Transformation abrogates an early G1-phase arrest point required for specification of the Chinese hamster DHFR replication origin.

The origin decision point (ODP) was originally identified as a distinct point during G1-phase when Chinese hamster ovary (CHO) cell nuclei experience a transition that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts. Passage of cells through the ODP requires a mitogen-independent protein kinase that is activated prior to restriction point control. Here we show that inhibition of an early G1-phase protein kinase pathway by the addition of 2-aminopurine (2-AP) prior to the ODP arrests CHO cells in G1-phase. Transformation with simian virus 40 (SV40) abrogated this arrest point, resulting in the entry of cultured cells into S-phase in the presence of 2-AP and a disruption of the normal pattern of initiation sites at the DHFR locus. Cells treated with 2-AP after the ODP initiated replication specifically within the DHFR origin locus. Transient exposure of transformed cells to 2-AP during the ODP transition also disrupted origin choice, whereas non-transformed cells arrested in G1-phase and then passed through a delayed ODP after removal of 2-AP from the medium. We conclude that mammalian cells have many potential sites at which they can initiate replication. Normally, events occurring during the early G1-phase ODP transition determine which of these sites will be the preferred initiation site. However, if chromatin is exposed to S-phase-promoting factors prior to this transition, mammalian cells, like Xenopus and Drosophila embryos, can initiate replication without origin specification.     

Lovastatin arrests CHO cells between the origin decision point and the restriction point

Asynchronously growing Chinese hamster ovary (CHO) cells treated with the pro-drug, beta-lactone ring form of lovastatin were arrested in G(1)-phase. Subsequent removal of lovastatin resulted in the synchronous entry of cells into S-phase regardless of the presence of mevalonic acid. Lovastatin-arrested cells contained hypophosphorylated retinoblastoma protein (Rb) and required serum mitogens to enter S-phase after lovastatin removal, indicating that cell-cycle arrest is prior to the restriction point (R-point). However, in contrast to quiescent cells, intact nuclei prepared from lovastatin-arrested cells were competent for DNA replication when introduced into Xenopus egg extracts. Initiation of replication by Xenopus egg cytosol took place specifically within the dihydrofolate reductase (DHFR) origin locus, demonstrating that cells were arrested after the origin decision point (ODP). We conclude that the beta-lactone ring form of lovastatin is an effective reagent with which to synchronize CHO cells between the ODP and R-point, without resulting in the withdrawal of cells from the cell-cycle into a quiescent state.     

Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts

Xenopus egg extracts initiate DNA replication specifically at the dihydrofolate reductase (DHFR) origin locus with intact nuclei from late G1-phase CHO cells as a substrate, but at nonspecific sites when purified DNA is assembled by the extract into an embryonic nuclear structure. Here we show that late G1-phase CHO nuclei can be cycled through an in vitro Xenopus egg mitosis, resulting in the assembly of an embryonic nuclear envelope around G1-phase chromatin. Surprisingly, replication within these chimeric nuclei initiated at a novel specific site in the 5' region of the DHFR structural gene that does not function as an origin in cultured CHO cells. Preferential initiation at this unusual site required topoisomerase II-mediated chromosome condensation during mitosis. Nuclear envelope breakdown and reassembly in the absence of chromosome condensation resulted in nonspecific initiation. Introduction of condensed chromosomes from metaphase- arrested CHO cells directly into Xenopus egg extracts was sufficient to elicit assembly of chimeric nuclei and preferential initiation at this same site. These results demonstrate clearly that chromosome architecture can determine the sites of initiation of replication in Xenopus egg extracts, supporting the hypothesis that patterns of initiation in vertebrate cells are established by higher order features of chromosome structure.     

