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Replication origins in eukaroytes
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Replication origins in eukaroytes Shane Donovan* and John FX Diffleyt Recent experiments in budding yeast and Xenopushave provided new insights into the regulation of eukaroytic DNA replication. The multi-subunit origin recognition complex plays a key role in initiation, remaining bound at origins of replication during most of the cell cycle. Early in the cell cycle, Cdc6 and the Mcm proteins 'reset' chromatin for another round of DNA replication. Cyclin-dependent kinases appear to play a dual role, both in activating replication origins and blocking the formation of new pre-replicative complexes; thus limiting replication to once per cell cycle.
Address *tlCRF Clare Hall Laboratories, Chromosome Replication Laboratory, South Mimms, Hertfordshire EN6 3LD, UK; re-mail: [email protected] Current Opinion in Genetics & Development 1996, 6:203-207 © Current Biology Ltd ISSN 0959-437X Abbreviations ACS ARS consensus sequence ARS autonomouslyreplicating sequence CDK cyclin-dependentkinase LCR locus control region ORC origin recognition complex pre-RC pre-replicativecomplex
Introduction Eukaryotic cells face an interesting problem: they must precisely duplicate their genomes before they divide. T h e complexity of this problem is underscored by several features of eukaroytic DNA replication: bidirectional replication begins from hundreds to thousands of 'origins' dispersed throughout their chromosomes, initiation must be restricted to a 'window' of the cell cycle called S phase, origins that have already fired need to be distinguished from those that have not yet done so, and mitosis itself must be restrained until the entire genome is replicated [1,2]. More than 30 years have elapsed since Jacob, Brenner and Cuzin [3] presented the replicon model to explain D N A replication in Escherichia coli [3]. In this model, a c/s-acting sequence, the 'replicator', directs a trans-acting factor, the 'initiator', to drive initiation from an origin. T h e identification of replicator sequences and initiator proteins in prokaryotes and eukaryotic viruses has led to an understanding of how replication is regulated in these organisms. Among eukaryotes, replicators in the budding yeast Saccharomyces cerevisiae have been the best characterized. Some of the trans-acting factors that interact with these sequences have now been identified and analysis of the replicator-protein interactions during the
cell cycle has suggested how replication can be limited to occur only once per cell cycle. Structure of yeast replicators and interactions with the origin recognition complex In budding yeast, replicators were initially identified as sequences that allowed plasmids to be maintained extrachromosomally and became known as autonomously replicating sequences (ARSs). Many ARSs have been shown to act as replicators both in plasmids and in chromosomes [4]. Extensive mutational analysis has revealed that yeast replicators all contain an exact, or a close, match to an 11 bp consensus sequence (A/T)TTTA(T/c)(A/G)qT~T(A/T), known as the ARS consensus sequence (ACS), that is essential for origin function [5,6]. T h e six subunit origin recognition complex (ORC) specifically recognizes the ACS, both in vivo and in vitro [7,8]. T h e ACS is found within a slightly larger element, domain A. Although the A element is essential for replicator function, it is not by itself sufficient; a flanking region called the B domain, 3' to the T-rich strand of the ACS, is also required for replicator function. A comparison of B domains from different yeast replicators reveals little sequence conservation. However, two general statements can be made: the sequences tend to be A+T rich and they are sensitive to alterations in distance and orientation relative to the ACS. One yeast replicator, ARS1, has previously been examined in considerable detail [6]. ARS1 is composed of an A element containing the ACS and auxiliary elements B1, B2, B3 and C.
