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Archaeal DNA Replication

Lori M. Kelman1and Zvi Kelman2

1Program in Biotechnology, Montgomery College, Germantown, Maryland 20876;

email: lori.kelman@montgomerycollege.edu

2National Institute of Standards and Technology and Institute for Bioscience and Biotechnology

Research, Rockville, Maryland 20850; email: zkelman@umd.edu

Annu. Rev. Genet. 2014. 48:71–97

The Annual Review of Genetics is online at

genet.annualreviews.org

This article’s doi:

10.1146/annurev-genet-120213-092148

2014 by Annual Reviews.

Keywords

Archaea, initiation of DNA replication, protein-DNA interactions,

protein-protein interactions, regulation of DNA replication, protein

structure

Abstract

DNA replication is essential for all life forms. Although the process is funda-

mentally conserved in the three domains of life, bioinformatic, biochemical,

structural, and genetic studies have demonstrated that the process and the

proteins involved in archaeal DNA replication are more similar to those in

eukaryal DNA replication than in bacterial DNA replication, but have some

archaeal-speciﬁc features. The archaeal replication system, however, is not

monolithic, and there are some differences in the replication process between

different species. In this review, the current knowledge of the mechanisms

governing DNA replication in Archaea is summarized. The general features

of the replication process as well as some of the differences are discussed.

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REVIEWS

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INTRODUCTION

In 1977, Carl Woese and colleagues recognized that the rRNA sequences and other physiological

features of a group of prokaryotes were different enough from those in Bacteria to classify them as a

separate domain: the Archaebacteria (now Archaea) (177). The archaeal domain is currently divided

into six different phyla (or kingdoms): Aigarchaeota, Crenarchaeota, Euryarchaeota, Korarchaeota,

Nanoarchaeota, and Thaumarchaeota (50).

Early bioinformatic studies using complete archaeal genome sequences suggested that although

Archaea contain circular chromosomes, as do Bacteria, the proteins and complexes that participate

in DNA replication are more closely related to those of Eukarya than to those of Bacteria (Table 1).

Since then, phylogenetic, biochemical, structural, and genetic studies have demonstrated the rela-

tionship between the archaeal and eukaryal DNA replication systems (47, 50, 60, 61, 71, 127, 143).

Here, the current state of knowledge on the mechanism of DNA replication in Archaea, the

similarities and differences between species, and the properties of individual proteins and com-

plexes are discussed. Because of space limitations, only representative references are provided, and

when appropriate the reader is referred to other reviews.

DNA replication takes place during the S phase of the cell cycle and is tightly coordinated with

other cell-cycle events and cell division. This review concentrates on replication, and the reader

is referred to several reviews for information on other aspects of the archaeal cell cycle (103, 113,

153). Topoisomerases play a major role in DNA replication by untwisting the DNA in front of and

behind the moving replication fork. Topoisomerases are not covered here but have been reviewed

(21, 42).

OVERVIEW OF DNA REPLICATION

Chromosomal DNA replication is an essential process that ensures the accurate and timely dupli-

cation of the genetic information. It is a complex and highly regulated process that is functionally

and structurally conserved in all life forms (127). In all organisms, the process, which occurs during

S phase, begins at a speciﬁc region on the chromosome called the origin of replication. Origin

binding proteins (OBPs) bind to the origin and locally unwind it (35) and, together with the he-

licase loader, assemble the two replicative helicases around the DNA to form the prereplication

complex (pre-RC) (Figure 1). Additional proteins bind to the pre-RC to form the preinitiation

complex (pre-IC), in which the DNA duplex is unwound and single-stranded (ss) DNA binding

protein (SSB) coats the ssDNA. DNA primase, DNA polymerase, and the rest of the replication

machinery are recruited to the SSB-ssDNA nucleoﬁlament at the origin. Upon activation, the

two helicases begin to unwind the duplex in opposite directions using energy derived from nu-

cleoside triphosphate hydrolysis. The moving helicases form the two replication forks to initiate

bidirectional DNA synthesis. The ssDNA exposed behind the helicase is coated with SSB. Ow-

ing to the antiparallel nature of DNA and the unidirectionality of DNA polymerase, the leading

strand is synthesized continuously, whereas the lagging strand is copied discontinuously as a se-

ries of Okazaki fragments (Figure 2,Figure 3a). A number of enzymes, including a nuclease,

a DNA polymerase, and DNA ligase, process the Okazaki fragments to form the mature duplex

DNA (Figure 3b). At the end of the replication process, the two replication forks (on a circular

chromosome, or adjacent forks on a linear chromosome) collide and DNA synthesis terminates.

ORIGIN OF REPLICATION

In all organisms, DNA replication starts at speciﬁc sites on the chromosome known as origins

of replication. All origins contain similar characteristics, including one or more A/T-rich regions

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Table 1 A summary of DNA replication features in the three domains of lifea

Archaeab

Attribute Bacteria Eukarya Euryarchaea Crenarchaea

Chromosome Circular Linear Circular Circular

Replication origin Single Multiple Single or Multiple Single or Multiple

Prereplication complex (pre-RC)

Origin recognition DnaA (1) ORC (6) Cdc6c(≥1) Cdc6 (≥1)

WhiPd(1)

HelicaseeDnaB (1) MCM (6) MCM (≥1) MCM (1)

Helicase loadereDnaC (1) Cdc6 (1)

Cdt1 (1)

Cdc6 (≥1) Cdc6 (≥1)

Preinitiation complex (pre-IC)

Cdc45 -Cdc45 (1) Cdc45 (1) Cdc45 (1)

GINS -GINS (4) GINS (1–2) GINS (1–2)

Single-stranded DNA binding

protein (SSB)

SSB (1) RPA (3) RPA (1–3) SSB (1)f

Elongation complex

Primase DnaG (1) Polα/primase (4) Primase (2) Primase (2)

Sliding clamp β-subunit (1) PCNA (1) PCNA (1) PCNA (3)

Clamp loader τ-complex (5) RFC (5) RFC (2) RFC (2)

DNA polymerase PolC (3) PolBg(1) PolB/PolD (1h/2)PolB (2–3)

Okazaki fragment maturation

Primer removal PolI (1) Fen1 (1) Fen1 (1) Fen1 (1)

Gap ﬁlling PolI (1) PolBi(1) PolB/PolD (1h/2)PolB (2–3)

DNA ligase NAD+-dependent (1) ATP-dependent (1) ATP-dependentj(1) ATP-dependent (1)

aBacteria-like features and proteins are in red, eukaryal-like features and proteins are in blue, and archaeal-speciﬁc proteins are in green. The range of

homologs identiﬁed in different species is shown in parentheses.

bTwo representative kingdoms are shown.

cThe genomes of species belonging to Methanococcales and Methanopyrales do not contain genes encoding for Cdc6 homologs.

dAn archaeal homolog of the eukaryotic Cdt1 protein, WhiP, was identiﬁed in some species.

eIn bacteria, the helicase and helicase loader are not considered to be part of the pre-RC but rather the pre-IC. Because this paper is about archaea, these

proteins are included under pre-RC.

fThe crenarchaeal SSB shares some features with the bacterial SSB and other features with the eukaryotic replication protein A (RPA).

gAll three replicative DNA polymerases in eukarya (Polα,Polδ,andPolε) belong to family B.

hIn some archaeal species, PolB is not essential for cell viability.

iPolδ, the lagging-strand DNA polymerase, participates in Okazaki fragment maturation.

jSome archaeal DNA ligases use NAD+as a cofactor.

Abbreviations: MCM, minichromosome maintenance; ORC, origin recognition complex; PCNA, proliferating cell nuclear antigen; RFC, replication

factor C.

referred to as duplex unwinding elements (DUE), and speciﬁc sequences that facilitate the binding

of OBPs. Upon binding to the origin, the proteins melt the duplex DNA to form the initial

replication bubble (Figure 1) (15).

Origins of replication in Archaea were ﬁrst identiﬁed using in silico skew analysis (74, 105). This

method made use of an observation in Bacteria that strand-speciﬁc biases in nucleotide, oligomer,

and codon frequencies could be identiﬁed along the chromosome, with an abrupt change in the

bias at the origin of replication and termination regions.

The origin of Pyrococcus abyssi was the ﬁrst to be mapped in vivo using pulsed-ﬁeld gel elec-

trophoresis and two-dimensional gel analysis (118, 126). As predicted by skew analysis (105),

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Origin

Cdc6

MCM

Primase

RNA primer

DNA polymerase

PCNA

Figure 1

Initiation of DNA replication. The (a) origin of replication is (b) recognized by Cdc6 ( yellow). (c) The binding

of Cdc6 to the origin region forms the initial replication bubble. (d) Cdc6 participates in the assembly of the

two m inichromosome maintenance (MCM) helicases ( green) around the DNA to form the two replication

forks. (e) The helicases move in opposite directions, enlarging the bubble. Primase ( pink) is recruited to the

DNA and synthesizes the RNA primers (light green). ( f) The rest of the replication machinery assembles and

initiates bidirectional DNA synthesis. For simplicity, the single-stranded DNA binding protein is omitted

and only the DNA polymerase (orange) and proliferating cell nuclear antigen (PCNA) (blue)areshown.

