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The Control of Mammalian DNA Replication

2001; Cell Press; Volume: 104; Issue: 5 Linguagem: Inglês

10.1016/s0092-8674(01)00260-4

1097-4172

Daniel Cimbora, Mark Groudine,

Bacterial Genetics and Biotechnology

Replication of the eukaryotic genome initiates at specific locations, termed origins, and progresses in a defined temporal order during S phase of the cell cycle. In higher eukaryotes our understanding of how origins are selected, and how replication timing is controlled, is far from complete. Recent experiments using a system that involves the incubation of intact mammalian nuclei in replication-competent Xenopus egg extracts have revealed roles for nuclear organization in origin selection, differences in the mechanism of origin specification between mammals and yeast, and an intriguing correlation between the repositioning of chromosomal domains after mitosis and the programming of replicating timing. Interestingly, these experiments have also shown that the specification of origins and programming of replication timing during S phase are independent events that occur during the preceding G1 phase. Biochemical analyses of known origins in higher eukaryotes have revealed common sequence and structural features such as DNA unwinding elements, binding sites for replication proteins and transcription factors, and sites of attachment to the nuclear matrix/scaffold. Moreover, genetic analyses have demonstrated that a discrete DNA segment containing an origin is necessary, and in some chromosomal contexts sufficient, for origin activity, and that sequences far from an origin can influence origin activity (Aladjem et al. 1998Aladjem M.I. Rodewald L.W. Kolman J.L. Wahl G.M. Science. 1998; 281: 1005-1009Crossref PubMed Google Scholar, Cimbora et al. 2000Cimbora D.M. Schübeler D. Reik A. Hamilton J. Francastel C. Epner E.M. Groudine M. Mol. Cell. Biol. 2000; 20: 5581-5591Crossref PubMed Scopus (98) Google Scholar and references therein). However, we are still far from understanding how such elements specify an initiation site within a chromosomal context. Recent experiments have begun to unravel the biochemical events that lead to origin selection, and suggest that epigenetic factors such as chromatin structure can influence origin choice. According to current models of replication in yeast, the origin recognition complex (ORC), composed of six conserved Orc proteins, associates with DNA in a sequence-specific manner throughout the cell cycle (references in Mizushima et al. 2000Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). During G1 phase, ORC recruits other factors including Cdc6 and the Mcm proteins to form a prereplication complex. Initiation of replication is then triggered by the association of additional factors and cyclin-dependent kinase (CDK) activity at the G1/S transition. Upon S phase entry, the prereplication complex is partially disassembled with the release of Cdc6 and Mcm proteins, preventing reinitiation of DNA replication until the next cell cycle. The association of an intact yeast ORC with origins throughout the cell cycle suggests that the first regulated step in prereplication complex assembly in yeast is the association of Cdc6 with ORC. Because of similarities in structure and function of ORC and other replication proteins, models of metazoan replication are based in large part on the yeast model. However, recent experiments demonstrate that in contrast to yeast, mammalian ORC is not a constitutive chromatin-bound complex, but rather partially disassembles during the cell cycle. The first hint of this came from the observation that the initiation of replication at specific origins in mammalian nuclei is a property acquired during the preceding G1 phase (references in Gilbert 1998Gilbert D.M. Curr. Opin. Genet. Dev. 1998; 8: 194-199Crossref PubMed Scopus (57) Google Scholar). In this experiment, when nuclei isolated from early G1 phase Chinese hamster ovary (CHO) cells were incubated in Xenopus replication extract, the hamster dihydrofolate reductase (DHFR) locus replicated without apparent initiation site preference. In contrast, in nuclei isolated at least 3–4 hr after metaphase, the DHFR locus replicated from the same specific origin (ori-β) used by hamster cells in culture. The point during G1 at which origin specificity is acquired was termed the origin decision point (ODP; Figure 1) . Immunodepletion experiments revealed that at the ODP, replication in CHO nuclei ceases to be dependent on Xenopus Orc proteins in the extract (Natale et al. 2000Natale D.A. Li C.