T h e past year has seen the use of linker-scan substitution for detailed mutagenesis of a second yeast replicator, ARS307 [9",10*'], which shows a similar organisation to ARS1. In addition to an A element containing the ACS, it contains two elements in a similar position to the B1 and B2 elements of ARS1. T h e two elements are, as with their ARS1 analogues, important for efficient origin function both on plasmids and in their normal chromosomal location. Although these two elements show little sequence similarity to the B1 and B2 elements of ARS1, they can be functionally substituted by their respective counterparts. This finding suggests that the ACS flanking region of ARS307 contains two sequence elements that are equivalent to the B1 and B2 elements of ARS1. T h e B1 and B2 elements could act as binding sites for trans-acting factors or could have structural roles, such as in origin unwinding. It has recently been demonstrated that at least one role of the B1 element is to cooperate with the ACS in efficiently binding ORC, both in vitro and in vivo [11°',12"]. Because domains A and B1 constitute
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a functional ARS, albeit an inefficient replicator, these results also argue that a major determinant of replicator function is the efficient binding of ORC. T h e role of the B2 element is still unknown; the failure to find any single point mutations within the B2 element that affect plasmid replication may indicate either a structural role for this element or an interaction with a factor with relaxed binding specificity [10"*].
ORC's role in the initiation of DNA replication ORC was suggested to have an important role in DNA replication because it binds to the essential ACS [7] and the B1 element [11"*,12"*]. Furthermore, a mutation in the ORC2 gene, which encodes the 72kDa subunit, causes plasmids to be lost at a high rate and defective chromosomal replication [13-15]. This conclusion has been supported by two papers in the past year reporting the isolation and characterization of the ORC5 gene, which encodes the 53kDa subunit of ORC [16"',17"]. Strains containing conditional mutations in the ORC2 and ORC5 genes lose plasmids at a high rate, even at the permissive temperatures. These plasmid-loss rates can be overcome by the addition of multiple copies of origins to the plasmids, indicating that the defect is due to a failure in some aspect of origin function. Using two-dimensional electrophoretic techniques that can distinguish between replication intermediates resulting from initiation at an active origin and intermediates derived from replication through an inactive origin, it has been directly shown that orc2 and orc5 mutants exhibit a clear reduction in the efficiency of initiation from chromosomal origins [16°%18"]. These results indicate that ORC has an essential role in initiating DNA replication. ORC binding by itself, however, is not sufficient to drive initiation as ORC remains bound at replicators after initiation takes place [ 19].
Pre-replicative complexes and Cdc6 at replicators In a classic series of experiments, Rao and Johnson used HeLa cell fusions to show that G1, but not GZ, nuclei could be promoted into S phase by a diffusible activity from S phase cells that induces DNA synthesis [20]. One important conclusion from this set of experiments is that progression through mitosis is required to 'reset' G2 nuclei to become competent to undergo S phase in the subsequent cell cycle. Thus, entry into S phase is dependent upon the passage through mitosis. T h e biochemical mechanism of the resetting event is presently not known (see SE Kearsey, K Labib and D Maiorano, this issue [pp 208-214]). One possible clue, however, comes from the study of protein-DNA interactions at yeast replicators during the cell cycle. Yeast origin chromatin alternates between post-replicative and pre-replicative states in the cell cycle [19]. T h e post-replicative state appears after initiation takes place
