Annual Reviews - Replication

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Annu. Rev. Biochem. 2005. 74:283315doi: 10.1146/annurev.biochem.73.011303.073859

Copyright c 2005 by Annual Reviews. All rights reserved

CELLULAR DNA REPLICASES: Componentsand Dynamics at the Replication Fork

Aaron Johnson2 and Mike ODonnell1,21Howard Hughes Medical Institute, 2The Rockefeller University,

New York City, New York 10021-6399; email: [email protected],

[email protected]

Key Words DNA replication, DNA sliding clamps, DNA polymerase, processivityclamp loader, protein-DNA interactions

Abstract Chromosomal DNA replicases are multicomponent machines that haveevolved clever strategies to perform their function. Although the structure of DNAis elegant in its simplicity, the job of duplicating it is far from simple. At the heartof the replicase machinery is a heteropentameric AAA+ clamp-loading machine thatcouples ATP hydrolysis to load circular clamp proteins onto DNA. The clamps encircleDNA 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, reflecting the circularshape of their respective clamp substrates. Clamps and clamp loaders also functionin other DNA metabolic processes, including repair, checkpoint mechanisms, and cellcycle progression. Twin polymerases and clamps coordinate their actions with a clamploader and yet other proteins to form a replisome machine that advances the replicationfork.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284THE E. coli REPLICASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

Pol III Core . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

The Sliding Clamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

The Complex Clamp Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

The Holoenzyme Particle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

Replisome Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

Protein Trafficking on DNA Sliding Clamps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

THE EUKARYOTIC REPLICASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

Proliferating Cell Nuclear Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

The RFC Clamp Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300Eukaryotic DNA Polymerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

The Eukaryotic Replisome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

Alternate RFC Complexes and Other Roles for Clamp Loaders . . . . . . . . . . . . . . . . 307

CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307

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284 JOHNSON ODONNELL

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 componentsin the well-defined 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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CELLULAR REPLICASES 285

The eukaryotic replication factor C (RFC) and the prokaryotic ( complex) clamp-

loader subunits are arranged in a circle and are each members of the AAA+ protein

family [briefly reviewed in (4)].

The replicases of all cells function within a greater context, involving many otherproteins. 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 defined 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 specific functional connection to

single-stranded DNA-binding protein (SSB), which is present at the fork to protectsingle-stranded DNA (ssDNA) and melt hairpins.

The eukaryotic replisome, in contrast, involves many more proteins and is less

defined at the current time [reviewed in (911)]. The eukaryotic helicase, primase,

and SSB are all heterooligomers. The helicase is thought to be the heterohexam-

eric MCM complex (12), and the primase is the four-subunit DNA polymerase

/primase that makes a hybrid RNA/DNA primer (13). The heterotrimeric repli-

cation protein A (RPA) functions as the SSB (14). Several additional eukaryotic

proteins without prokaryotic counterparts are thought to act during replication ini-

tiation and fork progression as well (e.g., Cdc45, GINS complex, Dpb11, Sld2,and Sld3) (1517). The functions of most of these components are presently un-

known. 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 modification, 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 specific target sites or, in one case, load an alternatePCNA-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 continuesto 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. coliREPLICASE

The first 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 identified in a mutant strain ofE. 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 specific 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 efficient enzyme having the highest specific activity

of any E. coli DNA polymerase (1).

Pol III core is a 1:1:1 heterotrimer of the polymerase, 3-5 proofreading ex-onuclease, 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 holoen-

zyme 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 holEgene can be deleted with little consequence (44).

Little structural information is available for Pol III core. The subunit is amember 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 fingers domains and two-metal mechanism of catalysis present



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TABLE 1 Escherichia coli replisome components and associated functionsa

in all other polymerases (46). Deletion mutagenesis suggests that the active site islocated 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 eukary-

otic 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, ex-

tending 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 becomean efficient replicase, the core polymerase requires the clamp. Cou-pled to , core becomes exceedingly fast (750 ntd/s) and processive (>50 kb).

