Mechanisms involved in DNA repair and genome maintenance are essential for all organisms on Earth and have been studied intensively in bacteria and eukaryotes. Their analysis in extremely thermophilic archaea offers the opportunity to discover strategies for maintaining genome integrity of the relatively little explored third domain of life, thereby shedding light on the diversity and evolution of these central and important systems. These studies might also reveal special adaptations that are essential for life at high temperature. A number of investigations of the hyperthermophilic and acidophilic crenarchaeote Sulfolobus solfataricus have been performed in recent years. Mostly, the reactions to DNA damage caused by UV light have been analysed. Whole-genome transcriptomics have demonstrated that a UV-specific response in S. solfataricus does not involve the transcriptional induction of DNA-repair genes and it is therefore different from the well-known SOS response in bacteria. Nevertheless, the UV response in S. solfataricus is impressively complex and involves many different levels of action, some of which have been elucidated and shed light on novel strategies for DNA repair, while others involve proteins of unknown function whose actions in the cell remain to be elucidated. The present review summarizes and discusses recent investigations on the UV response of S. solfataricus on both the molecular biological and the cellular levels.
All living organisms, from bacteria to archaea and eukaryotes, have an impressive number of proteins and pathways that seem to help to maintain the integrity of the genome and its replication with high fidelity during growth. The complexity of these repair and maintenance systems reflects their importance, particularly since DNA is constantly affected by chemical or physical attacks. The studies of UV reactions in model organisms, such as Escherichia coli and Saccharomyces cerevisiae, have contributed to a detailed understanding of DNA repair and maintenance mechanisms in bacteria and eukaryotes. More recently, investigations have been expanded to the domain of archaea (reviewed in [1–6]). In the present article, we review novel studies on the UV reaction of Sulfolobus solfataricus, a hyperthermophilic and acidophilic archaeon that is increasingly used as a model organism for hyperthermophilic archaea. It has been shown that the rate of spontaneous mutation frequencies in Sulfolobus is comparable with that of other micro-organisms, indicating that hyperthermophiles have efficient repair systems to maintain genomic stability even under these extreme growth conditions . Similarly, mutational analyses after exposure to short-wavelength UV light revealed that Sulfolobus was as sensitive and equally UV-mutable as E. coli and exhibited effective photoreactivation under visible light .
The study of UV reactions in Sulfolobus gives the opportunity to analyse strategies for maintaining genome integrity of the relatively little explored third domain of life, thereby shedding light on the diversity and evolution of such mechanisms. It might also expose special adaptations essential for life under particularly harsh environmental conditions.
The first hint of the existence of a UV-specific reaction in Sulfolobus stemmed from the early analysis of a genetic element , SSV1 (Sulfolobus spindle-shaped virus 1), that later turned out to be a virus infecting several species of this genus . Replication and propagation of the virus is strongly UV-inducible [9,10], and the first reaction after UV treatment of the host cells is the production of a small transcript, whose gene does not seem to contain the conserved boxes of archaeal promoters [11,12]. Furthermore, the replication inhibitor mitomycin C was the only known SSV1-inducing factor besides UV light, which led to the speculation that a specific SOS-like response similar to that in E. coli might exist in archaea [13,14]. A more recent genome-wide DNA microarray study showed that transcription of SSV1 is highly regulated and that the first transcript, T-ind, is probably activated via a DNA-damage-responsive host factor .
Recently, two genome-wide transcription studies have been performed with Sulfolobus species. A study by Götz et al.  characterized the transcriptional reactions of S. solfataricus and Sulfolobus acidocaldarius to a UV dose of 200 J/m2. Fröls et al.  compared the transcriptional reactions of S. solfataricus lysogenic for SSV1 with a strain free from virus, at a UV dose of 75 J/m2, which leads to optimal virus induction. Although the experimental set-up, the UV dose and the DNA microarrays used were different in the two studies, the UV-dependent regulated genes identified overlapped extremely well. The comparison of the UV-dependent regulated genes after UV treatment with 75 J/m2 compared with 200 J/m2 [16,17] showed >90% overlap in the genes. This means, for example, that 17 out of 19 most strongly induced genes in the study by Fröls et al.  are found among the top induced genes in a study by Götz et al.  (Figure 1).