P. abyssi contains a single origin of replication (referred to as oriC) located in an intergenic region

of the chromosome. Bidirectional DNA synthesis initiates from the origin and terminates in a

region of the chromosome opposite the origin (119, 126).

The presence of a single origin in a circular archaeal genome was not surprising, because

bacterial circular chromosomes contain a single origin. However, skew analysis failed to identify a

clear origin of replication in some archaeal species, and the use of more reﬁned in silico algorithms

suggested the presence of multiple origins in certain species (184). The presence of multiple

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MCM

Lagging strand

Leading strand

Primase

Primer

DNA polymerase

GINS

RPA

RFC

PCNA

Figure 2

A schematic representation of the archaeal replisome. MCM (minichromosome maintenance) is green, DNA

polymerase is orange, primase is pink, RFC (replication factor C) is purple, GINS is gray, RPA (replication

protein A) is yellow, and PCNA (proliferating cell nuclear antigen) is blue. Primers on the lagging strand are

shown in light green.

origins could confound skew analysis and explain the failure to identify an origin. Studies using

two-dimensional gel analysis, marker frequency analysis, and replication initiation point (RIP)

mapping of two Sulfolobus species revealed that these organisms contain three origins of replication

(72, 106, 149). Subsequent studies showed that other archaeal species also contain multiple origins.

Archaeal origins have characteristics similar to origins of replication from other organisms

(15). They are located in intergenic regions, are rich in A and T nucleotides, contain one or

more DUEs, and contain binding sites for OBPs [referred to as origin recognition boxes (ORBs)].

Many origins are located in regions of the chromosome that encodes DNA replication proteins.

Many archaeal origins are located upstream from genes that encode the archaeal homolog of the

eukaryotic Cdc6 protein. The Cdc6 proteins are the archaeal OBPs and were shown to bind to

ORBs. The proximity of the origin of replication to the OBPs is also common in Bacteria (89).

Several hypotheses have been proposed to explain the proximity of the gene that encodes the OBP

to the origin, such as the ability of the proteins to associate with the origin as soon as they are

synthesized or to reduce the probability of loss of the gene that encodes the OBP due to genomic

rearrangement (71, 103).

In several archaeal species with multiple origins of replication, the origin is located in the

vicinity of the homolog of another eukaryotic replication protein, Cdt1. This protein binds to the

origin of replication in its vicinity (148, 154). Several origins are not located upstream of a known

initiator protein (138), and the proteins that bind these origins have not yet been identiﬁed.

The origins of replication from most Archaea studied contain clear ORBs. ORBs are inverted

repeats located on both sides of the DUE region(s) and were shown to be the binding site for

the Cdc6 proteins (36, 45, 174). The ORBs from different species share sequence similarity with

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III

DNA polymerase

PCNA

Fen1

DNA ligase

RPA

III

Primase

RNA primer

PCNA

RFC

DNA polymerase

RPA

Figure 3

Okazaki fragment synthesis and maturation. (a) Okazaki fragment synthesis. I. Primase ( pink) synthesizes an RNA primer (light green)

on RPA (replication protein A) ( yellow)-coated single-stranded DNA. II. This primer is recognized by RFC (replication factor C)

(purple). III. RFC assembles PCNA (proliferating cell nuclear antigen) (blue) around the primer. IV. The DNA polymerase (orange)

associates with PCNA. V. This association initiates processive DNA synthesis. (b) Okazaki fragment maturation. I. The lagging-

strand DNA polymerase (orange) elongates the Okazaki fragment with the assistance of PCNA (blue). II. When the DNA polymerase

reaches the previous Okazaki fragment, its strand displacement activity forms a ﬂap structure. III. The ﬂap is removed by the activity of

Fen1 (ﬂap endonuclease 1) (teal) and PCNA. IV. The nick remaining on the DNA is sealed by DNA ligase ( yellow) and PCNA.

a consensus sequence referred to as the mini-ORB, followed, in some origins, by a string of

G nucleotides (a G-string) to form the intact ORB (10, 27). It was shown that the mini-ORB

is sufﬁcient to bind Cdc6 proteins and that Cdc6 from one organism can bind an ORB from

another species (149). Some origins do not have a clear ORB or mini-ORB consensus sequence,

although they contain other inverted repeats (138). It is not clear whether Cdc6, Cdt1, or other

species-speciﬁc initiator proteins can bind those repeats.

Interestingly, in some species the origins of replication could be deleted with no loss of viability

(53). However, in the absence of a canonical origin, the cellular recombination machinery appears

to be required for cell viability. The mechanism of initiation in this case is not clear. It is also not

known how widespread this phenomenon is among the Archaea.

PREREPLICATION COMPLEX

The pre-RC is the protein complex that assembles at the origin of replication during the G1 phase

of the cell cycle and is responsible for the regulation of the initiation process. In Eukarya, the pre-

RC is composed of four main components: the six-subunit origin recognition complex (ORC),

Cdc6 and Cdt1 proteins, and the six-subunit minichromosome maintenance (MCM) helicase. In

Archaea, pre-RC formation includes the assembly of the Cdc6 protein and the MCM helicase. In

some Archaea, a Cdt1 homolog (WhiP) or other, not yet identiﬁed, proteins may also be part of

the pre-RC.

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Cdc6

The OBP in Bacteria is the DnaA protein, which binds the DnaA box (the bacterial ORB) at the

origin of replication. In Eukarya, the OBP is the hexameric ORC complex composed of the Orc1–6

proteins. ORC associates with the origin and recruits additional components of the pre-RC, such

as the MCM helicase and the Cdc6 protein, to participate in MCM assembly at the origin. The

eukaryotic Orc1–5 proteins, Cdc6, and the bacterial DnaA protein all belong to the initiator clade

of the AAA+family of ATPases (27), and Orc1–5 and Cdc6 were proposed to be paralogs (110).

In nearly all Archaea, at least one clear homolog of the eukaryotic Orc or Cdc6 proteins has

been identiﬁed, and most contain two or three genes encoding for the protein (143). Owing to

similarity to both the eukaryotic Orc and Cdc6 proteins, the archaeal enzyme has been called Cdc6,

Orc, Cdc6/Orc, and Cdc6/Orc1. In this review, the protein is referred to as Cdc6. Although the

Cdc6 protein is essential for cell viability in most species studied (154), it appears to be dispensable

in others (T. Santangelo & Z. Kelman, unpublished results).

Because of their similarity to both the ORC complex and the Cdc6 protein, the archaeal

enzymes were suggested to be involved in both origin recognition and helicase loading (Table 1;

Figure 1). The role of Cdc6 protein in origin recognition has been established. The ﬁrst suggestion

of the association of Cdc6 with the origin of replication came from chromatin immunoprecipitation

(ChIP) analysis with P. abyssi (118). Subsequent in vivo and in vitro studies illustrated that although

the proteins can bind both ssDNA and double-stranded (ds) DNA, they show clear preference for

the ORB dsDNA (10, 27).

Although the archaeal Cdc6 proteins bind origins, the mechanism by which they initiate DNA

replication is not clear. The bacterial DnaA protein unwinds origin duplex DNA to facilitate the

assembly of the replicative helicase DnaB on the exposed ssDNA (27). In contrast, binding of the

eukaryotic ORC to the origin does not induce unwinding. Furthermore, there is accumulating

evidence to suggest that the eukaryotic MCM assembles at the origin around dsDNA and not

around ssDNA, as suggested for Bacteria (27). The similarities between the archaeal Cdc6 and the

eukaryotic ORC and Cdc6 proteins and the lack of clear data on the ability of the archaeal Cdc6

protein to melt the origin may suggest that, as in Eukarya, during pre-RC formation the MCM

helicase is assembled on dsDNA and origin melting occurs during pre-IC formation.

Eukaryotic Cdc6 plays an essential role in the assembly of the MCM helicase at the origin,

and thus it was suggested that the archaeal Cdc6 plays a similar role. Direct interaction between

Cdc6 and MCM has been reported in several archaeal species, and the interaction was capable of

regulating helicase activity (152). Studies showed that the interaction between Cdc6 and MCM

inhibited helicase activity (158), which is likely due to the dissociation of the MCM hexamers in

the presence of Cdc6 (79, 159). The inhibitory effect of Cdc6 on helicase activity is reminiscent

of the observation made in bacteria, where binding of the helicase loader DnaC to the helicase

DnaB inhibits helicase activity (172). Thus, this observation may support the hypothesis that the

archaeal Cdc6 functions as a helicase loader. Thermoplasma acidophilum Cdc6, however, stimulates

the activity of the MCM helicase (51, 52), which may suggest a different mechanism of helicase

loading in that species. Nevertheless, the mechanism of helicase assembly at the archaeal origin

and the role of Cdc6 protein in the process are not known, although several mechanisms have

been proposed (151).

Cdc6, DnaA, and Orc1-5 belong to the AAA+superfamily of ATPases (27, 34). The structural

organization of the Cdc6 protein is similar to that of the bacterial DnaA proteins in which the

N-terminal AAA+catalytic unit is followed by a DNA binding domain. However, although in

DnaA the DNA binding domain is a helix-turn-helix (HTH), the Cdc6 protein contains a winged-

helix domain (WHD) (27). Biochemical studies demonstrated that an intact WHD is required for

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ORB binding (10, 27, 35). The WHD also participates in the interaction between the Cdc6 and

MCM proteins (30, 66).

