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236 Biochemical Society Transactions (2004) Volume 32, part 2 DNA replication in thermophiles A.I. Majernı́k, E.R. Jenkinson and J.P.J. Chong1 Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K. Abstract DNA replication enzymes in the thermophilic Archaea have previously attracted attention due to their obvious use in methods such as PCR. The proofreading ability of the Pyrococcus furiosus DNA polymerase has resulted in a commercially successful product (Pfu polymerase). One of the many notable features of the Archaea is the fact that their DNA processing enzymes appear on the whole to be more like those found in eukaryotes than bacteria. These proteins also appear to be simpler versions of those found in eukaryotes. For these reasons, archaeal organisms make potentially interesting model systems to explore the molecular mechanisms of processes such as DNA replication, repair and recombination. Why archaeal DNA-manipulation systems were adopted over bacterial systems by eukaryotic cells remains a most interesting question that we suggest may be linked to thermophily. The ‘replicon’ model revisited The process of DNA replication appears to take place in a number of steps that were originally proposed in a model for understanding the regulation of DNA replication in bacteria. The replicon model proposed by Jacob, Brenner and Cuzin [1] has a number of features that can be obviously related to the situation found in both bacteria and eukaryotes, where the study of DNA replication is well advanced. It seems likely that these features will also be present in the archaeal paradigm, which is discussed further below. The salient features of the replicon model were that (i) an initiator protein could control DNA synthesis by acting on a replicator element and (ii) the activated replicator would allow replication of the DNA attached to the replicator (the replicon) to occur (Figure 1A). These features can be identified in bacteria such as Escherichia coli where DnaA acts as an initiator protein by specifically recognizing and binding to the origin sequence, oriC [2] (Figure 1B). The process whereby DNA replication is allowed to occur is then completed through a number of steps: additional DnaA proteins are recruited to the origin, where binding to both DNA elements and other DnaA molecules occurs [3]. The multimerization of DnaA while bound to DNA causes torsional stresses in nearby sequences, and a localized unwinding of an A/T-rich DNA element. The DnaA multimer together with this single-stranded DNA allow the recruitment of a DnaB–DnaC complex, which results in the loading of a hexameric DnaB complex on to the unwound DNA [4]. DnaB in this multimeric form displays DNA helicase (unwinding) activity that precedes the replication fork proteins required to duplicate the DNA Key words: Archaea, Cdc6, cell cycle, DNA replication, mini-chromosome maintenance (MCM), origin recognition complex (ORC). Abbreviations used: ORC, origin recognition complex; MCM, mini-chromosome maintenance. 1 To whom correspondence should be addressed, at the present address: Department of Biology (Area 5), University of York, P.O. Box 373, York Y010 5YW, U.K. (e-mail jpjc1@ york.ac.uk). C 2004 Biochemical Society duplex. The same steps must take place in eukaryotic DNA replication, although different proteins are involved in the process. In this case the ORC (origin recognition complex) binds to specific DNA sequences known as origins of replication (or ‘origins’) [5]. Whether localized duplex unwinding occurs is currently not clear. The ORC then appears to act as a landing pad for other proteins involved in DNA replication [6], most notably the recruitment of the MCM (mini-chromosome maintenance) proteins to the DNA via the Cdc6 and Cdt1 proteins [7,8]. MCM proteins have been shown to form hexameric complexes and are thought to provide the DNA helicase activity required for eukaryotic DNA replication (Figure 1C) [9]. Initial parallels in the replication processes of bacteria and eukaryotes can therefore be drawn thus: both mechanisms show recognition of a specific DNA element through the recruitment of a multimeric protein complex. This complex acts to recruit other proteins, and in particular the DNA helicase. In both cases the helicase is hexameric. Since Archaea are the remaining major evolutionary ‘domain’ of life, consideration of the components likely to be involved in archaeal DNA replication allows us to consider the universal applicability of the replicon model to DNA replication. DNA replication in Archaea: roles for Cdc6 Analysis of whole archaeal genome sequences has allowed the identification of sequences related to eukaryotic Cdc6 and MCM proteins but not to DnaA/B/C [10]. As is often the case with the Archaea, it is difficult to make all but the broadest of generalizations concerning this process. In most cases, Archaea contain one or more Cdc6-like sequence and one MCM sequence (see [11] for specific deviations from this situation). It has been recently shown that the archaeal Cdc6 protein from Pyrobaculum aerophilum and a thermophilic bacterial DnaA protein from Aquifex aeolicus have a very high level of structural similarity although they show no Thermophiles 2003 Figure 1 The replicon model updated (A) Simplified schematic of the replicon model. A replicator protein binds to the DNA initiator sequence and causes the replicon to be duplicated. DNA replication initiates at an origin. (B) Extended replicon scheme applied to E. coli. The replicator protein DnaA binds to the initiator oriC sequence. Multimerization of DnaA leads to localized unwinding of the DNA at an AT-rich origin and recruitment of the hexameric DnaB helicase via a DnaB–DnaC intermediate. (C) The replicon scheme in eukaryotes. Autonomously replicating sequences (ARS) containing an origin of replication and an ORC-binding site are occupied by the ORC protein complex, which may cause localized duplex unwinding. Hexameric