Molecular Biology replication

1. Current Protein and Peptide Science, 2000, 1, 139-154 139
1389-2037/00 $25.00+.00 © 2000 Bentham Science Publishers Ltd.
DNA Replication in the Third Domain (of Life)
Zvi Kelman*
Department of Molecular Biology, Memorial Sloan-Kettering Cancer Center, 1275 York
Avenue/Box 97, New York, NY 10021, USA
Abstract: DNA replication is the process underlying evolution and the propagation
of living organisms. Since the discovery of DNA-dependent DNA polymerases more
than 40 years ago, the mechanisms governing DNA replication have been
extensively studied in bacteria and eukarya. During the last several years, these
studies have been extended to the third domain of life, the archaea. Although archaea
are prokaryotes, their replication machinery and the proteins participating in the
initiation of DNA replication are more similar to those found in eukarya than bacteria. It appears,
however, that replication in archaea is a simpler version of the eukaryotic one as fewer polypeptides
participate in each phase of the replication process. The archaeal replication apparatus also has several
unique features not found in eukaryotic organisms. Furthermore, like bacteria, members of this domain
thrive under a broad range of environmental conditions including extreme temperature, high salt, pH, etc.
Thus, the replication machinery had to adapt to these extreme conditions. This article summarizes our
current understanding of the mechanisms governing DNA replication in archaea and highlights similarities
and differences between archaeal replication and that of bacteria and eukarya.
INTRODUCTION
For many decades, living organisms were
thought to be divided into two domains, eubacteria
and eukaryotes. In 1977, Woese and Fox suggested
that a third domain of life exists, the archaebacteria
[1]. Although only a few members of this domain
were known at that time, the distinct form of their
ribosomal RNAs (rRNA) and other cellular features
were the basis for their designation as a unique
domain [1,2]. In 1990, Woese and colleagues
suggested changing the names of the three domains
to eukarya (formally eukaryotes), bacteria
(eubacteria) and archaea (archaebacteria) [3]. The
reader is referred to several reviews published on the
biochemical and genetic characteristics of archaea
that led to their recognition as an independent
domain and the history of these studies [2,4,5].
The archaea domain can be divided into three
kingdoms: euryarchaeota, crenarchaeota and
korarchaeota (Fig. 1) [6]. Euryarchaeota, the most
diverse group, contains members that live in a broad
range of environmental conditions and include
hyperthermophiles, methanogens, halophiles and
*Address correspondence to this author at Department of Molecular
Biology, Memorial Sloan-Kettering Cancer Center, 1275 York
Avenue/Box 97, New York, NY 10021, USA; Phone: (212) 639-5895; Fax:
(212) 717-3627; E-mail: z-kelman@ski.mskcc.org
thermophilic methanogens. Crenarchaota is less
diverse, and its members include hyperthermophiles,
psychophiles and thermoacidophiles. To date only a
few members of the korarchaeota kingdom have
been identified and the extent of their diversity is not
yet clear.
In archaea the proteins that participate in DNA
replication, transcription and translation (so called
information processes) are more closely related to
eukaryotic proteins than to those found in bacteria.
This conclusion is based in large part on the amino
acid sequences of several archaea for which the
complete genome are known; Methanococcus
jannaschii [7], Archaeoglobus fulgidus [8],
Methanobacterium thermoautotrophicum ∆H [9],
Pyrococcus horikoshii OT3 [10] and Aeropyrum
pernix K1 [11]), and the properties of many of the
replication proteins that have been isolated.
This review aims to summarize our current
understanding of archaeal DNA replication and the
individual proteins participating in the process. It
will also discuss the similarities and differences
between DNA replication in archaea and eukarya.
For similarities in the transcription and translation
systems between archaea and eukarya, the reader is
referred to several recent reviews on these subjects
[12-17]. Because the termination of DNA
replication has not been studied in archaea or

2. 140 Current Protein and Peptide Science, 2000, Vol. 1, No. 2 Zvi Kelman
eukarya, this review will concentrate only on the
initiation and elongation stages.
DNA REPLICATION: AN OVERVEIW
DNA replication is the basis for the propagation
and evolution of living organisms. The mechanism
of DNA replication ensures the duplication and
transfer of genetic information during cell division.
In all organisms, DNA replication is a complex
event involving dozens of proteins and enzymes to
ensure the accurate duplication of the genetic
information. The mechanism that underlies the
DNA replication process is functionally (and often
structurally) conserved from bacteria to mammals
(reviewed [18-20]). In an overview, the process
starts at a specific sequence called an origin of
replication at which proteins bind and locally
unwind the duplex DNA allowing invasion of a
helicase. The helicase hydrolyzes ATP to melt the
duplex, and then the exposed single stranded DNA
(ssDNA) is coated with ssDNA binding protein
(SSB). The SSB-ssDNA nucleofilament lacks
secondary structure and serves as the template for
the replicase. Chromosomal replicases are
multiprotein machines, characterized by rapid and
highly processive DNA synthesis, that are
responsible for the replication of chromosomal
DNA. One strand of the chromosome is
synthesized continuously (leading strand), but due
to the antiparallel nature of the duplex and the
unidirectional movement of the polymerase, the
other strand is copied discontinuously (lagging
strand) as a series of fragments (Okazaki
fragments). This discontinuous mode of replication
requires frequent reinitiation of DNA chains, which
are primed by short RNA primers synthesized by a
primase. Only one primer is needed on the leading
strand. The RNA primer is recognized by the
replicase accessory complex which assembles a
ring shaped protein, the processivity factor (also
referred to as a sliding clamp), around the primer.
The clamp on DNA binds to the polymerase
catalytic unit and tethers it to the DNA for rapid and
processive DNA synthesis. Several proteins
participate in connecting the Okazaki fragments to
form a consecutive duplex DNA.
ARCHAEAL ORIGIN OF REPLICATION
Sites for the initiation of chromosomal DNA
replication are quite different between bacteria and
Fig. (1). Universal phylogenetic tree in rooted form. Branch lengths do not represent exact evolutionary distances, but the
branching order is correctly represented. Adapted from [133].

3. DNA Replication Current Protein and Peptide Science, 2000, Vol. 1, No. 2 141
eukarya. In bacteria, initiation occurs at a unique
site, oriC (250bp region), and the two forks emanate
from this site in opposite directions (reviewed in
[21]). In eukarya, on the other hand, initiation takes
place at multiple sites along the genome (reviewed in
[22]). Defined origins have been identified in
bacteria, bacteriophages and viruses (reviewed in
[23]). In eukaryotes, to date, defined origins have
been documented only in yeasts, and have a length
of 100-1000bp. Thus when the complete genome
sequences for several archaea were determined it
was expected that an archaeal chromosomal origin
would be identified readily using a search for
similarities to known bacterial, eukaryal and viral
origins. However, this has not been the case and an
unambiguous bona fide chromosomal origin has not
yet been identified in archaea.
