- Email: [email protected]
Mapping of a chromosome replication origin in an archaeon
COMMENT
References 1 Konninger, U.W. et al. (1999) The haemolysin-secreting ShlB protein of the outer membrane of Serratia marcescens: determination of surface-exposed residues and formation of ion-permeable pores by ShlB mutants in artificial lipid bilayer membranes. Mol. Microbiol. 32, 1212–1225
2 Vinion-Dubiel, A.D. et al. (1999) A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation. J. Biol. Chem. 274, 37736–37742 3 Maurer, J. et al. (1999) Characterization of the essential
transport function of the AIDA-I autotransporter and evidence supporting structural predictions. J. Bacteriol. 181, 7014–7020 4 Henderson, I.R. et al. (1998) The great escape: structure and function of the autotransporter proteins. Trends Microbiol. 6, 370–378
Mapping of a chromosome replication origin in an archaeon Rolf Bernander and Kirsten Skarstad
C
hromosome replication is fundamental to all cells, as complete duplication of the genetic material is required to ensure that both daughter cells receive a full genome complement during cell division. The organisms on Earth are currently divided into three main evolutionary lineages: the Archaea, Bacteria and Eukarya domains1. Chromosome replication mechanisms have been extensively investigated in bacteria and eukaryotes, whereas significantly less is known about archaeal replication, although this field is advancing rapidly2–4. The principal stages in the initiation of chromosome replication are similar in most organisms and replicons5, although the mechanistic details and participating proteins differ (Fig. 1; Table 1). DNA elements within the minimal replication origin are recognized by an initiator protein, and the two DNA strands are then separated at AT-rich regions within the origin. A helicase is loaded, which facilitates further unwinding, and the segregated DNA strands are stabilized and protected by singlestrand-DNA-binding proteins. The entire replication machinery is loaded, and held in place by a processivity factor (sliding clamp), which ensures that the replisome remains firmly attached to the template during DNA polymerization. The replication proteins identified in archaea are highly similar
to those of eukaryotic organisms2–4, whereas the bacterial replication machinery is considerably more distantly related (Table 1). Therefore, it might be anticipated that DNA sequence similarities should exist between archaeal and eukaryal minimal replication origins; however, similarity studies of complete archaeal genome sequences did not identify any putative replication origins. Consequently, the question of whether archaeal chromosomes contain multiple or single origins, and even whether replication is uni- or bidirectional, remained open. First mapping of a chromosome replication origin in an archaeon As the name indicates, the archaeon Pyrococcus abyssi thrives in deep-sea environments; the type strain was isolated from a hydrothermal vent at a depth of 2000 meters in the south-west Pacific Ocean6. The organism belongs to the Thermococcales order within the Euryarchaeota subdomain, and is an anaerobic hyperthermophile that grows optimally
R. Bernander* is in the Dept of Cell and Molecular Biology, Box 596, Biomedical Center, Uppsala University, SE-751 24 Uppsala, Sweden; K. Skarstad is in the Dept of Cell Biology, Institute for Cancer Research, 1310 Oslo, Norway. *tel: 146 18 471 40 58, fax: 146 18 53 03 96, e-mail: [email protected]
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
IN
MICROBIOLOGY
535
VOL. 8
NO. 12
around 96–100°C, depending on pressure. In a recent article7, Myllykallio et al. reported the mapping of a chromosome replication origin in P. abyssi. The origin was first identified by theoretical analysis of complete genome sequences8 – so-called skew analyses – in which replication origin and terminus regions are evident as abrupt shifts in strand-specific nucleotide, oligomer or codon biases in the DNA sequence. In this latest report, P. abyssi cultures were synchronized by puromycin treatment, and an 80-kb chromosome fragment that was replicated early after release of the replication block was identifed. Significantly, this fragment coincides with the putative origin region identified in the skew analyses. The chromosome fragment identified in both types of analyses was aligned between P. abyssi, Pyrococcus horikoshii and Pyrococcus furiosus and an intergenic region was shown to be highly conserved. The conserved region contains several features commonly found in replication origins9, including repeated DNA sequences and AT-rich regions. In terms of the methods used and the interpretation of the results in this report, we have the following comments. The detailed in vivo effects of puromycin on P. abyssi have not been reported. All cells might not be able to PII: S0966-842X(00)01878-3 DECEMBER 2000
COMMENT
Initiatorbinding elements
high enough to exclude the possibility that the origin might actually be located elsewhere in the 80-kb fragment, or that more than one origin might be present, although the skew analysis supports the location proposed by the authors.
