the eukaryotic replicative dna polymerases take shape

The eukaryotic replicative DNApolymerases take shapeErik Johansson1 and Stuart A. MacNeill2

1 Department of Medical Biochemistry and Biophysics, Umea University, SE-901 87 Umea, Sweden2 Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9ST, UK

Review

Three multi-subunit DNA polymerase enzymes lie at theheart of the chromosome replication machinery in theeukaryotic cell nucleus. Through a combination ofgenetic, molecular biological and biochemical analysis,significant advances have been made in understandingthe essential roles played by each of these enzymes atthe replication fork. Until very recently, however, littleinformation was available on their three-dimensionalstructures. Lately, a series of crystallographic and elec-tron microscopic studies has been published, allowingthe structures of the complexes and their constituentsubunits to be visualised in detail for the first time. Takentogether, these studies provide significant insights intothe molecular makeup of the replication machinery ineukaryotic cells and highlight a number of key areas forfuture investigation.

Assembly and composition of the replisomeDNA polymerases are central to the processes of genomereplication in all cells [1,2]. High-fidelity replication iscrucial for avoidance of mutations and the maintenanceof genome integrity. To achieve high-fidelity replicationand to meet the challenges posed in replicating through orin repairing damaged DNA, cells employ a large repertoireof specialized DNA polymerases. In eukaryotes alone, fivefamilies of DNA polymerase are represented, with humancells encoding as many as 15 different DNA polymeraseenzymes [1,2]. Additional polymerase families are founduniquely in bacteria and archaea (Box 1). Despite thiscomplexity, three DNA polymerases (Pol a–primase, Pold and Pol e) have been identified by genetic analysis inyeast as being essential for DNA replication in the eukar-yotic cell nucleus [3]. Each of these enzymes plays a keyrole as part of the molecular machinery (the replisome)present at replication forks.

All replicative DNA polymerases use a single-strandedtemplate to synthesize a new, complementary strand, andpolymerase activity is therefore dependent on priorunwinding of double-stranded DNA [1,2]. The elaborateseries of events leading to duplex DNA unwinding and theassembly of a functional replication fork is tightlyregulated. In eukaryotic cells, chromosome replicationis initiated at multiple sites on the chromosomes calledreplication origins. Replication initiation is a stepwiseprocess that is best understood in the budding yeastSaccharomyces cerevisiae [4]. In this organism, the origins

Corresponding author: MacNeill, S.A. ([email protected]).

0968-0004/$ – see front matter � 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2010.

are first bound by the origin recognition complex (ORC),which then recruits Cdc6 and Cdt1. Together, these threefactors then catalyse the loading of the MCM complex,comprising Mcm2–7, to form the pre-replicative complex(pre-RC) [4]. Pre-RC formation is restricted to the late M/early G1 phases of the cell cycle (when cyclin-dependentkinase (CDK) activity is low) to ensure replication occursonly once per cell cycle [5]. Activation of the pre-RC at theG1–S transition is triggered by S-phase CDK (S-CDK) andDbf4-dependent protein kinase (DDK) activity. S-CDKphosphorylates Sld2 and Sld3, allowing both proteins tointeract with Dpb11 [6,7]. Sld3 also interacts with Cdc45,whereas Dpb11 and Sld2 are reportedly part of a complexcalled the pre-loading complex (pre-LC), which also con-tains Pol e and GINS [5]. Once replication is initiated, theMCM helicase is believed to unwind DNA ahead ofthe replication fork as part of the CMG complex(Cdc45–MCM–GINS) [8]. The CMG complex appearsto form the core of a larger macromolecular assemblycalled the replisome progression complex (RPC) [9], whichcomprises a large number of essential and non-essentialfactors including, albeit in rather loose association,Pol a–primase [10]. The components of the RPC movebidirectionally with the replication forks as replicationproceeds.

