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The Arabidopsis gene PROLIFERA is required for proper cytokinesis during seed development.

The Arabidopsis thaliana (L.) Heynh. gene PROLIFERA (PRL) is a member of the MCM family of genes that are required for DNA replication during the S phase of the cell cycle. PRL is expressed in dividing cells throughout plant development. During reproductive development, PRL is expressed in both the developing megaspore mother cells and microspore mother cells, but is not expressed in the developing microgametophyte, suggesting that it does not function in the final haploid divisions leading to the production of a mature pollen grain. Disruption of PRL leads to megagametophyte and embryo lethality. prl mutant embryos arrest at a variety of stages, and often show defects in cytokinesis. Multinucleate cells and non-stereotypical cell division planes are commonly observed in developing prl mutant embryos, although mcm mutations in other organisms have not been reported to affect cytokinesis. These observations suggest that PRL may play a role in cytokinesis that is distinct from its role in regulating DNA replication. Additionally, a novel cytokinesis checkpoint that monitors cell cycle progression may exist in Arabidopsis.

Arabidopsis↗

Cloning of a cDNA encoding a human homologue of CDC47, a member of the MCM family.

A complementary DNA (cDNA) clone encoding a 719-amino acid (aa) protein was isolated, which has a 49% aa identity with budding yeast CDC47, a member of the MCM family. Antiserum raised against a C-terminal polypeptide bacterially produced from the cDNA clone detected a cellular protein about 80 kDa, which coincides with the size of the in vitro transcription/translation product of the cDNA. These results indicate that the cDNA covers the entire coding region of a human homologue of CDC47.

Amino Acid Sequence↗

Sequence of mouse CDC47, a member of the minichromosome maintenance (Mcm) family involved in the DNA replication licensing system.

The cDNA encoding a mouse protein, mCDC47, belonging to the minichromosome maintenance (Mcm) family possibly involved in the DNA replication licensing system, was cloned and sequenced. Its deduced amino acid (aa) sequence contained a putative DNA-dependent ATPase motif commonly observed in the family, and a zinc-finger-like domain found in some members of the family.

Adenosine Triphosphatases↗

The cell cycle regulator p27Kip1 interacts with MCM7, a DNA replication licensing factor, to inhibit initiation of DNA replication.

The G1/S phase restriction point is a critical checkpoint that interfaces between the cell cycle regulatory machinery and DNA replicator proteins. Here, we report a novel function for the cyclin-dependent kinase inhibitor p27Kip1 in inhibiting DNA replication through its interaction with MCM7, a DNA replication protein that is essential for initiation of DNA replication and maintenance of genomic integrity. We find that p27Kip1 binds the conserved minichromosome maintenance (MCM) domain of MCM7. The proteins interact endogenously in vivo in a growth factor-dependent manner, such that the carboxyl terminal domain of p27Kip1 inhibits DNA replication independent of its function as a cyclin-dependent kinase inhibitor. This novel function of p27Kip1 may prevent inappropriate initiation of DNA replication prior to S phase.

Binding Sites↗

Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 helicase.

Loading of the Mcm2-7 DNA replicative helicase onto origin-proximal DNA is a critical and tightly regulated event during the initiation of eukaryotic DNA replication. The resulting protein-DNA assembly is called the prereplicative complex (pre-RC), and its formation requires the origin recognition complex (ORC), Cdc6, Cdt1, and ATP. ATP hydrolysis by ORC is required for multiple rounds of Mcm2-7 loading. Here, we investigate the role of ATP hydrolysis by Cdc6 during pre-RC assembly. We find that Cdc6 is an ORC- and origin DNA-dependent ATPase that functions at a step preceding ATP hydrolysis by ORC. Inhibiting Cdc6 ATP hydrolysis stabilizes Cdt1 on origin DNA and prevents Mcm2-7 loading. In contrast, the initial association of Mcm2-7 with the other pre-RC components does not require ATP hydrolysis by Cdc6. Importantly, these coordinated yet distinct functions of ORC and Cdc6 ensure the correct temporal and spatial regulation of pre-RC formation.

