- Email: [email protected]
ORC proteins: marking the start
Available online at www.sciencedirect.com
ORC proteins: marking the start Dale B Wigley The DNA replication apparatus of archaea is more closely related to that of eukaryotes than eubacteria. Furthermore, recent work has shown that archaea, like eukaryotes, have multiple replication origins. Biochemical data are starting to reveal how archaeal origin binding proteins recognise and remodel origin DNA sequences. Crystal structures of archaeal replication origin binding proteins complexed with their DNA targets revealed details of how they interact with origins and showed that they introduce significant deformations of the DNA. Although these recent advances provide insight about the initial interactions of proteins at archaeal replication origins, the molecular mechanisms of origin assembly and firing still remain elusive. Address Cancer Research UK Clare Hall Laboratories, The London Research Institute, Blanche Lane, South Mimms, Potters Bar, Herts, UK Corresponding author: Wigley, Dale B ([email protected])
Current Opinion in Structural Biology 2009, 19:72–78 This review comes from a themed issue on Protein-nucleic acid interactions Edited by Elena Conti and Daniela Rhodes
0959-440X/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2008.12.010
Introduction It has been realised for some time that the proteins associated with archaeal DNA replication and repair processes share closer similarity to eukaryotes than to eubacteria [1]. Archaeal homologues of many eukaryotic replication proteins (including Origin Recognition Complex (ORC) proteins and MCM helicase) have been identified and characterised (reviewed in [2]). Proven archaeal replication origins vary in number from one in some species such as Pyrococcus [3] to three in Sulfolobus [4] and at least five in Haloferax, and although several of these are on extra chromosomal elements at least two are on the main chromosome [5]. Similarly, the number of Cdc6/ORC proteins (for brevity, Cdc6/ORC will be referred to hereafter as ORC) is most commonly one or two proteins but can be as many as 14 proteins [5]. Crystal structures of the single ORC protein from Pyrobaculum aerophilum [6] and the ORC2 protein from Aeropyrum pernix have been determined [7]. The proteins Current Opinion in Structural Biology 2009, 19:72–78
comprise two domains; an N-terminal AAA+ domain [8] and a C-terminal Winged Helix (WH) domain [9]. Consistent with these structures, the isolated AAA+ domain has been shown to retain ATPase activity and the WH domain binds to DNA albeit with a lower affinity than full length protein [7,10]. Sequence comparisons reveal that there are two classes of ORC proteins [7].
Organisation of archaeal replication origins Archaeal replication origins vary considerably, not only between species but even within an organism (Figure 1). Analysis of archaeal genome sequences revealed a series of short (13 bp) conserved repeats located close to ORC genes in several organisms [11]. Subsequent experimental data confirmed the putative origin in Pyrococcus abysii [12,13]. The later study also revealed that two of the repeats were longer and were located at either side of a region with a strong A:T base pair bias expected for a site at which unwinding of DNA is initiated, referred to as a Duplex Unwinding Element (DUE). Similar repeats were also found flanking a DUE in a region of chromosomal DNA from Halobacterium sp. NRC-1 that conveys autonomous replication to plasmids in that species [14]. These extended repeat sequences were the Origin Recognition Box (ORB) elements later identified in Sulfolobus solfataricus [15]. This subsequent study also showed that the ORC1 protein of S. solfataricus footprints at these ORB elements in both P. abyssi and Halobacterium demonstrating their conservation across species. Another well characterised, but quite different, archaeal replication origin system is that of Sulfolobus species. There are three ORC proteins in S. solfataricus [16] and also three replication origins [4]. These origins have been analysed at a biochemical level [15,17] and are surprisingly different to one another. All three origins contain binding sites for multiple ORC proteins but it is unclear whether the levels of the proteins might alter during the cell cycle or indeed if post-translational modifications such as phosphorylation might affect their activities. The origin named oriC1 is located adjacent to the Orc1 gene and contains three binding sites for the ORC1 protein (ORB1–ORB3) with an AT-rich DUE located between two of them (Figure 1). There are also three binding sites for the ORC2 protein, one of which overlaps with the ORB2 element. Replication Initiation Point (RIP) mapping indicates a replication start point is located within the ORB3 element. The second origin, referred to as oriC2, is located proximal to the Orc3 gene and contains multiple binding sites for all three ORC proteins, with two regions containing overlapping binding sites for ORC1/ORC3 and ORC2/ORC3 proteins, www.sciencedirect.com
ORC proteins: marking the start Wigley 73
Figure 1
Organisation of archaeal replication origins. Schematic diagram showing the distribution and orientation of ORB elements in several archaeal species at what have been shown in vitro to be bona fide replication origins [5,13,15,17,18,20]. Replication origins in other organisms have also been proposed on the basis of sequence analyses but are not included here [11,15,17,30]. The grey box denotes the AT-rich region of each origin that is presumed to be the duplex unwinding element (DUE). The open arrows indicate the location and orientation of the ORB elements, with the ORB4 sequence of A. pernix shown above. Other arrows show known binding sites for additional ORC proteins in S. solfataricus. Many of these organisms have additional origins that are less well characterised. Although highly variable, a common feature within these proven origins appears to be a DUE flanked by a pair of ORB elements, usually with inverted polarity.