Origin regions have been identified, however, in
several archaeal plasmids and viruses. These include
an origin from the Halobacterium salinarium
plasmid pHH1 [24], Halobacterium halobium
plasmid pNRC100 [25], Methanococcus
maripaludis plasmid pURB500 [26], Haloferax
plasmid pHK2 [27] and a Sulfolobus virus SSV1
[28]. No common sequences, however, have been
identified among these origins. This is reminiscent
of the eukaryal origins identified in Saccharomyces
cerevisiae which failed to identify a common
sequence element present in the origins of
Schizosaccaromyces pombe. Furthermore, in
bacteria, plasmid origins are different from those
that constitute oriC, and thus it is not surprising that
archaeal plasmid and viral origin sequences have
failed to help define the archaeal chromosomal
origin.
The inability to archaea has led to numerous
hypotheses regarding archaeal origin sequences and
the mode of replication. It was not clear whether
replication is uni- or bidirectional [29], or if archaea
replicate chromosomal DNA in a rolling circle-like
fashion. The latter mechanism was demonstrated for
several archaeal plasmids [30,31].
Recently, however, putative origins have been
identified in archaea. In some bacteria, careful
examination of the variation of the base composition
revealed an excess of G over C in the leading strand,
permitting the detection of an origin by plotting the
skew in the GC content along the genome [32].
Similar and improved methods (using four base best
words) have been recently applied for the analysis
of archaeal genomes. These in
silico considerations suggested a single putative
origin for M. jannaschii, M. thermoautotrophicum,
P. horikoshii, and Pyrococcus furiosus [33-36].
Interestingly, these regions are located close to the
gene encoding CDC6, a protein involved in initiation
of DNA replication (discussed below). The putative
origins range in size from 700-1000 bp and thus
many resemble eukaryotic origins. Only future in
vitro and in vivo studies will determine whether
these putative origins, or other regions of the
chromosomes, function as authentic origins.
THE INITIATION PHASE OF DNA
REPLICATION
Initiation of bacterial and eukaryal replication
can be divided into three steps. The first step
involves the binding of one or more origin
recognition proteins to the origin of replication.
These proteins are responsible for the regulation of
the initiation timing and promote the initiation
process by recruiting the replication machinery to
the origin. In the second step, DNA unwinding
begins at easily unwound sites within the origin
(usually A-T rich sequences) forming a replication
bubble. The origin recognition complex of proteins
may contribute to the unwinding process by
distorting the DNA, but in conjunction with other
proteins they lead to the recruitment of the helicase
to unwind the duplex. In the third step the replicative
polymerase is brought to the replication bubble and
bidirectional DNA synthesis is initiated.
Homologues of proteins participating in the
initiation of replication in eukaryotic cells have been
identified in archaea. Interestingly, each archaeal
species has a slightly different subset of these
proteins. This is different from the observation in
eukarya in which the homology and structure of the
initiation proteins of different organisms from yeast
to human are similar. This difference between
archaea and eukarya is probably due to the greater
genetic diversity among the various species in the
archaea domain.
The isolation and biochemical characterization of
archaeal initiation proteins has begun only recently.
Therefore, in the following section the proteins and
complexes required for the initiation process will be
discussed only briefly. For a comprehensive review
on the known function of the initiation proteins in
eukaryotic cells, the reader is referred to several
reviews on the subject [22,37,38].

4. 142 Current Protein and Peptide Science, 2000, Vol. 1, No. 2 Zvi Kelman
Origin Recognition Complex (ORC)
Based on genetic and biochemical studies, the
origin recognition complex (ORC) is believed to be
the eukaryotic factor that binds to the origin and is a
key element responsible for the initiation process
(reviewed in [37,38]). Since its first identification
and isolation from the yeast S. cerevisiae [39],
homologues of the ORC complex have been
identified and isolated from a wide range of
eukaryotic organisms. The complex contains six
distinct subunits that are named according to their
size (ORC1-6), with the largest subunit referred to
as ORC1.
In archaea, to date, homologues of ORC1 have
been identified only in the genome of P. furiosus
and in a plasmid from M. thermoformicicum. No
homologues of other ORC subunits have been
identified. Several archaea, however, have
homologues of the CDC6 protein (discussed
below). CDC6 shows sequence similarities to
ORC1 and thus may assume the functions of the
ORC protein. In the future, however, when more
archaeal sequences become available, new ORC
homologues may be identified. Nevertheless, only
ORC1 or CDC6 homologues have been identified
to date in archaea. There are several possibilities that
may explain the observed differences between
archaea and eukarya ORC. Different origins may
require different initiation processes. Archaea may
have other proteins (previously unknown) that are
not found in eukaryotes that associate with the
ORC1 homologue and participate in the initiation
process. Alternatively, the single ORC-like subunit
may oligomerize, making it more similar to the
multimeric eukaryotic origin recognition complex.
Future studies, including the isolation and
biochemical characterization of an archaeal ORC1
homologue, should shed light on its role in the
initiation of DNA replication in archaea.
CDC6 Protein
cdc6 was originally identified in yeast as a gene
needed for cell cycle progression in a screen
designed to identify genes encoding proteins needed
during the cell cycle [40]. The proteins identified in
this screen are called cell division cycle (CDC)
proteins. Since its identification in S. pombe and S.
cerevisiae, CDC6 homologues have been identified
in many eukaryotic organisms. Genetic and
biochemical studies, conducted mainly in the yeasts,
suggest an essential role for CDC6 in early S phase
prior to the initiation of DNA replication. The
CDC6 gene is cell cycle regulated with peak
expression during the G1 phase of the cell cycle
prior to the initiation of DNA replication. The
protein is rapidly degraded at the beginning of S
phase immediately after the initiation of DNA
replication. It is believed that CDC6, in conjunction
with ORC, regulates the timing of initiation and
participates in the loading of the helicase onto the
DNA.
CDC6 homologues have been identified in
several archaea. The number of CDC6 related
proteins, however, differs widely between the
different species. For example, in the genome of M.
thermoautotrophicum, two CDC6-related proteins
have been identified whereas none is found in the
genome of M. jannaschii. The reason for these
differences and their effect on the organism cell
cycle and the initiation process are currently
unknown.