and is due to ORC being bound at replication origins. At the end of mitosis a pre-replicative complex (pre-RC) assembles at origins and persists until S phase begins. Identifying components of pre-RCs should provide some insight into how S phase is regulated. Cdc6 seemed a good candidate for having a role in pre-RCs for various reasons. First, Cdc6 has an essential role early in S phase [21]; second, cdc6 mutants exhibit a high plasmid loss rate, which is suppressible by the addition of multiple origins to the plasmids, suggesting that Cdc6 is involved in origin function [22]; and third, CDC6 is normally transcribed at the end of mitosis [23*%24°° ] when pre-RCs form and de novo synthesis of both CDC6 and its Schizosaccharomyces pombe homologue cdcl 8 + is required for entry into S phase [24°%25]. Recent experiments have demonstrated that de novo CDC6 synthesis is required for the formation of preRCs. Moreover, pre-RCs are thermolabile in a strain containing a temperature-sensitive mutation in CDC6, indicating that CDC6 is essential for maintenance of the pre-replicative state [26"1. These results suggest that CDC6 may be a component of pre-RCs. The involvement of C d c 6 - - w h i c h is essential for DNA replication--in pre-RCs provides genetic evidence that pre-RCs may be genuine preinitiation complexes essential for DNA replication. Several lines of evidence suggest that ORC is also a component of pre-RCs. T h e footprint associated with the pre-RC includes protection of the A element similar to that seen in vitro with purified ORC [19]. ORC is present in G! and is capable of binding to origins because when pre-RCs are lost in GI in the cdc6ts strain, origins return to their post-replicative state [26**]. Recombinant CDC6 interacts with ORC in crude cell extracts and CDC6 acts as a high copy suppressor of a conditional mutant in the ORC5 gene, restoring the efficiency of initiation from the ARS1 origin [18°*1. Pre-RCs and cyclin-dependent kinases (CDKs) may also be involved in limiting replication to one round per cell cycle. It has been previously shown in S. pombe that the major CDK, encoded by cdc2+, has a crucial role in preventing re-replication: cdc2+ mutants can undergo a round of endoreduplication [27] and over-expression of the specific Cdc2/Clb kinase inhibitor encoded by rum1+, or deletion of the mitotic B cyclin encoded by cdc13+, both lead to multiple rounds of replication without an intervening mitosis [28,29]. In budding yeast, the major cell cycle CDK is Cdc28, which acts with G1 cyclins and B-type cyclins, Clbs, at several critical points in the cell cycle. Over-expression of p4ff ~1C1, a specific inhibitor of Cdc28/Clb kinase [30], in cells arrested at G2/M is sufficient to drive assembly of pre-RCs [31"*], indicating that Cdc28/CIb kinase acts
Replication origins in eukaryotes Donovan and Diffley
as an inhibitor of pre-RC formation. As Cdc28/Clb kinase activity is also necessary for entry into S phase [32], these results indicate that re-replication within a single cell cycle is blocked because the activity required for origin firing, provided by Cdc28/Clb kinase, also blocks formation of new pre-RCs (Fig. 1).
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(and perhaps Cdc6 in budding yeast) possibly represents the critical downstream target of CDK/Clb inhibition. Over-expression of this protein might then titrate out the inhibitory activity leading to unregulated assembly of pre-RCs in S phase, resulting in repeated initiation events. Further experiments are needed to test this hypothesis.
Other eukaroytic replication origins
Figure 1
G2
W
ORC
Mitosis
Competence to
~
Cdc6
form pre-replicative complexes
~
ORC ? Mcms ? G 1
Cdc28 / CIb5 6 \1l ,~jCdc7 / Dbf4 '
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S/G 2
<:: f 996 Current Opinion in Genetics & Development
A model of protein-DNA interactions at yeast replicators to explain how re-replication is prevented during the cell cycle. Origins are in their post-replicative state (i.e. only ORC is bound), after initiation takes place until the end of mitosis. At the end of mitosis, Cdc6-dependent pre-RCs appear at origins. Pre-RC assembly is restricted to a 'window' in the cell cycle, as indicated by the vertical bar, where Cdc28/Cyclin B kinase activity is absent; during the rest of the cell cycle, the assembly of pre-RCs is blocked by Cdc28/Cyclin B kinase activity. In this model, Cdc6 is recruited directly to replicators; as the figure indicates, it is presently unclear whether ORC or the Mcm family of proteins are components of pre-RCs. Initiation is triggered by Cdc?/Dbf4 kinase (Dbf4, the regulatory subunit of the kinase, has been shown to interact with initiation complexes in vivo [45]) and Cdc28/CIb5,6 protein kinases that act either directly or indirectly on pre-RCs. Replication displaces pre-RCs, which are incapable of rebinding until the end of mitosis. ORC binds to nascent replicator sequences during S phase.
