Saying a firm 'no' to DNA re-replication.
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Animals↗
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G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus.
Cyclin-dependent kinases (CDKs) activate the firing of replication origins during the S phase of the cell cycle. They also block re-initiation of DNA replication within a single cell cycle, by preventing the assembly of prereplicative complexes at origins. We show here that, in budding yeast, CDKs exclude the essential prereplicative-complex component Mcm4 from the nucleus. Although origin firing can be triggered by the B-type cyclins only, both G1-phase and B-type cyclins cause exit of Mcm4 from the nucleus. These results suggest that G1 cyclins may diminish the cell's capacity to assemble prereplicative complexes before B-type cyclins trigger origin firing during S phase.
CDC28 Protein Kinase, S cerevisiae↗
XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis.
In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.
Animals↗
The Cdt1 protein is required to license DNA for replication in fission yeast.
To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle. This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin. The control is overridden in fission yeast by overexpressing Cdc18 (ref. 11) which leads to continued DNA synthesis in the absence of mitosis. Other factors acting in this control have been postulated and we have used a re-replication assay to identify Cdt1 (ref. 14) as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants (A. Whitaker, I. Roysman and T. Orr-Weaver, personal communication), suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes.
Cell Cycle↗
Mitotic replication initiation proteins are not required for pre-meiotic S phase.
Initiation of mitotic DNA replication in eukaryotes requires conserved factors, including Cdc18/CDC6 and minichromosome maintenance (MCM) proteins. We show here that these proteins are not essential for meiotic DNA replication or subsequent meiotic divisions in fission yeast. In addition, vegetative replication checkpoint genes are not required for the arrest of meiotic divisions in response to pre-meiotic S-phase delays. Genes essential for other aspects of vegetative DNA replication, however, including polymerases and DNA ligase, are also required for pre-meiotic DNA synthesis. Our results indicate that the process of replication initiation and checkpoint control may be fundamentally different in mitotic and meiotic cells.
Cell Cycle Proteins↗
Chaotic license for genetic instability and cancer.
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Animals↗
Structural lessons in DNA replication from the third domain of life.
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Archaeal Proteins↗
Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis.
The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex. In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-dependent kinase activities. High cyclin A-dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins. Cyclin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei. In contrast, high cyclin E/cdk2 was not detected on chromatin, but was found soluble in the nucleoplasm. High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM directly. We conclude that chromatin-bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.
Animals↗
Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-gamma.
In response to IFN-gamma, the latent cytoplasmic Stat1 (signal transducer and activator of transcription) proteins translocate into the nucleus and activate transcription. We showed previously that Stat1 recruits a group of nuclear proteins, among them MCM5 (minichromosome maintenance) and MCM3, for transcription activation. MCM5 directly interacts with the transcription activation domain (TAD) of Stat1 and enhances Stat1-mediated transcription activation. In this report, we identified two specific residues (R732, K734) in MCM5 that are required for the direct interaction between Stat1 and MCM5 both in vitro and in vivo. MCM5 containing mutations of R732/K734 did not enhance Stat1-mediated transcription activation in response to IFN-gamma. In addition, it also failed to form complexes with other MCM proteins in vivo, suggesting that these two residues may be important for an interaction domain in MCM5. Furthermore, MCM5 bearing mutations in its ATPase and helicase domains did not enhance Stat1 activity. In vitro binding assays indicate that MCM3 does not interact directly with Stat1, suggesting that the presence of MCM3 in the group of Stat1TAD-interacting proteins is due to the association of MCM3 with MCM5. Finally, gel filtration analyses of nuclear extracts from INF-gamma-treated cells demonstrate that there is a MCM5/3 subcomplex coeluting with Stat1. Together, these results strongly suggest that Stat1 recruits a MCM5/3 subcomplex through direct interaction with MCM5 in the process of IFN-gamma-induced gene activation.
Binding Sites↗
Intra-G1 arrest in response to UV irradiation in fission yeast.
