The archaeal domain of life is as diverse in its population as are prokaryotic and eukary-otic. Archaea are classified into two kingdoms, euryarchaea and crenarchaea, based on differences in their 16S rRNA [69]. Relevant for this study is the archaeon Thermococcus kodakaraensis (Tk), which belongs to the kingdom of euryarchaea.
The machinery responsible for archaeal DNA replication appears to have fewer compo-nents than their eukaryotic counterparts, but the archaeal and eukaryotic proteins strik-ingly resemble one another in both sequence and structure (reviewed in [71]). The reduced complexity makes the archaeal system attractive for experimental studies.
The mode of archaeal DNA replication, however, resembles that used by prokaryotes.
The genome is organized in a circular chromosome and DNA replication emanates from one origin bi-directionally around the circular template [72, 73]. However, some archaea
1.2. Archaeal DNA replication 5
Fig. 1.2: Schematic representation of a rolling circle reaction. DnaB unwinds the primer that is annealed to an 100-nucleotide small minicircle and thereby generates single-stranded template for replication. The polymerase extends the primer that the helicase unwinds, and thereby provides double-stranded template for helicase unwinding. Addition of primase initiates lagging strand synthesis on the single-stranded tail that emerges in the leading strand polymerization. The minicircle includes only the nucleotides dCMP, dGMP and dAMP. Leading strand polymerization can therefore be monitored by the incorporation of dTTP and lagging strand polymerization by the incorporation of dATP. Adapted from [70].
species possess two or more putative origins [74–76]. It is tempting to speculate that these origins are used at different times during DNA replication, resembling the eukaryotic usage of early and late origins. Though origins of several archaea have been identified, the origin of the archaea T. kodakaraensis still has yet to be identified.
Little is known about the initiation of replication in archaea. Most sequenced archaeal genomes possess at least one origin binding protein variant, Orc/Cdc6 (reviewed in [77]), named because of its similarity to the eukaryotic proteins Orc and Cdc6. In Pyrococ-cus abyssi the Orc/Cdc6 protein is stably expressed throughout the cell cycle and is associated with chromatin [72]. The replicative helicase, minichromosome maintenance protein (MCM), localizes to the Orc/Cdc6-primed origins at the onset of DNA replica-tion [72]; MCM interacts with Cdc6 [78]. On DNA, both hexamers and double-hexamers have been observed by electron microscopy [79]. In solution, the MCM complex from the euryarchaea Methanothermobacter thermautotrophicus (Mt) was shown to form double hexamers, while the MCM from the crenarchaeal Sulfolobus solfataricus (Ss) exists as a single hexamer [71, 80]. The MCM complexes from the two organisms also differ in their ATPase activity. While the ATPase activity of Mt MCM is stimulated by ss DNA, the ac-tivity of Ss MCM is unaffected by DNA [80]. The zinc-binding motifs in the euryarchaeal MCM may explain the difference of properties. Euryarchaeal MCMs possess a zinc-finger motif of the C4 type, that the crenarchaeal MCMs lack [81]. The crenarchaeal MCMs, however, provide a C3 type of putative zinc finger domain [80]. The efficient binding of ssDNA requires the C4 type of zinc-finger domain [82]. The double- and single-hexameric forms of mtMCM both show helicase activity [83]. In contrast to the bacterial helicase DnaB, but like the eukaryotic MCM complex, the archaeal MCM complex has 3’→5’
helicase directionality, thus it moves along the leading strand template (see fig. 1.3) [82].