respectively. A DUE is again located between two of the sites and RIP mapping places the replication start point in this region. Curiously, the binding sites for the ORC1 protein are smaller versions of ORB elements referred to as ‘mini ORB’s’ and lack the conserved run of G residues at the 30 -end of full length ORB elements [15]. ORC1 protein binds to these mini-ORB sites several fold less tightly than to full length ORB elements [15]. The two binding sites for ORC3, although identical to one another, are completely different to canonical ORB elements and appear to represent a species-specific origin sequence. For the third origin, oriC3 (which is not located close to any of the ORC genes), all three proteins were again shown to bind with overlapping binding sites that flanked a DUE. Protein binding was much weaker at this origin than at the others and, curiously, the binding sites appear to share little similarity with the binding sites at the other replication origins [17]. www.sciencedirect.com
Using the sequence of the conserved ORB elements, the location of a replication origin was proposed for A. pernix [15]. At this origin (termed Ori1) there are four full length ORB elements arranged in pairs at either side of a DUE (Figure 1). Biochemical analysis has confirmed this to be an origin and RIP mapping revealed that replication is initiated within the A:T rich DUE [18]. Only one of the two ORC proteins in this organism (ORC1) binds at the origin. Once all four ORB elements have bound ORC1 proteins, a higher order assembly of the origin takes place that initates unwinding of the DUE [18]. Recent analysis identified a second replication origin in A. pernix [19] but this has not been characterised biochemically. Recent work has also identified replication origins in H. volvanii [5] and Methanothermobacter thermoautotrophicus [20] (Figure 1).
Structural information Complementary crystal structures of archaeal ORC proteins bound to ORB sequences have revealed important Current Opinion in Structural Biology 2009, 19:72–78
74 Protein-nucleic acid interactions
Figure 2
Crystal structures of ORC proteins bound to DNA. (a) The A. pernix ORC1 protein bound to an ORB element [21]. The AAA+ domain is depicted in magenta and the WH domain in cyan. The bound ADP is in black and the DNA is gold. (b) The S. solfataricus ORC1 (green) and ORC3 (blue) proteins bound to the overlapping sites found at a site within the oriC2 replication origin [22]. (c) Model for a replication origin showing a DUE flanked by inverted ORB elements. The crystal structure of the A. pernix ORC1/ORB4 complex and the EM reconstruction of the M. thermoautotrophicus MCM dodecamer [25] are shown above the DNA, that are all to scale. The WH domains shown to interact with MCM proteins [24] are cyan.
aspects of origin assembly [21,22] (Figure 2). The A. pernix ORC1 protein binds as a monomer to a single ORB element [21]. As expected, the WH domain forms extensive contacts with the ORB element but, quite unexpectedly, the AAA+ domain also contacts the DNA in an unprecedented fashion with a helical element inserting into the minor groove. This helical element is an insertion into the canonical AAA+ fold and is found in all proteins of the so-called ‘Initiator Clade’ that, in addition to archaeal ORC proteins, includes eukaryotic ORCs, Cdc6 and bacterial DnaA proteins [23]. The structure of S. solfataricus ORC1 and ORC3 bound to overlapping recognition sites revealed an additional level of complexity [22]. In this structure, the two ORC proteins bind adjacent to one another on the DNA and form contacts reminiscent of those in canonical AAA+ proteins. This allows a putative arginine finger from ORC1 to be positioned close to the ATPase site of the ORC3 suggesting that an adjustment of the subunit interface might position the arginine to promote ATP hydrolysis by the ORC3 subunit. One surprising aspect of both crystal structures was the paucity of sequence-specific contacts despite the conservation of ORB element sequences, suggesting that it is some feature of the DNA rather than sequence per se that Current Opinion in Structural Biology 2009, 19:72–78
is important for ORC binding. Examination of the DNA revealed a likely explanation. In both cases, the DNA is highly distorted, underwound across the whole binding site and also bent by 20–358. Presumably, these structural changes relate to origin assembly and MCM loading but at the present time these details remain unclear. The data presented in Figure 1 suggest that the minimal unit for an origin is a DUE with a pair of (usually) inverted ORB sites on either side. The crystal structures reveal that this orientation places the WH domains of the bound ORC proteins towards the DUE. It has been shown that archaeal ORC proteins interact with MCM through their WH domains [24]. Electron microscopy [25] has shown that the archaeal MCM dodecamer has a thickness of 240 A˚, equivalent to 70 bp of DNA, which is easily accommodated between the central inverted pair of ORB elements (Figure 2).