Once the duplex DNA at the origin is unwound, replica-tion on both the leading and lagging strands (Box 2) isinitiated by Pol a–primase [3]. This tetrameric complexcomprises two subunits of Pol a together with two primasesubunits (Figure 1). Primase acts by synthesising a short(� 10 nucleotides) RNA primer. The 30 end of the nascentstrand then translocates from the primase active site to thepolymerase active site to allow synthesis of � 20 nucleo-tides of DNA. After this initiation event, the clamp loadercomplex replication factor C (RFC) loads the sliding clampprocessivity factor PCNA onto double-stranded DNA [3].Either Pol d or Pol e is then loaded on to the PCNA–primer–

template ternary complex. The sliding clamp encircles thedouble-stranded DNA, and tethers Pol d and Pol e to thetemplate to increase enzyme processivity, although themechanism by which this occurs and the extent of theapparent stimulation varies greatly [11].

The precise functions of Pol d and Pol e at the replicationfork have been debated [12,13], but a growing body ofevidence suggests that Pol e participates in the synthesisof the leading strand [14] and Pol d in the synthesis of thelagging strand [15] (Box 2). Notably, the three polymerasescannot be co-purified from eukaryotic cells, suggesting that

01.004 Trends in Biochemical Sciences 35 (2010) 339–347 339

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Box 1. Polymerase families

DNA polymerases are the enzymes responsible for DNA synthesis in

all forms of life. Based on primary sequence and structural

similarity, seven distinct families of DNA polymerase have been

identified with diverse roles in DNA replication and repair [1,2].

These families share a common core catalytic architecture (the

palm, fingers and thumb domains) (Box 3) but frequently also

harbour additional activities tailored to their specific cellular

functions. The seven families are designated A, B, C, D, X, Y and

reverse transcriptase (RT) [1,2]. Family A enzymes include the

bacterial Pol I polymerases (involved in nucleotide excision repair

and Okazaki fragment processing during lagging strand replication;

these typically also carry both 50!30 and 30!50 exonuclease

activities) and the mitochondrial replication and repair polymerase

Pol g, whose structure was recently solved [75]. Family B enzymes

include the RB69 gp43, archaeal PolB and eukaryotic Pol a, Pol d and

Pol e replicative polymerases that are the subject of this review. Pol z

also falls into this family; this enzyme performs translesion DNA

synthesis (TLS). Family C includes the multi-subunit bacterial

replicative polymerase Pol III, whereas family D contains the

heterodimeric PolD enzymes found exclusively in archaea (but not

in all archaeal lineages) and currently believed to play a role in

chromosome replication. Enzymes of families X and Y perform

diverse roles in DNA repair and TLS. Family X includes, among

others, eukaryotic polymerases Pol b, Pol m and Pol l, and terminal

deoxynucleotidyl transferase, whereas family Y includes bacterial

Pol IV and Pol V (also known as DinB and UmuC, respectively) and

the eukaryotic Pol h, Pol k, Pol i and Rev1 enzymes. The RT family

includes retroviral reverse transcriptases and eukaryotic telomerase.

Structures are now available for many of these enzymes [1,2].

Review Trends in Biochemical Sciences Vol.35 No.6

the major components of the eukaryotic replisome are onlyloosely associated, in contrast to the situation in bacteria[16].

To fully understand how DNA is replicated at themolecular level, structures are required to explain bio-chemical and genetic experiments. In this paper, we reviewthe current knowledge of the molecular structures of repli-cative polymerases in the eukaryotic nucleus, focusing on

Box 2. DNA polymerases at the replication fork

The precise roles of the processive DNA polymerases Pol d and Pol eat the replication fork in eukaryotic cells have long been debated.

Over the years, evidence has accumulated for an asymmetrical

replication fork at which Pol d primarily replicates the lagging strand

and Pol e participates in the synthesis of the leading strand

[12,13,76], but what is the evidence for this? Initially, genetic

experiments with 30!50 proofreading exonuclease-deficient poly-

merase mutants showed that the two enzymes proofread opposite

DNA strands. In addition, several genetic studies in both budding

and fission yeast have repeatedly linked Pol d with the enzymes and

mechanisms of Okazaki fragment processing, and in these reports

there is no evidence for a role of Pol e in lagging strand synthesis

[12,77].