In addition to all known motifs present in other members of the AAA+family of ATPases,

an α-helix inserted into the canonical AAA+fold was also revealed by the structure of Cdc6 and

DnaA. This helical insertion is found in all proteins belonging to the initiator clade of the AAA+

family and is referred to as the initiator speciﬁc motif (ISM) (27).

The three-dimensional structure of Cdc6 bound to the ORB has been determined (36, 45). In

addition to extensive contact between the WHD and the DNA, the ISM also contacts the DNA.

However, although ORB sequences are conserved and in vivo and in vitro studies showed the

speciﬁcity of Cdc6 binding to the ORBs, only a few sequence-speciﬁc contacts were observed in

the co-crystals (36, 45). The mechanism by which binding speciﬁcity is achieved is not yet known

and may involve the helicase or other proteins (10, 27, 174).

Cdc6 is expressed during the G1 phase and degraded after initiation (107, 149). However,

the regulation of expression and of subsequent degradation is not understood. In Eukarya, phos-

phorylation of Cdc6 following the initiation process marks the enzyme for degradation. A similar

mechanism may regulate Cdc6 in Archaea. A number of archaeal protein kinases are cell-cycle

regulated (107), and in vivo phosphorylation of Cdc6 protein has been reported (144).

Alternatively, the archaeal Cdc6 proteins can autophosphorylate in vitro using the γ-phosphate

of ATP (46). Studies suggest that autophosphorylation may be regulated by MCM binding to Cdc6

and by the association of Cdc6 with DNA (46, 66). Although the role of autophosphorylation in

Cdc6 function is not known, it may play a regulatory role during pre-RC formation (47). It will

be important to determine whether Cdc6 can autophosphorylate in vivo as a ﬁrst step to establish

whether the process has a physiological role.

In bacteria, the activity of DnaA is regulated by a mechanism referred to as regulatory inactiva-

tion of DnaA (RIDA), which ensures that the chromosome replicates only once per cell cycle (98).

RIDA is dependent upon interaction between the bacterial processivity factor of DNA polymerase

III (PolIII) holoenzyme (the β-subunit), DnaA, and a homologous-to-DnaA (Hda) protein. Inter-

action between Hda and DnaA stimulates ATP hydrolysis by DnaA, resulting in an ADP-bound

DnaA protein that cannot bind to the origin of replication. However, Hda must associate with

the β-subunit on DNA to stimulate ATP hydrolysis by DnaA. Proliferating cell nuclear antigen

(PCNA) is the archaeal functional homolog of the β-subunit (80). In several species, the Cdc6

protein contains a PCNA-interacting protein (PIP) motif required for interactions with PCNA,

and PCNA and Cdc6 can form a complex in vivo (102) and in vitro (3). It was therefore proposed

that regulation of Cdc6 function in Archaea might resemble the bacterial RIDA process (100).

However, no homolog of Hda has been identiﬁed in Archaea. It is possible that another unidenti-

ﬁed protein is the archaeal functional homolog of Hda, or that an Hda-like protein is not needed

in Archaea as Cdc6 can interact directly with PCNA.

Other mechanisms may also regulate Cdc6 function. Archaeal Cdc6 proteins crystallized as

monomers with ADP tightly bound at the active site, and denaturation of Cdc6 was required for

removal of the bound ADP. As it is thought that the Cdc6 protein is active in the ATP-bound

form, several hypotheses for the role of the tight ADP binding for regulating the enzyme have

been proposed (10).

Winged-Helix Initiator Protein

Cdt1 is a eukaryotic protein that participates in the initiation of DNA replication as a part of the

pre-RC (27). Structurally, the middle and the C-terminal portions of the eukaryotic Cdt1 protein

have a WHD (97). When the three origins of replication in Sulfolobus solfataricus were identiﬁed, it

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was noted that only two origins are located upstream to a gene encoding Cdc6 (106). However, the

protein encoded by the gene upstream of the third S. solfataricus origin also contains two WHD,

although at the N and C termini of the molecule. This suggested that the protein might be the

archaeal homolog of Cdt1 (148). Because amino acid similarity between the archaeal and eukaryal

enzymes is low, the archaeal protein is called WhiP (winged-helix initiator protein). It was found

that the WhiP protein could bind to the origin of replication near its gene (148). To date, WhiP

homologs have been identiﬁed only in a small subset of species belonging to the crenarchaeota

branch. However, as many archaeal genomes contain proteins with a single WHD that is similar

to that found in WhiP, these proteins may play a role in the initiation process in those species.

Minichromosome Maintenance Helicase

The MCM proteins are the replicative helicases in Archaea responsible for the separation of the

dsDNA in front of the replication fork. The archaeal MCM proteins have been extensively studied

using structural, biochemical, and genetic tools (11, 16, 28, 93, 123, 152, 164).

In Eukarya, MCM is a complex of six different but related proteins (Mcm2–7). Most archaeal

species contain a single gene that encodes MCM. To date, in archaeal species with multiple MCM

genes, only one is essential for viability (59, 133). The single MCM homolog from the archaeon

Methanothermobacter thermautotrophicus was the ﬁrst to be biochemically characterized (23, 79,

156), but others have since been studied. As with other helicases, the MCM utilizes energy from

ATP hydrolysis to translocate along one strand of the DNA and displace the other. The enzyme

binds to and translocates along ssDNA and dsDNA in the 3to 5direction, and in vitro the enzyme

is processive on its own and able to displace several hundred bases (16, 28, 93, 152, 164). However,

on dsDNA the enzyme probably interacts with one strand, resulting in the observed 3to 5direc-

tionality. Biochemical and biophysical studies with mutated enzymes suggested that ssDNA and

dsDNA are located in the central hole of the hexameric ring. During DNA unwinding, the helicase

may encounter proteins on DNA (such as histones) or short RNA molecules, forming R-loops.

Archaeal MCM can displace proteins from DNA and unwind DNA-RNA hybrids (161, 162).

Early studies suggested that archaeal MCM proteins form dodecameric structures in solution

as judged by size-exclusion chromatography and sedimentation methods (23, 79, 156). Further

support for dodecameric structures came from the three-dimensional structure of the N-terminal

portion of the enzyme (40, 104) and the full-length protein (91). Electron microscope reconstruc-

tion studies, however, indicated that the enzyme could form other structures, including hexamers,

heptamers, octamers, ﬁlaments, and open rings (28, 152, 164).

Although the ability of MCM to form both hexamer and head-to-head dodecameric structures

was ﬁrst reported with an archaeal enzyme, it was later shown for the eukaryotic MCM (145) and

the bacterial DnaB (166). It is thought that the replicative helicases are assembled as dodecameric

rings at the origin. It is not clear whether the two hexamers associate during elongation or are

separated during DNA synthesis and move away from each other (151, 168). However, in vitro

studies indicate that the archaeal enzymes are active as single hexamers (160), suggesting that upon

loading the two rings work independently.

Structurally, archaeal MCM proteins can be divided into three main parts: an N-terminal part,

a catalytic region, and a C-terminal HTH domain (40, 152, 164). The N-terminal part of MCM

forms hexamers and dodecamers on its own (40, 67, 92). This part of the molecule contains a C4-

type zinc ﬁnger and a β-hairpin motif shown to be involved in MCM interaction with DNA (78,

141). The structure also revealed the presence of a long loop shown to play a role in communication

between the N-terminal DNA binding region and the catalytic part of the molecule (6, 150). The

central part of MCM contains the catalytic domains and all the conserved motifs found in other

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AAA+family members (28, 152, 164). The structure also revealed the presence of three β-hairpins

in the catalytic part; all are needed for helicase activity (17, 63, 122). The C-terminal part of MCM

was suggested to fold into an HTH domain (2), although the domain was not seen clearly in any

of the structures of the molecule. Biochemical studies suggested that the domain might play a

regulatory role, as removal resulted in increased helicase activity in vitro (7).

PREINITIATION COMPLEX

During S phase of the cell cycle, the pre-RC recruits additional factors, including Cdc45 and

GINS, to the origin to establish the pre-IC. Following pre-IC formation, the dsDNA is unwound

and SSB associates with the exposed ssDNA. The replisome is assembled at the origin and DNA

replication initiates.

Single-Stranded DNA Binding Protein

SSB plays an essential role in DNA replication by coating ssDNA behind the helicase to prevent

reannealing and protect the ssDNA from attack by nucleases and chemical modiﬁcation (32).

In Bacteria and Eukarya, SSB also stimulates the activity of DNA polymerases by generating a

uniform substrate without secondary structure (142). SSBs were also shown to play an important

role in coordinating DNA synthesis on the lagging strand in Bacteria and Eukarya (182, 183), but

this activity has not yet been reported in archaea.

In all domains of life, SSB binds to ssDNA via an oligonucleotide/oligosaccharide binding (OB)

fold (32). In bacteria, the SSB protein forms homotetramers in which each monomer contains a

single OB fold at the N terminus and a ﬂexible acidic C terminus, which is needed for interaction

with other proteins (82).