Implications for origin assembly To date, it has not been possible to initiate replication at a eukaryotic or archaeal origin in vitro. However, progress has been made towards that aim. For archaea, DNA footprinting has revealed binding sites for ORC proteins in a variety of organisms [15,17,18]. For A. pernix, origin assembly seems to be able to progress further in vitro than for other archaeal systems. Once www.sciencedirect.com
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Figure 3
Interactions between ORC proteins and ORB elements. (a) DNase I footprints in the region of the A. pernix ORB4 element (modified from [18]). As the ORC1 protein concentration increases, the footprint at ORB3 becomes clear and simultaneously the hypersensitivity at the DUE becomes evident (open arrows). In addition, the footprint corresponding to the AAA+ domain contact of the ORC1 protein bound at ORB4 moves from the region indicated by the black arrow to that shown by the grey arrow. The changes observed at the ORB4 element are representative of all four ORB elements [18]. (b) DNA contacts made by S. solfataricus ORC1 (grey) and ORC3 (black) in the crystal structure [22]. (c) DNA contacts made by the A. pernix ORC1 protein (black) as ORB4 as present in the crystal structure [21]. Remodelling of the ORC1 contacts (grey) occurs as the origin assembles and the contacts then resemble those of the ORC1 in panel (b). The DNA sequence present in the crystal structure is shown in black and the flanking sequence in grey.
ORC is bound at all four ORB elements, further structural changes are observed in the DNA, particularly within the DUE. These changes are consistent with some kind of higher order assembly [18], but what is this higher order assembly? Examination of the structural data together with these biochemical data begin to shed light on that process. The crystal structure of the A. pernix ORC1/ORB complex revealed two sets of interactions: the AAA+ domain www.sciencedirect.com
inserts into the minor groove at the G-string at one end of the ORB element while the WH domain inserts into the minor groove at a sequence at the other end (Figure 3). These interactions are consistent with footprinting data [18]. The structure of S. solfataricus ORC1/ORC3 complexed with an overlapping site is a little more complex. In this case, which is unique to this particular ORB element within this origin, the ORC3 protein binds to a non-canonical sequence that is specific for ORC3 while ORC1 binds to an adjacent ‘mini-ORB’ element that Current Opinion in Structural Biology 2009, 19:72–78
76 Protein-nucleic acid interactions
lacks the G-string. ORC3 binds about 10-fold more tightly than ORC1 [22] so it appears that the ORC3/ DNA complex recruits ORC1 to the site. This may also help to orient the ORC1 because the mini-ORB is almost palindromic. Despite binding to a different sequence, ORC3 binds in a similar manner to the A. pernix ORC1 protein, spanning 18 base pairs with the AAA+ domain inserting into the minor groove at one end of the sequence and the WH domain interacting at the other. By contrast, owing to its interaction with the ORC3 protein, the ORC1 protein is forced to adopt a stretched conformation causing the AAA+ domain to skip the minor groove and instead insert into the major groove a little further along the duplex (Figure 4). This extended conFigure 4
Differing conformations of the ORC1 proteins in the two structures. (a) In the A. pernix ORC1 structure, the WH domain (green) binds at one end of the ORB element in the minor groove and the AAA+ domain (magenta) inserts into the minor groove at the G-rich sequence about two turns further along the DNA. (b) In the S. solfataricus structure, the WH domain of ORC1 (green) binds to the mini-ORB sequence in a similar way to that of the A. pernix protein (panel (a)) but because of its contacts with the ORC3 protein (pale blue) the AAA+ domain (magenta) is pulled over by about half a turn and now inserts into the adjacent major groove (indicated by the arrow). The AAA+ domain of the ORC3 protein is inserted into the minor groove. Current Opinion in Structural Biology 2009, 19:72–78
formation of the ORC1 protein produces an increased contact region spanning 25 base pairs. The complex between the ORC1 and ORC3 proteins can be interpreted in either of two ways. The first is that this structure represents the initial stages in filament formation akin to those seen in eubacterial DnaA [26]. However, the DNA footprint data [15,18] do not support this suggestion for most origins although filaments on DNA have been observed at high ORC concentrations under certain circumstances [17,18]. An alternative is that ORC3 recruits and remodels the ORC1 protein serving to ‘activate’ the complex in some way. The DNA footprint data from the A. pernix system may shed some light on this second possibility. As described above, once all four ORB elements at the A. pernix Ori1 origin have ORC1 bound there is a remodelling of the whole region that results in distortion of the origin with partial unwinding across the DUE [18]. The ORC1 footprints prior to the remodelling are in very close agreement with the A. pernix crystal structure and show protection of a region extending from the WH domain contacts to those made by the AAA+ domain. However, after remodelling, although the footprint at the WH end of the contact region is essentially unaltered, there are large changes in the protection afforded by the AAA+ domain at all four ORB sequences (Figure 3). These changes can be interpreted as a shift in the binding of the AAA+ domain from the minor to the major groove, precisely the contacts made by ORC1 in the ORC1/ORC3 structure. By contrast, these changes are not seen when the footprinting experiments are carried out with the WH domain alone that does not remodel the origin. These data are consistent with the remodelling events observed in the structure of the ORC1/ORC3 complex. However, the footprint data are not consistent with filament formation or with binding of a second ORC1 at an ORB element. Consequently, it would appear that some other form of interaction between the ORC1 proteins, presumably involving the AAA+ domains, is required for the remodelling process. The direction of DNA bending is also altered after the remodelling (Figure 4) that may reflect changes in the topology of the origin DNA during this process. Consequently, although details of this remodelling across the whole origin remain a mystery, these structures together with the biochemical data appear to provide insight into the initial stages of that process. Despite remodelling the origin, the A. pernix origin assembly is still unable to load MCM. However, recent electron microscopy data have provided tantalising glimpses of that process [27]. These data revealed a second DNA binding surface on the MCM protein that wraps around the protein complex. The authors propose that this initial interaction with DNA facilitates www.sciencedirect.com
ORC proteins: marking the start Wigley 77
ring opening to allow DNA to access the central channel of the ring. Such a mechanism has parallels with that suggested for the loading of the Rho helicase [28,29].