Biochemical studies initially revealed that binds DNA topologically (57), im-

plying 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 of35 A in diameter that mayeasily 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 37C

(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 ofD. 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) Superposi-

tion of one subunit from the dimer structure (purple) and the monomer from the 11crystal structure (yellow), using domain II as a reference. (d) Model of an open clamp made

by arranging two monomer structures from 11 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 abilityof 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 getsagripon via the hydrophobic pocket and pushes on the loop to crack theinterface or to hold the interface open. Mutational analysis of these contact points

has confirmed 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 significant rigid body motions

between the domains, leading to a shallower crescent shape in the monomer (67)

(seeFigure2c).Thisimpliesthatthe ringisunderspringtensionintheclosedstate

until disrupts one interface, allowing tension between the domains to relax and

producing a gap for DNA-strand passage (see Figure 2d). The strong interactionsat the dimer interfaces likely maintain this tension.

The Complex Clamp Loader

The E. coli clamp loader is a complex composed of five different subunits in a

defined stoichiometry: 31

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 ,thedomainsof 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 311 complex is termed the minimal clamp loader, as it is sufficient 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 affinity

for the clamp (62). The subunit, which binds tightly to , is likely sequestered

by the other subunits without ATP present. Upon ATP binding, the complexundergoes 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 affinity 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-affinity DNA-binding state, presumably

until ADP dissociates and the complex can be recharged with triphosphatenucleotides (80, 8284).

The crystal structure of the 3 complex provided deeper insight into clamp-

loader 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 filled

in many details that will be discussed later. The five subunits ofE. coli 3 are

arranged in a circular fashion (70) (Figure 3b). The three subunits are adjacent

to one another, flanked 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 five 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 supple-

mented 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 1of 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 in-

teraction 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 clamp-

loading mechanism, suggesting regulation of substrate binding by conformationalchanges in distinct ATP sites during nucleotide binding and hydrolysis.

The C termini (domains III) of the five 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 oc-

cur 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 es-sential for function (93). The structure of3 lacks ATP and thus is in the inactive

state. Consistent with this, a dimer cannot be docked onto the complex (replac-

ing with -) without significant 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 311 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 of3 (left). The

-interacting helix of is marked in yellow. View from the C termini of3 (right)

[adapted from (70), copyright 2001, with permission from Elsevier]. (c) The ATP sitesof 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 dnaXgene, 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, 21, 12, 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 21 clamp loader

and that a specific 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 define an inherent asymmetry, and thus by definition, 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 flexible connection. However, DnaB or other holoenzyme

subunits may hold the cores in defined asymmetric positions (103). It has been

proposed that the asymmetric structure imposes distinct properties onto the two

polymerases, modeling their behavior to fit 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 extendsDNA 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 fits and starts that re-

peats 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 requiresinteraction 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 inter-

action 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 com-

pete 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 confirmed (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 specific mechanism for this

release step, which disengages core from , leaving the clamp behind on the

finished fragment. The release step occurs only at a nick, thus ensuring completionof 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 fivefold excess of free subunit in the E. coli cell (64).

Protein Trafficking on DNA Sliding Clamps

Many different proteins function with sliding clamps. An understanding of the

way proteins coordinate their traffic flow on clamps is now emerging. To illustrate



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how proteins may switch positions to utilize the clamp, we briefly describe three

important switches on that occur during the progression of theE. coli replication

fork.

The / clamp loader and Pol III core are known to bind the same face of inessentially 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 trafficking 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, takespossession 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 clampafter 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 trafficking 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, andthis 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 turnson 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 trafficking of different proteins on sliding clamps to ensure progression of

replication.

THE EUKARYOTIC REPLICASE

Although the complexity of architecture, interaction, and regulation of DNA repli-cation 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 trafficking on sliding clamps. Interactions with

the clamp fromE. coli. (a)The complex clamploader associates tightly with when

bound to ATP. DNA triggers ATP hydrolysis, resulting in low affinity for and DNA.

(b) When Pol III, the replicative polymerase, encounters a lesion in the DNA template,

it stalls, unable to overcome its inherent fidelity 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.].