To determine the role of Pol e, a mutation was created in its active

site that caused the enzyme’s mutation rate to increase and a

specific molecular signature to be left behind in regions of the DNA

synthesised by Pol e [14]. By placing a reporter gene in different

orientations on opposite sides of two replication origins and

analysing the mutations that resulted from replication, Pol e was

shown to participates in the synthesis of the leading strand. When

the same approach was taken to study Pol d function, the results

were consistent with Pol d primarily synthesising DNA on the

lagging strand [15]. However, it is likely that Pol d also synthesises

DNA on the leading strand under special circumstances, as the

replication fork requires some plasticity to meet different chal-

lenges, such as unrepaired DNA lesions, during replication

[12,76,77].

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recent advances in our understanding of the structuralbasis for their functions and highlighting key questions forfuture investigation.

Structure of the catalytic subunitEach of the three eukaryotic replicative polymerases is amulti-subunit entity, comprising a large catalytic subunitand several smaller subunits, most of which have beenshown to be essential for enzyme function in vivo in yeast[3] (Figure 1). The catalytic subunits are each members ofthe B-family of DNA polymerases [1,2] found in all threedomains of life (Box 1).

The first insights into the structures of B-familyenzymes came from studies of phage RB69 gp43 polymer-ase [17–19] and the replicative PolB enzymes from variousthermophilic archaeal species [20–23]. The structures ofthese monomeric enzymes were solved in a number ofdifferent states: in the apo form (without DNA) [19–23],with DNA in both polymerising [17] and editing [18]modes, and bound to a uracil-containing DNA template[24]. The structures of the proteins are broadly similar toone another (Figure 2). In each case, the proteins fold intofive distinct domains arranged in a ring-like structure: anN-terminal domain, a 30!50 exonuclease domain, and thepalm, fingers and thumb domains that make up the righthand-like catalytic core characteristic of DNA polymerasesfrom diverse polymerase families (Box 3) [1,2]. The palmdomain contains conserved acidic amino acid residues thatcatalyse the nucleotidyl transferase reaction whereas theprimer-template DNA binds in the cleft formed betweenthe fingers and thumb domains. Incorporation of a mis-matched base results in polymerase stalling and displace-ment of the primer template from the polymerase site tothe exonuclease site some 30-50 A distant [18], where themismatched nucleotide is removed (edited) in a proofread-ing reaction.

Although the structures of the prokaryotic B-familyenzymes provide a useful framework for understandingthe eukaryotic polymerases, their usefulness is limited byevolutionary distance and by the monomeric nature of theRB69 gp43 and archaeal PolB proteins. Thus, obtaininghigh-resolution structures of the multi-subunit eukaryoticenzymes is an important objective. Five structures areavailable to date, covering seven of the 12 subunits ofthe three polymerases, although for technical reasonsfew of the structures are full-length proteins (Figure 1).At present, the crystal structure of a ternary complexcomprising the catalytic subunit of Pol d with primedtemplate DNA and an incoming dCTP nucleotide [25]offers the only high-resolution view of the catalytic centreof any of the three replicative polymerases. The structureof yeast Pol3 (truncated at both its N- and C-terminal endsand crystallised in the absence of its associated subunits)resembles that of the gp43 and PolB enzymes with theircharacteristic palm–fingers–thumb, exonuclease and N-terminal domains (compare parts a–c of Figure 2). Thepalm domain carries the polymerase active site residues(Asp608 and Asp764), the fingers drape over the nascent G-dCTP base pair, part of the thumb interacts with thesugar–phosphate backbones of the primer template, andthe triphosphate moiety of the dCTP lies between the

Figure 1. Schematic summary of the subunit compositions of the three eukaryotic replicative polymerases: (a) Pol a, (b) Pol e and (c) Pol d. Each enzyme consists of a large

catalytic subunit (blue), a B-subunit (green) and a number of unrelated additional proteins with diverse structures and functions (various colours). Binding of the catalytic to

the B-subunits is mediated via the zinc binding modules (Zn) present at the C-termini of the catalytic subunits. The remaining subunits are positioned correctly where data

are available (see text for details); otherwise the locations shown are for illustrative purposes only.


fingers and palm in a manner similar to that previouslyobserved with the RB69 gp43 (Figure 2) [17].

