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Nat Rev Mol Cell Biol. Author manuscript; available in PMC 2005 October 17. Published in final edited form as: | PMCID: PMC1255919 NIHMSID: NIHMS3646 |
In search of the holy replicator David M. Gilbert David M. Gilbert is at the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, NewYork 13210, USA. e-mail: gilbertd/at/upstate.edu After 40 years of searching for the eukaryotic replicator sequence, it is time to abandon the concept of ‘the’ replicator as a single genetic entity. Here I propose a ‘relaxed replicon model’ in which a positive initiator–replicator interaction is facilitated by a combination of several complex features of chromatin. An important question for the future is whether the positions of replication origins are simply a passive result of local chromatin structure or are actively localized to coordinate replication with other chromosomal activities. In the summer of 1962, François Jacob and Sydney Brenner sketched in the sand of France’s La Tranche-sur-Mer a model () to explain the regulation of DNA synthesis in bacteria 1. Their ‘replicon model’ proposed that replication was regulated by a positive interaction between an initiator protein, which is encoded by a structural gene, and a specific genetic element of recognition, which is known as the replicator. Although originally intended to explain the replication of circular bacterial chromosomes, the replicon model was quickly adopted as the paradigm for eukaryotic chromosomes as well (). Proteins with the properties of initiators have been identified in species from Escherichia coli to man. By contrast, genetically defined replicators have yet to be identified in most eukaryotes and are clearly dispensable for replication in many eukaryotic systems. Even in its shortcomings, though, the model has been useful. Indeed, the replicon model was the driving force behind the discovery of bacterial and budding yeast replicators as well as initiator proteins — from dnaA in prokaryotes 2 to the origin-recognition complex (ORC) in eukaryotes 3. Indeed, all cellular chromosomes can be thought of as replicons, or clusters of replicons — the replication of which is regulated, at least in part, by a positive initiator–replicator interaction. In this historical perspective, I will summarize the salient findings over the course of these four decades (), with a focus on strategies to identify metazoan replicators and the debate over their existence. I propose that we replace the concept of the replicator as a specific DNA sequence with that of a context-dependent element that is recognized by the initiator through several features, which can include DNA sequence. By embracing the complexity of replicators as being akin to that of promoter elements, future experiments can define these various features that influence the initiator–replicator interaction and address the biological significance of specifying replication-origin sites, given that site-specific initiation is dispensable for regulated genome duplication 30. One of the beauties of the replicon model was the conceptually straightforward predictions that it provided. A replicator can confer the ability to replicate onto any DNA segments that are linked to it, and this became relatively simple to test after the development of recombinant-DNA technology in the early 1970s. By linking various segments of E. coli plasmids to a non-replicating DNA fragment that contained a selectable marker, Stanley Cohen and colleagues identified sequences that could confer, in cis, the ability of these molecules to replicate autonomously 4. This straightforward assay ( BOX 1) was later used to identify many different replicators, including oriC in E. coli5. Once a replicator sequence is defined, the initiator can be identified as a protein that binds to the replicator, and the dnaA protein in E. coli fulfilled this criterion 2. Allowing for a few minor adjustments, it is clear that the replicon model is not only a useful paradigm, but that it accurately describes all bacterial replication systems under normal physiological selective pressures. Moreover, the replicator DNA sequence and the initiator protein are highly conserved across different bacterial species 6. Box 1 | The autonomously replicating sequence (ARS) assay The ARS assay is used to identify replicators in genomes or DNA sequences that can range from those that are as simple as small bacterial plasmids and animal viruses to those that are as complex as budding and fission yeast genomes — the principle is very similar in all cases. The test genome is fragmented and the fragments are cloned into a vector that contains a selectable marker that can function in the appropriate host. The appropriate host is a cell type that is known to contain all the proteins that are necessary to support replication of the desired replicator, including the initiator. The collection of plasmids containing these genome fragments is then introduced into the host in such a way that each cell obtains one or fewer plasmids. Selection for the presence of the plasmid is applied, and the resulting recipient clonal colonies are expanded and evaluated for the