Minichromosome Maintenance (MCM)
Proteins
The minichromosome maintenance (MCM)
genes encode a family of proteins first identified by
their essential role in the maintenance of ARS
containing plasmids in S. cerevisiae [38,41]. MCM
homologues have been identified in all eukaryotes
from yeast to mammals and have highly conserved
amino acid sequences. The MCM family consists of
at least six distinct polypeptides (MCM2-7), and it
was demonstrated that complexes containing
different polypeptide compositions can be formed
within the cell [42]. Although the proteins play an
important role in the initiation of DNA replication as
well as during the elongation phase, the precise
functions of the MCM complexes during DNA
replication are not yet fully understood. Several
observations, however, have been made regarding
their function. A hexameric complex composed of a
dimer of a heterotrimer consisting of MCM4, 6 and
7, has been shown to possess ATP-dependent DNA
helicase activity both in human [43] and in S. pombe
[43a]. The complex was also shown to posses a
DNA dependent ATPase activity and to bind single
stranded DNA. The helicase activity observed with
the complex of MCM4, 6 and 7 can be inhibited in
vitro by MCM2. Thus, although there are six MCM
homologues in eukaryotic cells, they may assume
different functions. MCM2 may have a regulatory
role. In contrast to replicative helicases from
bacteria, the helicase activity associated with the
MCM4, 6 and 7 complex has a 3' to 5' polarity
similar to that of Simian Virus 40 (SV40) large T
antigen (T-Ag) [43,43a]. The discovery of a helicase
activity with properties similar to those of a
eukaryotic replicative helicase, together with the

5. DNA Replication Current Protein and Peptide Science, 2000, Vol. 1, No. 2 143
genetic studies demonstrating the important role of
MCMs in DNA replication, suggest that a subset of
the MCM proteins may function as the replicative
helicase recruited to the origin by ORC and CDC6.
At least one MCM homologue has been
identified in all sequenced archaeal genomes.
Similar to the findings with CDC6, however, the
number of individual MCM genes vary between
species. For example, in M. jannaschii four putative
homologues have been identified whereas only one
has been identified in others.
Studies with the archaeal MCM, however, have
only recently been initiated. Experiments carried out
with the single MCM protein homologue from the
archaeon M. thermoautotrophicum revealed that it
forms a double hexamer with a ring shaped
structure [43b]. This structure is similar to that of
DNA helicases isolated from bacteria and viruses. It
was also demonstrated that the archaeal protein
possesses DNA-dependent ATPase and DNA
helicase activities [43b,43c]. These results support
the notion that the archaeal MCM may be the
replicative helicase.
THE ELONGATION PHASE OF DNA
REPLICATION
Following the opening and unwinding of the
duplex DNA at the origin, the replicase is recruited
Table 1. Replication Proteins in Bacteria (E. coli), Eukarya (Human and Yeast) and Archaea
Bacteria Eukarya Archaea
Origin recognition DnaA
(one subunit)
Origin recognition complex (ORC)
(six subunits, ORC1-6)
ORC1 homologue
(not in all archaea)
Helicase loader DnaC
(one subunit)
CDC6
(one subunit)
CDC6 homologue
(zero to several homologues)
Replicative helicase DnaB
(one subunit)
MCM (?)
(six subunits)1
MCM homologue
(one to four homologues)
Topoisomerase Type I and II
Reverse gyrase2
Type I and II Type I and II
Reverse gyrase2
Single stranded DNA
binding protein (SSB)
SSB
(one subunit)
Replication protein A (RPA)
(three subunits)
RPA homologue
(one subunit)
Primase DnaG
(one subunit)
polα/primase complex
(four subunits)
primase homologue
Polymerase/
exonuclease
PolIII core
(three subunits, α, ε, θ)
Polδ
(three or four subunits)3
Polε
(at least five subunits)5
Family B DNA
polymerase
DP2-like polymerase (?)4
Clamp loader γ-complex
(five subunits,γ,δ,δ',χ,ψ)
RFC
(five subunits, RFC1-5)
RFC homologue
(two subunits, RFC1, RFC3)
Sliding clamp β PCNA PCNA homologue
Removal of primers PolI
RNase H
FEN-1
RNase H
FEN-1 homologue
RNase H homologue
Lagging strand
maturation
DNA ligase
(NAD-dependent)
DNA ligase I
(ATP-dependent)
DNA ligase I homologue
(ATP-dependent)
1 It is not yet clear if MCMs form the replicative helicase (see text for details)
2 Only in thermophilic organisms (see text for details)
3 In human and S. cervesiae polδ contains three subunits while in S. pombe four subunits have been identified.
4 It is not yet clear if DP2-like polymerases are replicative (see text for details)
5 Not all the subunits of polε have been identified.

6. 144 Current Protein and Peptide Science, 2000, Vol. 1, No. 2 Zvi Kelman
to the replication bubble to initiate rapid and
processive bidirectional DNA synthesis.
Chromosomal replicases are multiprotein complexes
that synthesize thousands of nucleotides without
dissociating from the DNA template. The replicases
of bacteria and eukarya appear to be similar in
function, overall organization, and structure
(reviewed in [18,20]). The high processivity of the
replicases is achieved by a ring shaped factor
("sliding clamp") that encircles DNA and upon
binding the polymerase catalytic unit and tethers it
to the DNA. The sliding clamp alone has no affinity
for DNA and other proteins are needed to assemble
the clamp around the DNA. The accessory protein
complex ("clamp loader"; also referred to as a
"molecular matchmaker") binds to the primer
terminus and couples ATP hydrolysis to the
assembly of the ring around the DNA primer.
Therefore, the replicase can be considered to
possess three components: the catalytic unit that
contains the DNA polymerase and proofreading 3'-
5' exonuclease activities, and two types of
polymerase accessory proteins, a clamp loader
complex that assembles the clamp around DNA and
a DNA sliding clamp. Replicases are aided by
several other proteins to accomplish the replication
and maturation of a duplex DNA. These include the
topoisomerases that relieve tension created in the
duplex DNA in front of and behind the replication
fork by the action of the helicase, polymerase, and
SSB, that coats exposed ssDNA regions formed
following strand separation by the helicase. DNA
ligase, RNase H and Fen-1 are required for
maturation of Okazaki fragments.
The most studied component of the archaeal
DNA replication complex has been the DNA
polymerases. These studies were stimulated after the
importance of thermostable enzymes for the
polymerase chain reaction (PCR) was determined.