Replication fidelity is determined largely by the abilityof the enzyme to discriminate between correct and incor-rect bases before these are incorporated into the nascentDNA; misincorporation occurs only once per � 105 repli-cated base pairs [26]. Analysis of the Pol3 structuresuggests a key role for a conserved binding pocket formedby residues from the fingers and palm domains of theenzyme in discriminating between correct (Watson–Crick)and incorrect base pairing [25]. Once an incorrect base isincorporated into the nascent (primer) DNA strand, thisstrand is displaced from the polymerase to the exonucleasedomain to facilitate proofreading, but what triggers this isunclear. Interestingly, the Pol3 structure reveals that theenzyme remains in contact with up to five base pairs of theduplex portion of the primer template. Disruption of thesecontacts as a consequence of nucleotide misincorporationcan result in destabilisation of primer template binding bythe polymerase domain, and favour binding by the exonu-clease domain. Slowing of the rate of polymerisation as aresult of misincorporation might also play a part here, byallowing extra time for the active site switch to occur [25].

The function of the Pol3 N-terminal domain (NTD) isunclear. In the structure, this domain appears to interactwith the unpaired segment of the template DNA(Figure 2d), but whether this interaction is essential forthe function of the enzyme is not known. The structure ofthe Pol3NTD ismore complex than that of the gp43 or PolBpolymerases, and displays closer similarity to the NTD ofherpes simplex virus I polymerase, another family Benzyme [27]. The NTD comprises three structural motifs.The first of these resembles to some extent the single-stranded DNA binding folds seen in the bacterial single-stranded binding protein [28] and eukaryotic replicationprotein A [29]. By analogy with the bacterial replicativepolymerase PolC [30], this motif has been proposed [25] toplay a role in guiding the single-stranded template strandto the polymerase active site. By contrast, motif IIresembles an RRM RNA-recognition motif typical ofdiverse ribonucleoproteins. It is not known if the NTDcan bind RNA, nor is it apparent what function this wouldperform. As noted previously [25], during lagging strandsynthesis. Pol3would be expected to encounter the 50 end ofthe previously synthesised Okazaki fragment, in whichcase it is not inconceivable that RNA recognition by the

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Figure 2. Replicative polymerase catalytic subunit structures. Family B polymerase structures from the three domains of life. (a) Bacteriophage RB69 gp43 polymerase (PDB

code 1IG9) was the first family B polymerase to have its structure solved [17]. Five structural domains characteristic of this family of proteins are shown: the palm, fingers

and thumb polymerase domains (Box 3), the proofreading exonuclease domain and the N-terminal domain. The shaded circles indicate the location of the exonuclease

(green) and polymerase (grey) active sites. (b) Tgo polymerase from the archaeon Thermococcus gorgonarius (PDB code 1TGO) has a very similar structure [21]. Archaeal

PolB enzymes possess the ability to recognise uracil at position +4 in the template strand and stall replication until the damaged (deaminated) base is repaired, thus

preventing incorporation of adenine into the nascent strand instead of guanine. Blue circle indicates the location of the uracil binding pocket in the N-terminal domain [24]

(c) Structure of an N- and C-terminally truncated form of the budding yeast Pol d catalytic subunit Pol3 (PDB code 3IAY) [25]. Again the characteristic palm, fingers and

thumb, exonuclease and the N-terminal domains are present. (d) Position of primer-template DNA (strands shown in cyan and magenta respectively) in the Pol3

polymerase active site. The four parts of the figure were drawn from the corresponding PDB files using MacPyMol (DeLano Scientific). In part (a), the primer-template DNA

is omitted for clarity.


NTD could have a role in the subsequent strand displace-ment and primer processing. In archaea, an additionalfunction of the PolB NTD is its ability to bind to uraciland hypoxanthine present in the template strand, causingthe polymerase to stall until the damage is repaired [31].This ‘read-ahead’ recognition probably plays an importantrole in mutation avoidance.

Catalytic subunit evolution: the curious case of the Pol eC-terminal domain

All three eukaryotic replicative polymerase catalytic sub-units are presumed tohave evolved froma commonancestorby gene duplication. However, recent bioinformatic analysis

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of the Pol e catalytic subunit has revealed an unexpectedtwist to this process [32]. The Pol e catalytic subunit (yeastPol2) is considerably larger than the corresponding subunitsof Pol a and Pol d [33], largely as a result of the presence ofadditional sequences inserted between the exonuclease–

polymerase module and the C-terminal zinc-bindingmodules. These inserted sequences correspond to a secondexonuclease–polymerase module distantly related to thefirst. The second module is inactive and presumably playsonly a structural role. Intriguingly, an echo of this situationis observed in archaea also, in which the same authors haveuncovered a class of conserved inactive PolB enzymes thatare likewise thought to perform a structural role [34].