Comparison of the UV-dependent up-regulated genes of S. solfataricus as found by two independent genome-wide transcriptomic studies [16,17]
Both genome-wide transcriptional studies observed that Sulfolobus exhibits a complex and specific transcriptional reaction to UV light that is paralleled by a phase of marked growth retardation. However, no genes involved in direct repair of DNA damage were found to be up-regulated strongly, indicating that a typical SOS response as characterized for E. coli is not found in these archaea (see below).
The global UV-specific reactions of Sulfolobus
The UV-reactive genes identified by transcriptomics were quite diverse in function, involved in, e.g., transcription, replication, repair, cell cycle and secretion processes (Figure 1). However, the majority of UV-regulated genes were of unknown function. The most dominant reaction was observed for the ORC1 (origin recognition complex 1)/Cdc6 (cell division cycle 6)-encoding genes. These proteins bind to the ORBs (origin-recognition boxes) at the origin of replication and mediate the loading of the MCM (mini-chromosome maintenance) helicases . Whereas Cdc6-1 and Cdc6-3 were proposed to promote replication, Cdc6-2 may act as a negative regulator for replication . The strong transcriptional up-regulation of cdc6-2 and the simultaneous down-regulation of cdc6-1 indicated a repression of the replication initiation during the UV response of Sulfolobus. In line with this finding, genes involved in cell-cycle regulation  were found to be down-regulated. Of genes involved in the process of replication, only Dpo (DNA polymerase) 2, showed a strong transcriptional induction after UV-treatment [16,17]. Whereas Dpo3 and Dpo1 are supposed to be involved in Okazaki fragment processing  and leading strand synthesis respectively , the role of the Crenarchaeota-specific Dpo2 [23,24] is still unclear. Its UV-specific induction indicates a possible function in DNA repair.
Sulfolobus spp. possess three copies of the basic transcription factor TFB, a homologue of the eukaryotic TFIIB [25,26]. One of these, tfb3 (KEGG accession number sso0280), showed a strong UV-dependent up-regulation comparable with the reaction of cdc6-2 [16,17]. In comparison with other TFBs, TFB-3 is truncated and lacks the B-finger domain for transcriptional initiation and HTH (helix–turn–helix) domain for binding to the BRE (TFB-recognition element) . Therefore it is unclear whether TFB-3 is able to act as a transcriptional regulator. in vitro, it competes with TFB-1 for interaction with the RNA polymerase , and stimulates TFB-1-dependent transcription initiation (M.F. White, unpublished work).
UV-specific reaction of genes involved in DNA repair of Sulfolobus spp.
After UV-irradiation, the first mechanism likely to be active is the photoreactivation that mediates the light-dependent direct reversion of the UV products in DNA [CPDs (cis-syn-cyclobutane pyrimidine dimers)] by the action of the photolyase (Phr) . The recently analysed crystal structure of the first archaeal photolyase from Sulfolobus tokodaii showed that the structure is highly similar to those of E. coli, Anacystis nidulans and Thermus thermophilus . A recent study demonstrated that S. solfataricus is able to remove 50% of the CPD within the first 30 min after UV-irradiation, whereas the removal rate was decreased 3–5-fold without light . No transcriptional induction of the corresponding phrB gene (KEGG accession number sso2472) was observed [16,17]. Both microarray studies suggest that the PhrB protein is constantly present in the cell and that its transcriptional regulation is not coupled to UV damage.
The Dpo4 of Sulfolobus belongs to the Y-family of polymerases, which is able to bypass DNA lesions such as CPDs and 6–4 photoproducts . It was demonstrated in vitro that the Dpo4 is able to insert bases opposite to CPD lesions, suggesting a close relation to the eukaryotic polymerase η involved in translesion DNA synthesis . Dpo4 was postulated to have an accessorial function during replication, therefore constitutive expression would be expected . In fact, no specific transcriptional induction was observed [16,17].
One of the biggest open questions in archaea is the existence and character of an NER (nucleotide excision repair) system, which removes photoproducts from DNA . Whereas genes homologous with the bacterial-like UvrABC and mismatch repair pathway are only present in mesophilic archaea ( and S. Fröls and C. Schleper, unpublished work), most archaea, including Sulfolobus spp. , have homologues of the eukaryotic NER nucleases XP (xeroderma pigmentosum) F (Rad1)/XPG (Rad2) and helicases XPB (Rad25)/XPD (Rad3). Although this system is little understood, the archaeal proteins are so closely related to their eukaryotic homologues that crystallographic and biochemical studies can have direct relevance for understanding the phenotypes of mutations in humans .