Conclusions Considerable progress has been made with understanding the molecular details of archaeal replication in recent years, but we are still a long way from understanding the process. Crystal structures coupled with biochemical analyses have provided insight into the initial stages of replication origin and assembly. However, many questions remain. How do ORC proteins interact with one another during origin assembly? Do ORC proteins form filaments equivalent to those of DnaA in eubacterial sytems or are they forming more discrete complexes like those in eukaryotes? Finally, how does ORC load MCM at origins and initiate unwinding? Only when we understand these processes will we start to begin how one of the most fundamental biological processes, the replication of DNA, begins.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Edgell DR, Doolittle WF: Archaea and the Origin(s) of DNA replication proteins. Cell 1997, 89:995-998.
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Myllykallio H, Lopez P, Lopez-Garcia P, Heilig R, Saurin W, Zivanovic Y, Philippe H, Forterre P: Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon. Science 2000, 288:2212-2215.
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Lundgren M, Andersson A, Chen L, Nilsson P, Bernander R: Three replication origins in Sulfolobus species: synchronous initiation of chromosome replication and asynchronous termination. Proc Natl Acad Sci U S A 2004, 101:7046-7051.
11. Lopez P, Philippe H, Myllykallio H, Forterre P: Identification of putative chromosomal origins of replication in Archaea. Mol Microbiol 1999, 32:883-886. 12. Matsunaga F, Forterre P, Ishino Y, Myllykallio H: In vivo interactions of archaeal Cdc6/Orc1 and minichromosome maintenance proteins with the replication origin. Proc Natl Acad Sci U S A 2001, 98:11152-11157. 13. Matsunaga F, Norais C, Forterre P, Myllykallio H: Identification of short Okasaki fragments synthesized from a prokaryotic replication origin. EMBO Rep 2003, 4:154-158. 14. Berquist BR, DasSarma S: An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobacterium sp. strain NRC-1. J Bacteriol 2003, 185:5959-5966. 15. Robinson NP, Dionne I, Lundgren M, Marsh VL, Bernander R, Bell SD: Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus. Cell 2004, 116:25-38. 16. She Q, Singh RK, Confalonieri F, Zivanovic Y, Allard G, Awayez MJ, Chan-Weiher CC, Clausen IG, Curtis BA, De Moors A et al.: The complete genome sequence of the crenarchaeaon Sulfolobus solfataricus P2. Proc Natl Acad Sci U S A 2001, 98:7835-7840. 17. Robinson NP, Blood KA, McCallum SA, Edwards PAW, Bell SD: Sister chromatid junctions in the hyperthormophilic archaeaon Sulfolobus solfataricus. EMBO J 2007, 26:816-824. A third replication origin in Sulfolobus is described. 18. Grainge I, Gaudier M, Schuwirth BS, Westcott SL, Sandall J, Atanassova N, Wigley DB: Biochemical analysis of a DNA replication origin in the archaeon Aeropyrum pernix. J Mol Biol 2006, 363:355-369. Biochemical data show that binding of ORC proteins to a replication origin remodels and unwinds the DNA. 19. Robinson NP, Bell SD: Extrachromosomal element capture and the evolution of multiple replication origins in archaeal chromosomes. Proc Natl Acad Sci U S A 2007, 104:5806-5811. A second replication origin in A.pernix is identified. 20. Majernı´k AI, Chong JP: A conserved mechanism for replication origin recognition and binding in archaea. Biochem J 2008, 409:511-518. First biochemical evidence for a replication origin in a methanogen. 21. Gaudier M, Schuwirth BS, Westcott SL, Wigley DB: Structural basis of DNA replication origin recognition by an ORC protein. Science 2007, 317:1213-1216. Crystal structure of ORC1 bound to an origin sequence reveals the DNA distortion caused in the initial stages of origin assembly.