Figure 3. Replicative polymerase non-catalytic subunit structures. (a) Structure of

the CTD of the budding yeast Pol a catalytic subunit Pol1 (amino acids 1263–1468)

complexed with the B-subunit Pol12 (amino acids 246–705) (PDB code 3FLO) [36].

The Pol1 CTD contains two C4 zinc-binding modules and interacts with Pol12

largely through the a2 helix binding the PDE-like domain of Pol12 and the second

zinc binding (Zn 2) module binding the Pol12 OB-fold domain. Blue spheres are

bound zinc ions. (b) Structure of the full-length human Pol d B-subunit p50

complexed with the N-terminal globular wHTH domain of the Pol d C-subunit p66

(amino acids 1–144) (PDB code 3E0J) [39]. Like Pol12 in part (a), p50 possesses a

PDE-like domain, but one in which the PDE catalytic residues are replaced,

rendering the domain inactive; shaded circle indicates the location of this inactive

catalytic centre (c). Structure of the heterodimeric primase from the archaeon

Sulfolobus solfataricus [46] formed from full-length catalytic subunit PriS (amino

acids 1–330, indicated as the small subunit) and a C-terminally truncated form of

the non-catalytic subunit PriL (residues 1–212, indicated as the large subunit) (PDB

code 1ZT2). The shaded circle indicates the position of the active site formed

around the catalytic triad (Asp101, Asp103, Asp235); the blue sphere is the bound

zinc ion. Note that the archaeal primase, unlike its eukaryotic counterpart, does not

form a stable complex with a replicative polymerase, and that the C-terminal [4Fe–

4S] domain of PriL is absent. FL, full-length proteins; T, truncated proteins. The

three parts of the figure were drawn from the corresponding PDB files using

MacPyMol (DeLano Scientific).

Box 3. Palm, fingers and thumb

The first DNA polymerase structure to be solved crystallographically

was that of the Klenow fragment of E.coli DNA polymerase I (Pol I)

almost 25 years ago [78]. Pol I is a multidomain protein with an N-

terminal 50!30 nuclease domain that can be proteolytically cleaved

to leave a stable polypeptide (named the Klenow fragment after its

co-discoverer) with 30!50 proofreading nuclease and polymerase

activities. Crystallisation of the Klenow fragment revealed a

structure that was likened to the palm, fingers and thumb of a right

hand, with the deep cleft formed by the fingers and thumb

suggested at the time to be of the correct size and shape to bind

duplex DNA. Later work with the Klenow fragment [1,2] showed that

DNA was indeed bound within the cleft, and subsequent crystal-

lographic studies, too numerous to describe in detail here, have

shown the palm, fingers and thumb architecture to be conserved in

many of the polymerase families despite extremely limited primary

sequence conservation and, in the case of the family X enzymes,

strong evidence suggestive of independent evolution [1,2]. The

polymerase active site residues are located in the palm domain, the

fingers are important for nucleotide binding, and the thumb domain

binds the DNA.


Bridging to the non-catalytic subunits: the role of the C-

terminal zinc-binding modules

The C-terminal ends of all three eukaryotic replicativepolymerase catalytic subunits (but not their prokaryoticcounterparts) harbour two C4 zinc-binding modules(Figure 1). Several studies have demonstrated the import-ance of these domains for in vivo function and for inter-action with the B-subunit [35]. The crystal structure of theyeast Pol1 C-terminal domain (CTD) complexed with itscognate B-subunit Pol12 was recently solved [36], provid-ing the first structural view of the interaction between thecatalytic and non-catalytic subunits. The CTD adopts anextended structure comprising two lobes (each containinga zinc-binding module) connected by a three-helix bundle(Figure 3a) and makes extensive contacts with the oligo-nucleotide/oligosaccharide binding (OB) fold and inactivephosphodiesterase (PDE)-like domains of the B-subunit.Mutations affecting the zinc-binding modules or the three-helix bundle are sufficient to disrupt B-subunit bindingand in vivo function. Given the degree of conservationdisplayed by the CTDs of the B-family catalytic subunits,it is likely that the structure of the Pol a CTD is a validmodel for the CTDs of Pol d and Pol e [36].