ability of the (usually circular) plasmid to be maintained and to replicate as an independent unit, autonomously, within the host cell. In some variations of this assay, replicating plasmids can be detected or enriched without cell-growth selection. Myriad permutations of this assay have been applied to metazoan systems, but have generally failed to identify any specific DNA sequences that confer ARS activity. Together, these studies show that initiation of replication in mammalian cells requires specific DNA sequences that are both close to and distant from the site of initiation, but no single consensus DNA-sequence motif is either necessary or sufficient for replicator activity. The simplest interpretation of these studies is that there is specificity to the initiator–replicator interaction, but the features that are recognized by the initiator are only indirectly determined by DNA sequences. |
Eukaryotes: a different problem It did not take long for investigators to appreciate that eukaryotic chromosomes had fundamentally different problems to solve, which could not be accommodated by the replicon model in its simplest form. Whereas bacteria usually have a single circular chromosome with only one replicator per chromosome, eukaryotes have several linear chromosomes that are each many times larger. Early autoradiography studies of DNA synthesis along the lengths of isolated DNA fibres showed that these chromosomes replicate using hundreds to thousands of replication origins 7,8 (). Moreover, there was clearly flexibility in the usage of replication origins at different developmental stages 9–11 and under different growth conditions 12,13. Also, replication origins were shown to fire asynchronously throughout the DNA-synthesis phase of the cell cycle 14,15. So, even though the existence of a common eukaryotic replicator sequence was proposed (), it was already clear that additional layers of regulation would be needed to explain such flexibility. Given these known complexities, perhaps it was not fair to hold the bacterial replicon model to the standard of eukaryotic replication. But the advent of budding yeast genetics in the 1970s provided the ideal model system in which to use the autonomous-replication assay, which had been so successful in bacterial systems, to identify eukaryotic replicators. The immediate success of this approach 16, which resulted in the cloning of several ‘autonomously replicating sequences’ (ARS elements), all but confirmed the replicon model as a useful paradigm for eukaryotes. Indeed, ARS elements have since been shown to be sites of replication initiation in their native context 17–19, and to contain a common DNA sequence that is required for replication initiation both on plasmids 20 and within chromosomes 21. Hence, this proof-of-principle experiment that was carried out 25 years ago set the stage for the hunt for replicators in higher eukaryotic organisms. Ironically, budding yeast has turned out to be an exceptional eukaryote with respect to the importance of a specific consensus replicator sequence. The mystery of the metazoa As they have genomes that are up to 100 times larger in size than those of yeast, mapping replication origins in higher eukaryotes was expected to be difficult. But no one could have predicted the degree of disparity that was uncovered between different systems, and even within individual systems that were studied using different methodologies. In 1980, Richard Harland and Ron Laskey demonstrated that any of several DNA fragments that were microinjected into Xenopus laevis eggs, even those containing mostly bacterial DNA, replicated with equal efficiency and in a regulated once-per-cell-cycle fashion 22. The results were compelling, and they were reproducible 23. In stark contrast, Nick Heintz and Joyce Hamlin demonstrated shortly thereafter that initiation of replication could be localized to specific sites in mammalian chromosomes 24. So, the great debate had begun. The X. laevis experiments showed that once-per-cell-cycle replication could be accomplished without the need for specific replicator sequences. But mammalian cells clearly did not initiate replication at random sites, and X. laevis embryos are a specialized system, which is designed to duplicate the entire genome within 20 minutes. For most, this was sufficient reason to disregard the X. laevis embryo results, and the quest for the ‘holy grail’ of the human replicator shifted into high gear. ARS assays: all or nothing The obvious first strategy of choice was the autonomously replicating sequence (ARS) assay that had worked so well in bacteria and yeast ( BOX 1). It is impossible to say how many clever permutations of this approach were tried and failed or found to be non-reproducible 25. In most cases, very poor transient replication activity 26 quickly resulted in selection for plasmids that had stably integrated into chromosomes 25. Then, in the late 1980s, Michelle Calos and colleagues demonstrated that the efficiency of ARS assays could be greatly improved by preventing the loss of plasmids from the nucleus using DNA sequences that tether plasmids to segregating chromosomes. The results of these experiments