-J. Sun W.-H. DePamphilis M.L. EMBO J. 2000; 19: 2728-2738Crossref PubMed Scopus (72) Google Scholar), suggesting that the ODP reflects the appearance of functional hamster ORCs. This is not due to differential expression of either of two hamster Orc proteins examined: Orc1 and Orc2 are expressed at similar, constant levels during M and G1. However, in contrast to Orc2, which is stably associated with chromatin during M and G1, the affinity of Orc1 binding to chromatin was found to vary from low during mitosis and early G1 to high in mid-G1. The stable association of Orc1 with chromatin coincides temporally with the ODP, suggesting that origin specification in hamster nuclei is due, at least in part, to the assembly of an intact ORC at origins. The dynamic behavior of ORC evident in hamster nuclei seems to be a general phenomenon in mammals. In human cells, Orc1 is released from chromatin during a subset of the cell cycle while the bulk of Orc2 remains bound to chromatin (Kreitz et al. 2000Kreitz, S., Ritzi, M., Baack, M., and Knippers, R. (2000). J. Biol. Chem., in press. Published online December 1, 2000. 10.1074/jbc.M009473200.Google Scholar), although there are conflicting reports regarding human Orc1 behavior in the literature. In addition, in vivo footprinting reveals the presence of an ORC-like complex bound to the human lamin B2 origin during G1 but not mitosis (Abdurashidova et al. 1998Abdurashidova G. Riva S. Biamonti G. Giacca M. Falaschi A. EMBO J. 1998; 15: 2961-2969Crossref Scopus (57) Google Scholar). Furthermore, ORC behavior is not the only difference between mammals and yeast: recent experiments with elutriated human cells demonstrate that a significant fraction of Cdc6 protein associates with chromatin throughout the cell cycle, in contrast to the regulated dissociation of yeast Cdc6 from prereplication complexes during S phase (Mendez and Stillman 2000Mendez J. Stillman B. Mol. Cell. Biol. 2000; 20: 8602-8612Crossref PubMed Scopus (731) Google Scholar). Together, these results suggest a model for mammalian origin specification in which Orc2 and Cdc6 remain associated with origins throughout the cell cycle, while Orc1 cycles on and off chromatin (Figure 2). Other Orc proteins may remain associated with Orc2 on chromatin, or may dissociate along with Orc1. The selection of sites for ORC assembly during G1 may be influenced by Cdc6, as it has recently been shown that yeast Cdc6 inhibits nonspecific DNA binding of yeast ORC in vitro (Mizushima et al. 2000Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). The partial disassembly and reassembly of mammalian ORC during the cell cycle is a potential control step not evident in yeast, providing an opportunity to vary origin choice from one cell cycle to the next. Origin usage in metazoans is dynamic, undergoing changes during embryonic development, upon alteration of gene activity or chromatin structure, and during gametogenesis. Regulating the assembly of an intact ORC at specific chromosomal sites is one mechanism by which these changes in origin use might occur. Disrupting nuclear integrity has been shown to abolish origin specificity in late G1 (post-ODP) CHO nuclei: permeabilization in such a way that nuclei are unable to exclude large molecules results in replication of the DHFR locus without a preferred initiation site (references in Gilbert 1998Gilbert D.M. Curr. Opin. Genet. Dev. 1998; 8: 194-199Crossref PubMed Scopus (57) Google Scholar). This likely reflects a requirement for an intact nuclear envelope in restricting the access of Xenopus factors (perhaps ORC, which is highly abundant in egg extracts) to chromatin. Studies in Xenopus embryos at the mid-blastula transition (MBT) reveal a progressive shift from promiscuous, high frequency origins to widely spaced, defined origins as the number of nuclei increases. The necessity for regulating the intranuclear concentration of critical factors is revealed by experiments showing a requirement for the nuclear envelope and a direct relationship between the origin spacing and the concentration of nuclei in Xenopus extracts (references in Walter et al. 1998Walter J. Sun L. Newport J. Mol. Cell. 1998; 1: 519-529Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Similarly, the concentration of CHO nuclei in Xenopus extract has an effect on the preference for DHFR ori-β, with maximal use of ori-β at a concentration of nuclei similar to that found at the MBT (Dimitrova and Gilbert 1998Dimitrova D.S. Gilbert D.M. J. Cell Sci. 1998; 111: 2989-2998Crossref PubMed Google Scholar). More recently it has been shown that a single round of replication can occur in vitro in the absence of nuclear structure by the sequential addition of Xenopus cytoplasmic extract and a nucleoplasmic extract of sufficiently high concentration (Walter et al. 1998Walter J. Sun L. Newport J. Mol. Cell. 1998; 1: 519-529Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Taken together, these results suggest that the role of the nuclear envelope is to maintain a low enough concentration of factors (perhaps ORC) in the nucleus to prevent nonspecific binding to chromatin, while maintaining a sufficiently high concentration of other nuclear factors to ensure efficient activation of prereplication complexes and their disassembly from chromatin after initiation. Chromatin structure appears to play a role in origin selection. The transition to specific origins at the Xenopus MBT is accompanied by a variety of changes including the onset of zygotic transcription, changes in chromatin structure, and changes in the attachment of chromatin to the nucleoskeleton. Although it has long been observed that nascent DNA is associated with the nucleoskeleton and that matrix/scaffold attachment sites are often found near replication origins, changes in nucleoskeletal attachment have been ruled out as a determinant of origin specificity at the MBT (Maric and Hyrien 1998Maric C. Hyrien O. Chromosoma. 1998; 107: 155-165Crossref PubMed Scopus (8) Google Scholar). In contrast, altered histone H4 acetylation and the incorporation of histone H1 into chromatin at the MBT suggest a relationship between chromatin structure and origin selection; this relationship is supported by recent experiments demonstrating that histone H1 has a direct inhibitory effect on the assembly of prereplication complexes on Xenopus sperm chromatin (Lu et al. 1998Lu Z.H. Sittman D.B. Romanowski P. Leno G.H. Mol. Biol. Cell. 1998; 9: 1163-1176Crossref PubMed Scopus (48) Google Scholar and references therein). Furthermore, when condensed metaphase chromosomes from hamster cells are added to Xenopus extracts, the DHFR locus is replicated from a novel origin, and activity of this novel origin is dependent on topoisomerase II–mediated chromatin condensation (references in Gilbert 1998Gilbert D.M. Curr. Opin. Genet. Dev. 1998; 8: 194-199Crossref PubMed Scopus (57) Google Scholar). Likewise, in yeast, an origin that is not normally active becomes active when the silent chromatin component Sir3 is mutated (Stevenson and Gottschling 1999Stevenson J.B. Gottschling D.E. Genes Dev. 1999; 13: 146-151Crossref PubMed Scopus (144) Google Scholar). Covalent modifications of DNA also appear to play a role in origin activity, as the methylation status of sequences at DHFR ori-β correlates with origin activity in hamster cells (references in Gilbert 1998Gilbert D.M. Curr. Opin. Genet. Dev. 1998; 8: 194-199Crossref PubMed Scopus (57) Google Scholar). Taken together, these observations point to a role for chromatin structure in determining sites of replication initiation in eukaryotes, perhaps by restricting the access of Orc proteins to certain chromosomal sites. The observations that transcriptional control elements are often found near origins and that transcription factors can stimulate origin activity have led to the suggestion that transcription per se may play a role in origin specification. However, the ability of transcription factors to recruit histone deacetylase activity and chromatin remodeling factors is compatible with an indirect role for transcription, mediated by chromatin structure. In contrast to the role of chromatin structure in specifying origin activity suggested by the previous examples, activity of the human β-globin origin does not vary with global changes in chromatin structure in the β-globin gene cluster or β-globin gene transcription in different cell types (Cimbora et al. 2000Cimbora D.M. Schübeler