Stability, chromatin association and functional activity of mammalian pre-replication complex proteins during the cell cycle

We have examined the behavior of pre-replication complex (pre-RC) proteins in relation to key cell cycle transitions in Chinese Hamster Ovary (CHO) cells. ORC1, ORC4 and Cdc6 were stable (T1/2 >2 h) and associated with a chromatin-containing fraction throughout the cell cycle. Green fluorescent protein-tagged ORC1 associated with chromatin throughout mitosis in living cells and co-localized with ORC4 in metaphase spreads. Association of Mcm proteins with chromatin took place during telophase, approximately 30 min after the destruction of geminin and cyclins A and B, and was coincident with the licensing of chromatin to replicate in geminin-supplemented Xenopus egg extracts. Neither Mcm recruitment nor licensing required protein synthesis throughout mitosis. Moreover, licensing could be uncoupled from origin specification in geminin-supplemented extracts; site-specific initiation within the dihydrofolate reductase locus required nuclei from cells that had passed through the origin decision point (ODP). These results demonstrate that mammalian pre-RC assembly takes place during telophase, mediated by post-translational modifications of pre-existing proteins, and is not sufficient to select specific origin sites. A subsequent, as yet undefined, step selects which pre-RCs will function as replication origins.     

The replication timing program of the Chinese hamster beta-globin locus is established coincident with its repositioning near peripheral heterochromatin in early G1 phase

We have examined the dynamics of nuclear repositioning and the establishment of a replication timing program for the actively transcribed dihydrofolate reductase (DHFR) locus and the silent beta-globin gene locus in Chinese hamster ovary cells. The DHFR locus was internally localized and replicated early, whereas the beta-globin locus was localized adjacent to the nuclear periphery and replicated during the middle of S phase, coincident with replication of peripheral heterochromatin. Nuclei were prepared from cells synchronized at various times during early G1 phase and stimulated to enter S phase by introduction into Xenopus egg extracts, and the timing of DHFR and beta-globin replication was evaluated in vitro. With nuclei isolated 1 h after mitosis, neither locus was preferentially replicated before the other. However, with nuclei isolated 2 or 3 h after mitosis, there was a strong preference for replication of DHFR before beta-globin. Measurements of the distance of DHFR and beta-globin to the nuclear periphery revealed that the repositioning of the beta-globin locus adjacent to peripheral heterochromatin also took place between 1 and 2 h after mitosis. These results suggest that the CHO beta-globin locus acquires the replication timing program of peripheral heterochromatin upon association with the peripheral subnuclear compartment during early G1 phase.     

Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.     

The spatial position and replication timing of chromosomal domains are both established in early G1 phase

Mammalian chromosomal domains replicate at defined, developmentally regulated times during S phase. The positions of these domains in Chinese hamster nuclei were established within 1 hr after nuclear envelope formation and maintained thereafter. When G1 phase nuclei were incubated in Xenopus egg extracts, domains were replicated in the proper temporal order with nuclei isolated after spatial repositioning, but not with nuclei isolated prior to repositioning. Mcm2 was bound both to early- and late-replicating chromatin domains prior to this transition whereas specification of the dihydrofolate reductase replication origin took place several hours thereafter. These results identify an early G1 phase point at which replication timing is determined and demonstrate a provocative temporal coincidence between the establishment of nuclear position and replication timing.     

A Protein Kinase-Dependent Block to Reinitiation of DNA Replication in G2 Phase in Mammalian Cells

Eukaryotic cells normally replicate their DNA only once between mitoses. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubation inXenopusegg extract. However, artificial permeabilization of the nuclear membrane of G2 nuclei allows induction of new initiations byXenopusegg extract. This is consistent with the action of a replication licensing factor which is believed to enter the nucleus when the nuclear membrane breaks down at mitosis. Here, we show that G2 nuclei will initiate a new round of replication in the absence of nuclear membrane permeabilization, if they are preexposed to protein kinase inhibitorsin vivo.Competence to rereplicate is generated within 30 min of drug treatment, well before the scheduled onset of mitosis. This demonstrates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity in mammalian cells. Kinase inhibition in G2 cells causes nuclear accumulation of replication protein A. Rereplication of kinase-inhibited G2 nuclei also depends on factors supplied byXenopusegg extract, which are distinct from those required for replication licensing.     