16/36


activity with other DNA metabolic machineries. Many of these details are only

now coming into focus.

Proliferating Cell Nuclear AntigenPCNA derives its name from the early finding that the protein is abundant in pro-

liferating 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 do-

mains 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 first to demonstrate that these clamp-binding

Figure 6 Structures of the eukaryotic clamp and clamp loader from Saccharomycescerevisiae. (a) View of the C-terminal face of PCNA. The ring-shaped PCNA is a head-

to-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 five 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)withthe5

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 finding 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 IIand III (24, 67, 150).

The RFC Clamp Loader

The eukaryotic clamp loader was first 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 RFCandE. coli complex was apparent early on. RFC consists of five different proteins

that are homologous to each other and to and ofE. 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. cere-

visiae are referred to as RFC15 (157) (see Table 2 for human RFC nomenclature).

RFC25 share a similar molecular weight and three-domain architecture charac-teristic 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 re-

sults 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 five-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 sitesbecause 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



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TABLE 2 Eukaryotic replisome components

Replisome

component

Saccharomycescerevisiae

(kDa) Human (kDa)

Function and remarks[Schizosaccharomyces pombe

name (S.p.)]

RFCa RFC (277.7)a RFC (314.9)a Pentameric clamp loadera

RFC1 (94.9) p140 (128.2) Binds ATP; phosphorylated

RFC2 (39.7) p37 (39.2) Binds ATP

RFC3 (38.2) p36 (40.6) Binds ATP

RFC4 (36.1) p40 (39.7) Binds ATP

RFC5 (39.9) p38 (38.5) Binds ATP or ADP

PCNA PCNA (28.9) PCNA (28.7) 87 kDa homotrimeric processivity

sliding clampa

Pol a Pol (220.2)a Pol (238.7)a Replicative DNA polymerasea

Pol3 (124.6) p125 (123.6) DNA polymerase, 3-5 exonuclease,

binds PCNA; subunit A (S.p. Pol3)

Pol31 (55.3) p50 (51.3) Structural subunit; subunit B (S.p. Cdc1)

Pol32 (40.3) p66 (51.4) Binds PCNA; subunit C (S.p. Cdc27);

binds Pol large subunit

p12 (12.4) Structural, stimulates processivity; subunit

D (S.p. Cdm1)

Pol a Pol (378.7)a Pol (350.3)a Replicative DNA polymerasea

Pol2 (255.7) p261 (261.5) DNA polymerase, 3

-5

exonuclease(S.p. Pol2/cdc20)

Dpb2 (78.3) p59 (59.5) Binds polymerase subunit (S.p. Dpb2)

Dpb3 (22.7) p17 (17.0) Binds Dpb4

Dpb4 (22.0) p12 (12.3) Present in ISW2/yCHRAC chromatin-

remodeling complex (S.p. Dpb4)

Pol a Pol (355.6)a Pol (340.6)a DNA polymerase/primasea

Pol1 (166.8) p180 (165.9) DNA polymerase

Pol12 (78.8) p68 (66.0) Structural subunit

Pri2 (62.3) p55 (58.8) Interacts tightly with p48

Pri1 (47.7) p48 (49.9) RNA primase catalytic subunit

MCMa MCM (605.6)a MCM (535)a Putative 3-5 replicative helicasea

Mcm2 (98.8) Mcm2 (91.5) Phosphorylated by Dbf4-dependent kinase

Mcm3 (107.5) Mcm3 (91.0) Ubiquitinated, acetylated

Mcm4 (105.0) Mcm4 (96.6) Helicase with MCM6,7; phosphorylated

by CDK; aka Cdc54

Mcm5 (86.4) Mcm5 (82.3) Aka Cdc46; Bob1 is a mutant form

Mcm6 (113.0) Mcm6 (92.3) Helicase with MCM4,7

Mcm7 (94.9) Mcm7 (81.3) Helicase with MCM4,6; ubiquitinated

RPAa

RPA (114)a

RPA (100.5)a

Single-stranded DNA-binding proteina

RPA70 (70.3) RPA70 (70.3) Binds DNA, stimulates Pol

RPA30 (29.9) RPA30 (29) Binds RPA70 and 14, phosphorylated

RPA14 (13.8) RPA14 (13.5) Binds RPA30

aInformation 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 first encountering DNA and subsequently recruiting PCNA. ATP site mu-