Recently, it has been shown that over-expression of cdc18 + in S. pombe also results in multiple rounds of
endoreduplication [33",34"']. One interesting explanation for this phenomenon is that the cdcl8 + encoded protein
The study of initiation in higher eukaryotcs has been hampered by a lack of knowledge of the exact sequence requirements of replicators. In fact, the issue of whether higher eukaryotes use defined discrete loci as origins remains controversial [35]. Recently, two human cell replication origins have been mapped to specific regions encompassing less than 2 kb of sequence [36,37]. One of these, mapped to a region between the 8-globin and 13-globin genes of the 13-globin cluster, has been the subject of genetic analysis. In a cell line derived from a thalassemia patient, where the region containing the origin was deleted, the 13-globin origin was inactivated and the region was replicated from an unidentified distant origin. It has been shown using a similar approach, recently, that sequences in the locus control region (LCR), >50 kb upstream from the 13-globin gene locus are essential for the 13-globin origin to fire. This might help to explain both the failure of a region just containing the 13-globin gene origin to confer autonomous replication and the difficulties in identifying small cloned DNA sequences capable of conferring autonomous replication [38"]. Initiation from the 13-globin origin occurs independently of gene expression from within the locus, although gene expression determines the timing of initiation from the origin [37,38"']. Thus, the LCR plays at least two distinct roles at the [3-globin locus: it is required for efficient gene expression and for replication origin utilization. In early Xenopus embryos, rDNA replication initiates at regular 9-12kb intervals, with no apparent dependence on specific sequences. In early embryos, the rDNA genes are not transcribed. When transcription of these genes resumes in the late blastula and early gastrula, however, the pattern of initiation site usage changes. Initiation no longer occurs within transcription units, but now becomes confined to the intergenic region [39"]. It will be interesting to know whether this change in specification is a direct consequence of transcription or the result of a change in the organization of chromatin that accompanies the resumption of transcription. Thus, events not necessarily located in the vicinity of an origin may influence whether an origin is active or inactive. The chromosomal location of yeast origins is also known to influence origin activity [40,41] and an important goal for the future will be to understand how overall chromatin context and nuclear organization, in which an origin resides, influence origin utilization and the timing of origin firing during S phase.
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Conclusions T h e study of yeast replicators is enabling both the identification of proteins that are directly involved in initiating DNA replication and the characterization of how replication from those origins is limited to one round per cell cycle. Studies of higher eukaryotic origins have demonstrated the importance of both chromosomal location and distal sequences for origin function. Finally, one of the highlights of the past year has been the characterization of 'licensing' in Xenopus egg extracts (for a review of licensing, see SE Kearsey, K Labib and D Maiorano in this issue [pp 208-214]). Licensing has been shown to be dependent on the binding of Mcm proteins to chromatin at the end of mitosis [42"°,43"°,44°°]. As licensing and pre-RCs both constitute possible molecular candidates for the mitotic nuclear resetting event, it will be important to determine whether they represent different aspects of the same phenomenon or whether they reflect separate but parallel mechanisms, both occurring at the end of mitosis and which are necessary for S phase.
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Using gel binding and a footprinting assay the authors show that sequences in the B1 element are required for efficient ORC binding. They also show that mutations within the B1 element reduce ORC binding and the efficiency of plasmid replication. Interestingly, they also demonstrate that a point mutation in B1 reduces plasmid replication but does not affect ORC binding, indicating that the B1 element may have another role aside from the efficient binding of ORC. Rowley A, Cocker JH, Harwood J, Diffley JFX: Initiation complex assembly at budding yeast replication origins begins with the recognition of a bipartite sequence by limiting amounts of the initiator, ORC. EMBO J 1995, 14:2631-2641. The authors show that ORC is present at levels corresponding to little more than one complex per replication origin - indicating that, in vivo, origin binding by ORC is very efficient. They also show that both the ACS and the B1 element are required for efficient ORC binding, both in vitro and in vivo. Mutations within both the B2 and B3 elements have no effect on ORC binding. 12. oo
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Replication origins in eukaryotes Donovan and Diffley
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42. •.