MCM complexes are recruited to the origin by the loading factors Cdc6 and Cdt1. Loaded MCMs act as a replication licensing factor, allowing the origin of fire once per cell cycle, ensuring replciation of one complete genomes’-worth of DNA. similarity at the level of amino acid sequence [12,13]. It is therefore very tempting to speculate that archaeal Cdc6 may be acting as the initiator protein and binding specifically to archaeal origin DNA sequences. This is supported by data from Forterre and co-workers who have shown not only that the loci of a number of Cdc6-like genes are adjacent to putative and actual origins of replication (a situation also seen in bacteria with DnaA genes), but also physical interaction of the Pyrococcus abyssi Cdc6 protein with DNA containing specific origin sequences [14–16]. It is of further note that in eukaryotes Cdc6 and a number of the ORC subunits have significant sequence similarity [17,18]. Thus the appealing scenario presented is that the single Cdc6-like protein found in some Archaea may multimerize on origins in a similar way to bacterial DnaA, causing similar torsional stresses and localized DNA melting, and ultimately that the Cdc6 sequences found in Archaea may have evolved into the sixsubunit ORC complex of eukaryotes. However, it could also be the case that the Cdc6-like homologues are ‘guilty by association’, just as is the case in eukaryotic systems where Cdc6 can also be detected at origins [19], and the Archaea may use a completely different protein to fulfil the role of ORC/DnaA. An alternative role for the archaeal Cdc6 proteins is as the helicase loading proteins. In eukaryotic DNA replication initiation the presence of both Cdc6 and Cdt1 is required for ‘loading’ of the MCMs on to DNA [7,8]. Again this possibility is complicated by the lack of homogeneity between species. Archaeal genomes with between 1 and 13 Cdc6-like genes have been reported. Biochemical studies of the two Cdc6-like proteins in Methanothermobacter thermautotrophicus have shown that the Cdc6-like proteins have ATPase activity and can autophosphorylate [20]. It has also been shown that ATP-bound Cdc6 can modulate the DNA helicase activity of MCM complexes [21]. Functional differences between the two Cdc6 proteins in this organism have yet to be demonstrated. Thus in species where more than one Cdc6-like gene exists, the simplest explanation is that one gene plays the initiator role of ORC and DnaA, while another acts as the helicase loader (equivalent to eukaryotic cdc6 and cdt1 or bacterial DnaC). The role of the additional Cdc6-like sequences found in species such as C 2004 Biochemical Society 237 238 Biochemical Society Transactions (2004) Volume 32, part 2 Sulfolobus and Halobacterium then arises. One possibility is that these organisms may replicate their genomes from more than one origin of replication, and that these proteins show different specificities for particular DNA sequences. Another possibility is that these multiple Cdc6-like proteins are snapshots in the evolution of the eukaryotic ORC, and that a heterologous Cdc6 complex more reminiscent of the current eukaryotic ORC is formed in these organisms. In species that have only a single Cdc6-like sequence (notably Pyrococcus species), it will be interesting to determine whether this protein plays a dual role as origin marker and helicase loader, or whether another, non-homologous protein plays one of these roles. A final mystery is the apparent lack of a Cdc6 gene in the genomes of Methanococcus jannaschii and Methanopyrus kandleri. Archaeal MCM: a model protein A number of biochemical studies have characterized archaeal MCMs as producing hexamers or double hexamers [22–24]. These proteins have provided a good system for initial forays into understanding the molecular mechanism by which the MCMs may act as a DNA helicase. The homomeric nature of these multimeric complexes provides a much simpler model for both structural and functional studies. A recent crystal structure of the N-terminal 286 amino acids of the M. thermautotrophicus protein has provided the impetus for further interest in the way in which these proteins might cause duplex DNA to be unwound [25]. This structure reinforces the idea that the MCM complex is hexameric (which contrasts with one study suggesting that heptamers may form [26]), and also provides data to support the notion that the central channel in this complex is sufficiently large to accommodate double-stranded DNA. It may therefore still be the case that the helicase activity of the MCMs is provided in the form of a rotary pump as suggested by Laskey and Madine [27]. What is certainly clear is that the archaeal system provides a great asset to our understanding of what is a key protein in eukaryotic DNA replication. Why make things more complicated? The massive increase in the amount of DNA contained in eukaryotic cells compared with prokaryotic ones is undoubtedly a major factor in the increased complexity found in the eukaryotic DNA replication process. The replicon was originally defined as “a genetic element such as an episome or a chromosome (of a bacterium or of a phage) . . . [that] . . . can only replicate as a whole” [1]. While this has been shown to be the case in Bacteria and some Archaea, where replication of a circular genomic DNA molecule is initiated from a single origin of replication by a pair of replication forks that travel in opposite directions around the chromosome, the replicon is a far more malleable entity in eukaryotes. Eukaryotic genomes are contained on multiple chromosomes that replicate from thousands of different origins simultaneously. This necessitates not only a requirement for a ‘replication C 2004 Biochemical Society licensing factor’ – a marker of un-duplicated DNA in the replicating cell [28] – but also more sophisticated regulation of the process of cell division: DNA replication should certainly not be re-initiated until sister chromatids have been safely segregated