Methanococcus maripaludis S2 is a methanogenic archaeon with a well-developed genetic system. Its mesophilic nature offers a simple system in which to perform complementation using bacterial and eukaryotic genes. Although information-processing systems in archaea are generally more similar to those in eukaryotes than those in bacteria, the order Methanococcales has a unique complement of DNA replication proteins, with multiple MCM (minichromosome maintenance) proteins and no obvious originbinding protein. A search for homologues of recombination and repair proteins in M. maripaludis has revealed a mixture of bacterial, eukaryotic and some archaeal-specific homologues. Some repair pathways appear to be completely absent, but it is possible that archaeal-specific proteins could carry out these functions. The replication, recombination and repair systems in M. maripaludis are an interesting mixture of eukaryotic and bacterial homologues and could provide a system for uncovering novel interactions between proteins from different domains of life.
- DNA repair
- DNA replication
The chimaeric nature of archaeal genomes has been commented on at some length. In general, the information-processing pathways in these organisms are perceived as simplified versions of their eukaryotic counterparts. DNA replication, repair and recombination (‘the three Rs’) have all been examined using archaea as models for the equivalent eukaryotic processes . A requirement to manipulate DNA has inevitably resulted in a level of similarity between how the three Rs occur in all domains of life. However, it is clear that the proteins and mechanisms involved in these processes are generally more similar between archaea and eukaryotes. For example, key DNA replication proteins have little sequence or structural similarity between eukaryotic and bacterial equivalents. Archaeal models have undoubtedly increased our understanding of eukaryotic information-processing pathways. However, with the accumulation of results, it is becoming increasingly difficult to generalize about these pathways even within the archaea. Differences at the species level are presumably due to horizontal transfer. Variation in the components of the three Rs is particularly striking in the order Methanococcales, which provide a good illustration of the innate plasticity of DNA-processing pathways.
How do the Methanococcales replicate their DNA?
Methanococcales are an extreme example of the variation noted in DNA replication in archaea. Flow cytometric studies have indicated that the DNA content in these cells is atypical of archaea, and more similar to what is seen in bacteria [2,3]. Consistent with this observation, there is no clear archaeal/eukaryotic Cdc6/ORC (origin recognition complex)-like protein in these species, which recognizes and binds origins of replication. Equally, there is nothing that looks like the bacterial equivalent (DnaA). The Methanococcales are also unusual among archaea in possessing multiple MCM (minichromosome maintenance) homologues [4,5]. MCMs are functionally equivalent to the bacterial DnaB helicase, and form the replicative helicase in both eukaryotes and archaea. Eukaryotic MCM complexes consist of heterohexamers with six different, but related, subunits. All archaea examined to date possess a single MCM homologue that forms homohexamers. These simplified complexes have provided mechanistic insight into how the eukaryotic MCM complex is likely to function. Unusually, Methanococcus maripaludis S2 possesses four MCM homologues, all of which contain the motifs that have so far been identified as being essential for function as a DNA helicase [4,5].
All four M. maripaludis MCMs co-purified when co-expressed, indicating that they form a heteromeric complex in vitro . This system may provide insights into subunit–subunit interactions and specializations associated with individual MCMs in the eukaryotic complex. Two of the four M. maripaludis MCMs appear to have arisen via an ancient duplication event and are conserved throughout the Methanococcales. The other two MCM homologues in M. maripaludis appear to have arisen via a duplication involving mobile elements [5,6]. Whether the MCMs in this organism have evolved specific roles in the helicase complex remains to be established.
Repair, recombination and how these pathways interact with the replication machinery in the Methanococcales have not been studied. There are no obvious homologues of damage-induced checkpoint triggers such as the eukaryotic ATM (ataxia telangiectasia mutated)/ATR (ATM- and Rad3-related) genes in M. maripaludis, so how damage is recognized and whether DNA damage checkpoint mechanisms exist is currently unknown. M. maripaludis has been reported to be highly sensitive to UV damage , suggesting that some repair pathways may be compromised. Given that these organisms are strict anaerobes, and therefore unlikely to encounter direct sunlight, loss of these pathways should not be surprising. On the other hand, Methanococcales exist at a wide range of temperatures, and therefore general DNA damage repair mechanisms must exist.