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Chong JPJ, Mahbubani HM, Khoo C-Y, Blow JJ: Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature 1995, 375:418-421. Licensing activity is separated into two biochemical fractions. One of these fractions contained at least three pelypeptides and one of these polypeptides is identified as the Xenopus homologue of yeast MCM3, Xenopus Mcm3 is shown to bind to chromatin during G1 and to dissociate from it during replication. G 2 nuclei were shown to require nuclear envelope breakdown to allow Mcm3 chromatin binding. The components of the other fraction are unknown though it is postulated that they are required for the binding of MCM proteins to chromatin. 44. .-
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Dowetl S J, Romanowski P, Diffley JFX: Interaction of Dbf4, the Cdc7 protein kinase regulatory subunit, with yeast replication odgins in vivo. Science 1994, 265:1243-1246.
Replication origins in eukaroytes Shane Donovan* and John FX Diffleyt Recent experiments in budding yeast and Xenopushave provided new insights into the regulation of eukaroytic DNA replication. The multi-subunit origin recognition complex plays a key role in initiation, remaining bound at origins of replication during most of the cell cycle. Early in the cell cycle, Cdc6 and the Mcm proteins 'reset' chromatin for another round of DNA replication. Cyclin-dependent kinases appear to play a dual role, both in activating replication origins and blocking the formation of new pre-replicative complexes; thus limiting replication to once per cell cycle.
Address *tlCRF Clare Hall Laboratories, Chromosome Replication Laboratory, South Mimms, Hertfordshire EN6 3LD, UK; re-mail: [email protected] Current Opinion in Genetics & Development 1996, 6:203-207 © Current Biology Ltd ISSN 0959-437X Abbreviations ACS ARS consensus sequence ARS autonomouslyreplicating sequence CDK cyclin-dependentkinase LCR locus control region ORC origin recognition complex pre-RC pre-replicativecomplex
Introduction Eukaryotic cells face an interesting problem: they must precisely duplicate their genomes before they divide. T h e complexity of this problem is underscored by several features of eukaroytic DNA replication: bidirectional replication begins from hundreds to thousands of 'origins' dispersed throughout their chromosomes, initiation must be restricted to a 'window' of the cell cycle called S phase, origins that have already fired need to be distinguished from those that have not yet done so, and mitosis itself must be restrained until the entire genome is replicated [1,2]. More than 30 years have elapsed since Jacob, Brenner and Cuzin [3] presented the replicon model to explain D N A replication in Escherichia coli [3]. In this model, a c/s-acting sequence, the 'replicator', directs a trans-acting factor, the 'initiator', to drive initiation from an origin. T h e identification of replicator sequences and initiator proteins in prokaryotes and eukaryotic viruses has led to an understanding of how replication is regulated in these organisms. Among eukaryotes, replicators in the budding yeast Saccharomyces cerevisiae have been the best characterized. Some of the trans-acting factors that interact with these sequences have now been identified and analysis of the replicator-protein interactions during the
cell cycle has suggested how replication can be limited to occur only once per cell cycle. Structure of yeast replicators and interactions with the origin recognition complex In budding yeast, replicators were initially identified as sequences that allowed plasmids to be maintained extrachromosomally and became known as autonomously replicating sequences (ARSs). Many ARSs have been shown to act as replicators both in plasmids and in chromosomes [4]. Extensive mutational analysis has revealed that yeast replicators all contain an exact, or a close, match to an 11 bp consensus sequence (A/T)TTTA(T/c)(A/G)qT~T(A/T), known as the ARS consensus sequence (ACS), that is essential for origin function [5,6]. T h e six subunit origin recognition complex (ORC) specifically recognizes the ACS, both in vivo and in vitro [7,8]. T h e ACS is found within a slightly larger element, domain A. Although the A element is essential for replicator function, it is not by itself sufficient; a flanking region called the B domain, 3' to the T-rich strand of the ACS, is also required for replicator function. A comparison of B domains from different yeast replicators reveals little sequence conservation. However, two general statements can be made: the sequences tend to be A+T rich and they are sensitive to alterations in distance and orientation relative to the ACS. One yeast replicator, ARS1, has previously been examined in considerable detail [6]. ARS1 is composed of an A element containing the ACS and auxiliary elements B1, B2, B3 and C.