In Eukarya, the SSB is the heterotrimeric replication protein A (RPA) and comprises the RPA70,

RPA32, and RPA14 proteins (142). RPA70 contains four OB folds, whereas RPA32 and RPA14

each contain one OB fold; however, only three OB folds from RPA70 and the OB fold of RPA32

are involved in ssDNA binding. The RPA70 protein also contains a C4-type zinc-ﬁnger motif and

RPA32 contains a WHD, and both are found near the C-terminus. These motifs, however, do

not participate in DNA binding but rather in complex stability and protein-protein interactions

(142).

The single RPA homolog from Methanocaldococcus jannaschii was the ﬁrst archaeal SSB to be

characterized (70). Other archaeal SSB proteins show signiﬁcant variation in structure, domain

organization, and subunit composition. In most Aigarchaea and Crenarchaea, the SSBs are similar

to those in Bacteria in that they have a single OB fold at the N terminus followed by a ﬂexible, acidic

C terminus, which is not required for DNA binding (171), whereas the genomes of Korarchaea and

Thaumarchaea contain homologs of both SSB and RPA (143). The structure of the Crenarchaea

OB fold, however, is more similar to that of the eukaryotic RPA70 than to that of the bacterial

SSB (84).

The SSB proteins from the other archaeal lineages are diverse in structure and domain organi-

zation, although overall they are similar to the eukaryotic RPA. Whereas some organisms encode a

single RPA with multiple OB folds (70), others contain multiple RPA homologs (88). It was found

that some organisms with multiple RPAs form trimeric complexes similar to the eukaryotic RPA

complex (88). Genetic studies also showed that in some cases when multiple RPAs are present,

only a subset is essential for cell viability (163). In addition to the OB fold, several archaeal RPA

proteins also contain a zinc-ﬁnger motif (147, 163).

In silico approaches identiﬁed SSBs in most archaeal species either by similarity to the eu-

karyotic or bacterial SSB or by searches for putative OB folds. However, to date, no canonical

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SSB has been detected in one crenarchaeal order, Thermoproteales. Screening of a Thermoproteus

tenax extract for proteins capable of binding to ssDNA revealed a new type of SSB [referred to as

ThermoDBP (Thermoproteales-speciﬁc DNA binding protein)] (136). The protein was shown

to form a homodimer with a novel DNA binding fold at the N terminus and a leucine zipper at

the C terminus that mediates dimerization (136).

The Cdc45-MCM-GINS Complex

In Eukarya, the MCM helicase is not active on its own but is activated by association with two

accessory factors: the GINS complex and the Cdc45 protein. The resulting complex is referred to

as the CMG (Cdc45-MCM-GINS) complex and is thought to function as the replicative helicase

(129). However, in contrast to the eukaryotic Mcm2–7 complex, in vitro studies showed that the

archaeal MCM is active on its own without association with other proteins. Nevertheless, a CMG

complex may also be present in Archaea and may play a role in vivo.

The GINS complex. In Eukarya, the GINS complex is a ring-shaped heterotetramer of four

related proteins, Sld5, Psf1, Psf2, and Psf3 (GINS is derived from the Japanese go-ichi-ni-san,

meaning 5-1-2-3), that plays an essential role in establishment and maintenance of replication forks

(65, 108). In addition to interacting with MCM and Cdc45, the GINS complex also interacts with

the DNA polymerase α(Polα)-primase complex that synthesizes primers on the lagging strand

and with the leading-strand DNA polymerase Polε(65, 108). It is thought that the GINS complex

may be the eukaryotic functional homolog of the bacterial τ-subunit that couples the helicase,

primase, and polymerases at the replication fork and thus plays an important scaffolding role in

replisome formation and in coordinating leading- and lagging-strand synthesis (64).

The ﬁrst archaeal GINS homologs were identiﬁed using in silico approaches that detected

their weak similarity to the eukaryotic proteins (112). This study was followed by the isolation and

characterization of the GINS complex from S. solfataricus and other species (9, 114). Although

some species contain a single homolog, designated GINS15 (also called GINS51) for its similarity

to the eukaryotic Psf1 and Sld5 proteins, other species contain two homologs, a GINS15 and

another referred to as GINS23 for its similarities to the eukaryotic Psf2 and Psf3 proteins (108,

110). However, it has been shown that in all cases studied, the GINS proteins form tetrameric

complexes, either homotetramers of GINS15 (128) or a heterotetramer of two subunits of GINS15

and two of GINS23 (114). As is the case in Eukarya, the archaeal GINS proteins are essential for

cell viability (155).

Supporting evidence for GINS’s function as a scaffolding complex comes from the observation

that the archaeal complex interacts with a number of replisome components, including primase,

MCM, Cdc45, DNA polymerase D (PolD), and PCNA (102, 109, 114, 140). The genes encoding

GINS15 and GINS23 are often in an operon with the genes encoding PCNA and/or the small

subunit of primase (PriS) and MCM, respectively (14, 108).

Structural studies revealed that each of the two GINS subunits is composed of two distinct

domains: a large A domain and a smaller B domain. The order of the two domains, however, is

different in the different subunits. In the GINS15 protein the A domain is at the N terminus and

the B domain is at the C terminus (AB type), whereas the orientation is reversed in GINS23 (BA

type) (65).

The structure of the Thermococcus kodakarensis GINS revealed that two GINS15 proteins form

a top layer and two GINS23 proteins form a bottom layer with an overall structure that resembles

a trapezoid with a narrow cavity in the center (130). The structure suggests that the B domain

of GINS15 does not participate in tetramer formation (130). It is possible that the B domain

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is needed for GINS interaction with other components of the CMG complex or with other

replication proteins.

The structure also revealed similarities between the B domain and the C-terminal part of PriS

(65, 167). This C-terminal domain, however, is not present in PriS from all species. Because the

gene encoding GINS15 in several archaeal species is in an operon with the gene encoding PriS

(14, 108), it has been suggested that the C-terminal domain of PriS may have been acquired from

the GINS15 protein via tandem duplication (167).

Cdc45. The ﬁrst suggestion that Archaea might contain a Cdc45 homolog came from a study that

identiﬁed a nuclease in T. kodakarensis associated with the GINS complex (101, 102). A structural

prediction analysis suggested that the protein, referred to as GAN (GINS-associated nuclease),

has limited similarity to the bacterial RecJ and the eukaryotic Cdc45 proteins (90, 101). Detailed in

silico analyses have shown that all archaeal species contain a protein with similarity to Cdc45 and

the bacterial RecJ (111). Although the T. kodakarensis protein possesses nuclease activity, this is not

the case for all archaeal Cdc45 proteins. When the GINS complex was puriﬁed from S. solfataricus,

a protein with similarity to the RecJ DNA binding domain copuriﬁed with the complex (114).

This protein does not include the RecJ nuclease domain and does not possess nuclease activity.

Similarly, the eukaryotic Cdc45 protein does not contain key residues for nuclease activity and no

nuclease activity could be detected in vitro with the eukaryotic enzyme ( J. Hurwitz & Z. Kelman,

unpublished results). Therefore, the function of the archaeal Cdc45 in DNA replication probably

does not depend on its nuclease domain. Nevertheless, it is likely that the archaeal and eukaryal

Cdc45 evolved from the bacterial RecJ (110), and the archaeal Cdc45 and the bacterial RecJ have

similar three-dimensional structures (S. Nair & Z. Kelman, unpublished results).

The role of Cdc45 in archaeal DNA replication is not clear. The protein interacts with GINS

and PolD, which may suggest a role at the replication fork. However, although the eukaryotic

Cdc45 is essential for cell viability (115), the genes encoding the archaeal enzyme can be deleted

from the chromosome without a major effect on cell growth (T. Santangelo & Z. Kelman, un-

published results). This may suggest that the protein is not essential for DNA replication or that

another factor can replace it. Future studies are needed to determine the role, if any, for the

archaeal Cdc45 protein in vivo.

ELONGATION COMPLEX

Following MCM activation, the replication bubble is formed, and the other components of the

replisome are recruited to the DNA to establish the two replication forks and initiate bidirectional

DNA synthesis.

Primase

DNA polymerases are incapable of initiating DNA synthesis de novo and require a 3-hydroxyl-

primed template in order to elongate DNA chains. DNA primases synthesize short RNA primers

on template DNA, which are subsequently extended by DNA polymerase (Figure 2;Figure 3a)

(44). In bacteria, DNA primase consists of a single subunit, the DnaG protein, which associates

with both the DnaB helicase and PolIII. It synthesizes a 10–12 oligoribonucleotide primer

that is transferred to the bacterial clamp loader. In Eukarya, DNA primase is a heterodimer

containing a catalytic p48 subunit in tight association with a regulatory p58 subunit. In vivo, the

heterodimer is found in a complex with two other proteins, the Polαcatalytic subunit (p180)

and the B subunit (p70), to form the Polα-primase complex (44). The primase component

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synthesizes 10–15 oligoribonucleotide primers that are elongated by the Polαcomponent,

generating covalently linked RNA-DNA oligonucleotide chains of 30–40 nucleotides (44). These

pre-Okazaki fragments are recognized by the clamp loader.