Many DNA polymerases have been identified and
isolated from different species and their biochemical
properties have been extensively characterized.
Recently, however, several studies on the
polymerase accessory proteins, SSB,
topoisomerases and proteins that support Okazaki
fragment maturation have also been reported. These
studies demonstrated that, as is the case with the
initiation phase, the elongation phase of DNA
replication in archaea is more similar to eukarya
than to bacteria (Table 1). In the following section,
the properties of proteins participating in the
elongation phase of DNA replication in archaea are
summarized.
DNA Polymerases
DNA replication is achieved by a DNA-
dependent DNA polymerase (pol) that uses single
stranded DNA as a template to synthesize the
complementary strand [19]. Most organisms
possess several DNA polymerases that differ in
their catalytic properties such as processivity,
fidelity and rate of chain extension. Different
polymerases are used for replication, repair and
recombination, and have distinct polypeptide
compositions. For example, the polymerases that
replicate the chromosomes of bacteria and eukarya
are multisubunit complexes, while those involved in
repair are composed of only one or a few subunits
[19]. It was also shown that the replicative
polymerase forms a dimer for the simultaneous
replication of the leading and lagging strand [44-
48]. Dimeric structures are not required of enzymes
that participate in DNA repair. There are unique
DNA polymerases that replicate the DNA of
organelles (mitochondria and chloroplasts). Based
on their amino acid sequences, DNA polymerases
can be classified into at least five distinct groups
[49-51]. Type (or family) A polymerases are named
for their homology to Escherichia coli polI and
include eubacterial, mitochondrial (polγ ) and
bacteriophage polymerases. Type-B polymerases
are named for their homology to E. coli polII. This
family is more diverse than family A; it includes
bacterial, bacteriophage, archaea, and viral
polymerases and the catalytic subunits of the
eukarya replicative polymerases polα, polδ and polε.
The eubacterial replicative polymerase (polIII,
DnaE) is the prototype of the type-C group, and the
type-X group includes proteins with homology to
the eukaryal β polymerase involved in DNA repair
with some members also identified in bacteria and
archaea. A new group of polymerases, with little
homology to any of the above families, has been
identified in archaea [51,52]. This family is named
after the first member identified, the DP2
polymerase from P. furiosus. These five groups
appear only distantly related and members in each
group can be further subdivided by their function
and sequence similarities. In the following section
the different polymerases (divided by subgroups)
found in archaea are described.
B-Type Polymerases
Members of family B have been identified in all
archaea examined to date including at least one
member of this family in each of the archaea for
which a complete genome sequence is available.

7. DNA Replication Current Protein and Peptide Science, 2000, Vol. 1, No. 2 145
This observation makes this group the most diverse
in archaea. The number of B-type polymerases,
however, varies between the organisms [53]. In M.
jannaschii, for example, there is only one member
of the family [7] while in A. pernix there are three
[11]. All archaeal members of this family are similar
in amino acid sequence, domain organization and
overall three dimensional structure [54,55]. In
eukarya some members of family B lack 3'-5'
exonuclease activity (e.g. polα). All archaeal type-B
polymerases identified to date possess a potent 3'-5'
proofreading exonuclease activity [56-58]. The 3'-5'
exonuclease activity endows polymerases with high
fidelity thus making them more suitable for PCR
reactions compared to thermostable enzymes from
thermophilic bacteria which lack this activity [e.g.
Thermus aquaticus (Taq) polymerase].
Similar to B-type polymerases isolated from
eukarya and bacteria, the polymerases from different
species vary in their biochemical properties
(summarized in [50]). The differences includes
processivity, preferred templates for in vitro activity
(activated DNA, homopolymers, single stranded
plasmid DNA, etc.), inhibition by
dideoxynucleotides and Km for nucleotides and
DNA. Several of the biochemical properties are
widely diverse among archaeal polymerases. These
differences are, for the most part, due to the extreme
conditions under which many archaea grow. One
obvious and readily explained difference is the
temperature needed for optimal polymerase activity.
For example, thermophiles are expected to be more
active at higher temperatures compared to their
mesophile and psychrophile homologues that
denature under similar conditions. Similarly, the salt
requirements should also vary as several species
grow at salt concentrations that are as high as 5M
NaCl [59]. There are several examples, however,
where conditions for maximal enzymatic activity in
vitro differ from the optimal growth conditions for
the organism [50]. These differences may be the
result of the inability to mimic the conditions within
the cell that are required for its viability.
Another notable difference between archaeal and
eukarya B-type polymerases is the effect of
aphidicolin on their enzymatic activity. Eukaryal
replicative polymerases (polα, polδ and polε) are
sensitive to aphidicolin [19]. Early studies on
archaeal DNA replication demonstrated that like the
eukarya polymerases, the archaeal enzymes were
also sensitive to aphidicolin ([60-62] see also
[63,64]). In later studies, however, polymerases
that are aphidicolin resistant were identified [60,65-
69]. Based on these observations, it was assumed
that the latter polymerases belong to family A, which
is aphidicolin resistant. When the genes encoding
these enzymes were cloned and sequenced it became
apparent that both the aphidicolin resistant and
aphidicolin sensitive polymerases belong to family
B. Further studies demonstrated that a wide range of
aphidicolin sensitivity can be found among the B-
type polymerases from archaea (summarized in
[64]).
Several members of family B polymerases
contain inteins (protein introns). Inteins are internal
protein fragments within another protein. The extein
(the polypeptide portion that is retained after
splicing) and the intein are translated together as one
polypeptide chain. The intein is then excised post-
translationally while the exteins are spliced together
to form a mature extein protein. No additional
proteins are needed for the splicing event [70]. The
excised intein is also a stable protein and many
inteins have been shown to be homing-
endonucleases [71]. The endonuclease encoded by
the intein cuts the DNA at a specific site (the intein
integration site), resulting in a double strand break
in the allele that does not contain the intein. If the
intein sequence is present in the DNA, the
recognition site for the endonuclease is absent since
it is interrupted by the intein DNA. Recombination
between the DNA allele containing the intein
sequence and the double strand break on the other
allele can result in duplication of the intein sequence
in a homing fashion. For a more comprehensive
description of inteins, their discovery, function and
biochemical properties, the reader is referred to
several recent reviews on the subject [70-74].
Not all B-type polymerases from archaea contain
inteins. It was noted, however, that to date all
polymerases that contain inteins are from the
euryarchaeota kingdom. The number of inteins
present in each polymerase varies between species.
In several there is only one intein e.g. Pyrococcus
sp. GB-D [75], in others there are two e.g.