were astonishingly clear. Plasmids could be maintained for many generations, but replication was independent of DNA sequence and was initiated at random sites within these plasmids 27. The dirty ‘random’ word had reared its ugly head again, placing a nasty stigma on the ARS assay. For those bent on finding the holy replicator, it was clear that some nuance of mammalian cell culture precluded the ability of ARS assays to reveal physiological replicator activity. Religion, politics and replication origins By the early 1990s, most investigators had conceded that classic genetic approaches, including those that employed promising viral models ( BOX 2), were insufficient for identifying metazoan replicators, and many sought a more complete characterization of replicators in their native context. Finding replication intermediates in a genome of 3 billion basepairs required new technological developments, and several new methods for mapping replication origins emerged. Rather than clarifying things, however, the ‘random versus site specific’ debate escalated to a whole new level. In 1990, almost simultaneously, two reports in which different methods were used to map initiation sites within the Chinese hamster dihydrofolate reductase ( DHFR) locus arrived at diametrically opposed conclusions. One study claimed that >80% of initiations within a 27-Kb region emanated from a single site 28, whereas the other claimed that replication initiated at sites distributed virtually at random throughout the very same region 29. These results shifted the debate from a philosophical comparison of different biological systems to a detailed technical evaluation of different methodological approaches, leaving investigators unable to reach a consensus as to the basic structure of metazoan replication origins nor the best means to evaluate this structure. Box 2 | The contribution of viral replicators The small double-stranded DNA viruses (for example, polyoma and papilloma viruses) were the preferred model for studying eukaryotic replication in the 1980s, and because they use the host’s machinery for replication, they were particularly promising models for mammalian chromosome replication. In fact, most of our knowledge of the events that occur downstream of replication-origin unwinding comes from studies with simian virus-40 (SV40), in which defined in vitro replication systems identified the proteins that are essential for DNA synthesis in human cells 83,84. Initiation of replication clearly requires the interaction of a virally encoded initiator protein with a defined replicator sequence that could be genetically dissected with classic autonomously replicating sequence (ARS) assays 85. But these viruses do not replicate once per cell cycle, they supply their own virally encoded initiator, and they are not subjected to the same selective pressures as cellular chromosomes, so they are limited in their use as models for mammalian replicator function. By contrast, the large Epstein–Barr virus (EBV) does not supply its own initiator, but instead requires ORC 86. Latent (non-lytic) replication of EBV is restricted to one round per cell cycle 87, making this virus a good potential model for cellular replication. Interestingly though, a region known as oriP, which was shown to have ARS activity in the presence of a virally encoded protein, is dispensable for latent viral replication 87, and origin-mapping studies have shown that initiation-site selection in the full-sized EBV genome is just as degenerate as it is within cellular chromosomes, whether or not oriP is present 82. |
Nearly 15 years have passed and the distribution of initiation sites at this and many other metazoan loci has still not been resolved. As more replication origins were mapped, some were localized to very defined sites, whereas others mapped indiscriminately throughout ‘initiation zones’ of 10–50 Kb (reviewed in ref. 30). Most of these sites have been mapped by only one method, so the extent to which these differences can be accounted for by methodological considerations is not clear. On the other hand, nearly every replication-origin mapping method has been applied to the DHFR locus, and there is still no consensus on the pattern of initiation at this locus. Some methods detect specific ‘replicator’ sites within the non-transcribed region downstream of the DHFR locus 31, whereas other methods reveal initiation sites throughout every interrogated sequence within the same region 32. At present, the best explanation for these discrepancies is that initiation can take place at many sites throughout this 50-Kb region, but that some methods are only capable of detecting a subset of these sites. In the meantime, people who worked with the X. laevis system had all but proven that any DNA sequence can function as a replicator. After the development of cell-free replication systems that were derived from X. laevis eggs, many investigators were able to demonstrate the promiscuity of replication in this system, and the apparently random choice of initiation sites within each DNA molecule 33–35. The same was found to be true within the native chromosomes of early X. laevis36 and Drosophila melanogaster37 