G1 is a crucial phase of cell growth because the decision to begin another mitotic cycle is made during this period. Occurrence of DNA damage in G1 poses a particular challenge, because replication of damaged DNA can be deleterious and because no sister chromatid is present to provide a template for recombinational repair. We therefore have studied the response of Schizosaccharomyces pombe cells to UV irradiation in early G1 phase. We find that irradiation results in delayed progression through G1, as manifested most critically in the delayed formation of the pre-replication complex. This delay does not have the molecular hallmarks of known checkpoint responses: it is independent of the checkpoint proteins Rad3, Cds1, and Chk1 and does not elicit inhibitory phosphorylation of Cdc2. Irradiated cells eventually progress into S phase and arrest in early S by a rad3- and cds1-dependent mechanism, most likely the intra-S checkpoint. Caffeine alleviates both the intra-G1- and intra-S-phase delays. We suggest that intra-G1 delay may be widely conserved and discuss significance and possible mechanisms.
Adenosine Triphosphatases↗
Sequential MCM/P1 subcomplex assembly is required to form a heterohexamer with replication licensing activity.
Replication licensing factor (RLF) is a multiprotein complex involved in ensuring that chromosomal DNA replicates only once in a single cell cycle. It comprises two components, termed RLF-M and RLF-B. Purified RLF-M consists of a mixture of complexes containing all six members of the MCM/P1 family of minichromosome maintenance proteins. The precise composition of these different complexes and their contribution to RLF-M activity has been unclear. Here we show that in Xenopus extracts, MCM/P1 proteins mainly form heterohexamers containing each of the six proteins. This heterohexamer is readily split into subcomplexes, whose interactions and subunit composition we characterize in detail. We show for the first time an ordered multistep assembly pathway by which the heterohexamer can be reformed from the subcomplexes. Importantly, this novel pathway is essential for DNA replication, since only the full heterohexamer can bind productively to chromatin and provide RLF-M activity.
Biopolymers↗
The MCM3 acetylase MCM3AP inhibits initiation, but not elongation, of DNA replication via interaction with MCM3.
Minichromosome maintenance (MCM) proteins are essential components of pre-replication complexes, which limit DNA replication to once per cell cycle. MCM3 acetylating protein, MCM3AP, binds and acetylates MCM3 and inhibits cell cycle progression. In the present study, we examined inhibition of the cell cycle by MCM3AP in a cell-free system. We show here that wild type MCM3AP, but not the acetylase-deficient mutant, inhibits initiation of DNA replication, but not elongation. Both wild type and acetylase-deficient mutant MCM3AP, however, can bind to chromatin through interaction with MCM3. These results indicate that MCM3 acetylase activity of MCM3AP is required to inhibit initiation of DNA replication and that association of MCM3AP to chromatin alone is not sufficient for the inhibition. We also show that interaction between MCM3 and MCM3AP is essential for nuclear localization and chromatin binding of MCM3AP. Furthermore, the chromatin binding of MCM3AP is temporally correlated with that of endogenous MCM3 when cells were released from mitosis. Hence, MCM3AP is a potent natural inhibitor of the initiation of DNA replication whose action is mediated by interaction with MCM3.
Acetyltransferases↗
Phosphorylation of Mcm4 at specific sites by cyclin-dependent kinase leads to loss of Mcm4,6,7 helicase activity.
Mcm proteins that play an essential role in eukaryotic DNA replication are phosphorylated in vivo, and cyclin-dependent protein kinase is at least in part responsible for the phosphorylation of Mcm4. Our group reported that the DNA helicase activity of Mcm4,6,7 complex, which may be involved in initiation of DNA replication, is inhibited following phosphorylation by Cdk2/cyclin A in vitro. Here, we further examined the interplay between mouse Mcm4,6,7 complex and cyclin-dependent kinases and determined the sites required for the phosphorylation of Mcm4. Six Ser and Thr residues, in all, were required for the phosphorylation. Inhibition of Mcm4,6,7 helicase activity by Cdk2/cyclin A was largely relieved by introducing mutations in these residues of Mcm4. Anti-phosphothreonine antibodies raised against one of these sites reacted with Mcm4 prepared from HeLa cells at mitotic phase but did not bind to those at G(1) and G(1)/S, suggesting that this site is mainly phosphorylated in the mitotic phase. Mcm4,6,7 complex purified from HeLa cells at the mitotic phase exhibited a low level of DNA helicase activity, compared with the complexes prepared from cells at other phases. These results suggest that phosphorylation of Mcm4 at specific sites leads to loss of Mcm4,6,7 DNA helicase activity.