Spatial distribution and specification of mammalian replication origins during G1 phase

We have examined the distribution of early replicating origins on stretched DNA fibers when nuclei from CHO cells synchronized at different times during G1 phase initiate DNA replication in Xenopus egg extracts. Origins were differentially labeled in vivo versus in vitro to allow a comparison of their relative positions and spacing. With nuclei isolated in the first hour of G1 phase, in vitro origins were distributed throughout a larger number of DNA fibers and did not coincide with in vivo origins. With nuclei isolated 1 h later, a similar total number of in vitro origins were clustered within a smaller number of DNA fibers but still did not coincide with in vivo origins. However, with nuclei isolated later in G1 phase, the positions of many in vitro origins coincided with in vivo origin sites without further change in origin number or density. These results highlight two distinct G1 steps that establish a spatial and temporal program for replication.     

Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex.

Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1-mediated recruitment of RPA into foci on chromatin. We have investigated whether both of these pre-RCs can be detected in Chinese hamster ovary (CHO) cells. Early- and late-replicating chromosomal domains were pulse-labeled with halogenated nucleotides and prelabeled cells were synchronized at various times during the following G1-phase. The recruitment of Mcm2 and RPA to these domains was examined in relation to the formation of a nuclear envelope, specification of the dihydrofolate reductase (DHFR) replication origin and entry into S-phase. Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throughout G1-phase. During S-phase, detectable Mcm2 was rapidly excluded from PCNA-containing active replication forks. By contrast, detergent-resistant RPA foci were undetectable until the onset of S-phase, when RPA joined only the earliest-firing replicons. During S-phase, RPA was present with PCNA specifically at active replication forks. Together, our data are consistent with a role for Mcm proteins, but not RPA, in the formation of mammalian pre-RCs during early G1-phase.     

Cdt1 downregulation by proteolysis and geminin inhibition prevents DNA re-replication in Xenopus

In late mitosis and G1, Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other cell cycle stages, licensing is inhibited, thus ensuring that origins fire only once per cell cycle. Three additional factors--the origin recognition complex, Cdc6 and Cdt1--are required for origin licensing. We examine here how licensing is regulated in Xenopus egg extracts. We show that Cdt1 is downregulated late in the cell cycle by two different mechanisms: proteolysis, which occurs in part due to the activity of the anaphase-promoting complex (APC/C), and inhibition by a protein called geminin. If both these regulatory mechanisms are abrogated, extracts undergo uncontrolled re-licensing and re-replication. The extent of re-replication is limited by checkpoint kinases that are activated as a consequence of re-replication itself. These results allow us to build a comprehensive model of how re-replication of DNA is prevented in Xenopus, with Cdt1 regulation being the key feature. The results also explain the original experiments that led to the proposal of a replication licensing factor.     

An analysis of the regulation of DNA synthesis by cdk2, Cip1, and licensing factor