embryos. Importantly, these latter studies revealed that replication initiation is eventually focused to specific sites after the blastula stage of development. These results were interpreted along existing party lines. Proponents of site-specific initiation assumed that random site selection was a special adaptation for rapid replication in early embryos, which was mediated either by a specialized embryonic initiator protein or by the high concentration of maternally accumulated initiator proteins that become diluted during development. These possibilities were ruled out by reconstituting ORC-depleted X. laevis egg extracts with human ORC, which restored sequence-independent initiation of replication in a concentration-independent manner 38. The other party declared that developmentally regulated features of chromatin, rather than a specific DNA sequence, dictate origin specification. This interpretation was supported by the fact that X. laevis egg extracts are capable of initiating replication at specific sites when provided with chromatin from somatic cells as a template 35, and that the defining epigenetic structure is re-assembled early in G1 phase of each cell cycle 39. In fact, simply pre-loading transcription factors onto naked DNA templates is sufficient to direct site-specific initiation in pre-blastula X. laevis embryos 40. DNA sequence: a clear contributor New hope in the hunt for the holy replicator arrived when Howard Cedar and colleagues demonstrated that a naturally occurring 8-Kb deletion that encompassed a region of replication-initiation activity within the human β-globin locus ( HBB) eliminated bidirectional replication in this region 41. Hailed as a triumph that would deliver us from the dark ages, this result inspired further deletion studies that soon revealed a more complex picture. At both the HBB and the DHFR locus, deletions 25–50 Kb away from the region of replication-initiation activity also completely eliminated its activity 42,43. Deletions within the large region of activity at the DHFR locus actually augmented activity at the remaining sites 44, implying a high degree of redundancy of potential replicators throughout this region. A critical test of ‘replicator’ activity is to show that a DNA segment can direct the initiation of replication when placed in different sequence contexts. In the absence of convenient ARS assays, the next best option was to move putative replicators to other locations within cellular chromosomes. The first such study provided evidence that a 16-Kb segment from within the Chinese hamster ovary (CHO) DHFR replication-initiation zone could function as a replicator when integrated at random ectopic chromosomal sites 45. However, this result was not universally accepted because negative-control fragments could not be directly compared at the same chromosomal sites. This limitation was overcome by using microbial recombinases to integrate test fragments into the same site, which provided persuasive evidence that specific DNA sequences are required for replicator activity 46. More recent studies have revealed conceptually similar results, whether the sequences are derived from highly localized replication origins 47–49 or from the broad DHFR initiation zone 50. In all cases, ‘replicator’ activity can be transferred to many (but not all) ectopic sites. Importantly, however, these studies have not identified any specific DNA sequences or structural motifs that are shared by different replicators. Replicator–initiator interactions The discovery of a hetero-hexameric ORC in all eukaryotes from budding yeast to man 3,51 provided a whole new strategy to hunt for replicators. Instead of searching for poorly defined sequences with laborious methods, the sequences to which the initiator binds, logically, should define the replicator. In fact, budding yeast ORC binds to replication-origin DNA sequences substantially better than to random templates 52, and fission yeast ORC has evolved a specialized domain to recognize asymmetric stretches of adenines and thymidines that define its origins of replication 53. So, it was quite revealing to learn that purified human and D. melanogaster ORC have little or no sequence-specific binding activity 38,54. Interestingly though, D. melanogaster ORC has a strong preference for binding to negatively supercoiled DNA 54. A preference for superhelical tension could explain why both local and distant sequences can affect initiation activity without the requirement for a specific consensus-sequence recognition element. Chromatin immunoprecipitation (ChIP) experiments can potentially identify ORC-binding sites regardless of their DNA sequence, and recent studies suggest that ORC occupies replication origins in all eukaryotes, although its relative binding affinity compared to other sites is unknown. As expected, budding yeast 55 and fission yeast 56 ORCs show significant enrichment at origins, but some preference for origin occupation is also seen in flies 57,58 and humans 59. One drawback of most ChIP studies is that they interrogate only a few carefully chosen sites, which does not provide an unbiased view of the sequences to which ORC binds. Hybridization of DNA