AT-rich region
Minimal origin
1 Recognition and binding Initiator protein
2 Unwinding
Helicase
3 Loading of helicase (× 2) Helicase loader
Clamp loader
4 Further unwinding Loading of complete replisome including sliding clamp
Sliding clamp
5 Priming; initiation of bidirectional DNA synthesis
Fig. 1. General scheme of initiation of chromosome replication. Although the mechanistic details differ, the principal stages are common to all organisms. Only a subset of the proteins involved have been included in the drawing, and the precise spatial distribution of the various components is not intended to be accurately represented. Proteins that have been suggested to be involved in the various initiation stages in archaea, eukaryotes and bacteria are listed in Table 1. In addition to the indicated features, replication origins usually contain other repeat sequences9, both direct and inverted.
complete chromosome replication after the addition of puromycin, and the proportion of the cell population that is able to restart replication after drug removal is unknown. However, even if it
TRENDS
IN
would be possible to increase the signal strength, this would presumably not affect the qualitative result (i.e. which DNA fragment is replicated first). Also, the resolution of the in vivo mapping is not
MICROBIOLOGY
536
VOL. 8
NO. 12
Perspectives The identification of an archaeal replication origin has opened up the possibility of its functional cloning. Laboratory manipulations of extremophilic organisms, such as P. abyssi, are associated with technical difficulties regarding growth conditions, efficient transformation procedures and reliable selectable markers, which complicate cloning attempts. An additional possible complication is incompatibility: that is, difficulties in establishing an extrachromosomal element that is under the same replication control as the host chromosome. The mechanisms behind incompatibility are still not fully understood; the origin of Escherichia coli turned out to display a low degree of incompatibility, whereas minichromosomes initially could not be established in Bacillus subtilis. Whether incompatibility will complicate attempts to establish archaeal minichromosomes in vivo, and thereby the functional cloning of a replication origin, is difficult to predict. However, whether difficult or not, cloning of an archaeal origin will give important information about the mechanisms of replication control. When replication origins are eventually functionally characterized from a variety of archaeal species, it will be interesting to investigate whether they all display a similar organization, or if differences might exist, for example between extremophilic and nonextremophilic species, between eury- and crenarchaea or perhaps between those that display clear biases in skew analyses and those that do not. All of the archaeal replication proteins in Table 1 have been purified, in most cases from Methanobacterium thermoautotrophicum, and investigated in vitro3,10. Thus, with the present mapping of an archaeal origin,
DECEMBER 2000
COMMENT
Table 1. Replication proteins in Archaea, Eukarya and Bacteriaa Stage in Function Figure 1
Domain Archaea
Eukarya
Bacteria
1
Origin recognition
Orc1/Cdc6b
ORC
DnaA
2–4
Single-strandbinding protein
Rpa
RPA
Ssb
3
Helicase loading
Cdc6b
CDC6
DnaA and DnaC
3
Helicase
Mcm
MCM
DnaB
4
Clamp loader
Rfc
RFC
g complex
4
Sliding clamp
Pcna
PCNA
DnaN
5
Main replicative DNA polymerase
Family B Family B (possibly unique)
Family C
a Abbreviations: Cdc6, cell-division-cycle protein 6; DnaA (B, C, N), mutants affected in different steps of bacterial DNA replication; Mcm, minichromosome-maintenance protein; Orc, origin-recognition complex; Pcna, proliferating cell nuclear antigen; Rfc, replication factor C; Rpa, replication protein A; Ssb, single-strand-binding protein. b Not experimentally verified in Archaea.