B-subunitThe B-subunits of the three replicative polymerases arerelated to one another, conserved across evolution, andessential for chromosome replication. The B-subunits ofboth Pol a and Pol e are phosphorylated in vivo in a cellcycle-dependent manner [37,38]. Structures for two B-sub-units are available: the N-terminally truncated Pol12 fromyeast Pol a in complex with the Pol1 CTD [36] (Figure 3a)and p50 from human Pol d in complex with the N-terminaldomain of the C-subunit p66 (p66N) [39] (Figure 3b). Pol12and p50 each contain an OB domain and an inactive calci-neurin-like PDE domain. Both the PDE and OB domains ofPol12 are involved in interactions with the Pol1 CTD, withthe PDE domain contacting the first zinc-binding moduleand the OB domain contacting the C-terminal zinc-bindingmodule (Figure 3a). Interestingly, there is no overlap be-tween the two interacting regions [36].

TheB-subunits have an interesting evolutionary history:B-subunit homologues are found as components of PolDpolymerase present in some, but not all, archaeal lineages(Box 1) [40,41]. PolD is a two-subunit enzyme that isessential for viability [42] and presumably for chromosome

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replication, the precise role of which remains unclear. Thelarge subunit (DP2) possesses DNA polymerase activitywhereas the small subunit (DP1) contains a PDE-likedomain and is active as a 30!50 exonuclease, conferringproofreadingactivity on the enzyme.ThePDEdomain in theeukaryoticB-subunits is inactiveasa result of theabsenceofkey catalytic residues; these subunits now appear to play astructural role only.

In addition to the OB and PDE domains, the structure ofthe N-terminal domain of the human Pol e B-subunit hasalso been solved by nuclear magnetic resonance and iscomposed of four a-helices and two b-strands that form afour-helix bundle and a short parallel b-sheet [43]. Inter-estingly, although the function of this domain is unknown,it shows structural similarity with the helical domains ofAAA+ (ATPases associated with various cellular activities)superfamily members [44], including replication clamploaders.

Additional subunitsIn addition to the catalytic B-subunit core, the three repli-cative polymerases possess additional subunits (Figure 1)that are not evolutionarily related to one another and thatperform diverse and often essential functions in vivo. Un-derstanding the functions of these proteins, their effect oncatalysis and how they are regulated, is extremely import-ant for understanding overall enzyme function.

Primase

Primase is a heterodimeric enzyme comprising a small(catalytic) and large (non-catalytic) subunit that forms astable association with the Pol a catalytic B-subunit core,resulting in the tetrameric Pol a–primase complex. Thelarge primase subunit interacts with the Pol1 CTD inin-dependently of the latter’s zinc binding motifs [45]. Bothprimase subunits (Pri1 and Pri2) (Figure 1) are essentialfor replication in yeast.

Although no eukaryotic primase structures are avail-able, the two subunits of archaeal primase (designatedPriS and PriL for small and large subunit, respectively)are related to the eukaryotic primase proteins (although inthese organisms, primase does not form a stable complexwith a DNA polymerase), and the structure of a dimericarchaeal primase comprising full-length PriS and a C-terminally truncated PriL from the archaeon Sulfolobussolfataricus has been solved [46]. Overall, the PriS–PriLcomplex has a curved shape, with the primase active sitebeing located in the PriS subunit (Figure 3c). The structureof Pyrococcus horikoshii PriS with UTP bound in the activesite is also available, providing further information onnucleotide binding and the likely catalytic mechanismsinvolved [47]. The PriL protein is located distal to theactive site and apparently does not play a direct role incatalysis. Instead PriL has been proposed to have a role inlimiting the length of the nascent primer, with contactsbetween PriL and the RNA primer possibly resulting in apausing of primer synthesis that could present an oppor-tunity for the 30 end of the primer to be transferred to apolymerase active site [46].