The crystal structure of the double-hexameric Mt MCM shows a central cavity in the com-plex that can encircle double-stranded DNA. Mutations in the central cavity abolish DNA binding [83]. MCM proteins are members of the AAA+ family. Proteins of the AAA+ are remodelers of macromolecular structures whose ATPase domains require amino acid side chains of two adjacent complex subunits. Biochemical studies have established that Mt MCM contains DNA-dependent ATPase activity [84], translocates along ss and ds DNA (reviewed in [85]) and displaces DNA bound proteins [86]. In vivo, the different archaeal species have various copy numbers of MCM variants (reviewed in [77]). Thermococcus kodakaraensis has three different MCM proteins [87]. Two of the MCMs, Tk MCM2 and Tk MCM3, form hexameric structures and display in vitro helicase activity. While the genes for Tk MCM1 and Tk MCM2 could be deleted from the organism, attempts to delete the Tk MCM3 gene were unsuccessful, suggesting an essential character of Tk MCM3. Interactions of MCM to other replicative proteins have been shown. The MCM proteins interact with the GINS complex (Go, ichi, nii, san; Japanese for five, one, two, three) bothin vivo and in vitro (see fig. 1.3). The GINS proteins have been identified in a yeast-two-hybrid screen to MCM inS. solfataricus [88]. The GINS complex is a dimer of a dimer, consisting of two copies of GINS23 and GINS15 [89]. While the GINS complex itself has no enzymatic activity, it interacts with the MCM complex and DNA primase [88–93]. Thus, the GINS complex has been proposed to act as a bridge between one of the lagging strand polymerases and the replicative helicase (fig. 1.3). The GINS complex also interacts with a protein that is a homologue of the bacterial RecJ and the human Cdc45, RecJdbh, inS. solfataricus [88].
PCNA
DNA pol
MCM
GINS
Primase
Primer Lig1
FEN1
SSB
Fig. 1.3: Model of the archaeal replisome. The helicase, MCM, unwinds the duplex DNA ahead of the polymerase (DNA Pol) on the leading strand. The polymerase achieves its processivity due to PCNA. PCNA interacts with lagging strand processing factors, FEN1 and Ligase 1. MCM interacts over GINS indirectly with the DNA primase. Adapted from [77].
The study described in the paper ’A novel DNA nuclease is stimulated by association with the GINS complex’ characterized a nuclease, tk1252p, that localizes at the replication fork [91]. This exonuclease digests ss DNA in the 5’→3’ direction and shares homology with the human Cdc45 protein [94]. Tk1252p interacts with the GINS complex, specifically with
1.2. Archaeal DNA replication 7 the GINS15 subunit and has been named GAN (GINS associated nuclease). Based on the GAN-GINS interaction and the GAN-Cdc45 homology, Makarova and colleagues proposed that a complex of MCM, GINS and RecJdbh/GAN is a homologue of the human CMG complex (consisting of Cdc45, MCM2-7 and GINS complex), the eukaryotic replicative helicase [94].
After an initial unwinding of the DNA at the origin, the single stranded DNA is bound by the single strand binding (SSB) protein to protect the DNA from damage. Crenarchaeotes cover DNA with an E. coli SSB-like protein complex [95, 96]. The crenarchaeal SSB re-sembles the structure of the bacterial SSB more than the eukaryotic RPA. Euryarchaeotes possess an RPA-like protein complex. The RPA complex of Pyrococcus furiosus is a het-erotrimer containing subunits of 41, 32 and 14 kDa [97]. Overall, the RPA complexes found in euryarchaea are diverse in size and composition. However, all studied euryarchaea pos-sess at least one p41 subunit and many contain a p32 subunit homologue [98–100]. The structural differences between the RPA- and SSB-like complexes are attributed for the differences in properties. The SSB ofSulfolobus solfataricus interacts with Ss MCM [80], while such interactions have not yet been found for euryarchaeal RPA. On the other side, RPA ofMethanothermobacter thermautotrophicus inhibits the Mt PolB DNA synthesis in the absence of processivity factors [101].
Upstream of the helicase at the replication fork, the leading and lagging strand poly-merases duplicate the DNA template. The polypoly-merases require a processivity factor, PCNA (proliferating cell nuclear antigen) that stimulates the polymerase processivity of Thermococcus kodakaraensis polymerase B (Tk PolB) from about 0.5 to about 7 kbp.