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22. Deuber EL, Corn JE, Bell SD, Berger JM: Replication origin recognition and deformation by a heterodimeric archaeal Orc1 complex. Science 2007, 317:1210-1213. Crystal structure of ORC1 and ORC3 proteins bound to DNA showing how interactions between the proteins remodels the contacts with DNA.
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23. Erzberger JP, Berger JM: Evolutionary relationships and structural mechanisms of AAA+ proteins. Ann Rev Biophys Biomol Struct 2006, 35:93-114.
Norais C, Hawkins M, Hartman AL, Eisen JA, Myllykallio H, Allers T: Genetic and physical mapping of DNA replication origins in Haloferax volcanii. PLoS Genet 2007, 3:e77. Biochemical and genetic analyses of origins in H. volcanii. Liu J, Smith CL, DeRyckere D, DeAngelis K, Martin GS, Berger JM: Structure and function of cdc6/Cdc18: implications for origin recognition and checkpoint control. Mol Cell 2000, 6:637-648.
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Singleton MR, Morales R, Grainge I, Cook N, Isupov MN, Wigley DB: Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix. J Mol Biol 2004, 343:547-557.
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Neuwald AF, Aravind L, Spouge JL, Koonin EV: AAA+: a class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res 1999, 9:27-43.
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Gajiwala KS, Burley SK: Winged helix proteins. Curr Opin Struct Biol 2000, 10:110-116.
10. De Felice M, Esposito L, Pucci B, De Falco M, Manco G, Rossi M, Pisani FM: Modular organisation of a Cdc6-like protein from the crenarchaeon Sulpholobus solfataricus. Biochem J 2004, 278:46424-46431. www.sciencedirect.com
24. Kasiviswanathan R, Shin J, Kelman Z: DNA binding by the Methanothermobacter thermoautotrophicus Cdc6 protein is inhibited by the minichromosome maintenance helicase. J Bacteriol 2006, 188:4577-4580. Biochemical data showing an interaction between archaeal ORC and MCM proteins. 25. Costa A, Pape T, van Heel M, Brick P, Patwardhan A, Onesti S: Structural basis of the Methanothermobacter thermautotrophicus MCM helicase activity. Nucleic Acids Res 2006, 34:5829-5838. 26. Erzberger JP, Mott ML, Berger JM: Structural basis for ATPdependent DnaA assembly and replication-origin remodeling. Nat Struct Mol Biol 2006, 13:676-683. 27. Costa A, van Duinen G, Medagli B, Chong J, Sakakibara N, Kelman Z, Nair SK, Patwardhan A, Onesti S: Cryo-electron microscopy reveals a novel DNA-binding site on the MCM helicase. EMBO J 2008, 27:2250-2258. Current Opinion in Structural Biology 2009, 19:72–78
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Electron microscopy data reveal a novel DNA interaction mode for MCM helicase that may have implications for loading of hexameric helicase rings onto DNA.
29. Skordalakes E, Berger JM: Structure of the Rho transcription terminator: mechanism of mRNA recognition and helicase loading. Cell 2003, 114:135-146.
28. Kim DE, Patel SS: The kinetic pathway of RNA binding to the Escherichia coli transcription termination factor Rho. J Biol Chem 2001, 276:13902-13910.
30. Zhang R, Zhang C: Identification of replication origins in archaeal genomes based on the Z-curve method. Archaea 2005, 1:335-346.
Current Opinion in Structural Biology 2009, 19:72–78
www.sciencedirect.com
ORC proteins: marking the start Dale B Wigley The DNA replication apparatus of archaea is more closely related to that of eukaryotes than eubacteria. Furthermore, recent work has shown that archaea, like eukaryotes, have multiple replication origins. Biochemical data are starting to reveal how archaeal origin binding proteins recognise and remodel origin DNA sequences. Crystal structures of archaeal replication origin binding proteins complexed with their DNA targets revealed details of how they interact with origins and showed that they introduce significant deformations of the DNA. Although these recent advances provide insight about the initial interactions of proteins at archaeal replication origins, the molecular mechanisms of origin assembly and firing still remain elusive. Address Cancer Research UK Clare Hall Laboratories, The London Research Institute, Blanche Lane, South Mimms, Potters Bar, Herts, UK Corresponding author: Wigley, Dale B ([email protected])
Current Opinion in Structural Biology 2009, 19:72–78 This review comes from a themed issue on Protein-nucleic acid interactions Edited by Elena Conti and Daniela Rhodes
0959-440X/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2008.12.010
Introduction It has been realised for some time that the proteins associated with archaeal DNA replication and repair processes share closer similarity to eukaryotes than to eubacteria [1]. Archaeal homologues of many eukaryotic replication proteins (including Origin Recognition Complex (ORC) proteins and MCM helicase) have been identified and characterised (reviewed in [2]). Proven archaeal replication origins vary in number from one in some species such as Pyrococcus [3] to three in Sulfolobus [4] and at least five in Haloferax, and although several of these are on extra chromosomal elements at least two are on the main chromosome [5]. Similarly, the number of Cdc6/ORC proteins (for brevity, Cdc6/ORC will be referred to hereafter as ORC) is most commonly one or two proteins but can be as many as 14 proteins [5]. Crystal structures of the single ORC protein from Pyrobaculum aerophilum [6] and the ORC2 protein from Aeropyrum pernix have been determined [7]. The proteins Current Opinion in Structural Biology 2009, 19:72–78
comprise two domains; an N-terminal AAA+ domain [8] and a C-terminal Winged Helix (WH) domain [9]. Consistent with these structures, the isolated AAA+ domain has been shown to retain ATPase activity and the WH domain binds to DNA albeit with a lower affinity than full length protein [7,10]. Sequence comparisons reveal that there are two classes of ORC proteins [7].