attempts can be made to establish in vitro systems for archaeal chromosomal replication. It remains to be seen, however, whether M. thermoautotrophicum or P. abyssi will win the race. Both organisms are anaerobic thermophiles and, in the long term, the use of nonextremophilic archaea could facilitate development of in vitro assays and contribute systems more suitable for direct comparisons with the most thoroughly characterized bacterial and eukaryotic replicons. Both archaea and bacteria lack nuclear membranes and most species have single, small, circular chromosomes. The Myllykallio report indicates that further similarities include bidirectional replication from a single origin, a higher rate of replication-fork movement than eukaryotes, and a genetic organization in which the origin is situated next to genes involved in replication initiation. Similarities between archaea and eukaryotes include a homologous set of replication proteins (Table 1) and, by inference, a closer mechanistic similarity at the molecular level between the replication machineries. Whether the Archaea should be regarded as a mosaic of the other two evolutionary lineages depends upon the evolutionary relationships between the three domains of
life, an issue which presently is under intense debate. Eukaryotic cells contain membrane-surrounded nuclei and a multitude of organelles. The principal cell-cycle regulators in eukaryotes – cyclins and cyclindependent kinases – have not been identified in archaea or bacteria, and it could be that this is an additional layer of control that has only evolved in eukaryotic cells. It then becomes an interesting question whether similarities in replication control between pro- and eukaryotes might still exist at a more basal level. Studies of archaea will provide insights into this and other important cell-cycle issues, and the Myllykallio report
IN
Acknowledgements This work was supported by The Swedish Natural Science Research Council, the Swedish Institute, the Research Council of Norway and the Norwegian Cancer Society. References 1 Woese, C.R. and Fox, G.E. (1977) Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc. Natl. Acad. Sci. U. S. A. 74, 5088–5090 2 Bernander, R. (1998) Archaea and the cell cycle. Mol. Microbiol. 29, 955–961 3 Bernander, R. (2000) Chromosome replication, nucleoid segregation and cell division in Archaea. Trends Microbiol. 8, 278–283 4 Edgell, D.R. and Doolittle, W.F. (1997) Archaea and the origin(s) of DNA replication proteins. Cell 89, 995–998 5 Baker, T.A. and Bell, S.P. (1998) Polymerases and the replisome: machines within machines. Cell 92, 295–305 6 Erauso, G. et al. (1993) Pyrococcus abyssi sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Arch. Microbiol. 160, 338–349 7 Myllykallio, H. et al. (2000) Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon. Science 288, 2212–2215 8 Lopez, P. et al. (1999) Identification of putative chromosomal origins of replication in Archaea. Mol. Microbiol. 32, 883–886 9 Boulikas, T. (1996) Common structural features of replication origins in all life forms. J. Cell. Biochem. 60, 297–316 10 Liu, J. et al. (2000) Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control. Mol. Cell 6, 637–648
Mapping of a chromosome replication origin in an archaeon: Response Hannu Myllykallio and Patrick Forterre
I
n their accurate and fair comment on our work, Bernander and Skarstad critically summarize the main findings of our contribution. We agree that, despite the fact that puromycin was clearly shown to block DNA replication in Pyrococcus abyssi, the detailed in vivo effects of this
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
constitutes an important step in this direction.