tational studies have demonstrated that, in general, disrupting any one of the fourconsensus ATP sites has a significant effect on the activity of RFC (168170),

although ATP binding may suffice 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 deficient 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, ATPS

was used, and RFC was produced with R to Q mutations in the SRC motifs. The

RFC-ATPS-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 hasrevealed 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-ATPS-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. Althoughthis mutation should ensure that no ATPS 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 sub-

units 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 trans-

lation. Overall, the helical operations that relate all five subunits have a pitch of5.6 A per 60 rotation, ending with RFC5, which lies 25 A above PCNA and is

significantly separated from the ATPase domain of RFC1. This right-handed helix

and pitch mimics that of duplex B-form DNA. Furthermore, there is sufficient

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 an-other crystal structure into the center of the ring shows that the specific 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 flexible 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 specific

primer/template recognition is in agreement with recent biophysical evidence ofRFC/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 specific 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-discontinuousmanner. 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 first synthesizing its own12-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 viacompetition 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,andDrosophila.

DNA POLYMERASE DNA polymerase (Pol ) in fission yeast, human, and other

eukaryotic organisms is composed of four essential subunits (178, 179). Interest-

ingly, in S. cerevisiae, Pol has only three subunits, and no apparent homologueexists for the fourth subunit (180). Furthermore, the third subunit can be deleted

from budding yeast, although cell growth is compromised (181).

A unified subunit nomenclature for Pol has recently been proposed (182). On

the basis ofSchizosaccharomyces pombe Pol subunits and their homologues in

other eukaryotes, the subunits have been renamed AD for the Pol3(A), Cdc1(B),

Cdc27(C), andCdm1(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 thatresulted 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-finger 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 fidelity, 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 proteincomplex stabilization rather than catalytic enhancement. Even the small D subunit

from the human four-subunit Pol complex significantly stimulated the A/B/C

subcomplex in assays with RFC and PCNA (179).

DNA POLYMERASE (POL ) 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 conflicting genetic studieson 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 higherfidelity 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).

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).

Onepossible 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 fills 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 aloneunder some conditions, are sufficient 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 move-

ment. 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 interest-

ing 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 het-erotrimer [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 firm. 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 ob-

served 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

findings 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 theMCM4/6/7 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 evo-

lutionary 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 ofE. 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



25/36


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 yetexists 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, assemblesat 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 fission 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 RPA-

stimulated 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 specific roles of these

complexes are not understood.

CONCLUDING REMARKS

Each passing year brings significant advances in our understanding of replica-

tion fork mechanisms. However, for each question answered, 10 more crop up.

Even in the relatively well-defined 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 hap-

pens 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 be-

fore it stops? To advance past a lesion, E. coli mounts the SOS response and

gets past the lesion by recombinative repair processes, collectively termed repli-

cation restart [reviewed in (113, 114)]. How does the replication machinery

coordinate its action with those of repair and recombination? How does the repli-

some deal with transcribing RNA polymerase and other proteins tightly bound to

DNA?The sliding clamps in both prokaryotes and eukaryotes interact with many dif-

ferent 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 fidelity but can by-

pass 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-fidelity enzymes restricted and handed back to the high-fidelity replicase af-

ter a lesion is bypassed? PCNA appears to be modified by ubiquitination and/orsumoylation to assist this process. How do these modifications control the traf-

ficking of different polymerases on PCNA? Eukaryotes use a variety of alternate

clamp loaders in which RFC1 is replaced by another protein. These RFC1 sub-

stitutes 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 andwhat 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 influence during ongoing

S-phase events?

The authors hope this review highlighted several important advances in ourknowledge 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 helpwith 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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