Thermococcus litoralis [76], M. jannaschii [7],
Pyrococcus kodakaraensis [77], and the polymerase
from Thermococcus sp. TY contains three inteins
[78]. In all polymerases studied,
inteins are located in highly conserved regions
within the polymerase catalytic unit. These
conserved motifs are known as regions I, II and III
[79] on the linear amino acid sequences of the
polymerase. The presence of inteins in these regions
prevents the enzyme from working unless they are
spliced out.

8. 146 Current Protein and Peptide Science, 2000, Vol. 1, No. 2 Zvi Kelman
Another unique feature of B-type polymerases
was identified in the enzyme isolated from M.
thermoautotrophicum. The polymerase catalytic
unit is split into two polypeptides encoded on two
different strands of DNA, 850kb apart on the M.
thermoautotrophicum chromosome [9]. Neither
subunit alone is active and both subunits must
associate to create an active polymerase [80].
Recently, the three dimensional structures of two
archaeal B-type polymerases were reported [54,55].
Although the two polymerases are from two
different organisms, Thermococcus gorgonarius
[54] and Desulfurococcus strain TOK [55], both
have similar structures. Their topology is also
similar to the structure of the B-type polymerase
from bacteriophage RB69 (gene product (gp) 43)
[81]. The overall similarities between the
thermophilic archaeal polymerases and the
mesophilic gp43 polymerase have revealed
structural features needed for thermostable
adaptation. These features include disulfide bonds
within the polymerase, clustering of positively
charged amino acid residues leading to an enhanced
electrostatic interaction between DNA and protein,
as well as closely packed loops.
An interesting structural feature identified in the
archaeal polymerase is the presence of a putative
RNA binding domain at the N-terminus [55]. The
three dimensional structures of several RNA
binding domains are almost superimposable with
the N-terminal region of the Desulfurococcus B-
type polymerase. Based on sequence similarities, it
was suggested that all archaeal B-type polymerases
have this domain and a similar DNA binding
domain is also found in eukaryotic polδ and polε
[55]. However, it is not yet clear whether this region
indeed binds RNA, and the biological significance
of the interactions between DNA polymerases and
RNA is unknown. Further studies are needed to
address these questions.
DNA polymerases are required for chromosomal
replication as well as for recombination and repair.
Thus, it is not clear whether members of the B-type
polymerases are the replicative polymerases or
needed for other processes. In the crenarchaeota the
answer is simple. To date, only members of the B-
type polymerases have been identified in organisms
from this kingdom based on the complete genome
sequence of A. pernix [11] and the isolation and
purification of individual polymerases from other
species. Do B-type polymerases also participate in
chromosomal replication in euryarchaeota? The
hallmark of replicative polymerases in eukarya and
bacteria is the stimulation of the polymerase by the
accessory proteins [18,20]. Homologues of the
eukaryotic accessory proteins, proliferating cell
nuclear antigen (PCNA) and replication factor C
(RFC) have been identified in all archaea studied to
date (discussed below). It was recently
demonstrated that the activity of a B-type
polymerase from the euryarchaeota M. thermoau-
totrophicum is stimulated by PCNA and RFC
[80,82]. PCNA was also shown to stimulate a B-
type polymerase from the crenarchaeota Sulfolobus
solfataricus [83]. Although the latter study was
performed in the absence of RFC (discussed
below), it is believed that RFC also participates in
the process. The stimulation of the polymerase by
these factors suggests that a B-type polymerase may
function as the replicative polymerase in
euryarchaeota. Alternatively, the B-type polymerases
in euryarchaeota may work in conjunction with the
DP2-like polymerase (discussed below) at the
replication fork.
DP2-Like Polymerases
Several years ago, a new family of DNA
polymerases was identified in archaea. This family
is named after the first member identified, the DP2
polymerase from P. furiosus [51]. Since their
identification in P. furiosus, homologues of DP1
and DP2 have been identified in all archaea from the
euryarchaeota kingdom for which the complete
genome sequence is available [52,84-86]. The
proteins from different organisms are also likely to
be similar in structure and function, as it was
demonstrated that the subunits of P. furiosus and
M. jannaschii are interchangeable for replication in
vitro [85]. Homologues of the DP2 polymerases
have not been identified in the complete genome of
A. pernix (crenarchaeota) [11]. This suggests that
the DP2 polymerase may be a lineage specific
enzyme.
Since the sequence of the DP2 polymerase is
poorly related to other DNA polymerases, it was not
initially identified as a polymerase. A unique
approach was used to identify this new family of
polymerases. A genomic library made from a
thermophilic organism (P. furiosus) was
transformed into E. coli cells. A cell extract was
analyzed for its ability to support in vitro DNA
replication at high temperatures in order to identify a
DNA fragment that encoded polymerase activity
[51]. Examination of the different open reading
frames on the cloned DNA fragment expressed in
E. coli identified the active polymerase as a dimer
of two distinct polypeptides, DP1 and DP2 [51].

9. DNA Replication Current Protein and Peptide Science, 2000, Vol. 1, No. 2 147
Based on sequence similarities and its
biochemical properties, it is believed that the
catalytic activity resides in the large subunit DP2,
and that DP1 serves as an accessory factor [84].
Only limited polymerase activity was detected in the
absence of DP1 [51]. Interestingly, it was recently
shown that the small subunit, DP1, has homology to
the small (non-catalytic) subunits of the eukaryotic
replicative polymerase; polα (p70 subunit), polδ
(CDC27) and polε (p55 subunit) [87].
Based on its biochemical properties (processivity,
3'-5' exonuclease activity) and the similarities
between DP1 and the small subunits of the
eukaryotic replicative polymerases, it was proposed
that the DP2 polymerase functions as the replicative
polymerase in euryarchaeota [51,84]. As described
above, in bacteria and eukaryotes the replicative
polymerases are stimulated by the accessory
proteins. To date, however, it has not been
demonstrated that the DP2 polymerase is stimulated
by archaeal RFC and PCNA. Such a stimulation
would strengthen the hypothesis that DP2 is indeed
involved in chromosomal replication. If the DP2
polymerase participates in chromosomal replication,
it probably does so in conjunction with a member of
the B-type polymerases, which have been shown to
be stimulated by RFC and PCNA (discussed above)
[80,83]. In such cases, one enzyme may replicate the
leading strand while the other replicates the lagging
strand.
Other Polymerases
To date, no member of the A family of DNA
polymerases has been identified in archaea. This is
similar to the case in eukarya where type-A
polymerases were shown to be essential only for
organelle replication such as mitochondrial polγ .