The archaeal primase is a two-subunit complex; a small subunit p41 (PriS) contains the catalytic

activity, and a large subunit p46 (PriL) regulates primase activity and contains an iron-sulfur

domain. Both subunits were shown to be essential for cell viability (155). Nanoarchaeota genomes

encode a shorter form of primase in which the iron-sulfur domain of PriL is fused to the catalytic

domain of PriS (143).

The ﬁrst report on the biochemical properties of the archaeal primase came from studies

on the small subunit from M. jannaschii (31), followed by studies on the dimeric enzyme from

other species (60). No homologs of the eukaryotic Polαor B subunit have been identiﬁed in

archaeal genomes (96). In contrast to DNA primases from Bacteria and Eukarya that can utilize

only ribonucleotides, in vitro studies with the archaeal catalytic subunit PriS and the two-subunit

complex PriS-PriL demonstrated that the archaeal primase is capable of initiating oligonucleotide

chains de novo from either ribonucleotide triphosphates (rNTPs) or deoxyribonucleotide

triphosphates (dNTPs) (60, 96).

Although the primase can utilize both ribo- and deoxynucleotides in vitro, several different

assays, including RNA unmasking, RNA labeling, and RIP mapping, strongly suggest that primers

are composed of oligoribonucleotides in vivo (119, 149). The much higher cellular levels of rNTPs

in comparison to dNTPs (89) may explain the discrepancies between primase activity in vitro and

primer synthesis in vivo.

Archaeal primases also have strand displacement, gap-ﬁlling, terminal transferase, and py-

rophosphatase activity (60), but the roles of those activities are not clear. In vitro studies also

showed that the enzymes are capable of generating nucleotide adducts in the presence of rNTPs,

dNTPs, and small molecules with −OH and −NH groups. The reaction results in the formation

of r/dNMP-O-R and r/dNMP-N-R (19, 20). It is not clear whether the enzyme makes similar

compounds in vivo and whether such products have a physiological role.

The three-dimensional structure of PriS from several species has been determined, revealing

a two-domain structure (95, 137): a large primase domain (62), which contains the catalytic part,

and a small domain that appears to have a species-speciﬁc fold. The catalytic domain also contains

a zinc-ﬁnger motif. Although the role of zinc binding is not clear, the proximity of this motif

to the active site may suggest a role in catalysis. DnaG also contains a zinc binding domain that

participates in ssDNA binding (25), and therefore it is possible that zinc binding has a similar role

in the archaeal primase.

The structures of the N-terminal part of PriL alone and a complex of the N-terminal part of

PriL and PriS have been determined (95). The N-terminal part of PriL interacts with PriS and

also forms an arm that connects the catalytic subunit to the DNA binding domain located at the

C terminus of PriL (116). The C-terminal part of PriL contains an iron-sulfur domain (87). The

structure and role of the iron-sulfur domain is not yet known, but this domain may play a role in

maintaining the correct three-dimensional structure of the C-terminal domain (137).

Polymerase Accessory Proteins

The replicative polymerase on its own has very low processivity. High processivity is conferred

by a ring-shaped processivity factor (sliding clamp) that encircles DNA and acts to tether the

polymerase catalytic unit to the template for processive DNA synthesis (Figure 2;Figure 3a) (58,

94, 131). The sliding clamps are stable rings and thus cannot assemble themselves around DNA

but must be loaded onto DNA by a clamp loader complex (69, 181). The clamp loader recognizes

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the 3end of the single strand–duplex (primer-template) junction and utilizes ATP hydrolysis

to assemble the clamp around the primer. The clamp encircles the primer and then binds the

polymerase for rapid and processive DNA synthesis. Upon completion of an Okazaki fragment,

the polymerase dissociates from the clamp, leaving it assembled around the duplex DNA. The

clamps left on the DNA interact with proteins needed for Okazaki fragment maturation, chromatin

remodeling, and other cellular processes (170).

Replication factor C. In eukarya, the clamp loader is the pentameric replication factor C (RFC)

complex composed of one large subunit (Rfc1) and four small subunits (Rfc2-5), all belonging

to the AAA+family of ATPases (69, 181). Most archaea contain two homologs of RFC (22).

One is similar to the small subunits of the eukaryotic RFC and thus is referred to as RFC small

(RFCS), and the other is similar to the large subunit of the eukaryotic RFC and is named RFC

large (RFCL). Like the eukaryotic RFC complex, the archaeal complex is pentameric, with four

subunits of RFCS associated with one subunit of RFCL (58, 127).

RFC must be bound to ATP in order to associate with and open the PCNA ring. Following

the assembly of PCNA around the primer, the interaction of RFC with the DNA and PCNA

stimulates its ATPase activity. Upon ATP hydrolysis, the afﬁnity of RFC to DNA and PCNA is

substantially reduced, resulting in the dissociation of RFC from the DNA and PCNA (58, 127).

In Bacteria and Eukarya, primase remains associated with the primer following primer synthesis.

It was shown that primases and the clamp loaders interact with SSB, and competition between the

clamp loader and primase for the interaction with SSB plays an important role in the handoff of the

primer from primase to the clamp loader (182, 183). A similar RPA-dependent handoff mechanism

may also exist in archaea, although it has not yet been reported. Studies have shown, however,

that the archaeal primase interacts directly with RFC (109, 178). It is thus possible that in Archaea

the handoff of the primers from primase to RFC does not require the involvement of RPA.

As mentioned above, PCNA remains on the DNA after the completion of an Okazaki fragment.

However, during replication there are more Okazaki fragments than PCNA trimers within the

cell, and so the clamps must be recycled. It was shown that the eukaryotic RFC can actively remove

PCNA rings from DNA (180) and a similar unloading activity was proposed for archaeal RFC (77).

Biochemical, biophysical, structural, and molecular modeling studies with RFC have shed light

on the mechanism by which RFC assembles the PCNA around DNA (68, 124). The data suggest

that during the assembly process, RFC, in the ATP-bound form, forms a right-handed spiral on

the top of the clamp with a pitch that is congruent with the helical geometry of duplex DNA.

Binding to the clamp results in clamp opening with an out-of-plane conﬁguration resembling a

right-handed spring washer (69), enabling assembly around the DNA.

Proliferating cell nuclear antigen. PCNA is a trimeric ring that encircles dsDNA and can slide

bidirectionally along it. All activities described for the PCNA proteins require them to encircle

the DNA duplex; no biochemical function for PCNA separate from DNA has been reported.

PCNA was ﬁrst reported as a processivity factor for replicative DNA polymerases, but subsequent

studies have established that PCNAs also associate with, and modulate the activity of, many other

proteins involved in nucleic acid metabolic transactions and cell-cycle regulation (131, 170). It

was proposed that the movement of PCNA along dsDNA might function as a moving platform

for enzymes that participate in DNA metabolic processes but have low afﬁnity for DNA (76).

Most of the proteins that interact with PCNA do so via a PIP motif (173). The PIP motif

interacts with the loop that connects the two domains in each PCNA monomer [referred to as

the interdomain connecting loop (IDCL)] (49). Biochemical and structural analysis elucidated the

requirement for an intact PIP motif for interaction with PCNA (60).

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The three-dimensional structures of PCNA proteins from several archaeal species were de-

termined (131), as were PCNA complexes with interacting enzymes, including DNA ligase, ﬂap

endonuclease 1 (Fen1), and DNA polymerase B (PolB) (60). Although the crenarchaeota PCNA

protein forms heterotrimers while PCNA from the other kingdoms form homotrimers, the ring

structures are very similar (131), and they are also similar to the bacterial β-subunit and the

eukaryotic PCNA (58, 80).

The structure revealed that three monomers interact in a head-to-tail manner to form a trimeric

ring sufﬁciently large to accommodate dsDNA. Each monomer is composed of two structurally

similar domains, so the trimer has a pseudo six-fold symmetry (75). The two domains in each

monomer are connected by the IDCL (49).

Although all PCNA proteins are acidic (80), the charge distribution on the ring surface is not

symmetrical. The outer surface is negatively charged, whereas there is a net positive electrostatic

potential in the central cavity where the dsDNA is located (131). The positive charge in the

central cavity was shown to be required for interaction with the DNA, and it was suggested that

the negatively charged surface might prevent nonspeciﬁc interaction with DNA. In contrast to the

structures of other PCNA proteins, the structures of the halophilic PCNA proteins revealed very

few positively charged residues within the central cavity (125, 176), which may suggest a different

mechanism of interaction for halophile PCNA with DNA (125).

When the structures of the sliding clamps were initially determined, it was hypothesized that the

DNA passes perpendicularly through the central hole, allowing bidirectional sliding along dsDNA

(180). However, structural studies of PCNA-DNA complexes from several archaeal species suggest

that the DNA has a substantial tilt while passing through the central hole of the ring (121).

Several mechanisms regulate the activity of eukaryotic PCNA. One involves the binding of

small PIP-containing regulatory proteins to PCNA, which in turn prevents other proteins from

binding and thus regulates (inhibits) the effect of PCNA on their activity (41). Archaea may

use similar mechanisms in which binding of small proteins to PCNA regulates its interaction

with other enzymes. Studies have shown that a small protein from T. kodakarensis,referredtoas

Thermococcales inhibitor of PCNA (TIP), binds to PCNA and prevents its interaction with DNA

polymerase and other enzymes. However, the mechanism of binding of TIP to PCNA does not

involve a canonical PIP motif (99).