Organisation of archaeal replication origins Archaeal replication origins vary considerably, not only between species but even within an organism (Figure 1). Analysis of archaeal genome sequences revealed a series of short (13 bp) conserved repeats located close to ORC genes in several organisms [11]. Subsequent experimental data confirmed the putative origin in Pyrococcus abysii [12,13]. The later study also revealed that two of the repeats were longer and were located at either side of a region with a strong A:T base pair bias expected for a site at which unwinding of DNA is initiated, referred to as a Duplex Unwinding Element (DUE). Similar repeats were also found flanking a DUE in a region of chromosomal DNA from Halobacterium sp. NRC-1 that conveys autonomous replication to plasmids in that species [14]. These extended repeat sequences were the Origin Recognition Box (ORB) elements later identified in Sulfolobus solfataricus [15]. This subsequent study also showed that the ORC1 protein of S. solfataricus footprints at these ORB elements in both P. abyssi and Halobacterium demonstrating their conservation across species. Another well characterised, but quite different, archaeal replication origin system is that of Sulfolobus species. There are three ORC proteins in S. solfataricus [16] and also three replication origins [4]. These origins have been analysed at a biochemical level [15,17] and are surprisingly different to one another. All three origins contain binding sites for multiple ORC proteins but it is unclear whether the levels of the proteins might alter during the cell cycle or indeed if post-translational modifications such as phosphorylation might affect their activities. The origin named oriC1 is located adjacent to the Orc1 gene and contains three binding sites for the ORC1 protein (ORB1–ORB3) with an AT-rich DUE located between two of them (Figure 1). There are also three binding sites for the ORC2 protein, one of which overlaps with the ORB2 element. Replication Initiation Point (RIP) mapping indicates a replication start point is located within the ORB3 element. The second origin, referred to as oriC2, is located proximal to the Orc3 gene and contains multiple binding sites for all three ORC proteins, with two regions containing overlapping binding sites for ORC1/ORC3 and ORC2/ORC3 proteins, www.sciencedirect.com
ORC proteins: marking the start Wigley 73
Figure 1
Organisation of archaeal replication origins. Schematic diagram showing the distribution and orientation of ORB elements in several archaeal species at what have been shown in vitro to be bona fide replication origins [5,13,15,17,18,20]. Replication origins in other organisms have also been proposed on the basis of sequence analyses but are not included here [11,15,17,30]. The grey box denotes the AT-rich region of each origin that is presumed to be the duplex unwinding element (DUE). The open arrows indicate the location and orientation of the ORB elements, with the ORB4 sequence of A. pernix shown above. Other arrows show known binding sites for additional ORC proteins in S. solfataricus. Many of these organisms have additional origins that are less well characterised. Although highly variable, a common feature within these proven origins appears to be a DUE flanked by a pair of ORB elements, usually with inverted polarity.
respectively. A DUE is again located between two of the sites and RIP mapping places the replication start point in this region. Curiously, the binding sites for the ORC1 protein are smaller versions of ORB elements referred to as ‘mini ORB’s’ and lack the conserved run of G residues at the 30 -end of full length ORB elements [15]. ORC1 protein binds to these mini-ORB sites several fold less tightly than to full length ORB elements [15]. The two binding sites for ORC3, although identical to one another, are completely different to canonical ORB elements and appear to represent a species-specific origin sequence. For the third origin, oriC3 (which is not located close to any of the ORC genes), all three proteins were again shown to bind with overlapping binding sites that flanked a DUE. Protein binding was much weaker at this origin than at the others and, curiously, the binding sites appear to share little similarity with the binding sites at the other replication origins [17]. www.sciencedirect.com
Using the sequence of the conserved ORB elements, the location of a replication origin was proposed for A. pernix [15]. At this origin (termed Ori1) there are four full length ORB elements arranged in pairs at either side of a DUE (Figure 1). Biochemical analysis has confirmed this to be an origin and RIP mapping revealed that replication is initiated within the A:T rich DUE [18]. Only one of the two ORC proteins in this organism (ORC1) binds at the origin. Once all four ORB elements have bound ORC1 proteins, a higher order assembly of the origin takes place that initates unwinding of the DUE [18]. Recent analysis identified a second replication origin in A. pernix [19] but this has not been characterised biochemically. Recent work has also identified replication origins in H. volvanii [5] and Methanothermobacter thermoautotrophicus [20] (Figure 1).