PCNA acts as a homotrimer. For its loading on DNA, PCNA requires a loading factor, RFC (replication factor C) [102] which is a pentamer of four copies of the small subunit and one copy of the large subunit, RFCS and RFCL, respectively. RFC loads PCNA preferably on primer-template junctions [1] and requires ATP for its action. Thermococ-cus kodakaraensis possesses two distinct PCNA variants, tk0582 and tk0535, that form homotrimers and stimulate the DNA synthesis activity of Tk PolB [102]. PCNA of S.
solfataricus has been shown to interact with different factors at the replication fork, such as ligase 1 and FEN1, which are both involved in Okazaki fragment maturation. Due to its interaction with the polymerases and downstream processing factors, PCNA has been suggested to act as an organizational platform in addition to its role as a processivity factor [103].
Euryarchaeotes, like T. kodakaraensis, possess two polymerases, D and B, while crenar-chaeotes contain only polymerase B [104, 105]. Polymerase D (PolD) is a dimer of the smaller subunit DP1 and the larger subunit DP2 [106] DP2 alone contains limited poly-merase activity [107]. DP1 shows sequence homology to the smaller, noncatalytic, sub-units of the eukaryotic replicative polymerases, with Polα(p70), Pol δ (Cdc27p) and Pol
!(p55) [108]. It also harbors a 3’→5’ exonuclease activity, which is highest on mismatched DNA template [109]. The excision of mismatches suggests a role in DNA repair or on the lagging strand in DNA replication [110]. Polymerase B is widely diverse across the differ-ent archaeal strains (summarized in [111, 112]). PolB of T. kodakaraensis is monomeric and is highly processive when bound to PCNA [102]. A couple of considerations suggest a role for PolB as the leading strand polymerase in DNA replication. In crenarchaea
PolB is the only polymerase identified to date [104, 105]. Also, the eukaryotic replicative polymerases belong to the B family of polymerases. Finally, PolB contains a potent 3’→5’
exonuclease proofreading activity [113]. However, which protein acts as the lagging strand polymerase remains unclear.
While the leading strand polymerase only requires one initiation event, the lagging strand polymerase requires priming to initiate each Okazaki fragment. DNA primase mediates priming. Archaea possess two DNA primases, a DnaG-like primase and a eukaryote-like primase. The DnaG-type primase has been demonstrated to play a role in RNA degradation and its gene is dispensable for cell growth [91, 114, 115]. The eukaryote-like primase is a heterodimer of a small p41 and larger p46 subunits. The enzymatic activity lies in the p41 subunit, while the p46 subunit regulates the activity and alters the properties of the catalytic subunit [116]. To date, no homologue of the eukaryotic polymeraseαhas been identified in archaea [117]. The absence of polymeraseαsuggests to that the primase may be responsible for Okazaki fragment initiation prior to the switch to the lagging strand polymerase occurs. The enzymatic properties of the different archaeal primases vary widely. In some archaea, priming with rNTPs has been reported [72, 118, 119]. For Pyrococcus furiosus (Pf), however, the primase initiates DNA synthesis with dNTPs. Also, Pf p41 was able to extend the DNA chains up to several kilobases. In complex with p46 the DNA chains were significantly shorter, the in vitro activity could however be stimulated with ATP. Also, the Pf p41/p46 complex was shown to initiate primers with rNTPs [116].
Readers are referred to the ’Characterization of the DNA primase complex isolated from the archaeon,Thermococcus kodakaraensis’ for the purification and characterization of the Tk primase. This section describes the primer initiation with both dNTPs and rNTPs by the primase complex and the catalytic subunit. The primase complex is required for the primer usage by Tk PolB, the putative replicative polymerase. Current in vitro meth-ods available allow the study of polymerases and helicases separately from each other.
However, under physiological conditions helicase and polymerases interact directly or in-directly, and probably influence each other’s properties. Thus, the second study introduces the rolling circle assay to study the link between replicative helicase, polymerase and pri-mase. In this study, the coupled action of Tk MCM2 and Tk PolB produced fragments longer than 10 kilobases. These long fragments, which mimic leading strands, provided the template for lagging strand synthesis, initiated by the p41/p46 primase complex. Thus, in the presence of all three protein fractions, leading and lagging strand synthesis were observed.