An active NER system to remove photoproducts was postulated for S. solfataricus, indicated by an efficient repair of CPD in the dark and a UV-caused transcriptional induction of the potentially NER-associated genes XPF, XPG and XPBI . UV-induction of these genes is in contrast with the global transcriptomic studies where no UV-dependent reaction was observed [16,17]. We therefore assume that, in the study of Salerno et al. , it was not possible to distinguish between transcriptional induction based on UV light as opposed to transcriptional enhancement of housekeeping genes that was based on some synchronization of the cells after UV-irradiation.
Two recently published studies confirmed a functional NER mechanism in S. solfataricus [27,33]. The removal of photoproducts during permanent dark incubation was demonstrated in vivo for S. solfataricus. Interestingly, no evidence for a TCR (transcription-coupled repair) system was observed. This is in contrast with analyses in E. coli, S. cerevisiae and human cells, where the TCR system is at least 2-fold more efficient than the global genomic repair system .
Of all genes potentially involved in DNA-repair systems, only those belonging to the archaeal rad50/mre11 operon showed slightly UV-dependent transcriptional induction [16,17]. In eukarya and bacteria, the related proteins Rad50/Mre11 and SbcC/SbcD are involved in DSB (double-strand break) repair via HR (homologous recombination) or NHEJ (non-homologous end joining), DNA-damage detection and cell-cycle checkpoint signalling . All archaeal genomes possess homologues of the proteins Rad50 and Mre11 . The core nuclease Mre11 exhibits strand-dissociation and strand-annealing properties, with an intrinsic DNA-binding activity. The nuclease activity is regulated by sequence homology of the DNA substrates and by the interaction with Rad50, which probably binds DNA ends and brings them together . In most thermophilic archaea, rad50 and mre11 are clustered with the two genes nurA and herA: NurA is a 5′→3′ exonuclease and HerA is a DNA helicase able to utilize both 3′ or 5′ single-stranded DNA extensions . The study by Quaiser et al.  showed recently that the proteins Rad50, HerA, Mre11 and RadA are constantly present in exponentially grown S. solfataricus cells, and 50% of Rad50 was found as DNA-bound molecules. It was also shown that Mre11 interacted with Rad50 and HerA. In addition, both γ-irradiation and inhibition of replication recruited Mre11 (and RadA) to the DNA . These results strongly suggest that, in S. solfataricus, the Rad50–Mre11–HerA protein complex is involved in DSB repair via homologous recombination. Such a role has recently been demonstrated in vitro for the proteins from Pyrococcus furiosus . In contrast with the genes of the rad50/mre11 operon, the radA gene (KEGG accession number sso0250), characterized to catalyse the process of homologous DNA pairing and strand exchange , did not show UV-dependent transcriptional induction [16,17]. These results are in line with earlier observations of Sandler et al. [39a], who did not see increased levels of radA mRNA after UV-irradiation (10 J/m2) in S. solfataricus.
Through comparison of the two DNA microarray studies, UV dose-dependent reactions can also be dissected. After treatment with a UV dose of 200 J/m2 used in the study of Götz et al.  (compared with 75 J/m2 in the study by Fröls et al. ), additional transcriptional reactions were observed. The sso2078–sso2080 genes (KEGG accession numbers), probably involved in the detoxification of DNA damage by ROS (reactive oxygen species), showed an up-regulation in both strains. Another difference caused by higher UV dose might be the up-regulation of the genes for β-carotene biosynthesis (KEGG accession numbers sso2905 and sso2906) that were observed by Götz et al. . The production of pigments probably represents an additional protection mechanism against UV light.
UV-specific cellular reactions of Sulfolobus
Although the presence of CPDs have been demonstrated in Sulfolobus as an immediate damage of the DNA after UV treatment , we have recently shown the fragmentation of genomic DNA as a secondary effect . This is most probably caused by cellular reactions, such as stalled or collapsed replication forks due to unremoved photoproducts or by secondary effects of repair mechanisms [17,40]. Similar observations have been made earlier in yeast, mouse cells and E. coli [41–43], indicating that DSBs represent a general secondary DNA-damage effect of UV-irradiation in all three domains of life. Induction of genes potentially involved in HR might be involved in repair of DSBs (see above). Interestingly, reverse gyrase, a topoisomerase specific to extremophilic and hyperthermophilic archaea that can introduce positive supercoil into DNA, was shown to be recruited to DNA upon UV treatment . Its activity might be crucial for DNA repair and chromosomal maintenance.