The activation of DNA replication appears to involve at least four steps. These include origin recognition, origin unwinding, primer synthesis, and a switching step to a continuous elongation mode. Moreover, in higher eukaryotes a number of studies have shown that much of the DNA replication which occurs is restricted to specific sites within the nuclei. It has been proposed that these replication foci are composed of a large number of origin sites which are clustered together into an aggregate. The molecular basis for this aggregation is currently not well understood. Regulation of the activation of DNA replication is a complicated process. The G1-S kinase cdk2 is a positive regulator of replication. The p21 protein is a negative regulator of replication both by inhibiting cdk2 kinase and the replication protein PCNA. Moreover, it has been proposed that origin usage is restricted to a single firing per cell cycle by a "licensing factor." Using a cell-free replication system derived from Xenopus eggs we have investigated at what step in the replication process these regulators participate. We present evidence that the clustered organization of DNA into foci is not a transient arrangement, but rather, it persists following DNA replication. We also find that foci form on both sperm chromatin and bacteriophage lambda DNA incubated in extracts depleted of cdk2 kinase. Therefore, our data support the conclusion that organization of chromatin into foci is an early event in the replication pathway preceding activation of cdk2 kinase. With respect to the role of cdk2 during activation of DNA replication we find that in cdk2-depleted extracts primer synthesis does not occur and RP-A remains tightly associated with foci. This strongly suggests that cdk2 kinase is required for activating the origin unwinding step of the replication process. Consistent with this interpretation we find that addition of rate limiting quantities of the cdk2 inhibitor p21 protein to an extract delays primer synthesis. Interestingly, in the presence of p21 primer synthesis does occur after a delay and then replication arrests. This is consistent with the published demonstration that p21 can inhibit PCNA, a protein required for replication beyond the priming step. Therefore, our results provide additional support to the proposal that the post-priming switching step is a key regulatory step in replication. With respect to the role of licensing factor during DNA replication it has recently been shown that treatment of mitotic extracts with kinase inhibitor DMAP inactivates "licensing factor."(ABSTRACT TRUNCATED AT 400 WORDS)     

The binding of ORC2 to chromatin from terminally differentiated cells

Nuclei from terminally differentiated Xenopus erythrocytes lack essential components of the prereplication complex, including the origin recognition complex (ORC) proteins XORC1 and XORC2. In Xenopus egg extract, these proteins are able to bind erythrocyte chromatin from permeable nuclei, but not from intact nuclei, even though they are able to cross an intact nuclear envelope. In this report we use both permeable and intact erythrocyte nuclei to investigate the role of cyclin-dependent kinase activity in modulating the binding of XORC2 to chromatin. We find that elevating the level of cyclin A-dependent kinase in egg extract prevents the binding of XORC2 to chromatin from permeable nuclei and that kinase inhibition reverses this effect. We also observe a nuclear transport-dependent accumulation of H1 kinase activity within intact nuclei incubated in the extract. However, inhibiting this kinase activity does not facilitate the binding of XORC2 to chromatin, suggesting that other molecules and/or mechanisms exist to prevent association of XORC proteins with replication origins within intact nuclei from terminally differentiated cells.     

Licensing of DNA replication by a multi-protein complex of MCM/P1 proteins in Xenopus eggs.

In eukaryotes, chromosomal DNA is licensed for a single round of replication in each cell cycle. Xenopus MCM3 protein has been implicated in the licensing of replication in egg extract. We have cloned cDNAs encoding five immunologically distinct proteins associated with Xenopus MCM3 as members of the MCM/P1 family. Six Xenopus MCM proteins formed a physical complex in the egg extract, bound to unreplicated chromatin before the formation of nuclei, and apparently displaced from replicated chromatin. The requirement of six XMCM proteins for the replication activity of the egg extract before nuclear formation suggests that their re-association with replicated chromatin at the end of the mitotic cell cycle is a key step for the licensing of replication.     

The nuclear membrane prevents replication of human G2 nuclei but not G1 nuclei in Xenopus egg extract.

We have used synchronized HeLa cells to investigate the role of the nuclear membrane in preventing rereplication in a single cell cycle. Nuclei were prepared with intact nuclear membranes using streptolysin-O or digitonin and assayed for replication in Xenopus egg extracts. Intact G1 nuclei replicate semiconservatively, but intact G2 nuclei do not replicate in egg extract. However, permeabilizing the nuclear membranes of G2 nuclei by treatment with NP-40 allows them all to replicate in egg extract under cell cycle control, suggesting that integrity of the nuclear membrane is required to distinguish G2 from G1 human nuclei and to prevent rereplication within a single cell cycle. The results are discussed in terms of the previously proposed licensing factor model.     