Structural information Complementary crystal structures of archaeal ORC proteins bound to ORB sequences have revealed important Current Opinion in Structural Biology 2009, 19:72–78
74 Protein-nucleic acid interactions
Figure 2
Crystal structures of ORC proteins bound to DNA. (a) The A. pernix ORC1 protein bound to an ORB element [21]. The AAA+ domain is depicted in magenta and the WH domain in cyan. The bound ADP is in black and the DNA is gold. (b) The S. solfataricus ORC1 (green) and ORC3 (blue) proteins bound to the overlapping sites found at a site within the oriC2 replication origin [22]. (c) Model for a replication origin showing a DUE flanked by inverted ORB elements. The crystal structure of the A. pernix ORC1/ORB4 complex and the EM reconstruction of the M. thermoautotrophicus MCM dodecamer [25] are shown above the DNA, that are all to scale. The WH domains shown to interact with MCM proteins [24] are cyan.
aspects of origin assembly [21,22] (Figure 2). The A. pernix ORC1 protein binds as a monomer to a single ORB element [21]. As expected, the WH domain forms extensive contacts with the ORB element but, quite unexpectedly, the AAA+ domain also contacts the DNA in an unprecedented fashion with a helical element inserting into the minor groove. This helical element is an insertion into the canonical AAA+ fold and is found in all proteins of the so-called ‘Initiator Clade’ that, in addition to archaeal ORC proteins, includes eukaryotic ORCs, Cdc6 and bacterial DnaA proteins [23]. The structure of S. solfataricus ORC1 and ORC3 bound to overlapping recognition sites revealed an additional level of complexity [22]. In this structure, the two ORC proteins bind adjacent to one another on the DNA and form contacts reminiscent of those in canonical AAA+ proteins. This allows a putative arginine finger from ORC1 to be positioned close to the ATPase site of the ORC3 suggesting that an adjustment of the subunit interface might position the arginine to promote ATP hydrolysis by the ORC3 subunit. One surprising aspect of both crystal structures was the paucity of sequence-specific contacts despite the conservation of ORB element sequences, suggesting that it is some feature of the DNA rather than sequence per se that Current Opinion in Structural Biology 2009, 19:72–78
is important for ORC binding. Examination of the DNA revealed a likely explanation. In both cases, the DNA is highly distorted, underwound across the whole binding site and also bent by 20–358. Presumably, these structural changes relate to origin assembly and MCM loading but at the present time these details remain unclear. The data presented in Figure 1 suggest that the minimal unit for an origin is a DUE with a pair of (usually) inverted ORB sites on either side. The crystal structures reveal that this orientation places the WH domains of the bound ORC proteins towards the DUE. It has been shown that archaeal ORC proteins interact with MCM through their WH domains [24]. Electron microscopy [25] has shown that the archaeal MCM dodecamer has a thickness of 240 A˚, equivalent to 70 bp of DNA, which is easily accommodated between the central inverted pair of ORB elements (Figure 2).
Implications for origin assembly To date, it has not been possible to initiate replication at a eukaryotic or archaeal origin in vitro. However, progress has been made towards that aim. For archaea, DNA footprinting has revealed binding sites for ORC proteins in a variety of organisms [15,17,18]. For A. pernix, origin assembly seems to be able to progress further in vitro than for other archaeal systems. Once www.sciencedirect.com
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Figure 3
Interactions between ORC proteins and ORB elements. (a) DNase I footprints in the region of the A. pernix ORB4 element (modified from [18]). As the ORC1 protein concentration increases, the footprint at ORB3 becomes clear and simultaneously the hypersensitivity at the DUE becomes evident (open arrows). In addition, the footprint corresponding to the AAA+ domain contact of the ORC1 protein bound at ORB4 moves from the region indicated by the black arrow to that shown by the grey arrow. The changes observed at the ORB4 element are representative of all four ORB elements [18]. (b) DNA contacts made by S. solfataricus ORC1 (grey) and ORC3 (black) in the crystal structure [22]. (c) DNA contacts made by the A. pernix ORC1 protein (black) as ORB4 as present in the crystal structure [21]. Remodelling of the ORC1 contacts (grey) occurs as the origin assembles and the contacts then resemble those of the ORC1 in panel (b). The DNA sequence present in the crystal structure is shown in black and the flanking sequence in grey.