Absent from the PriS–PriL complex structure is the C-terminal domain of the PriL protein (designated PriL-

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CTD) (Figure 1). This region of the protein is conservedin both archaeal and eukaryotic PriL proteins and har-bours an [4Fe–4S] iron–sulfur cluster coordinated by fourconserved cysteine residues [48,49]. Iron–sulfur clustershave recently been identified in a number of enzymesinvolved in DNA repair, including the xeroderma pigmen-tosum complementation group D family helicases, theDNA glycosylase MutY, and endonuclease III. Mutationsin the human and yeast primase [4Fe–4S] clusters result inthe inhibition of primer synthesis [48,49], yet the precisebiochemical function of the primase [4Fe–4S] clusterremains unknown.

C and D subunits of Pol d

DNA polymerase d contains two subunits in addition to thecatalytic B-subunit core. The C-subunit of the complex isdesignated Pol32 in budding yeast, Cdc27 in fission yeast,and p66 or p68 in human cells [3]. Whereas Cdc27 is anessential protein, Pol32 is not, although cells lacking Pol32display defects in replication, in translesion synthesis(TLS) [50,51] and break-induced replication [52–54]. TheC-subunit comprises a globular winged helix–turn–helix(wHTH) domain of � 150 amino acids that has beencrystallised in complex with the B-subunit p50 [39](Figure 3b) and an extended C-terminal region that con-tains at least three protein–protein interaction motifs [3]:one for interaction with the Pol a catalytic subunit, asecond for interaction with the sliding clamp PCNA, and(at least in budding yeast) a third for interaction with thePol z-associated protein Rev1 [55], this last interactionbeing important for Pol z-catalysed TLS. From a regulatorypoint of view, it could be significant that human p66 can bephosphorylated [56,57], ubiquitylated and SUMOylated[modified by small ubiquitin-like modifier (SUMO)] [58]in vivo. Phosphorylation near the Pol a binding siteappears to correlate with chromatin association of p66at the beginning of S-phase, and has been suggested tobe important for replisome assembly, whereas phosphoryl-ation of residues within the PIP (PCNA-interactingprotein) box motif might inhibit PCNA binding [57]. Thefunctions of p66 SUMOylation and ubiquitylation remainunknown. Two SUMOylation sites have been mapped tothe extended C-terminal domain of p66, but neither is closeto the Pol a or PCNA interaction motifs [58].

TheD-subunit of Pol d is the p12 protein. First identifiedin fission yeast as the non-essential Cdm1 protein [59], p12is apparently absent from many organisms (includingbudding yeast), and when present displays only limitedsequence conservation across evolution [3]. Believed tohave a role in stabilising and stimulating the activity ofPol d, downregulation of p12 expression in murine cells bysmall interfering RNA also leads to reduced cell prolifer-ation [60]. More recent work has highlighted an unex-pected role for p12 in the DNA damage response. Inhuman cells, p12 is ubiquitylated [58,61] and rapidlydegraded in response to various DNA damaging agentsincluding UV and methyl methanesulfonate [61]. Degra-dation of p12, which is dependent on the activity of theproteasome but in a ubiquitylation-independent manner[58], produces a heterotrimeric Pol d complex with alteredproperties including reduced ability to perform TLS on


mutagenic templates and enhanced proofreading activity[62]. Human p12 interacts directly with both the catalyticand B-subunits of Pol d, with PCNA via a highly degen-erate PIP box motif [63] and with the Bloom’s syndromehelicase BLM [64]. No structural information is availablefor p12, although a possible interaction surface for fissionyeast Cdm1 on the B-subunit Cdc1, adjacent to the cata-lytic–B-subunit interface [36], has been proposed [65].

Small subunits of Pol eDNApolymerase e possesses two small subunits, Dpb3 andDpb4, which interact with the C-terminal domain of Pol2 ina manner that does not require the zinc-binding modules(Figure 1b) [66]. The functions of Dpb3 and Dpb4 remainincompletely understood, and although neither protein isessential in budding yeast, Dpb3 is essential in fissionyeast [3]. In budding yeast, DPB3 deletion leads to anincreased mutation rate, whereas DPB4 deletion resultsin a delayed S-phase progression [3]. Both Dpb3 and Dpb4contain histone-fold motifs. The two proteins form a dimerthat interacts with double-stranded (ds)DNA to confer onPol e high affinity for dsDNA [67]. Dpb4 also forms acomplex with Dpb3-like subunit 1 (Dls1), and is importantfor the function of ISW2, a complex which slides mono-nucleosomes along the chromosome [68,69]. Dpb4 hasdifferent partners in the two complexes but appears tohave the same function, tethering ISW2 and Pol e to thedouble-stranded DNA [67,70,71]. The structure of theDro-sophila melanogaster Dpb4–Dls1 (CHRAC14–CHRAC16)complex has been solved [72], providing a model for Dpb3–

Dpb4 structure.