Besides the induced internal cellular reactions to UV light, a remarkable cellular aggregation of Sulfolobus cells was observed after UV-irradiation [17,45] (Figure 2). More than 80% of all cells in a culture formed aggregates of 10–20 cells or sometimes many more. This process which reaches its maximum at 6–8 h after irradiation is UV-dose-dependent and reversible . The cellular aggregation is mediated by pili formation. Pili are formed exclusively after UV treatment and are encoded by a type IV pili biogenesis operon, the ups operon (UV-inducible type IV pili operon of Sulfolobus), which is strongly induced upon exposure of the cells to UV light . Although the cellular aggregation was not inducible by other stressors, such as pH or temperature, treatment of the cells with DSB-inducing agents, i.e. mitomycin or bleomycin, caused the same phenotype .
Fluorescence micrograph of a cellular aggregate from S. solfataricus, formed 6 h after UV treatment (at 75 J/m2) at 254 nm
The biological function of the UV-induced cellular aggregation of S. solfataricus is not clear. The observation that even very low doses of UV light down to 5 J/m2 induced the reaction  implies that cellular aggregation may be a natural response of the cells and probably represents a protection mechanism like the stress-induced biofilm formation of Archaeoglobus fulgidus . Alternatively (or additionally), it might mediate enhanced DNA transfer between cells via a conjugation-like mechanism that could increase repair of UV-damaged chromosomes. Grogan and co-workers demonstrated an enhanced exchange of genetic marker in S. acidocaldarius of two to three orders of magnitude after UV-treatment [8,47–49]. Similarly, significantly enhanced frequency of conjugation in S. solfataricus was observed upon UV-irradiation (S. Fröls and C. Schleper, unpublished work).
Integrated model of UV-specific reactions in S. solfataricus
In conclusion, S. solfataricus exhibits a complex transcriptional regulation and also many cellular reactions to UV light. Besides the action of the DNA-repair mechanisms, the UV-dependent transcriptional-reactive genes involved many different cellular networks, such as the cell cycle, or hierarchic levels, such as the observed cellular aggregation, mediated by a UV-regulated pili secretion system that possibly enhances conjugation frequencies. It is also important to mention that the functions of the majority of genes that are specifically up- or down-regulated upon UV treatment remains currently unknown.
On the basis of the above summarized results, one could envisage the following scenario of the UV-caused reactions of S. solfataricus:
(i) After UV treatment, photoproducts are removed by the constitutively present photolyase and potentially by a still poorly understood NER system.
(ii) Non-repaired DNA lesions block ongoing replication; this results in a collapsing of the replication forks and in DSBs in DNA. Further DSBs are created through repair mechanisms.
(iii) This secondary effect of DNA lesions might induce a UV-dependent transcriptional reaction. The presence of the DSB DNA damage might, for instance, be sensed by the SSB (single-stranded DNA-binding protein) and might be mediated by the Mre11 and Rad50 proteins. The UV-dependent response causes: (a) a cell-cycle arrest, (b) a repression of initiation of replication (reaction of the cdc6 genes), (c) induction of TFB-3 (KEGG accession number sso0280) and potentially of other transcriptional regulators, (d) induction of additional genes, putatively involved in DNA-repair mechanisms (mre11/rad50 operon, dpo2, recB-like nucleases), and (e) an induction of a pili system (ups operon) and further genes involved in secretion.
(iv) The pili production mediates aggregation of S. solfataricus cells, thereby initiating enhanced conjugative exchange of DNA to increase repair of genomes via HR reminiscent of repair strategies used by Deinococcus radiodurans .
Molecular Biology of Archaea: Biochemical Society Focused Meeting held at University of St Andrews, U.K., 19–21 August 2008. Organized and Edited by Stephen Bell (Oxford, U.K.) and Malcolm White (St Andrews, U.K.).
cell division cycle 6
cis-syn-cyclobutane pyrimidine dimer
nucleotide excision repair
Sulfolobus spindle-shaped virus 1
operon, UV-inducible type IV pili operon of Sulfolobus
We apologize to colleagues whose work could not be cited owing to limited space.
S.F. was supported by the German Ministry, Bundesministerium für Bildung und Forschung, Metagenomics Cluster [grant number 4.1] of the Göttingen GenoMics Network and by the University of Bergen.