We have undertaken a search for M. maripaludis S2 genes involved in recombination and repair (Table 1). We have identified a mixture of both bacterial and eukaryotic homologues. Some repair pathways, notably MMR (mismatch repair), are apparently absent in most archaea, although it is possible that completely different proteins fulfil the same functions. Some pathways appear to consist of a mixture of bacterial and eukaryotic proteins, suggesting that studying these systems could reveal novel interactions. The fact that such a melange of proteins might function together is illustrative of the plasticity likely to be inherent in DNA-processing mechanisms. Understanding what changes need to be made to bacterial and eukaryotic proteins so that different systems can interact with each other and how these repair mechanisms are co-ordinated with replication systems remains to be determined.
NHEJ (non-homologous end-joining)
NHEJ is a process by which DSBs (double-stranded breaks) in DNA can be repaired and was first identified in eukaryotes (for a review, see ). It is the major pathway of DSB repair during G1-phase of the cell cycle in eukaryotes, when HR (homologous recombination) is not possible because only a single copy of each chromosome is present. NHEJ can result in both error-free and mutagenic repair, depending on whether DNA ends are re-ligated with or without any processing. Two highly conserved proteins, Ku70 and Ku80, recognize and bind to DNA ends at DSBs and act as a platform to recruit other proteins required for repair [9,10]. NHEJ pathways have been identified in some bacteria and appear to only require two proteins, Ku and LigD [11,12]. As in eukaryotes, Ku binds to DNA ends at DSBs and recruits LigD, which possesses exonuclease, gap-filling polymerase and DNA-ligase activities and allows completion of repair . We have been unable to identify any Ku homologues within the genome of M. maripaludis. In fact, with the exception of Archaeoglobus fulgidus, Ku appears to be absent from all archaea . It is possible that an archaeal-specific protein could play the role of Ku, or perhaps Mre11/Rad50 homologues are involved in DNA-binding to allow NHEJ. It is also possible that, as in many bacteria, the NHEJ pathway is absent from M. maripaludis, perhaps because multiple chromosomes are present in the cell throughout the cell cycle, allowing the more accurate process of HR to be used to repair DSBs.
NER (nucleotide excision repair)
The NER pathway recognizes and repairs damaged DNA bases and it is a major pathway for the repair of UV-induced damage in both bacteria and eukaryotes (for a review, see ). Damaged bases are recognized by proteins that detect distortions in DNA. The most common types of DNA damage caused by UV light are CPDs (cyclobutane–pyrimidine dimers) and 6–4 photoproducts. In both eukaryotes and bacteria, there are two types of NER: GGR (global genome repair) and TCR (transcription-coupled repair) . GGR is a genome-wide process, whereas TCR only occurs at sites where DNA is being transcribed . GGR and TCR differ only in the proteins used to recognize DNA damage, with the processes downstream of damage recognition being carried out by the same set of proteins .
In bacterial GGR, damage recognition is carried out by the UvrA/B proteins (for a review, see ). In bacterial TCR, stalled RNA polymerase is detected by Mfd, a protein which modulates RNA polymerase activity to allow binding of UvrA/B to the site of damage . After damage recognition, TCR and GGR processes are identical, with UvrB/C cutting the single strand of DNA either side of the damage to allow excision, which involves the DNA helicase UvrD . DNA polymerase I then fills the gap and DNA ligase A completes repair. The process of NER in eukaryotes occurs by the same basic mechanism as in bacteria, but many more proteins are involved .