ORC is bound at all four ORB elements, further structural changes are observed in the DNA, particularly within the DUE. These changes are consistent with some kind of higher order assembly [18], but what is this higher order assembly? Examination of the structural data together with these biochemical data begin to shed light on that process. The crystal structure of the A. pernix ORC1/ORB complex revealed two sets of interactions: the AAA+ domain www.sciencedirect.com
inserts into the minor groove at the G-string at one end of the ORB element while the WH domain inserts into the minor groove at a sequence at the other end (Figure 3). These interactions are consistent with footprinting data [18]. The structure of S. solfataricus ORC1/ORC3 complexed with an overlapping site is a little more complex. In this case, which is unique to this particular ORB element within this origin, the ORC3 protein binds to a non-canonical sequence that is specific for ORC3 while ORC1 binds to an adjacent ‘mini-ORB’ element that Current Opinion in Structural Biology 2009, 19:72–78
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lacks the G-string. ORC3 binds about 10-fold more tightly than ORC1 [22] so it appears that the ORC3/ DNA complex recruits ORC1 to the site. This may also help to orient the ORC1 because the mini-ORB is almost palindromic. Despite binding to a different sequence, ORC3 binds in a similar manner to the A. pernix ORC1 protein, spanning 18 base pairs with the AAA+ domain inserting into the minor groove at one end of the sequence and the WH domain interacting at the other. By contrast, owing to its interaction with the ORC3 protein, the ORC1 protein is forced to adopt a stretched conformation causing the AAA+ domain to skip the minor groove and instead insert into the major groove a little further along the duplex (Figure 4). This extended conFigure 4
Differing conformations of the ORC1 proteins in the two structures. (a) In the A. pernix ORC1 structure, the WH domain (green) binds at one end of the ORB element in the minor groove and the AAA+ domain (magenta) inserts into the minor groove at the G-rich sequence about two turns further along the DNA. (b) In the S. solfataricus structure, the WH domain of ORC1 (green) binds to the mini-ORB sequence in a similar way to that of the A. pernix protein (panel (a)) but because of its contacts with the ORC3 protein (pale blue) the AAA+ domain (magenta) is pulled over by about half a turn and now inserts into the adjacent major groove (indicated by the arrow). The AAA+ domain of the ORC3 protein is inserted into the minor groove. Current Opinion in Structural Biology 2009, 19:72–78
formation of the ORC1 protein produces an increased contact region spanning 25 base pairs. The complex between the ORC1 and ORC3 proteins can be interpreted in either of two ways. The first is that this structure represents the initial stages in filament formation akin to those seen in eubacterial DnaA [26]. However, the DNA footprint data [15,18] do not support this suggestion for most origins although filaments on DNA have been observed at high ORC concentrations under certain circumstances [17,18]. An alternative is that ORC3 recruits and remodels the ORC1 protein serving to ‘activate’ the complex in some way. The DNA footprint data from the A. pernix system may shed some light on this second possibility. As described above, once all four ORB elements at the A. pernix Ori1 origin have ORC1 bound there is a remodelling of the whole region that results in distortion of the origin with partial unwinding across the DUE [18]. The ORC1 footprints prior to the remodelling are in very close agreement with the A. pernix crystal structure and show protection of a region extending from the WH domain contacts to those made by the AAA+ domain. However, after remodelling, although the footprint at the WH end of the contact region is essentially unaltered, there are large changes in the protection afforded by the AAA+ domain at all four ORB sequences (Figure 3). These changes can be interpreted as a shift in the binding of the AAA+ domain from the minor to the major groove, precisely the contacts made by ORC1 in the ORC1/ORC3 structure. By contrast, these changes are not seen when the footprinting experiments are carried out with the WH domain alone that does not remodel the origin. These data are consistent with the remodelling events observed in the structure of the ORC1/ORC3 complex. However, the footprint data are not consistent with filament formation or with binding of a second ORC1 at an ORB element. Consequently, it would appear that some other form of interaction between the ORC1 proteins, presumably involving the AAA+ domains, is required for the remodelling process. The direction of DNA bending is also altered after the remodelling (Figure 4) that may reflect changes in the topology of the origin DNA during this process. Consequently, although details of this remodelling across the whole origin remain a mystery, these structures together with the biochemical data appear to provide insight into the initial stages of that process. Despite remodelling the origin, the A. pernix origin assembly is still unable to load MCM. However, recent electron microscopy data have provided tantalising glimpses of that process [27]. These data revealed a second DNA binding surface on the MCM protein that wraps around the protein complex. The authors propose that this initial interaction with DNA facilitates www.sciencedirect.com
ORC proteins: marking the start Wigley 77
ring opening to allow DNA to access the central channel of the ring. Such a mechanism has parallels with that suggested for the loading of the Rho helicase [28,29].
Conclusions Considerable progress has been made with understanding the molecular details of archaeal replication in recent years, but we are still a long way from understanding the process. Crystal structures coupled with biochemical analyses have provided insight into the initial stages of replication origin and assembly. However, many questions remain. How do ORC proteins interact with one another during origin assembly? Do ORC proteins form filaments equivalent to those of DnaA in eubacterial sytems or are they forming more discrete complexes like those in eukaryotes? Finally, how does ORC load MCM at origins and initiate unwinding? Only when we understand these processes will we start to begin how one of the most fundamental biological processes, the replication of DNA, begins.
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