sequences that co-immunoprecipitate with ORC in complete-genome arrays (ChIP-chip) can provide this information and have already proved successful in budding yeast 60. Interestingly, not all ARS consensus sequences are identified, which could be due to a lack of sensitivity of the method, to a requirement for further undefined sequences 61, or to features of chromatin that prevent ORC from binding to some sites. Another confounding issue with these studies is that, counter-intuitively, stronger ORC binding sites have been shown to be less efficient replicators 89. In any case, these studies require arrays that achieve complete unbiased coverage of the genome, which, at present, are available for very few species. An alternative approach is to clone and sequence DNA segments that co-immunoprecipitate with ORC, which was recently done for a human cell line 62. In both the human and yeast studies, many (but not all) ORC-binding sites coincide with replication-initiation activity ( BOX 3). Box 3 | Regulation downstream of the initiator–replicator interaction In addition to regulating the initiator–replicator interaction, the activity of a potential replication origin in metazoans is also strongly influenced by events downstream of the ORC–DNA interaction. In budding yeast, there are many origins that do not fire in every cell cycle and some that do not fire at all in a normal cell cycle, but these silent origins are occupied by ORC and other pre-RC proteins 88. In fact, ORC is even more tightly bound to silent origins 89. Many of these silent origins are activatable under the appropriate conditions, which implies that functional pre-RCs are assembled at these sites. The conditions that silence some budding yeast origins or activate others are beginning to be understood, and they include regulators of chromatin structure 77, nucleosome-free transcriptional-control elements 69, proximity to nucleosomes 76, or simply proximity to other replication origins 90,91. In X. laevis embryos, there are clearly many more pre-RCs assembled than are used 92, and the relative preference for initiating replication at specific sites can be influenced downstream of ORC binding by the presence of transcription factors 40. In D. melanogaster, binding of a protein complex that contains the oncoprotein Myb is required for efficient initiation of replication at the chorion gene cluster during selective gene amplification, but is not required for ORC localization to this origin 93. For the time being, it is important to appreciate that in many cases it has not been determined whether the factors that influence origin activity are upstream or downstream of ORC binding, and that this will be an important distinction for our future understanding of replication origins. |
If purified ORC does not preferentially bind to specific sequences, and replicators do not share a common sequence element, then what directs ORC to specific chromosomal sites in vivo? Several mechanisms can be imagined (). One possibility is that other proteins interact with ORC to direct it to specific sequences. This could occur through the direct interaction of ORC with chromatin proteins, or such proteins could alter the local topological state of DNA. For example, the EBNA1 protein of Epstein–Barr virus (EBV) interacts with ORC and might recruit ORC to the EBV origin 63. In D. melanogaster follicle cells, an interaction of ORC with E2F transcription factors 64,65 — possibly in conjunction with a developmentally regulated Myb-oncoprotein-containing complex 66 and the histone de-acetylase Rpd3 ( ref. 67) — seems to orchestrate a developmentally regulated redistribution of ORC to facilitate amplification of the chorion gene cluster. Chromosomal activities such as transcription can also destabilize nucleosomes and create superhelical tension, which might explain the transcription-dependent origin activity that has been described in several systems 68–70. In short, any mechanism that removes a nucleosome and results in negative supercoiling could create a high-affinity binding site for ORC 54. Another way to direct a nonspecific binding protein to specific sites is to prevent it from binding to other sites. Exclusion can occur at a global level, for example by transcriptional interference, which antagonizes the initiation of replication 68,71, or by the interaction with global repressors, as is suggested during amplification of the chorion gene cluster in D. melanogaster64–67. There can also be more localized exclusion effects. A recent study shows that methylated DNA will neither bind ORC nor initiate replication in the otherwise omnipotent X. laevis egg extracts 72. By cleverly constructing plasmid templates that contain a small segment of unmethylated DNA, ORC binding was focused to a specific segment of the plasmid and replication was initiated within 2 Kb of this segment. This intriguing result could explain an observed loss of localized initiation in methylation-deficient cell lines 73, as well as the correlation between unmethylated CpG islands and replication origins 74. Several less well-defined features of chromosomes also influence the usage of replication origins, but in most cases it