Another mechanism by which the eukaryotic PCNA is regulated involves modiﬁcation by

small proteins, such as ubiquitin and SUMO. These proteins, when binding PCNA, modulate

PCNA interaction with other enzymes (33). Although small modiﬁer proteins have also been

identiﬁed in Archaea (120), it remains to be determined whether they modify PCNA and whether

the modiﬁcation plays a role in regulating PCNA function.

A third mechanism used by Eukarya to regulate PCNA activity is phosphorylation of the

interacting enzymes, which results in the modulation of their interactions with PCNA (56). Protein

phosphorylation is common in Archaea (83), and in vivo phosphorylation of PCNA and several

replication proteins has been reported (144). Therefore, it is possible that phosphorylation may

regulate the activity of the archaeal PCNA.

DNA Polymerase

DNA replication is achieved by a DNA-dependent DNA polymerase that uses primed ssDNA as a

template to synthesize the complementary strand. Bacterial PolIII replicates both the leading and

lagging strands (127). In Eukarya, two different polymerases, Polεand Polδ, replicate the leading

and lagging strands, respectively (127). Two different DNA polymerases, PolB and PolD, have

been implicated in archaeal DNA replication.

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B-type polymerases. The archaeal PolB DNA polymerases have been extensively studied, mainly

for their importance in polymerase chain reaction. A large number of enzymes have been identiﬁed,

puriﬁed, and biochemically characterized (60, 110, 139). At least one PolB homolog has been

identiﬁed in every archaeal genome. In addition to polymerase activity, these enzymes possess a

3to 5exonuclease proofreading activity. Polymerases are not processive; processivity is achieved

by PolB interaction with PCNA.

For extended processivity, bacterial and eukaryal replicative polymerases require SSB. In vitro

studies showed, however, that at least in some archaea, RPA inhibited rather than stimulated the

primer extension activity of PolB (81), and direct interaction between RPA and PolB has also

been reported (81, 102). The presence of PCNA relieved, but did not eliminate, the inhibitory

effect of RPA. Why RPA inhibits PolB and what the role of the inhibition is in vivo is not clear.

It is possible that when a polymerase is a part of the replisome, RPA does not inhibit its activity.

Alternatively, PolB may not be the replicative enzyme in Archaea (29, 155) but instead is involved

in DNA repair and/or recombination. If this is the case, then inhibition by RPA may insure that

PolB will replicate only short DNA fragments, as expected for a repair enzyme. It is also possible

that PolB replicates only the leading strand, and therefore its ssDNA template is not coated with

RPA. Future studies are needed to determine how common the inhibition of PolB by RPA is, as

well as the physiological role of that inhibition.

The three-dimensional structures of several archaeal PolB enzymes have been determined

and revealed similar topology to other DNA polymerases, including the palm, ﬁnger, thumb,

and exonuclease domains (60, 73). One feature of the archaeal PolB not found in other family

members is an N-terminal domain involved in speciﬁc interactions with uracil and hypoxanthine

on the template strand (24). Copying of damaged DNA could result in irreversible mutations.

Therefore, the ability of a polymerase to recognize uracil or hypoxanthine, followed by replication

stalling, may enable the cell to repair the DNA damage prior to replication (24, 60).

D-type polymerases. The genomes of all Archaea except Crenarchaea contain the archaeal-

speciﬁc PolD in addition to PolB. PolD was originally isolated from Pyrococcus furiosus by screening

for DNA polymerase activity in cell extract (169). PolD is a dimeric enzyme, and the large subunit

(DP2) contains polymerase catalytic activity while the small subunit (DP1) contains the 3to 5

exonuclease activity. The DP2 protein does not display any sequence similarity with other protein

families (110), whereas DP1 shares amino acid sequence similarity with several of the small,

noncatalytic subunits of the eukaryotic Polα,Polδ,andPolε(1). Although each subunit alone has

low activity, the dimeric PolD complex exhibits polymerase and exonuclease activities (60).

The three-dimensional structures of the N-terminal parts of DP1 and DP2 have been solved

(117, 179). The structure of the N-terminal part of DP1 conﬁrmed the bioinformatic prediction

that it shares similarity with the small subunits of Polαand Polε(179). Although high-resolution

structures of the full-length proteins have not yet been reported, bioinformatic predictions provide

insight into the structure and domain organization, including the presence of an OB fold in DP1

and a zinc-ﬁnger motif in DP2 (110). Mutational analyses of conserved residues, regions, and

motifs have shed light on the regions required for activity, on interactions between DP1 and DP2,

and on interactions with DNA (157).

Which is the replicative polymerase in Archaea? The three replicative polymerases in Eukarya,

Polα,Polδ,andPolε, all belong to family B, and therefore it was presumed that members of

this family also replicate the archaeal genomes. Crenarchaea genomes encode only members of

PolB, and therefore PolB must be the replicative enzyme in this kingdom. In other archaeal

branches, however, both PolB and PolD are present. It is not clear whether both polymerases

are involved in chromosomal replication, as in Eukarya, or only one of the two is, as in Bacteria,

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with the other polymerase playing a different cellular role. Genetic studies with PolB from several

species suggested that both PolB and PolD are required for cell viability (13). In other organisms,

however, PolB is dispensable for cell growth (29, 155). These contradictory observations need

further evaluation. They may suggest, however, that at least in some species, PolD replicates both

the leading and lagging strands.

Aphidicolin is an inhibitor of the eukaryotic replicative polymerases and thus was thought to

inhibit all members of the family B enzymes. PolD, however, is not sensitive to aphidicolin (48,

169). Therefore, sensitivity to aphidicolin was suggested to serve as an additional tool to determine

whether the archaeal PolB is essential for cell growth. The picture, however, is not that clear. In

vitro studies have shown that although aphidicolin inhibits PolB from some species (48, 169), it

has no effect on PolB activity in others (81). In vivo studies did not provide much clariﬁcation.

Although aphidicolin was shown to inhibit the growth of Halobacterium halobium, there was no

effect on the growth of the crenarchaea Sulfolobus acidocaldarius (43). The S. acidocaldarius genome

contains only PolB, again demonstrating that some archaeal PolB enzymes are not sensitive to

the drug. Taking the in vivo and in vitro evidence together, it is clear that aphidicolin sensitivity

cannot be used as a tool to determine whether PolB is required for chromosomal replication.

Additional studies are needed to determine whether both PolB and PolD are a part of the

replisome or whether only PolD is involved in chromosomal replication and PolB has another

function. It is possible that although both polymerases are replicative enzymes, in the absence

of PolB, PolD can substitute for PolB activity. Although in Eukarya Polεis the leading-strand

polymerase (127), it was shown that cells harboring mutant Polεare viable and that Polδcan

replicate both DNA strands (85). It is possible that in archaea, PolD replicates the lagging strand

and PolB copies the leading strand. The role for PolD in lagging-strand synthesis may be supported

by the observation that PolD exhibits a strand displacement activity (54) that is required for Okazaki

fragment maturation. In addition, PolD can utilize RNA primers more efﬁciently than PolB (55),

whereas processivity of PolB, in the presence of PCNA, is greater than that of PolD (132). Similar

observations were made in Eukarya, where the leading-strand polymerase is more processive than

the lagging-strand enzyme (12). In addition, the inhibition of PolB by RPA may suggest that it

is not replicating the lagging strand because the lagging-strand polymerase probably needs to

displace RPA.

OKAZAKI FRAGMENT MATURATION

During chromosomal replication, the leading strand is replicated as a continuous strand, whereas

the lagging strand is replicated discontinuously as a series of Okazaki fragments (Figure 3). The

RNA primers that initiate each Okazaki fragment must be removed, the gap in the DNA must be

ﬁlled, and the newly synthesized Okazaki fragment must be ligated to the previous fragment to

create the mature dsDNA. This task is achieved by the concerted activity of several enzymes, in-

cluding DNA polymerase, Fen1, and DNA ligase (Figure 3b) (5). The lagging-strand polymerase,

upon reaching the previous Okazaki fragment, displaces the primer and provides the substrate for

Fen1. Following Fen1 removal of the primer, the lagging-strand DNA polymerase ﬁlls the gap

and DNA ligase joins the two adjacent Okazaki fragments.

Flap Endonuclease 1

Fen1 is a structure-speciﬁc nuclease that plays an important role in Okazaki fragment maturation

on the lagging strand (4). The lagging-strand DNA polymerase, using its strand displacement

activity, removes the RNA primer and forms a branched DNA molecule (ﬂap structure). The

5-ﬂap endonuclease activity of Fen1 removes the primer (Figure 3b).

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The three-dimensional structures of Fen1 from several archaeal species and co-complexes

with DNA have been determined (60). The structures reveal that the protein catalytic domain is

similar to other nucleases. In addition, Fen1 contains a helical clamp that encircles the 5-ﬂap and

interacts with the upstream and downstream duplex DNA. Upon binding to the substrate, the

protein undergoes a conformational change that closes the clamp around the single-stranded ﬂap.