M. maripaludis possesses homologues of bacterial proteins Mfd and UvrA, B and C, suggesting that damage recognition and incision of DNA could occur via a bacterial-like process. However, M. maripaludis does not possess a homologue of UvrD, the bacterial DNA helicase required for excision of ss (single-stranded) DNA. Furthermore, the UvrA/B/C genes are present in M. maripaludis in an operon that does not contain any helicase homologues that could substitute for UvrD. Interestingly, M. maripaludis also has homologues of eukaryotic NER proteins, including the endonucleases XPF (where XP is xeroderma pigmentation complementation group) and XPG, and the DNA helicase XPD. PCNA (proliferating-cell nuclear antigen), RFC (replication factor C) and ligase I are involved in the gap-filling and ligation steps at the end of NER in eukaryotes, and homologues of all of these proteins exist in M. maripaludis. However, the polymerase involved in NER in Methanococcus is probably archaeal-specific, as none of the eukaryotic or bacterial polymerases involved in NER have homologues in M. maripaludis.
The mixture of bacterial and eukaryotic homologues in M. maripaludis implies that NER could occur via several different pathways. Although damage recognition occurs via bacterial-like proteins, the downstream process of repair could utilize a mixture of bacterial and eukaryotic homologues (Figure 1). Perhaps the eukaryotic DNA helicase XPD is able to substitute for UvrD and interacts with UvrA/B/C. Alternatively, XPD could act with XPG and XPF in a separate pathway, with another unidentified helicase acting with UvrA/B/C. Another possibility is that the NER pathway in M. maripaludis is not functional. M. maripaludis has been reported previously to be unusually sensitive to UV damage , perhaps implying that such damage cannot be repaired efficiently, if at all, by the chimaeric mixture of NER genes present in this organism. M. maripaludis also lacks a photolyase homologue, which is required for light-activated repair of UV-induced damage. This could explain further the high sensitivity of M. maripaludis to UV light.
The strand-directed MMR system is a mechanism for identifying DNA replication errors that proofreading polymerase may have missed . An important feature is the ability to recognize the newly synthesized strand and repair it to match the original template. At the core of MMR are two proteins called MutS and MutL found in bacterial and eukaryotic cells. MutS binds specifically to the mismatched base pair and recruits MutL. MutL searches nearby DNA for a nick (indicative of the newly synthesized strand) and degrades the nicked strand back through the mismatch. The ss gap can then be repaired by a polymerase and a ligase. In bacteria, an additional protein called MutH creates nicks at unmethylated (newly replicated) GATC sites.
We were unable to identify MutS, L or H in M. maripaludis. In fact, these genes are absent from all class I methanogens (Methanococcales, Methanobacteriales and Methanopyrales) . Interestingly, they are also lacking in hyperthermophilic archaea . Perhaps MMR has been replaced in these organisms by an alternative system.
Base excision repair
DNA is susceptible to damage from spontaneous base changes due to oxidation, methylation or hydrolysis. The BER (base excision repair) pathway is largely responsible for repairing this type of damage . Removal of the damaged base is the first step in this process and is catalysed by a DNA glycosylase. There are various DNA glycosylases, each specific for a particular type of damaged base. The remaining sugar phosphate is removed by an AP (apurinic/apyrimidinic) endonuclease. This leaves a single nucleotide gap in one strand, which can be filled by a DNA polymerase and sealed by a DNA ligase.
It appears that the M. maripaludis BER system is made up of homologues of eukaryotic components. Methanococcus encodes a glycosylase/lyase of the bifunctional Nth family, which cleaves the phosphodiester backbone at the same time as removing the base . This leaves an aldehyde residue blocking the 3′-terminus of the damaged strand, which still has to be processed by an AP endonuclease. Methanococcus encodes an AP endonuclease of the APE1 type.