MICROBIOLOGY
537
VOL. 8
NO. 12
inhibitor have not been reported. Nevertheless, during our experiments, the same early replicating 80-kb segment was identified independently, whether or not cells were treated with puromycin1, although with a smaller signal in the absence of drug. Moreover, using neutral/neutral 2-D agarose PII: S0966-842X(00)01881-3 DECEMBER 2000
References 1 Konninger, U.W. et al. (1999) The haemolysin-secreting ShlB protein of the outer membrane of Serratia marcescens: determination of surface-exposed residues and formation of ion-permeable pores by ShlB mutants in artificial lipid bilayer membranes. Mol. Microbiol. 32, 1212–1225
2 Vinion-Dubiel, A.D. et al. (1999) A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation. J. Biol. Chem. 274, 37736–37742 3 Maurer, J. et al. (1999) Characterization of the essential
transport function of the AIDA-I autotransporter and evidence supporting structural predictions. J. Bacteriol. 181, 7014–7020 4 Henderson, I.R. et al. (1998) The great escape: structure and function of the autotransporter proteins. Trends Microbiol. 6, 370–378
Mapping of a chromosome replication origin in an archaeon Rolf Bernander and Kirsten Skarstad
C
hromosome replication is fundamental to all cells, as complete duplication of the genetic material is required to ensure that both daughter cells receive a full genome complement during cell division. The organisms on Earth are currently divided into three main evolutionary lineages: the Archaea, Bacteria and Eukarya domains1. Chromosome replication mechanisms have been extensively investigated in bacteria and eukaryotes, whereas significantly less is known about archaeal replication, although this field is advancing rapidly2–4. The principal stages in the initiation of chromosome replication are similar in most organisms and replicons5, although the mechanistic details and participating proteins differ (Fig. 1; Table 1). DNA elements within the minimal replication origin are recognized by an initiator protein, and the two DNA strands are then separated at AT-rich regions within the origin. A helicase is loaded, which facilitates further unwinding, and the segregated DNA strands are stabilized and protected by singlestrand-DNA-binding proteins. The entire replication machinery is loaded, and held in place by a processivity factor (sliding clamp), which ensures that the replisome remains firmly attached to the template during DNA polymerization. The replication proteins identified in archaea are highly similar
to those of eukaryotic organisms2–4, whereas the bacterial replication machinery is considerably more distantly related (Table 1). Therefore, it might be anticipated that DNA sequence similarities should exist between archaeal and eukaryal minimal replication origins; however, similarity studies of complete archaeal genome sequences did not identify any putative replication origins. Consequently, the question of whether archaeal chromosomes contain multiple or single origins, and even whether replication is uni- or bidirectional, remained open. First mapping of a chromosome replication origin in an archaeon As the name indicates, the archaeon Pyrococcus abyssi thrives in deep-sea environments; the type strain was isolated from a hydrothermal vent at a depth of 2000 meters in the south-west Pacific Ocean6. The organism belongs to the Thermococcales order within the Euryarchaeota subdomain, and is an anaerobic hyperthermophile that grows optimally
R. Bernander* is in the Dept of Cell and Molecular Biology, Box 596, Biomedical Center, Uppsala University, SE-751 24 Uppsala, Sweden; K. Skarstad is in the Dept of Cell Biology, Institute for Cancer Research, 1310 Oslo, Norway. *tel: 146 18 471 40 58, fax: 146 18 53 03 96, e-mail: [email protected]
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
IN
MICROBIOLOGY
535
VOL. 8
NO. 12
around 96–100°C, depending on pressure. In a recent article7, Myllykallio et al. reported the mapping of a chromosome replication origin in P. abyssi. The origin was first identified by theoretical analysis of complete genome sequences8 – so-called skew analyses – in which replication origin and terminus regions are evident as abrupt shifts in strand-specific nucleotide, oligomer or codon biases in the DNA sequence. In this latest report, P. abyssi cultures were synchronized by puromycin treatment, and an 80-kb chromosome fragment that was replicated early after release of the replication block was identifed. Significantly, this fragment coincides with the putative origin region identified in the skew analyses. The chromosome fragment identified in both types of analyses was aligned between P. abyssi, Pyrococcus horikoshii and Pyrococcus furiosus and an intergenic region was shown to be highly conserved. The conserved region contains several features commonly found in replication origins9, including repeated DNA sequences and AT-rich regions. In terms of the methods used and the interpretation of the results in this report, we have the following comments. The detailed in vivo effects of puromycin on P. abyssi have not been reported. All cells might not be able to PII: S0966-842X(00)01878-3 DECEMBER 2000
COMMENT
Initiatorbinding elements
high enough to exclude the possibility that the origin might actually be located elsewhere in the 80-kb fragment, or that more than one origin might be present, although the skew analysis supports the location proposed by the authors.