In Eukarya, it was shown that the preferred substrate of Fen1 contains an unpaired 3nucleotide

(3-ﬂap) overlapping with a variable length region of 5ssDNA (5-ﬂap), referred to as an overlap-

ﬂap (39). The structure of the archaeal Fen1 with DNA suggested that an overlap-ﬂap may also

be the preferred substrate for the archaeal enzyme (18).

As is the case with the eukaryotic Fen1, the archaeal enzyme is not essential for cell viability

(155). In Eukarya, Exonuclease I (ExoI), a homolog of Fen1 with similar catalytic activity, may

replace Fen1 activity in the deletion strain (4). However, in Archaea there are no clear homologs

of ExoI or other known nucleases with Fen1-like activity. Thus, it is not clear which protein(s)

replaces Fen1 activity in the deleted strain, although it was suggested that the archaeal Cdc45 might

substitute for Fen1 in the deleted strain (101). Support for this idea comes from the observation that

although Cdc45 or Fen1 can be readily deleted from T. kodakarensis cells, the double mutation is

lethal (T. Santangelo & Z. Kelman, unpublished results). Alternatively, RNase H, which degrades

RNA in a RNA-DNA hybrid, may be responsible for primer removal in the Fen1 deleted strain.

Future studies are needed, however, to identify the mechanism by which Okazaki fragments mature

in the absence of Fen1.

DNA Ligase

DNA ligase plays an essential role in chromosomal DNA replication by joining adjacent Okazaki

fragments on the lagging strand. DNA ligases can be divided into two groups according to the

cofactor required for activity: ATP or NAD+.NAD

+-dependent ligases are found predominantly

in Bacteria, whereas eukaryotic genomes contain ATP-dependent ligases (110).

Genes that encode DNA ligase have been identiﬁed in all archaeal genomes and were shown

to be essential for cell viability. The ﬁrst archaeal DNA ligase was identiﬁed in the genome of the

thermophilic archaeon Desulfolobus ambivalens (86). The ﬁrst characterized archaeal ligase from

M. thermautotrophicus demonstrated a strict requirement for ATP as a cofactor (165), but subse-

quent studies identiﬁed a small subset of archaeal ligases that are NAD+-dependent (185), whereas

others can utilize both cofactors (60).

The structures of several archaeal DNA ligases were determined (121, 135) and together

with the structure of the eukaryotic DNA ligase bound to DNA (134) revealed a three-domain

organization consisting of a DNA binding domain (DBD), a catalytic nucleotidyltransferase do-

main, and a C-terminal OB fold domain. In the absence of DNA substrate, the enzyme has an

open, extended form. Upon DNA binding, the enzyme forms a closed structure around the ds-

DNA with both the DBD and OB domains binding to the minor groove on both sides of the

nick, thus positioning the catalytic domain for catalysis. It was suggested that binding to PCNA

facilitates the transition from extended to closed structure (57).

The Role of Proliferating Cell Nuclear Antigen in

Okazaki Fragment Maturation

The enzymes required for Okazaki fragment maturation, including DNA polymerase, Fen1, and

DNA ligase, interact with PCNA via a PIP motif, and interaction with PCNA modulates their

enzymatic activities (60, 170). These interactions suggest that PCNA coordinates the Okazaki

fragment maturation process by sequentially recruiting factors to the lagging strand.

88 Kelman ·Kelman

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In crenarchaea, PCNA is a heterotrimer and each subunit of PCNA has speciﬁcity for binding to

DNA polymerase, Fen1, or DNA ligase (8). This speciﬁcity is likely to have arisen from differences

in the structure of the IDCL among the three different PCNA proteins (175), enabling them to

discriminate between small differences in the PIP motifs of the different interacting enzymes. The

simultaneous binding of all three proteins to PCNA provides an efﬁcient coupling of the three

enzyme activities. Upon ﬂap formation by a lagging-strand polymerase, Fen1 cleaves the ﬂap, the

gap is ﬁlled by the polymerase, and the Okazaki fragments are joined by DNA ligase.

PCNA proteins from all other archaeal species, however, form homotrimeric rings (131).

Therefore, it is not clear whether the role of PCNA in regulating Okazaki fragment maturation

in these species is similar to that in Crenarchaea. Although DNA polymerase, Fen1, and DNA

ligase all interact with PCNA, it is not clear whether they can bind simultaneously.

TERMINATION OF DNA REPLICATION AND

PRODUCT RESOLUTION

At the end of chromosomal replication, converging replication forks must complete replication

and the replisomes must dissociate from the DNA. It is essential that converging forks not pass each

other and continue replication, as this results in over-replication of part of the chromosome. In

Bacteria, termination occurs in a speciﬁc chromosomal region referred to as the termination region

(ter). The speciﬁc ter-binding protein Tus participates in the termination process by regulating

replisome movement, ensuring precise replication of the chromosome (146).

Limited information is available on the mechanism of replication termination in Archaea. The

circular nature of archaeal chromosomes may suggest a mechanism similar to bacterial termination,

but a study using S. solfataricus suggested that termination occurs by random collision of the two

replication forks and not at a speciﬁc site (37). In addition, the presence of ter-like sequences or

Tus-like proteins has not yet been reported in Archaea.

Bacterial chromosomes contain dif (deletion induced ﬁlamentation) regions near ter,where

concatemers formed during chromosome segregation are resolved by the Xer site-speciﬁc re-

combinases (XerC and XerD) in conjunction with FtsK (146). Only a few studies on archaeal

chromosome resolution have been published. Archaeal genomes contain a dif-like sequence and

Xer homologs (26, 37), but the location of the dif sequence in the chromosome is different in

different species. In some species the dif region is located in the termination zone (26), but it is

not near the termination region in others (37). The results to date, although limited, suggest that

Archaea may use a bacterial-like mechanism for chromosome resolution.

CONCLUDING REMARKS

Much progress has been made in understanding archaeal DNA replication, but there remains

much to be elucidated. Many factors have been identiﬁed and characterized, and the three-

dimensional structures of most proteins have been determined. New genetic tools (38) have

identiﬁed new proteins that may be involved in replication. These genetic tools, together with

ﬂuorescence and single-molecule approaches, should lead to new and exciting studies on the

replication process in vivo.

Future studies will concentrate on poorly understood aspects of the replication process. The

mechanism that regulates the initiation process is unknown, and the coordination between ini-

tiation and other cell-cycle events is yet to be explored. Similarly, the study of termination and

chromosome segregation is in its infancy and more research is needed.

The development of an in vitro replication system is one of the goals of future research.

Replication in bacteria, viruses, and phages was characterized after in vitro replication systems

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were developed, and it is anticipated that such a system will similarly serve as a catalyst for the

understanding of archaeal replication.

DISCLOSURE STATEMENT

The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that

might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank Drs. Jerard Hurwitz, Satish Nair, and Thomas Santangelo for sharing data prior to

publication. We thank Dr. Debra Weinstein for help with ﬁgures. We wish to apologize to

colleagues whose primary work was not cited because of space limitations.

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Annual Review of

Genetics

Volume 48, 2014

Contents

L1 Retrotransposons and Somatic Mosaicism in the Brain

Sandra R. Richardson, Santiago Morell, and Geoffrey J. Faulkner ppppppppppppppppppppppppp1

Factors Underlying Restricted Crossover Localization

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pENCODE: A Plant Encyclopedia of DNA Elements

Amanda K. Lane, Chad E. Niederhuth, Lexiang Ji, and Robert J. Schmitz pppppppppppppp49

Archaeal DNA Replication

Lori M. Kelman and Zvi Kelman pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp71

Molecular Genetic Dissection of Quantitative Trait Loci Regulating

Rice Grain Size

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Exploring Developmental and Physiological Functions of Fatty Acid

and Lipid Variants Through Worm and Fly Genetics

Huanhu Zhu and Min Han pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp119

Quality Control and Inﬁltration of Translation by Amino Acids

Outside of the Genetic Code

Tammy Bullwinkle, Beth Lazazzera, and Michael Ibba pppppppppppppppppppppppppppppppppp149

Vulnerabilities on the Lagging-Strand Template: Opportunities for

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Ashwana D. Fricker and Joseph E. Peters ppppppppppppppppppppppppppppppppppppppppppppppppp167

Self-Organization of Meiotic Recombination Initiation: General

Principles and Molecular Pathways

Scott Keeney, Julian Lange, and Neeman Mohibullah pppppppppppppppppppppppppppppppppppp187

Cancer: Evolution Within a Lifetime

Marco Gerlinger, Nicholas McGranahan, Sally M. Dewhurst, Rebecca A. Burrell,

Ian Tomlinson, and Charles Swanton ppppppppppppppppppppppppppppppppppppppppppppppppppp215

Diverse Epigenetic Mechanisms of Human Disease

Emily Brookes and Yang Shi pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp237

From Egg to Gastrula: How the Cell Cycle Is Remodeled During the

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Jeffrey A. Farrell and Patrick H. O’Farrell ppppppppppppppppppppppppppppppppppppppppppppppp269

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Cellular and Molecular Mechanisms of Single and Collective Cell

Migrations in Drosophila: Themes and Variations

Shirin M. Pocha and Denise J. Montell pppppppppppppppppppppppppppppppppppppppppppppppppppp295

The Structure and Regulation of Flagella in Bacillus subtilis

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Transcription-Associated Mutagenesis

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Gastrointestinal Microbiota–Mediated Control of Enteric Pathogens

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The Relations Between Recombination Rate and Patterns of Molecular

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The Genetics of Neisseria Species

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Regulation of Transcription by Long Noncoding RNAs

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Centromeric Heterochromatin: The Primordial Segregation

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Messenger RNA Degradation in Bacterial Cells

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The Archaeal Legacy of Eukaryotes: A Phylogenomic Perspective

Article

Full-text available

Jul 2014

The origin of the eukaryotic cell can be regarded as one of the hallmarks in the history of life on our planet. The apparent genomic chimerism in eukaryotic genomes is currently best explained by invoking a cellular fusion at the root of the eukaryotes that involves one archaeal and one or more bacterial components. Here, we use a phylogenomics approach to reevaluate the evolutionary affiliation between Archaea and eukaryotes, and provide further support for scenarios in which the nuclear lineage in eukaryotes emerged from within the archaeal radiation, displaying a strong phylogenetic affiliation with, or even within, the archaeal TACK superphylum. Further taxonomic sampling of archaeal genomes in this superphylum will certainly provide a better resolution in the events that have been instrumental for the emergence of the eukaryotic lineage.