Members of family X have been identified so far in
only one archaeon, M. thermoautotrophicum [9].
Members of family X have also been identified in
some thermophilic bacteria (Aquifex and Thermus).
This observation led to the suggestion that genes of
this family of polymerases were transferred
between bacteria and archaea [88]. Thus, it is
conceivable that members of this family will be
identified in other archaea.
Polymerase Accessory Proteins
Proliferating Cell Nuclear Antigen (PCNA)
In bacteria and eukarya, processive DNA
synthesis involves a ring-shaped protein, called a
DNA sliding clamp, that encircles DNA and acts to
tether the polymerase catalytic unit to the DNA
template (reviewed in [89,90]). The three
dimensional structures of the eukaryotic (PCNA)
and prokaryotic (the β subunit of polIII
holoenzyme) sliding clamps have been determined
[91-93]. Although PCNA is a trimer and the β
subunit forms a dimer, the overall structure of these
clamps is similar; the two clamps are
superimposable and each ring has similar
dimensions and a central cavity large enough to
accommodate duplex DNA [89]. Both proteins can
also slide bidirectionaly on duplex DNA [94,95].
PCNA, a ubiquitous protein with a highly
conserved amino acid sequence, has been identified
in all eukaryotes from unicellular organisms to
humans [89]. Homologues of PCNA have also been
identified in eukaryotic viruses and in archaea
(discussed below). In eukarya, PCNA also
participates in processes other than chromosomal
replication including repair, recombination, post-
replication DNA metabolism, and cell cycle control
(summarized in [96]).
In all archaea where the complete genome
sequence is known, homologues of PCNA have
been identified. The amino acid sequence, length,
charge distribution and aggregation state (a trimer)
are all similar to those of PCNA isolated from
eukaryotic cells. In the euryarchaeota kingdom, one
PCNA homologue has been identified [7-10]. In
members of the crenarchaeota kingdom multiple
homologues of PCNA have been identified. In
Sulfolobus solfataricus and Pyrobaculum
aerophilum two homologues of PCNA have been
identified [83] while in the complete genomic
sequence of the crenarchaeota A. pernix there are
three homologues ([11] S. Pietrokovski, personal
communication). It was suggested that the multiple
PCNA homologues in crenarchaeota result from an
early lineage-specific gene duplication [83]. In
eukarya, on the other hand, there are only limited
examples of two PCNAs in one organism
(summarized in [97]). There are also a few viruses
that contain two PCNA homologues. However, only
in carrot has it been shown that these two PCNA
homologues may be functional (unpublished
observation).
Replication Factor C (RFC)
In all organisms studied to date, the sliding clamp
can not assemble itself around DNA, but must be
loaded onto DNA by a protein complex referred to
as a clamp loader (reviewed in [18,20]). In bacteria
and eukarya, the clamp loader is a five subunit
complex called the γ -complex and RFC,

10. 148 Current Protein and Peptide Science, 2000, Vol. 1, No. 2 Zvi Kelman
respectively. The clamp loader recognizes the 3'
end of the single strand/duplex (primer-template)
junction and utilizes ATP hydrolysis to assemble
the clamp around the DNA primer. The sliding
clamp, encircling DNA, then interacts with the
polymerase catalytic unit for rapid and processive
DNA synthesis. Upon completion of an Okazaki
fragment, the polymerase dissociates from the clamp
leaving the clamp behind, assembled around the
duplex DNA. In bacteria and eukarya the clamp
loader has a dual function; in addition to its role as a
clamp loader, it also functions as a clamp unloader
to remove used clamps from DNA [95].
RFC is highly conserved in all eukarya from
yeast to humans in its subunit structure
(summarized in [98]). It contains five subunits
ranging in size between 36 and 140 kDa as revealed
by SDS-PAGE analysis. Genes encoding each of
these subunits have been cloned from both
mammals and S. cerevisiae and each subunit has
been shown to be essential using deletion analysis
in yeast. The predicted amino acid sequences of
each of the yeast and human RFC subunits reveal
significant homology in seven regions referred to as
RFC boxes (box II to VIII) [98]. The large subunit
(p140, RFC1) contains an additional box (box I)
within its N-terminal region which shares
homology with prokaryotic DNA ligases.
Homologues of RFC have been identified in
archaea that have beeen completely sequenced [7-
11]. Interestingly, only two proteins with
homology to RFC have been identified within these
genomes. The two subunits show homology with
RFC3 and the C-terminal region of RFC1. The
RFC1 homologue lacks the DNA ligase domain at
the N-terminus.
RFC and PCNA Stimulation of DNA
Polymerase Activity
In bacteria and eukarya, the sliding clamp
endows the polymerase with its high processivity.
Recently, an in vitro replication assay demonstrated
that RFC and PCNA from M. thermoautotrophicum
have similar functions in archaea. In the presence of
both RFC and PCNA, a member of the B-type
polymerases became a processive enzyme even in
the presence of RPA (discussed below) [80,82]. It
was also demonstrated that like its eukaryotic
counterpart, the M. thermoautotrophicum RFC can
load M. thermoautotrophicum PCNA onto DNA in
an ATP dependent reaction [82].
Due to its torodial structure, PCNA alone can
diffuse onto linear but not circular DNA templates
at the duplex end and can translocate by sliding
over the duplex region to a primer terminus [99]. By
doing so, PCNA can support processive replication
by the polymerase in the absence of RFC only when
the DNA is linear and has a double stranded end
[99]. Using such an approach, it was demonstrated
that the two PCNA homologues identified in
Sulfolobus solfataricus supported processive
replication by a B-type polymerase [83]. These
experiments were performed in the absence of RFC
(this complex has not yet been isolated from this
organism).
Replication Protein A (RPA)
In bacteria and eukarya, SSB is an essential
component of all replication systems (reviewed in
[100-102]). SSBs stimulate the activity of DNA
polymerases by removing secondary structures
(such as hairpins) that interfere with polymerase
movement. SSB also coats the single stranded DNA
behind the helicase and thus protects the DNA from
attack by nucleases and chemical modification.
In eukarya the SSB is called replication protein A
(RPA). RPA is a three subunit complex with
apparent molecular masses of 70, 34 and 14 kDa
(reviewed in [102]). The large subunit contains two
tandem single-stranded DNA binding domains
[103], and the middle subunit also contains an
additional domain [104]. In all archaea for which the
complete genome sequence is known, one RPA
homologue has been identified. The protein shows
homology to the large subunit of eukarya RPA
[105,106] and contains four tandem repeats of a
single stranded DNA binding domain. In
Archaeoglobus fulgidus, two proteins have been
identified [8], each of which contains two single
stranded DNA binding domains [106].