is not known whether they are upstream or downstream of the initiator–replicator interaction ( BOX 3). The proximity of nucleosomes can have both inhibitory 75 and stimulatory 76 effects on the activity of budding yeast replication origins, although, at least in some cases, these effects are known to be downstream of ORC binding 76. There is evidence that histone modifications can influence the usage of replication origins in budding yeast 77, D. melanogaster 67 and X. laevis40. However, the effects of histone acetylation are not likely to be straightforward, as different early firing origins at the chicken β-globin locus have different patterns of epigenetic histone modifications 78. Certainly, the fact that several origins are regulated during development 70,79,80 and under different growth conditions 81,82 implies that there are many complex factors that influence origin specification (). A preview of the 50th anniversary What can we expect in the next decade? First and foremost, the initiator–replicator interaction will become appreciated as an interaction with far greater complexity than was previously expected. The concept of the replicator as a specific genetic element will cease to be useful, and will be replaced by that of a context-dependent element akin to our present concept of promoters. New methodologies to map replicators more rapidly and confidently will continue to drive discovery. The structure of metazoan ORC and the mechanisms by which it recognizes DNA will be revealed. Proteins that interact with ORC will be identified and their effects on ORC binding to specific DNA sequences will be evaluated. Comparative analysis of the many different factors that influence ORC–replicator interactions will reveal unique and common features of recognition. Further factors downstream of ORC binding and/or pre-RC assembly will also be found to influence the usage of replication origins. Perhaps most importantly, we will begin to address the biological significance of origin localization. Ultimately, the regulation of replication-origin usage during development and in response to different growth conditions will become integrated into our knowledge of the cell cycle and cell differentiation. The findings of the past decade ( BOX 4) warrant a revision of the original concept of a replicator as it applies to eukaryotic cells. The idea that replication is initiated by the binding of an initiator to a specific DNA-sequence recognition element should be replaced by a ‘relaxed replicator’ model in which DNA sequence constitutes only one of several features that influence where replication will initiate. DNA topology, DNA methylation, transcriptional activity, ancillary protein chaperones, and long- and short-distance chromatin effects might each be absolutely critical or totally dispensable to origin specification under different conditions. This revision does not detract from the fundamental value and spirit of the replicon model. Jacob and Brenner intended the model to describe bacterial systems, and we should no more expect the replicon model to apply to metazoans than we do the operon model. Replication is clearly initiated by the positive interaction of an initiator with a replicator, but we must come to accept that the replicator can no longer be defined by a single specific DNA sequence. Our present, widely accepted, model for restricting replication to precisely once-per-cell-cycle does not invoke any requirement for specific DNA-sequence elements 30. So, it is arguably the presence of specific initiation sites that is the greater mystery. In this regard, a highly significant question now is whether the ‘relaxed replicator’ arose by default or by design. Is the replicator simply any available ORC-binding site, or are origins actively positioned, possibly to coordinate the direction of replication forks with other chromosomal functions 30? If so, selective pressures might have moulded a different blend of targeting mechanisms for each chromosomal site, and replicators might turn out to be no less complex than transcriptional promoters, where it is now a familiar concept that a common machinery can be targeted by many different mechanisms. Box 4 | The status quo: a list of acceptable statements Replicators - There are many more replicators (that is, potential replication origins that are occupied by the origin-recognition complex (ORC)) than are used during S phase.
- More replicators are used than are needed to complete S phase.
- Replicators fire asynchronously throughout S phase.
- Replicator usage can be regulated during development and under different metabolic conditions.
- Once-per-cell-cycle genome duplication does not require specific replicator sequences.
- Different replicators have different DNA-sequence requirements; some have no DNA-sequence requirements.
- Many different features of chromatin structure and function can influence the activity of replicators.
Initiators - ORC homologues are found in all eukaryotes.
- ORC occupies replicators in vivo.
- The factors that influence the DNA-binding activity of ORC are highly divergent.
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politics and replication origins