D. Reik A. Hamilton J. Francastel C. Epner E.M. Groudine M. Mol. Cell. Biol. 2000; 20: 5581-5591Crossref PubMed Scopus (98) Google Scholar and references therein). It is possible that the assays used to analyze locus-wide chromatin structure (nuclease sensitivity and histone acetylation) may not accurately reflect altered states of chromatin in the immediate vicinity of the origin, and further investigation of the structure of chromatin near the β-globin origin in different cell types will be required to resolve this issue. Taken together, these results suggest that chromatin structure can affect origin choice, but this relationship is likely to be complex and may vary from locus to locus. Chromosomal domains replicate at characteristic times during S phase that often correlate with gene activity: active loci typically replicate early in S phase and inactive loci replicate later. It is not yet clear whether gene activity is influenced by replication timing, or whether replication timing is a consequence of gene activity, but these possibilities are not mutually exclusive. One factor that may influence both gene activity and replication timing is position within the nucleus. The colocalization of inactive genes with Ikaros and HP1 proteins near centromeric heterochromatin in interphase nuclei, and the dynamic repositioning of active versus inactive genes relative to this compartment, suggest that nuclear position and the local protein environment of a chromosomal domain are determinants of gene activity (Francastel et al. 1999Francastel C. Walters M.C. Groudine M. Martin D.I.K. Cell. 1999; 99: 259-269Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar and references therein). Similarly, early- and late-replicating sequences occupy distinct nuclear positions during S phase, suggesting that nuclear position may dictate replication timing. This notion is further supported by the recent demonstration that replication timing of chromosomal domains in CHO nuclei is determined during early G1 at the same time that these domains are repositioned in the nucleus following mitosis. This step, termed the temporal decision point (TDP), is analogous to the origin decision point (ODP) but rather than marking the acquisition of origin specificity, the TDP marks the acquisition of the replication timing program. The existence of the TDP was demonstrated by an elegant series of experiments using CHO nuclei in Xenopus extracts (Dimitrova and Gilbert 1999Dimitrova D.S. Gilbert D.M. Mol. Cell. 1999; 4: 983-993Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Early-replicating sequences in CHO nuclei are distributed throughout the euchromatic regions of the nucleus, while later-replicating sequences are located at the nuclear periphery and perinucleolar regions; similar patterns are observed in hamster cells and other mammalian cell lines. Gilbert and colleagues isolated CHO nuclei at various points in G1 and compared the distribution of early- and late-replicating domains in the subsequent S phase in Xenopus extract (in vitro) to early- and late-replicating domains labeled independently during the previous S phase in hamster cells (in vivo). In nuclei isolated at least 2 hr after metaphase, early- and late-replicating domains are appropriately distributed. In contrast, nuclei isolated only 1 hr after metaphase replicate DNA, but the distribution of early and late replication domains appears random with respect to previously labeled early- and late-replicating sequences (Figure 3). These observations were confirmed by the molecular analysis of specific loci with known replication timing: the proper temporal order of replication was observed only in nuclei isolated after the TDP. Thus, replication timing is programmed in CHO nuclei between 1 and 2 hr after metaphase. Further experiments demonstrate that nuclei that have reached the TDP have not yet acquired the ability to recognize DHFR ori-β, indicating that the TDP and ODP are independent events. Furthermore, both the ODP and the TDP precede the restriction (R) point, a late G1 control point after which cells are committed to S phase entry independent of growth conditions (Figure 1; references in Gilbert 1998Gilbert D.M. Curr. Opin. Genet. Dev. 1998; 8: 194-199Crossref PubMed Scopus (57) Google Scholar). Analysis of cultured CHO cells demonstrates that chromosomal