T h e past year has seen the use of linker-scan substitution for detailed mutagenesis of a second yeast replicator, ARS307 [9",10*'], which shows a similar organisation to ARS1. In addition to an A element containing the ACS, it contains two elements in a similar position to the B1 and B2 elements of ARS1. T h e two elements are, as with their ARS1 analogues, important for efficient origin function both on plasmids and in their normal chromosomal location. Although these two elements show little sequence similarity to the B1 and B2 elements of ARS1, they can be functionally substituted by their respective counterparts. This finding suggests that the ACS flanking region of ARS307 contains two sequence elements that are equivalent to the B1 and B2 elements of ARS1. T h e B1 and B2 elements could act as binding sites for trans-acting factors or could have structural roles, such as in origin unwinding. It has recently been demonstrated that at least one role of the B1 element is to cooperate with the ACS in efficiently binding ORC, both in vitro and in vivo [11°',12"]. Because domains A and B1 constitute
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a functional ARS, albeit an inefficient replicator, these results also argue that a major determinant of replicator function is the efficient binding of ORC. T h e role of the B2 element is still unknown; the failure to find any single point mutations within the B2 element that affect plasmid replication may indicate either a structural role for this element or an interaction with a factor with relaxed binding specificity [10"*].
ORC's role in the initiation of DNA replication ORC was suggested to have an important role in DNA replication because it binds to the essential ACS [7] and the B1 element [11"*,12"*]. Furthermore, a mutation in the ORC2 gene, which encodes the 72kDa subunit, causes plasmids to be lost at a high rate and defective chromosomal replication [13-15]. This conclusion has been supported by two papers in the past year reporting the isolation and characterization of the ORC5 gene, which encodes the 53kDa subunit of ORC [16"',17"]. Strains containing conditional mutations in the ORC2 and ORC5 genes lose plasmids at a high rate, even at the permissive temperatures. These plasmid-loss rates can be overcome by the addition of multiple copies of origins to the plasmids, indicating that the defect is due to a failure in some aspect of origin function. Using two-dimensional electrophoretic techniques that can distinguish between replication intermediates resulting from initiation at an active origin and intermediates derived from replication through an inactive origin, it has been directly shown that orc2 and orc5 mutants exhibit a clear reduction in the efficiency of initiation from chromosomal origins [16°%18"]. These results indicate that ORC has an essential role in initiating DNA replication. ORC binding by itself, however, is not sufficient to drive initiation as ORC remains bound at replicators after initiation takes place [ 19].