HR allows the exchange of genetic material between two DNA duplexes of the same, or similar sequence . It is an essential process and its importance is illustrated by universal conservation of the key RecA family of recombinases (RecA in bacteria, Rad51 in eukaryotes and RadA in archaea). An important function of HR is to rescue stalled replication forks and repair DNA DSBs without any loss of information. Once a DSB is detected, an exonuclease degrades 5′-nucleotides leaving an ss 3′-overhang. This process is performed by the RecBCD complex in bacteria and by the MRN (Mre11/Rad50/Nbs1) complex in mammals. RecA proteins bind co-operatively to the ssDNA, forming a nucleoprotein filament that can intertwine with the recipient duplex. HR can also be used to repair ss breaks. In bacteria, the RecFOR pathway works in conjugation with the RecQ helicase and RecJ exonuclease to prepare the gap for RecA binding and filament formation. Strand exchange is a key step in HR, where the RecA-coated strand interacts with the recipient duplex DNA until a complementary region is located and a heteroduplex can be formed. The heteroduplex is then extended by branch migration, forming an Hj (Holliday junction). In bacteria, RuvA specifically recognizes Hj structures and recruits hexameric RuvB to move the branch point using ATP. The final step in HR is to cut two of the four strands, resolving the Hj and releasing two separate duplexes. The Hj resolvase is RuvC in bacteria and GEN1 in eukaryotes.
HR in M. maripaludis appears to be carried out by a mixture of universal, bacterial, eukaryotic and archaeal-specific proteins. M. maripaludis possesses clear homologues of eukaryotic DSB-recognition and end-processing proteins Mre11 and Rad50, although Nbs1 homologues could not be identified. M. maripaludis does not have homologues of RecBCD, but does have homologues of some of the ss break-processing components RecF, RecQ and RecJ. Another protein thought to act early on in HR is Hel308, which may remodel stalled forks . Nucleoprotein filaments are formed in archaea by RadA, which is more similar to eukaryotic Rad51 than bacterial RecA. Euryarchaeota also have a RadA paralogue called RadB, which does not cause strand invasion by itself, but stabilizes RadA on DNA . Branch migration in Methanococcus may be performed by a homologue of RuvB, although RuvA and C are lacking. M. maripaludis also possesses the archaeal Hj resolvase called Hjc, which is unrelated to bacterial RuvC or eukaryotic GEN1 . Methanococcus also has a homologue of a protein called Hef found in Euryarchaeota that was first identified in Pyrococcus furiosus . Interestingly, Haloferax volcanii Hef was recently shown to have dual roles in HR: (i) as a nuclease to resolve Hjs (such as Hjc); and (ii) as a helicase to reverse stalled replication forks and expose the causative lesion for repair by another pathway (e.g. BER) .
In M. maripaludis S2, on the basis purely of the presence or absence of known DNA repair proteins, MMR is lacking and BER is more similar to the eukaryotic pathway, whereas NER and HR combine components found in bacteria and eukaryotes with archaeal-specific proteins. All three domains of life suffer the same types of DNA damage, so perhaps it is not surprising that the enzymes that repair them are relatively transferable. However, those enzymes also have to interact with each other, and M. maripaludis may provide a useful model for studying what changes are needed to accommodate these interactions.
Work in our laboratory is supported by Cancer Research UK [grant number C23949/A7771].
Molecular Biology of Archaea II: A Biochemical Society Focused Meeting held at Robinson College, Cambridge, U.K., 16–18 August 2010. Organized and Edited by Stephen Bell (Oxford, U.K.) and Finn Werner (University College London, U.K.).
Abbreviations: AP, apurinic/apyrimidinic; ATM, ataxia telangiectasia mutated; BER, base excision repair; DSB, double-strand break; GGR, global genome repair; Hj, Holliday junction; HR, homologous recombination; MCM, minichromosome maintenance; MMR, mismatch repair; NER, nucleotide excision repair; NHEJ, non-homologous end-joining; ss, single-stranded; PCNA, proliferating-cell nuclear antigen; RFC, replication factor C; TCR, transcription-coupled repair; XP, xeroderma pigmentosum complementation group
- © The Authors Journal compilation © 2011 Biochemical Society