The archaeal RPA has biochemical properties
that are similar to the three subunit eukaryotic RPA
complex. These include high affinity for ssDNA
and a nearly similar binding site length (20
nucleotides for M. jannaschii compared to 30 for
eukaryotic RPA [106]). The differences between the
RPA of archaea and eukarya identified to date are
due in part to the growth conditions of the
organisms. Whereas the two archaeal RPA studied,
M. jannaschii [106] and M. thermoautotrophicum
(unpublished observation), bind more tightly to
DNA at high temperature (70°C), the eukarya RPA
binds poorly to DNA at similar temperatures
(unpublished observation). When more archaeal
RPAs are isolated and studied, it will be of interest

11. DNA Replication Current Protein and Peptide Science, 2000, Vol. 1, No. 2 149
to determine the effects of other factors on their
DNA binding properties such as low temperature
(psychrophile), pH (acidophile and alkaliphile) and
salt (halophile).
What is the role for RPA in archaeal replication?
In mesophilic organisms one important function of
RPA is to melt secondary structures in the DNA
that affect polymerase movement. In thermophiles,
however, this problem is likely to be reduced.
Therefore, RPA may have additional functions in
archaea. In mesophiles, RPA interacts with many
cellular proteins including several that participate in
DNA replication (summarized in [102]). It was
shown that the interactions between RPA and the
replication proteins play important roles during
replication. For example, it was demonstrated that
specific interactions between RPA and
polα/primase, polδ and RFC are important for
lagging strand replication [107].
In vitro replication studies conducted with the
RPA homologue isolated from M.
thermoautotrophicum have revealed some
interesting properties of the protein. In contrast to
bacteria and eukarya in which the SSB stimulates
polymerase activity, M. thermoautotrophicum RPA
inhibited DNA replication by a member of type B
DNA polymerases [80]. It was also demonstrated
that the inhibition, at least in part, is due to a direct
interaction between the polymerase and RPA. The
inhibition by RPA can be relieved by the addition of
the accessory proteins PCNA and RFC. The role of
this inhibition and whether it can be generalized to
RPA homologues from other archaea, however,
remains to be investigated. It was suggested that the
inhibitory effects of RPA may govern the activity of
the polymerase such that only the polymerase-clamp
complex (the processive form of the enzyme) is
active [80].
Topoisomerases
During replication (and transcription) the DNA
is supercoiled in front of and behind the moving
polymerase. Positive supercoiling occurs when the
two strands are coiled in the same sense as in B-
form DNA, while negative supercoiling occurs when
the two strands are coiled in the opposite direction.
Thus, positive supercoils are produced in front of
the replication fork while negative supercoils are
produced behind it [19,108]. The degree and type of
supercoiling are altered by topoisomerases that
prevent the accumulation and regulate the level of
supercoiling within the DNA molecule.
Topoisomerases are also required for DNA
supercoiling needed for DNA compaction after
replication is completed.
DNA topoisomerases change the number of
topological links between the two strands of the
double helix (the linking number (Lk)). Thus, they
can either relax or supercoil DNA. Topoisomerases
catalyze the change in Lk by introduced transient
breaks (nicks) in the phosphodiester backbone of
the DNA and passing the DNA through these
breaks. Topoisomerases are classified according to
their mechanism of action. Type I topoisomerases
introduce transient single-strand breaks, whereas
type II topoisomerases introduce double-strand
breaks (reviewed in [19,108]). Other differences
include the requirement for ATP (only type II) and
structure of the protein (type I is monomeric while
type II is dimeric). Although both types of enzymes
are present in eukarya and bacteria, the biochemical
properties of the enzymes between the two domains
differ. For example, only the bacterial type II
enzyme can introduce negative supercoils into
DNA. The differences in type I topoisomerases
include the observation that the eukaryotic enzyme
is transiently covalently linked to the 3'-end of the
DNA and relaxes both negative and positive
supercoils; in contrast, the bacterial enzyme is linked
covalently to the 5'-end and relaxes only negative
supercoils.
Several type I and type II topoisomerases have
been identified and studied in archaea (summarized
in [63,64,109]). One interesting observation
regarding topoisomerases, first made in archaea, was
the isolation of a novel enzyme, reverse
gyrase. This enzyme, first identified in Sulfolobus
acidocaldarius [110], has the unique ability to
introduce positive supercoils into DNA in the
presence of ATP. Since its discovery, reverse
gyrase has been identified in all hyperthermophilic
and many thermophilic bacteria and archaea (e.g.
[111-113]). The enzyme was named reverse gyrase
[110] because it catalyzed a reaction opposite to that
catalyzed by gyrase (the bacterial type II enzyme is
also known as DNA gyrase). Gyrase relaxes
positive supercoils (by introducing negative
supercoils) whereas reverse gyrase introduces
positive supercoils in DNA. Because reverse gyrase
is dependent on ATP and is able to introduce
supercoils, the enzyme was originally thought to be
a type II topoisomerase [110]. However,
subsequent studies demonstrated that reverse
gyrase is resistant to novobiocin (a ubiquitous
inhibitor of the type II enzymes) [114] and that the
sequence of the gene encoding reverse gyrase [115]
revealed that the enzyme is more similar to a type I
enzyme. DNA sequences of reverse gyrases from

12. 150 Current Protein and Peptide Science, 2000, Vol. 1, No. 2 Zvi Kelman
several other archaea (e.g. [116,117]) have
supported the hypothesis and established that
reverse gyrase is a type I enzyme. To date, all but
one of the enzymes studied are composed of a
single subunit. The exception is the enzyme isolated
from Methanopyrus kandleri, which contains two
subunits [118].
The presence of reverse gyrase in all
hyperthermophilic and many thermophilic
organisms suggests that this enzyme is essential for
life under extreme temperature conditions [119]. It
was hypothesized that reverse gyrase causes an
increase in the linking number which helps to
maintain the DNA structure and thermodynamic
parameters of DNA when exposed to high
temperature at values similar to those observed in
mesophiles [109]. It is not yet clear, however, if
positive supercoiling accounts for the stability of
DNA and the organisms ability to grow at high
temperature [120].