into separate compartments, and cytokinesis should not be completed until the genomes have been partitioned. This contrasts with the situation in Bacteria, where multiple initiation events may result in several replicons being duplicated simultaneously [29,30]. The bacterial mode of replication appears to be the case in at least some Archaea. Archaeal genomes are encoded on circular chromosomes. Replication studies in Pyrococcus have identified a single replication origin that is replicated by a pair of replication forks [15]. Flow cytometry studies in a number of archaeal species also indicate that several genomes’worth of DNA may be contained in a single cell [31,32]. Whether these observations are generalizable over the whole domain remains to be determined. As mentioned before, Pyrococcus species have only a single Cdc6-like sequence, and therefore a single origin may be expected. The situation with regards to functional origins in the species containing multiple Cdc6-like sequences has yet to be experimentally determined. In the same way, only a few species of Archaea have been examined by flow cytometry. It is interesting that flow cytometric analysis of the euryarchaeon Methanococcus jannaschii (which appears not to have a Cdc6-like sequence, but has multiple MCM sequences) also indicates the presence of multiple genomes of DNA in individual cells [31]. Whatever the actual situation may be, and however conserved it is between individual species whether Crenarchaeota or Euryarchaeota, it is clear that the mechanisms governing cell-cycle control and division are controlled by novel means. The sophisticated cell cycle of eukaryotes is controlled by a number of different kinase activities, most notably those of the cyclin-dependent kinases. The expansion of the complement of related proteins found in eukaryotic replication initiation may also arise from this necessarily complex co-ordination of events – multiple ORC and MCM subunits allow a more subtle control of DNA replication events. No homologues to the cyclin-dependent kinases are found in Archaea. Although in some cases there are FtsZ homologues (a key protein in bacterial cell-cycle regulation), interacting proteins appear to be non-homologous, and therefore novel (reviewed in [33]). The Archaea therefore present an interesting paradigm of replication proteins that most likely use mechanisms of great relevance to eukaryotic DNA replication studies, but which are controlled by novel means. The identification of cell-cycle control proteins in the Archaea will undoubtedly be of great interest to a wide audience and yield greater understanding not only of archaeal but also eukaryotic organisms. Archaeal DNA replication as the prototype for eukaryotic DNA replication One of the most interesting aspects of the Archaea is their position in evolution. The broadest of comparisons of the Thermophiles 2003 three domains of life gives us the previously mentioned observation that the proteins involved in eukaryotic DNA metabolism appear to be more related to those in Archaea than in Bacteria. Given that Bacteria are clearly the more ancient organisms, the question must arise as to why eukaryotic cells have adopted the archaeal DNA-processing machinery over the bacterial system? Why did the Archaea need to solve the problem of DNA replication a second time unless they coevolved with the Bacteria? Obviously producing new machinery for such a fundamental process is fraught with hazards. The archaeal proteins do not appear to have arisen out of a gradual evolution of bacterial proteins, but the wholesale adoption of a different system – for example, the DNA helicase in the bacterial system (DnaB) is processive in the opposite direction (5 to 3 ) to that of the archaeal MCM (3 to 5 ). What force could be exerted to effect such a change? One potential influence might be the presence of increased temperature. In order to become a thermophilic organism, a number of changes must be put in place to maintain the integrity of the genome [34]. DNA will breathe and even melt at higher temperatures and these elevated temperatures will also increase the frequency of DNA damage (undoubtedly a contributing factor to the presence of eukaryotic-like DNA repair pathways in many of the Archaea) [35,36]. One solution to the melting problem is to bind compacting proteins to the DNA. For example, the binding of histones to DNA increases the temperature at which a given sequence will melt. However, the addition of histones introduces further problems to the DNA replication machinery, which must cope with these additional obstacles before the duplex can be melted. Therefore, one possible explanation for the wholesale adoption of a different DNA replication system may be a need for the ancient nascent thermophile to be able to unwind histone-bound DNA. Having evolved DNA replication and repair systems able to deal with chromatin rather than ‘naked’ DNA allows further sophistication of the cell. As well as conferring thermophily, histones compact the DNA, allowing more genetic material to be stored in a given volume. More genetic material allows further complexity to arise and may therefore be the explanation for why upgraded archaeal DNA manipulation systems are found in modern-day eukaryotes. This line of reasoning would support the hypothesis that eukaryotic cells arose from a thermophile, and would suggest that rather than describing archaeal Cdc6 and MCM as eukaryotic-like, it might be more accurate to describe eukaryotic Cdc6 and MCMs as ‘archaeon-like’. Note added in proof (received 13 February 2004) A considerable understanding of the role of Cdc6 and potential multiple replication origins in the Archaea has been afforded recently [37]. We thank Dr Alex Jeffries for a critical reading of this review. J.P.J.C. is a BBSRC David Phillips Research Fellow. References 1 Jacob, F., Brenner, S. and Cuzin, F. (1963) Cold Spring Harbor Symp. Quant. 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