Complex structuresAlthough there has been considerable progress in deter-mining the structures of the various subunits of the repli-cative polymerases, no high-resolution structures of intactcomplexes have been obtained. Instead, both cryo-EM[36,73] and small angle X-ray scattering (SAXS) [74] havebeen used to visualise multi-subunit complexes at lowresolution. These structures provide a helpful overviewof the shape of the polymerase domains, and will allowdocking of discrete domains when high-resolution struc-tures are solved.

The first low-resolution structure to be reported was thecryo-EM structure of the intact yeast Pol e complex [73].The reconstruction of Pol e revealed two separate domainsseparated by a flexible hinge: a globular domain carryingthe catalytic subunit Pol2, and a tail domain that wassuggested to include the accessory subunits Dpb2, Dpb3and Dpb4. Primer-extension assays demonstrated that thetail domain was required for Pol e to be fully processive.Next, the EM structure of the complex formed by thecatalytic and B-subunits (Pol1 and Pol12 respectively)(Figure 1) of yeast Pol a was presented [36]. The high-resolution structure of the Pol1 CTD together with Pol12(Figure 3a) could be fitted into the volume of the EMstructure, and again, the two subunits appeared to beseparated by a flexible hinge. It was reported that theCTD–B subunit complex interacts with dsDNA, but thatthis did not alter the polymerase activity of the catalyticsubunit. This could be analogous to Pol e and Pol d, where

the presence of the small subunits does not affect catalyticactivity except where this is dependent on interaction withdsDNA or PCNA. Based on the comparison with the EMstructure of Pol e, the authors proposed that the separationof the catalytic domain and the small subunits could be ageneral feature of the architecture of the eukaryotic repli-cative DNA polymerases that allows multiple regulatoryinteractions between the small subunits and other mol-ecules at the replication fork [36]. SAXS analysis of yeastPol3 complexed to Pol31 and the N-terminal domain ofPol32 also revealed an extended structure [74].

Concluding remarks and future perspectivesFurther high-resolution structures of the catalytic subu-nits of the replicative polymerases will without doubtprovide a solid structural basis for understanding howhigh-fidelity DNA synthesis is accomplished and genomeintegrity maintained in eukaryotic cells. However, theimportance of the accessory subunits for the in vivo func-tion of the polymerases cannot be overstated. The currentEM structures suggest that the accessory subunits form adiscrete domain, separated by a flexible hinge from thecatalytic subunit. This makes the small subunits fullyaccessible to interactionswith additional proteins andwithduplex DNA. To fully understand how the small subunitsregulate the function of the catalytic subunits, high-resol-ution structures of intact polymerase complexes will beneeded. These will also be useful to design experimentsusing site-directed mutagenesis to explore interactionsbetween the accessory subunits and other proteins atthe eukaryotic replication fork.

Other questions remain. For example, although therehas been no systematic analysis of post-translationalregulation of the three replicative polymerases, evidencehighlighting potential roles for phosphorylation, ubiquity-lation, SUMOylation and proteolytic degradation in reg-ulating polymerase function already exists. We areentering an exciting period when the underlying molecularevents regulating DNA synthesis and replication fidelitycan be explored as the replicative DNA polymerases takeshape.

AcknowledgementsWe apologise to those colleagues whose work was not cited because offormatting and space restrictions. Research in our laboratories is fundedby Swedish Cancer Society, Swedish Research Council, the Fund for BasicScience-Oriented Biotechnology and the Medical Faculty at UmeaUniversity, Smartafonden and the Kempe Foundation (EJ) and by theScottish Universities Life Sciences Alliance (SM).

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the eukaryotic replicative dna polymerases take shape

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