Pre-replicative complexes and Cdc6 at replicators In a classic series of experiments, Rao and Johnson used HeLa cell fusions to show that G1, but not GZ, nuclei could be promoted into S phase by a diffusible activity from S phase cells that induces DNA synthesis [20]. One important conclusion from this set of experiments is that progression through mitosis is required to 'reset' G2 nuclei to become competent to undergo S phase in the subsequent cell cycle. Thus, entry into S phase is dependent upon the passage through mitosis. T h e biochemical mechanism of the resetting event is presently not known (see SE Kearsey, K Labib and D Maiorano, this issue [pp 208-214]). One possible clue, however, comes from the study of protein-DNA interactions at yeast replicators during the cell cycle. Yeast origin chromatin alternates between post-replicative and pre-replicative states in the cell cycle [19]. T h e post-replicative state appears after initiation takes place
and is due to ORC being bound at replication origins. At the end of mitosis a pre-replicative complex (pre-RC) assembles at origins and persists until S phase begins. Identifying components of pre-RCs should provide some insight into how S phase is regulated. Cdc6 seemed a good candidate for having a role in pre-RCs for various reasons. First, Cdc6 has an essential role early in S phase [21]; second, cdc6 mutants exhibit a high plasmid loss rate, which is suppressible by the addition of multiple origins to the plasmids, suggesting that Cdc6 is involved in origin function [22]; and third, CDC6 is normally transcribed at the end of mitosis [23*%24°° ] when pre-RCs form and de novo synthesis of both CDC6 and its Schizosaccharomyces pombe homologue cdcl 8 + is required for entry into S phase [24°%25]. Recent experiments have demonstrated that de novo CDC6 synthesis is required for the formation of preRCs. Moreover, pre-RCs are thermolabile in a strain containing a temperature-sensitive mutation in CDC6, indicating that CDC6 is essential for maintenance of the pre-replicative state [26"1. These results suggest that CDC6 may be a component of pre-RCs. The involvement of C d c 6 - - w h i c h is essential for DNA replication--in pre-RCs provides genetic evidence that pre-RCs may be genuine preinitiation complexes essential for DNA replication. Several lines of evidence suggest that ORC is also a component of pre-RCs. T h e footprint associated with the pre-RC includes protection of the A element similar to that seen in vitro with purified ORC [19]. ORC is present in G! and is capable of binding to origins because when pre-RCs are lost in GI in the cdc6ts strain, origins return to their post-replicative state [26**]. Recombinant CDC6 interacts with ORC in crude cell extracts and CDC6 acts as a high copy suppressor of a conditional mutant in the ORC5 gene, restoring the efficiency of initiation from the ARS1 origin [18°*1. Pre-RCs and cyclin-dependent kinases (CDKs) may also be involved in limiting replication to one round per cell cycle. It has been previously shown in S. pombe that the major CDK, encoded by cdc2+, has a crucial role in preventing re-replication: cdc2+ mutants can undergo a round of endoreduplication [27] and over-expression of the specific Cdc2/Clb kinase inhibitor encoded by rum1+, or deletion of the mitotic B cyclin encoded by cdc13+, both lead to multiple rounds of replication without an intervening mitosis [28,29]. In budding yeast, the major cell cycle CDK is Cdc28, which acts with G1 cyclins and B-type cyclins, Clbs, at several critical points in the cell cycle. Over-expression of p4ff ~1C1, a specific inhibitor of Cdc28/Clb kinase [30], in cells arrested at G2/M is sufficient to drive assembly of pre-RCs [31"*], indicating that Cdc28/CIb kinase acts
Replication origins in eukaryotes Donovan and Diffley
as an inhibitor of pre-RC formation. As Cdc28/Clb kinase activity is also necessary for entry into S phase [32], these results indicate that re-replication within a single cell cycle is blocked because the activity required for origin firing, provided by Cdc28/Clb kinase, also blocks formation of new pre-RCs (Fig. 1).
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(and perhaps Cdc6 in budding yeast) possibly represents the critical downstream target of CDK/Clb inhibition. Over-expression of this protein might then titrate out the inhibitory activity leading to unregulated assembly of pre-RCs in S phase, resulting in repeated initiation events. Further experiments are needed to test this hypothesis.