AT-rich region
Minimal origin
1 Recognition and binding Initiator protein
2 Unwinding
Helicase
3 Loading of helicase (× 2) Helicase loader
Clamp loader
4 Further unwinding Loading of complete replisome including sliding clamp
Sliding clamp
5 Priming; initiation of bidirectional DNA synthesis
Fig. 1. General scheme of initiation of chromosome replication. Although the mechanistic details differ, the principal stages are common to all organisms. Only a subset of the proteins involved have been included in the drawing, and the precise spatial distribution of the various components is not intended to be accurately represented. Proteins that have been suggested to be involved in the various initiation stages in archaea, eukaryotes and bacteria are listed in Table 1. In addition to the indicated features, replication origins usually contain other repeat sequences9, both direct and inverted.
complete chromosome replication after the addition of puromycin, and the proportion of the cell population that is able to restart replication after drug removal is unknown. However, even if it
TRENDS
IN
would be possible to increase the signal strength, this would presumably not affect the qualitative result (i.e. which DNA fragment is replicated first). Also, the resolution of the in vivo mapping is not
MICROBIOLOGY
536
VOL. 8
NO. 12
Perspectives The identification of an archaeal replication origin has opened up the possibility of its functional cloning. Laboratory manipulations of extremophilic organisms, such as P. abyssi, are associated with technical difficulties regarding growth conditions, efficient transformation procedures and reliable selectable markers, which complicate cloning attempts. An additional possible complication is incompatibility: that is, difficulties in establishing an extrachromosomal element that is under the same replication control as the host chromosome. The mechanisms behind incompatibility are still not fully understood; the origin of Escherichia coli turned out to display a low degree of incompatibility, whereas minichromosomes initially could not be established in Bacillus subtilis. Whether incompatibility will complicate attempts to establish archaeal minichromosomes in vivo, and thereby the functional cloning of a replication origin, is difficult to predict. However, whether difficult or not, cloning of an archaeal origin will give important information about the mechanisms of replication control. When replication origins are eventually functionally characterized from a variety of archaeal species, it will be interesting to investigate whether they all display a similar organization, or if differences might exist, for example between extremophilic and nonextremophilic species, between eury- and crenarchaea or perhaps between those that display clear biases in skew analyses and those that do not. All of the archaeal replication proteins in Table 1 have been purified, in most cases from Methanobacterium thermoautotrophicum, and investigated in vitro3,10. Thus, with the present mapping of an archaeal origin,
DECEMBER 2000
COMMENT
Table 1. Replication proteins in Archaea, Eukarya and Bacteriaa Stage in Function Figure 1
Domain Archaea
Eukarya
Bacteria
1
Origin recognition
Orc1/Cdc6b
ORC
DnaA
2–4
Single-strandbinding protein
Rpa
RPA
Ssb
3
Helicase loading
Cdc6b
CDC6
DnaA and DnaC
3
Helicase
Mcm
MCM
DnaB
4
Clamp loader
Rfc
RFC
g complex
4
Sliding clamp
Pcna
PCNA
DnaN
5
Main replicative DNA polymerase
Family B Family B (possibly unique)
Family C
a Abbreviations: Cdc6, cell-division-cycle protein 6; DnaA (B, C, N), mutants affected in different steps of bacterial DNA replication; Mcm, minichromosome-maintenance protein; Orc, origin-recognition complex; Pcna, proliferating cell nuclear antigen; Rfc, replication factor C; Rpa, replication protein A; Ssb, single-strand-binding protein. b Not experimentally verified in Archaea.