In addition to reverse gyrase, several other type I
and type II topoisomerases with unique properties
have been identified in archaea. For example, it was
thought that all type II topoisomerases possessed
similar amino acid sequences. The isolation and
characterization of a new member of the type II
enzyme from Sulfolobus shibatae [121]
demonstrated that this is not the case. Although the
amino acid sequence of the archaeal protein is
similar to homologous proteins from other archaea,
they differ from other members of the type II
family. This has led to the classification of the type
II topoisomerases into two evolutionarily distinct
protein families: type IIA and type IIB.
To date, many archaeal topoisomerases from
different species have been identified, purified and
biochemically characterized. The role of archaeal
topoisomerases in vivo is not yet clear. Some of
their functions during DNA metabolism are likely to
be similar to those of topoisomerases in eukarya
and bacteria. Since many archaeal organisms live
under extreme conditions [59], topoisomerases may
have additional functions in stabilizing the DNA
under these unique conditions (as suggested for
reverse gyrase at high temperature, discussed
above). Future studies may help to define the
precise roles of topoisomerases in this domain of
life.
Okazaki Fragment Maturation
During chromosomal replication the leading
strand is replicated as a continuous strand. The
lagging strand, on the other hand, is replicated
discontinuously as a series of Okazaki fragments.
In bacteria, these fragments are 2000 bp in length
while in eukarya they are several hundred bases.
The length of the Okazaki fragments in archaea,
however, is not yet known. Nevertheless, several
proteins that participate in the processing of the
Okazaki fragments to create a mature double
stranded DNA have been identified in this domain.
The archaeal proteins have been shown to be
homologues of eukaryotic enzymes. These proteins
include homologues of DNA ligase I, Flap
endonuclease (FEN-1) and RNase H. The
examination of the proteins involved in Okazaki
fragment maturation in archaea has only been
recently carried out. As yet, no studies with the
homologue of RNase H have been done.
Preliminary studies have been performed on DNA
ligase and FEN-1, and they will be briefly described
here.
The major difference between bacterial and
eukaryal DNA ligases is the coenzyme that
participates in the ligation reaction. While bacterial
ligases utilize NAD, eukaryotic ligases utilize ATP
[19]. The predicted amino acid sequence of the
DNA ligases identified in several archaea, and those
identified in the complete genome sequence of other
archaea, revealed that these enzymes are similar to
the eukaryotic form. These sequence similarities
suggest that the archaeal enzymes utilize ATP [122].
The isolation of the DNA ligase I homologue from
M. thermoautotrophicum and the characteri-zation
of its biochemical properties demonstrated that the
enzyme is indeed eukaryotic-like, utilizing ATP as
its source of energy [122a].
FEN-1, also called maturation factor 1 (MF-1)
and DNase IV, is a protein possessing 5'-flap
endonuclease activity on branched DNA molecules
(flap structures) and a 5' to 3' exonuclease activity
[123,124]. The 5' to 3' exonuclease activity of
FEN-1, together with the activity of RNase H, was
shown to be required for Okazaki fragment
maturation in eukarya [123-126]. FEN-1 proteins
identified in many archaeal organisms have been
shown to have amino acid sequences similar to the
eukaryotic enzyme [127,128]. Studies conducted
on the purified FEN-1 homologue from M.
jannaschii [129,130], P. horikoshii [128] and P.
furiosus [122] revealed that its biochemical
properties are similar to the eukaryotic enzyme with
respect to substrate specificity and the effects of salt
and pH. Because the archaeal homologues studied
were all from thermophilic organisms, the major
difference noted between the eukarya and archaea
protein was in the effect of temperature on optimal
enzyme activity [130-132]. The P. furiosus and M.

13. DNA Replication Current Protein and Peptide Science, 2000, Vol. 1, No. 2 151
jannaschii homologues of FEN-1 have also been
crystallized and their three dimensional structures
determined [129,132].
CONCLUDING REMARKS
The genetic information of the third domain of
life, the archaea, is contained within a circular
chromosome. Several archaea possess more than
one chromosome, but all are circular. This is similar
to the situation found in bacteria where the
chromosomal DNA is also circular. Another
apparent similarity to bacteria is the presence of a
single origin of replication. The mechanism of DNA
replication, however, is more similar to that found in
eukarya. This may be explained by the observation
that both the eukarya and archaea domains started
from the same branch in the universal phylogenetic
tree (Fig. 1).
Intensive studies on archaeal replication have
been initiated only in the past few years, motivated,
in part, by the determination of the complete genome
sequences of several members of this domain. In
analyzing these sequences, it was noted that
although archaeal replication proteins are similar to
their eukaryotic counterparts, the replication
machinery appears to be simpler than that in
eukarya. Many of the components that participate in
archaea replication are less complex in structure and
polypeptide composition than those of eukaryotic
cells. Furthermore, several unique replication
proteins have also been discovered (e.g. DP2-
polymerase). Future biochemical and structural
studies are needed before we will be able to fully
appreciate the similarities between the archaea and
eukarya replication systems.
Many archaea grow under extreme environmental
conditions. To date, most archaea studied are either
thermophilic or hyperthermophilic organisms. It is
of considerable interest to determine how the
replication machinery adapted to other
environmental conditions (low temperature, high/low
pH and high salt concentrations). The isolation and
characterization of replication proteins from
organisms that grow under these markedly different
conditions will help to elucidate these questions.
The archaea domain can be divided into three
kingdoms (euryarchaeota, crenarchaeota and
korarchaeota). Preliminary studies demonstrated
that the processes involved in DNA replication
among these three kingdoms may differ. For
example, it was demonstrated that the DP2
polymerase is present in euryarchaeota but not in
the complete genome sequence of one member of
the crenarchaeota. PCNA, on the other hand, has
several homologues in each crenarchaeota species,
but only one in euryarchaeote. The extent of the
differences and similarities between the three
archaeal kingdoms remains to be elucidated.
Archaea are fascinating organisms that grow in
the most extreme environments on earth. Because of
their diverse growth conditions, their biochemical
processes, including DNA replication, have changed
and adapted to the different environments. Future
studies will no doubt elucidate the adaptation
mechanism and should shed light on the basic
biological questions of how these different
conditions required for growth have affected the
proteins and their structure, function and
biochemical properties.
ACKNOWLEDGMENTS
I am grateful to Dr. Jerard Hurwitz for his
encouragement during the course of this work. Drs.
Jerard Hurwitz and Lori Kelman are thanked for
their invaluable suggestions on the manuscript. I
wish to thank Drs. Jerard Hurwitz, Shmuel
Pietrokovski and Bruce Stillman for sharing data
prior to publication. I also wish to apologize to
colleagues whose work was not cited due to space
limitations. This work was supported by the
National Institute of Health Grant GM34559.
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