Other eukaroytic replication origins
Figure 1
G2
W
ORC
Mitosis
Competence to
~
Cdc6
form pre-replicative complexes
~
ORC ? Mcms ? G 1
Cdc28 / CIb5 6 \1l ,~jCdc7 / Dbf4 '
t
G1/S V
S/G 2
<:: f 996 Current Opinion in Genetics & Development
A model of protein-DNA interactions at yeast replicators to explain how re-replication is prevented during the cell cycle. Origins are in their post-replicative state (i.e. only ORC is bound), after initiation takes place until the end of mitosis. At the end of mitosis, Cdc6-dependent pre-RCs appear at origins. Pre-RC assembly is restricted to a 'window' in the cell cycle, as indicated by the vertical bar, where Cdc28/Cyclin B kinase activity is absent; during the rest of the cell cycle, the assembly of pre-RCs is blocked by Cdc28/Cyclin B kinase activity. In this model, Cdc6 is recruited directly to replicators; as the figure indicates, it is presently unclear whether ORC or the Mcm family of proteins are components of pre-RCs. Initiation is triggered by Cdc?/Dbf4 kinase (Dbf4, the regulatory subunit of the kinase, has been shown to interact with initiation complexes in vivo [45]) and Cdc28/CIb5,6 protein kinases that act either directly or indirectly on pre-RCs. Replication displaces pre-RCs, which are incapable of rebinding until the end of mitosis. ORC binds to nascent replicator sequences during S phase.
Recently, it has been shown that over-expression of cdc18 + in S. pombe also results in multiple rounds of
endoreduplication [33",34"']. One interesting explanation for this phenomenon is that the cdcl8 + encoded protein
The study of initiation in higher eukaryotcs has been hampered by a lack of knowledge of the exact sequence requirements of replicators. In fact, the issue of whether higher eukaryotes use defined discrete loci as origins remains controversial [35]. Recently, two human cell replication origins have been mapped to specific regions encompassing less than 2 kb of sequence [36,37]. One of these, mapped to a region between the 8-globin and 13-globin genes of the 13-globin cluster, has been the subject of genetic analysis. In a cell line derived from a thalassemia patient, where the region containing the origin was deleted, the 13-globin origin was inactivated and the region was replicated from an unidentified distant origin. It has been shown using a similar approach, recently, that sequences in the locus control region (LCR), >50 kb upstream from the 13-globin gene locus are essential for the 13-globin origin to fire. This might help to explain both the failure of a region just containing the 13-globin gene origin to confer autonomous replication and the difficulties in identifying small cloned DNA sequences capable of conferring autonomous replication [38"]. Initiation from the 13-globin origin occurs independently of gene expression from within the locus, although gene expression determines the timing of initiation from the origin [37,38"']. Thus, the LCR plays at least two distinct roles at the [3-globin locus: it is required for efficient gene expression and for replication origin utilization. In early Xenopus embryos, rDNA replication initiates at regular 9-12kb intervals, with no apparent dependence on specific sequences. In early embryos, the rDNA genes are not transcribed. When transcription of these genes resumes in the late blastula and early gastrula, however, the pattern of initiation site usage changes. Initiation no longer occurs within transcription units, but now becomes confined to the intergenic region [39"]. It will be interesting to know whether this change in specification is a direct consequence of transcription or the result of a change in the organization of chromatin that accompanies the resumption of transcription. Thus, events not necessarily located in the vicinity of an origin may influence whether an origin is active or inactive. The chromosomal location of yeast origins is also known to influence origin activity [40,41] and an important goal for the future will be to understand how overall chromatin context and nuclear organization, in which an origin resides, influence origin utilization and the timing of origin firing during S phase.
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Conclusions T h e study of yeast replicators is enabling both the identification of proteins that are directly involved in initiating DNA replication and the characterization of how replication from those origins is limited to one round per cell cycle. Studies of higher eukaryotic origins have demonstrated the importance of both chromosomal location and distal sequences for origin function. Finally, one of the highlights of the past year has been the characterization of 'licensing' in Xenopus egg extracts (for a review of licensing, see SE Kearsey, K Labib and D Maiorano in this issue [pp 208-214]). Licensing has been shown to be dependent on the binding of Mcm proteins to chromatin at the end of mitosis [42"°,43"°,44°°]. As licensing and pre-RCs both constitute possible molecular candidates for the mitotic nuclear resetting event, it will be important to determine whether they represent different aspects of the same phenomenon or whether they reflect separate but parallel mechanisms, both occurring at the end of mitosis and which are necessary for S phase.
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Replication origins in eukaryotes Donovan and Diffley
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