domains reacquire their characteristic distributions in the nucleus 1 to 2 hr after mitosis, precisely the time at which replication timing is programmed (Dimitrova and Gilbert 1999Dimitrova D.S. Gilbert D.M. Mol. Cell. 1999; 4: 983-993Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). This coincidence suggests that nuclear positioning plays a role in replication timing control. Chromosomal repositioning in early G1 has been noted in other cell types, including human fibroblasts in which the position of chromosomal domains correlates with the proliferative state of the cells (Bridger et al. 2000Bridger J.M. Boyle S. Kill I.R. Bickmore W.A. Curr. Biol. 2000; 10: 149-152Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). These observations suggest that altering nuclear position may be a general mechanism for controlling replication in mammals; however, the mechanism by which this occurs is not yet clear. Asymmetric distribution of replication proteins in the nucleus suggests that some locations will favor early replication by allowing the rapid binding or activation of replication factors at the onset of S phase. Xenopus and Drosophila Orc proteins physically interact with the heterochromatin protein HP1 (Pak et al. 1997Pak D.T. Pflumm M. Chesnokov I. Huang D.W. Kellum R. Marr J. Romanowski P. Botchan M.R. Cell. 1997; 91: 311-323Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar), raising the possibility that HP1 may influence replication timing by altering the availability of replication factors. Consistent with this, replication timing of the human β-globin locus is correlated with the position of the locus relative to centromeric heterochromatin in interphase nuclei (Schübeler et al. 2000Schübeler D. Francastel C. Cimbora D.M. Reik A. Martin D.I.K. Groudine M. Genes Dev. 2000; 14: 940-950PubMed Google Scholar). A relationship between nuclear position and replication timing is also evident in yeast, and it has been demonstrated that the transcriptional silencing protein Sir3, which forms foci near the nuclear periphery, is required for late replication from a telomeric origin (Stevenson and Gottschling 1999Stevenson J.B. Gottschling D.E. Genes Dev. 1999; 13: 146-151Crossref PubMed Scopus (144) Google Scholar). Interestingly, the late timing of a yeast telomeric origin is also established during G1 (Raghuraman et al. 1997Raghuraman M.K. Brewer B.J. Fangman W.L. Science. 1997; 276: 806-809Crossref PubMed Scopus (134) Google Scholar), and early- and late-replicating yeast origins assume distinct nuclear distributions in G1 phase nuclei (Heun et al. 2001Heun, P., Laroche, T., Raghuraman, M.K., and Gasser, S.M. (2001). J. Cell Biol., in press.Google Scholar), suggesting that the mechanism that programs replication timing soon after metaphase may be evolutionarily conserved. The existence of G1 steps at which origin choice and replication timing are programmed in mammalian nuclei was demonstrated using a heterologous system based on Xenopus extracts. In this system, the origin decision point correlates with ORC assembly on chromatin, and the temporal decision point coincides with the postmitotic repositioning of chromosomal domains in the nucleus. It is likely that these control steps identified in vitro reflect regulatory events in living cells. However, although we do not yet have a complete picture, potential differences among metazoan replication control mechanisms are already apparent. For example, the number and spatial arrangement of replication foci differs between mammalian primary cells and cell lines (Kennedy et al. 2000Kennedy B.K. Barbie D.A. Classon M. Dyson N. Harlow E. Genes Dev. 2000; 14: 2855-2868Crossref PubMed Scopus (243) Google Scholar), and different profiles of Orc protein expression during the cell cycle are evident among metazoans (Natale et al. 2000Natale D.A. Li C.-J. Sun W.-H. DePamphilis M.L. EMBO J. 2000; 19: 2728-2738Crossref PubMed Scopus (72) Google Scholar and references therein), suggesting different replication control mechanisms may exist. These differences emphasize the need for caution when extrapolating results from one organism or experimental system to another. The identification of further similarities and the reconciliation of differences among these diverse systems will be important for refining models of replication control in higher eukaryotes.