Initiation of DNA replication in nuclei from quiescent cells requires permeabilization of the nuclear membrane

We have investigated the replication capacity of intact nuclei from quiescent cells using Xenopus egg extract. Nuclei, with intact nuclear membranes, were isolated from both exponentially growing and contact- inhibited BALB/c 3T3 fibroblasts by treatment of the cells with streptolysin-O. Flow cytometry showed that > 90% of all contact- inhibited cells and approximately 50% of the exponential cells were in G0/G1-phase at the time of nuclear isolation. Intact nuclei were assayed for replication in the extract by incorporation of [alpha- 32P]dATP or biotin-dUTP into nascent DNA. Most nuclei from exponential cells replicated in the egg extract, consistent with previous results showing that intact G1 nuclei from HeLa cells replicate in this system. In contrast, few nuclei from quiescent cells replicated in parallel incubations. However, when the nuclear membranes of these intact quiescent nuclei were permeabilized with lysophosphatidylcholine prior to addition to the extract, nearly all the nuclei replicated under complete cell cycle control in a subsequent incubation. The ability of LPC-treated quiescent nuclei to undergo DNA replication was reversed by resealing permeable nuclear membranes with Xenopus egg membranes prior to extract incubation demonstrating that the effect of LPC treatment is at the level of the nuclear membrane. These results indicate that nuclei from G1-phase cells lose their capacity to initiate DNA replication following density-dependent growth arrest and suggest that changes in nuclear membrane permeability may be required for the initiation of replication upon re-entry of the quiescent cell into the cell cycle.     

Origin licensing and p53 status regulate Cdk2 activity during G1

Origins of DNA replication are licensed through the assembly of a chromatin-bound prereplication complex. Multiple regulatory mechanisms block new prereplication complex assembly after the G1/S transition to prevent rereplication. The strict inhibition of licensing after the G1/S transition means that all origins used in S phase must have been licensed in the preceding G1. Nevertheless mechanisms that coordinate S phase entry with the completion of origin licensing are still poorly understood. We demonstrate that depletion of either of two essential licensing factors, Cdc6 or Cdt1, in normal human fibroblasts induces a G1 arrest accompanied by inhibition of cyclin E/Cdk2 activity and hypophosphorylation of Rb. The Cdk2 inhibition is attributed to a reduction in the essential activating phosphorylation of T160 and an associated delay in Cdk2 nuclear accumulation. In contrast, licensing inhibition in the HeLa or U2OS cancer cell lines failed to regulate Cdk2 or Rb phosphorylation, and these cells died by apoptosis. Co-depletion of Cdc6 and p53 in normal cells restored Cdk2 activation and Rb phosphorylation, permitting them to enter S phase with a reduced rate of replication and also to accumulate markers of DNA damage. These results demonstrate dependence on origin licensing for multiple events required for G1 progression, and suggest a mechanism to prevent premature S phase entry that functions in normal cells but not in p53-deficient cells.     

Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import

During late mitosis and early interphase, origins of replication become "licensed" for DNA replication by loading Mcm2-7 complexes. Mcm2-7 complexes are removed from origins as replication forks initiate replication, thus preventing rereplication of DNA in a single cell cycle. Premature origin licensing is prevented in metaphase by the action of geminin, which binds and inhibits Cdt1/RLF-B, a protein that is required for the loading of Mcm2-7. Recombinant geminin that is added to Xenopus egg extracts is efficiently degraded upon exit from metaphase. Here, we show that recombinant and endogenous forms of Xenopus geminin behave differently from one another, such that a significant proportion of endogenous geminin escapes proteolysis upon exit from metaphase. During late mitosis and early G1, the surviving population of endogenous geminin does not associate with Cdt1/RLF-B and does not inhibit licensing. Following nuclear assembly, geminin is imported into nuclei and becomes reactivated to bind Cdt1/RLF-B. This reactivated geminin provides the major nucleoplasmic inhibitor of origin relicensing during late interphase. Since the initiation of replication at licensed origins depends on nuclear assembly, our results suggest an elegant and novel mechanism for preventing rereplication of DNA in a single cell cycle.