCE: METABOLITES OF FATTY ACID SYNTHESIS AS HYPOTHALAMIC SENSORS, Paul Dowell, Zhiyuan Hu,
and M. Daniel Lane 515 535
STRUCTURE AND PHYSIOLOGIC FUNCTION OF THE LOW-DENSITY LIPOPROTEIN RECEPTOR, Hyesung Jeon and Stephen C. Blacklow COPPER-ZINC SUPEROXIDE DISMUTASE AND AMYOTROPHIC LATERAL SCLEROSIS, Joan Selverstone Valentine, Peter A. Doucette,
and Soshanna Zittin Potter
563 595 649 681 711 739
THE STRUCTURE AND FUNCTION OF SMC AND KLEISIN COMPLEXES,

Kim Nasmyth and Christian H. Haering
ANTIBIOTICS TARGETING RIBOSOMES: RESISTANCE, SELECTIVITY, SYNERGISM, AND CELLULAR REGULATION, Ada Yonath DNA MISMATCH REPAIR, Thomas A. Kunkel and Dorothy A. Erie GENE THERAPY: TWENTY-FIRST CENTURY MEDICINE, Inder M. Verma
and Matthew D. Weitzman
THE MAMMALIAN UNFOLDED PROTEIN RESPONSE, Martin Schr der o

and Randal J. Kaufman
THE STRUCTURAL BIOLOGY OF TYPE II FATTY ACID BIOSYNTHESIS, Stephen W. White, Jie Zheng, Yong-Mei Zhang,
and Charles O. Rock 791
STRUCTURAL STUDIES BY ELECTRON TOMOGRAPHY: FROM CELLS TO MOLECULES, Vladan Lu i , Friedrich F rster, cc o
and Wolfgang Baumeister 833 867
PROTEIN FAMILIES AND THEIR EVOLUTIONA STRUCTURAL PERSPECTIVE, Christine A. Orengo and Janet M. Thornton

Annual Reviews - Replication

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CELLULAR DNA REPLICASES: Components

THE E. coli REPLICASE

Pol III Core

The Sliding Clamp

The Complex Clamp Loader

The Holoenzyme Particle

Protein Trafcking on DNA Sliding Clamps

THE EUKARYOTIC REPLICASE

Proliferating Cell Nuclear Antigen

The RFC Clamp Loader

Information concerns a protein complex.

Eukaryotic DNA Polymerases

The Eukaryotic Replisome

Alternate RFC Complexes and Other Roles for Clamp Loaders

20. 21. 22. 23. 24.

91. 92. 93.

96. 97. 98. 99. 100.

102. 103. 104. 105. 106. 107.

108. 109. 110.

137. 138. 139.

147. 148. 149. 150.

212. 213. 214. 215.

237. 238. 239. 240. 241. 242.

Annual Review of Biochemistry Volume 74, 2005

1 29 53 83 115 129 179 199

THE BIOCHEMISTRY OF PARKINSONS DISEASE,

APPLICATIONS OF DNA MICROARRAYS IN BIOLOGY,

ZONA PELLUCIDA DOMAIN PROTEINS, Luca Jovine, Costel C. Darie,

PROLINE HYDROXYLATION AND GENE EXPRESSION,

STRUCTURAL INSIGHTS INTO TRANSLATIONAL FIDELITY,

ORIGINS OF THE GENETIC CODE: THE ESCAPED TRIPLET THEORY,

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