Characterization of Family D DNA polymerase from Thermococcus sp. 9°N

Article

Full-text available

May 2014
EXTREMOPHILES

Accurate DNA replication is essential for maintenance of every genome. All archaeal genomes except Crenarchaea, encode for a member of Family B (polB) and Family D (polD) DNA polymerases. Gene deletion studies in Thermococcus kodakaraensis and Methanococcus maripaludis show that polD is the only essential DNA polymerase in these organisms. Thus, polD may be the primary replicative DNA polymerase for both leading and lagging strand synthesis. To understand this unique archaeal enzyme, we report the biochemical characterization of a heterodimeric polD from Thermococcus. PolD contains both DNA polymerase and proofreading 3′–5′ exonuclease activities to ensure efficient and accurate genome duplication. The polD incorporation fidelity was determined for the first time. Despite containing 3′–5′ exonuclease proofreading activity, polD has a relatively high error rate (95 × 10−5) compared to polB (19 × 10−5) and at least 10-fold higher than the polB DNA polymerases from yeast (polε and polδ) or Escherichia coli DNA polIII holoenzyme. The implications of polD fidelity and biochemical properties in leading and lagging strand synthesis are discussed. Electronic supplementary material The online version of this article (doi:10.1007/s00792-014-0646-9) contains supplementary material, which is available to authorized users.

The archaeal DNA replication machinery: past, present and future

Article

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Dec 2013
GENES GENET SYST

Living organisms are divided into three domains: Bacteria, Archaea, and Eukarya. Whereas Bacteria and Archaea are both prokaryotes, proteins involved in information processes; replication, transcription, and translation, are more similar in Archaea and Eukarya. Here the history of the research on archaeal DNA replication is summarized and the future of the field is discussed.

Global Phylogenomic Analysis Disentangles the Complex Evolutionary History of DNA Replication in Archaea

Article

Full-text available

Jan 2014

The archaeal machinery responsible for DNA replication is largely homologous to that of eukaryotes and is clearly distinct from its bacterial counterpart. Moreover, it shows high diversity in the various archaeal lineages, including different sets of components, heterogeneous taxonomic distribution, and a large number of additional copies that are sometimes highly divergent. This has made the evolutionary history of this cellular system particularly challenging to dissect. Here, we have carried out an exhaustive identification of homologs of all major replication components in over 140 complete archaeal genomes. Phylogenomic analysis allowed assigning them to either a conserved and probably essential 'core' of replication components that were mainly vertically inherited, or to a variable and highly divergent 'shell' of extra copies that have likely arisen from integrative elements. This suggests that replication proteins are frequently exchanged between extra-chromosomal elements and cellular genomes. Our study allowed clarifying the history that shaped this key cellular process (ancestral components, horizontal gene transfers, gene losses), providing important evolutionary and functional information. Finally, our precise identification of core components permitted to show that the phylogenetic signal carried by DNA replication is highly consistent with that harbored by two other key informational machineries (translation and transcription), strengthening the existence of a robust organismal tree for the Archaea.

An Extended Network of Genomic Maintenance in the Archaeon Pyrococcus abyssi Highlights Unexpected Associations between Eucaryotic Homologs

Article

Full-text available

Nov 2013
PLOS ONE

In Archaea, the proteins involved in the genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of eukaryotes. Characterizations of components of the eukaryotic-type replication machinery complex provided many interesting insights into DNA replication in both domains. In contrast, DNA repair processes of hyperthermophilic archaea are less well understood and very little is known about the intertwining between DNA synthesis, repair and recombination pathways. The development of genetic system in hyperthermophilic archaea is still at a modest stage hampering the use of complementary approaches of reverse genetics and biochemistry to elucidate the function of new candidate DNA repair gene. To gain insights into genomic maintenance processes in hyperthermophilic archaea, a protein-interaction network centred on informational processes of Pyrococcus abyssi was generated by affinity purification coupled with mass spectrometry. The network consists of 132 interactions linking 87 proteins. These interactions give insights into the connections of DNA replication with recombination and repair, leading to the discovery of new archaeal components and of associations between eucaryotic homologs. Although this approach did not allow us to clearly delineate new DNA pathways, it provided numerous clues towards the function of new molecular complexes with the potential to better understand genomic maintenance processes in hyperthermophilic archaea. Among others, we found new potential partners of the replication clamp and demonstrated that the single strand DNA binding protein, Replication Protein A, enhances the transcription rate, in vitro, of RNA polymerase. This interaction map provides a valuable tool to explore new aspects of genome integrity in Archaea and also potentially in Eucaryotes.

DNA replication

Article

Jan 1992

Introduction and Historical Perspective

Article

Aug 2011

Patrick Forterre

The discoveries of DNA supercoiling and DNA topoisomerases have been two of the most important breakthroughs in biology in the last century. Negative supercoiling is a critical feature of bacterial genomes and transient supercoiling is produced in all organisms by DNA binding proteins and/or various DNA tracking processes. DNA topoisomerases are major elements in cellular life and the plethora of natural antibiotics and antitumor drugs that target these enzymes testify for their importance. DNA topoisomerases I and II, catalyzing DNA strand transfer via single or double-strand breaks in DNA molecules respectively, originated and evolved to solve topological problems raised by the plectonemic coiling of two DNA strands in the double helix, and specialized topoisomerases acquired the ability to produce supercoiling. The first DNA topoisomerases were discovered in Escherichia coli (protein ω, DNA gyrase, Topo III, and Topo IV) and eukaryotic cells (Topo IB, Topo II, Topo III). Later on, new families and subfamilies of DNA topoisomerases were discovered in Archaea, the third domain of life (reverse gyrase, Topo V, Topo VI), challenging the prokaryote/eukaryote dichotomy. DNA topoisomerases are now classified into five families of homologous proteins (Topo IA, IB, IC, Topo IIA, IIB) based on structural similarities. These families have been divided into subfamilies, some of them characterized by unique enzymatic properties (gyrase, reverse gyrase). The distribution of these families and subfamilies do not overlap with the universal tree of life, and some subfamilies are specific for viruses. This suggests that viruses played an important role in the origin and distribution of DNA topoisomerases among cellular organisms.

pENCODE: a plant encyclopedia of DNA elements

Article

Aug 2014

ENCODE projects exist for many eukaryotes, including humans, but as of yet no defined project exists for plants. A plant ENCODE would be invaluable to the research community and could be more readily produced than its metazoan equivalents by capitalizing on the preexisting infrastructure provided from similar projects. Collecting and normalizing plant epigenomic data for a range of species will facilitate hypothesis generation, cross-species comparisons, annotation of genomes, and an understanding of epigenomic functions throughout plant evolution. Here, we discuss the need for such a project, outline the challenges it faces, and suggest ways forward to build a plant ENCODE. Expected final online publication date for the Annual Review of Genetics Volume 48 is November 23, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

The solution structure of full-length dodecameric MCM by SANS and molecular modeling

Article

Oct 2014
PROTEINS

The solution structure of the full-length DNA helicase minichromosome maintenance (MCM) protein from Methanothermobacter thermautotrophicus was determined by small-angle neutron scattering (SANS) data together with all-atom molecular modeling. The data were fit best with a dodecamer (dimer of hexamers). The twelve monomers were linked together by the B/C domains, and the ATPase (AAA+) catalytic regions were found to be freely movable in the full-length dodecamer both in the presence and absence of Mg2+ and 50-meric ssDNA. In particular, the SANS data and molecular modeling indicate that all twelve AAA+ domains in the dodecamer lie approximately the same distance from the axis of the molecule, but the positions of the helix-turn-helix region at the C-terminus of each monomer differ. In addition, the A domain at the N-terminus of each monomer is tucked up next to the AAA+ domain for all twelve monomers of the dodecamer. Finally, binding of ssDNA does not lock the AAA+ domains in any specific position, which leaves them with the flexibility to move both for helicase function and for binding along the ssDNA. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.

A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein

Article

Apr 2014
NUCLEIC ACIDS RES

Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA.

Archaeal DNA replication

Article

November 2014

Lori M. Kelman · Zvi Kelman

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