attempts can be made to establish in vitro systems for archaeal chromosomal replication. It remains to be seen, however, whether M. thermoautotrophicum or P. abyssi will win the race. Both organisms are anaerobic thermophiles and, in the long term, the use of nonextremophilic archaea could facilitate development of in vitro assays and contribute systems more suitable for direct comparisons with the most thoroughly characterized bacterial and eukaryotic replicons. Both archaea and bacteria lack nuclear membranes and most species have single, small, circular chromosomes. The Myllykallio report indicates that further similarities include bidirectional replication from a single origin, a higher rate of replication-fork movement than eukaryotes, and a genetic organization in which the origin is situated next to genes involved in replication initiation. Similarities between archaea and eukaryotes include a homologous set of replication proteins (Table 1) and, by inference, a closer mechanistic similarity at the molecular level between the replication machineries. Whether the Archaea should be regarded as a mosaic of the other two evolutionary lineages depends upon the evolutionary relationships between the three domains of
life, an issue which presently is under intense debate. Eukaryotic cells contain membrane-surrounded nuclei and a multitude of organelles. The principal cell-cycle regulators in eukaryotes – cyclins and cyclindependent kinases – have not been identified in archaea or bacteria, and it could be that this is an additional layer of control that has only evolved in eukaryotic cells. It then becomes an interesting question whether similarities in replication control between pro- and eukaryotes might still exist at a more basal level. Studies of archaea will provide insights into this and other important cell-cycle issues, and the Myllykallio report
IN
Acknowledgements This work was supported by The Swedish Natural Science Research Council, the Swedish Institute, the Research Council of Norway and the Norwegian Cancer Society. References 1 Woese, C.R. and Fox, G.E. (1977) Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc. Natl. Acad. Sci. U. S. A. 74, 5088–5090 2 Bernander, R. (1998) Archaea and the cell cycle. Mol. Microbiol. 29, 955–961 3 Bernander, R. (2000) Chromosome replication, nucleoid segregation and cell division in Archaea. Trends Microbiol. 8, 278–283 4 Edgell, D.R. and Doolittle, W.F. (1997) Archaea and the origin(s) of DNA replication proteins. Cell 89, 995–998 5 Baker, T.A. and Bell, S.P. (1998) Polymerases and the replisome: machines within machines. Cell 92, 295–305 6 Erauso, G. et al. (1993) Pyrococcus abyssi sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Arch. Microbiol. 160, 338–349 7 Myllykallio, H. et al. (2000) Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon. Science 288, 2212–2215 8 Lopez, P. et al. (1999) Identification of putative chromosomal origins of replication in Archaea. Mol. Microbiol. 32, 883–886 9 Boulikas, T. (1996) Common structural features of replication origins in all life forms. J. Cell. Biochem. 60, 297–316 10 Liu, J. et al. (2000) Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control. Mol. Cell 6, 637–648
Mapping of a chromosome replication origin in an archaeon: Response Hannu Myllykallio and Patrick Forterre
I
n their accurate and fair comment on our work, Bernander and Skarstad critically summarize the main findings of our contribution. We agree that, despite the fact that puromycin was clearly shown to block DNA replication in Pyrococcus abyssi, the detailed in vivo effects of this
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
constitutes an important step in this direction.
MICROBIOLOGY
537
VOL. 8
NO. 12
inhibitor have not been reported. Nevertheless, during our experiments, the same early replicating 80-kb segment was identified independently, whether or not cells were treated with puromycin1, although with a smaller signal in the absence of drug. Moreover, using neutral/neutral 2-D agarose PII: S0966-842X(00)01881-3 DECEMBER 2000