Amino Acid Sequence↗
Sir Antagonist 1 (San1) is a ubiquitin ligase.
Mutations in Sir Antagonist 1 (SAN1) suppress defects in SIR4 and SPT16 in Saccharomyces cerevisiae. San1 contains a RING domain, suggesting that it functions by targeting mutant sir4 and spt16 proteins for degradation by a ubiquitin-mediated pathway. Consistent with this idea, mutant sir4 and spt16 proteins are unstable in SAN1 cells but are stabilized in san1Delta cells. We demonstrate that San1 possesses ubiquitin-protein isopeptide ligase activity in vitro, and the ubiquitin-protein isopeptide ligase activity of San1 is required for its function in vivo. Wild-type Sir4 has a half-life of about 21 min, and san1Delta increased Sir4 half-life to >90 min. In contrast, san1Delta did not affect the stability of wild-type Spt16, Sir3, Sir2, or the Spt16-associated proteins Pob3 and Nhp6. Loss of SAN1 also did not affect the stability of Ste6-166, a highly unstable protein in yeast. These results support the idea that San1 controls the turnover of a specific class of unstable nuclear proteins. Sir4 nucleates the assembly of silent chromatin at telomeres and the silent mating-type loci (HM) in S. cerevisiae. Sir4 can also affect silencing in the rDNA indirectly by sequestering limiting Sir2. Increasing the stability of wild-type Sir4 by deleting SAN1 had only subtle effects on silencing, suggesting that silent chromatin in yeast is robustly buffered against changes in Sir4 stability. Consistent with the idea that San1 participates as an accessory factor to regulate silent chromatin, including the silent mating-type loci, microarray analysis defined a small but statistically significant role for San1 in transcription of several mating pheromone-responsive genes.
Amino Acid Sequence↗
Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin.
Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.
Amino Acid Sequence↗
Multiple domains of fission yeast Cdc19p (MCM2) are required for its association with the core MCM complex.
The members of the MCM protein family are essential eukaryotic DNA replication factors that form a six-member protein complex. In this study, we use antibodies to four MCM proteins to investigate the structure of and requirements for the formation of fission yeast MCM complexes in vivo, with particular regard to Cdc19p (MCM2). Gel filtration analysis shows that the MCM protein complexes are unstable and can be broken down to subcomplexes. Using coimmunoprecipitation, we find that Mis5p (MCM6) and Cdc21p (MCM4) are tightly associated with one another in a core complex with which Cdc19p loosely associates. Assembly of Cdc19p with the core depends upon Cdc21p. Interestingly, there is no obvious change in Cdc19p-containing MCM complexes through the cell cycle. Using a panel of Cdc19p mutants, we find that multiple domains of Cdc19p are required for MCM binding. These studies indicate that MCM complexes in fission yeast have distinct substructures, which may be relevant for function.
Antibodies↗
Fission yeast Cdc23/Mcm10 functions after pre-replicative complex formation to promote Cdc45 chromatin binding.
Using a cytological assay to monitor the successive chromatin association of replication proteins leading to replication initiation, we have investigated the function of fission yeast Cdc23/Mcm10 in DNA replication. Inactivation of Cdc23 before replication initiation using tight degron mutations has no effect on Mcm2 chromatin association, and thus pre-replicative complex (pre-RC) formation, although Cdc45 chromatin binding is blocked. Inactivating Cdc23 during an S phase block after Cdc45 has bound causes a small reduction in Cdc45 chromatin binding, and replication does not terminate in the absence of Mcm10 function. These observations show that Cdc23/Mcm10 function is conserved between fission yeast and Xenopus, where in vitro analysis has indicated a similar requirement for Cdc45 binding, but apparently not compared with Saccharomyces cerevisiae, where Mcm10 is needed for Mcm2 chromatin binding. However, unlike the situation in Xenopus, where Mcm10 chromatin binding is dependent on Mcm2-7, we show that the fission yeast protein is bound to chromatin throughout the cell cycle in growing cells, and only displaced from chromatin during quiescence. On return to growth, Cdc23 chromatin binding is rapidly reestablished independently from pre-RC formation, suggesting that chromatin association of Cdc23 provides a link between proliferation and competence to execute DNA replication.
Anaphase-Promoting Complex-Cyclosome↗
Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18.
We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe. After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication. Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication.
Anaphase↗