Adenosine Triphosphate↗

Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication.

Little is known about the architecture and biochemical composition of the eukaryotic DNA replication fork. To study this problem, we used biotin-streptavidin-modified plasmids to induce sequence-specific replication fork pausing in Xenopus egg extracts. Chromatin immunoprecipitation was employed to identify factors associated with the paused fork. This approach identifies DNA pol alpha, DNA pol delta, DNA pol epsilon, MCM2-7, Cdc45, GINS, and Mcm10 as components of the vertebrate replisome. In the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45, GINS, and MCM2-7 are enriched at the pause site. The data suggest the existence of a large molecular machine, the "unwindosome," which separates DNA strands at the replication fork and contains Cdc45, GINS, and the MCM2-7 holocomplex.

Adenosine Triphosphatases↗

Expression of minichromosome maintenance proteins in vascular smooth muscle cells is ERK/MAPK dependent.

Proliferation of vascular smooth muscle cells (VSMC) represents a key event for the pathogenesis of postangioplasty restenosis. Minichromosome maintenance proteins (MCM) form essential components of the prereplicative complex at DNA replication origins and are regulated by E2F. The present studies were designed to investigate the signal transduction pathways controlling the expression of MCM6 and MCM7 in VSMC in response to mitogenic stimuli. MCM6 and MCM7 expression was substantially increased after stimulation with platelet-derived growth factor-BB and insulin. Pretreatment with PD98059, a specific inhibitor of the extracellular signal-regulated kinases (ERK)-mitogen-activated protein kinase (MAPK), competely inhibited the mitogen-induced MCM6 and MCM7 mRNA and protein expression, demonstrating a critical role for this pathway in transmitting transmembrane signals required for the initiation of DNA replication. The p38MAPK inhibitor SB203580, the phosphatidylinositol 3 kinase (PI3-kinase) pathway inhibitor wortmannin, and the protein kinase C pathway (PKC) inhibitor Gö 6976 did not significantly affect mitogen-induced MCM6 and MCM7 expression. Transient transfection experiments revealed that PD98059 inhibited mitogen-induced MCM6 and MCM7 transcriptional activation. In addition, blockade of ERK/MAPK signaling with PD98059 strongly inhibited phosphorylation of the retinoblastoma protein (Rb) and activity of a luciferase reporter plasmid driven by multiple E2F elements. Inhibition of mitogen-induced MCM6 and MCM7 expression by PD98059 was reversed by ectopic overexpression of E2F, indicating that ERK/MAPK signaling is required for events that occur upstream of E2F release from phosphorylated Rb. In combination, these data demonstrate that the ERK/MAPK signal transduction pathway plays a central role in regulating E2F-dependent MCM expression and DNA replication in VSMC.

Becaplermin↗

Conditional expression of MCM7 increases tumor growth without altering DNA replication activity.

The minichromosome maintenance (MCM) 2-7 complex is a putative DNA helicase complex that facilitates the initiation of DNA replication. Here, we generated a cell line MCM7(+/-)/MCM7-FLAG, in which one allele of MCM7 is mutated whereas a tetracycline-repressible promoter could manipulate the expression of exogenous MCM7 protein. Overexpressed MCM7 protein supports efficient DNA replication of Epstein-Barr virus oriP and rapid formation of tumors in nude mice without altering the activity of cellular DNA replication. This system provides a unique setting for studying the function of MCM7 and for screening for potential therapeutics for malignant tumors.

Animals↗

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells.

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.

Animals↗

Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs.

In eukaryotic cells, firing of DNA replication origins normally does not recur until after M phase. This characteristic is thought to be due to the properties of "initiation" proteins like Orc, Cdc6, and Mcms. Using formaldehyde cross-linking, we show that Cdc6p and Mcm7p associate specifically with replication origins during G1 but not during G2 in S. cerevisiae. Mcm7p's association with origins depends on Cdc6p. Ectopic expression of Cdc6p enables it to associate with origins during G2, but this fails to recruit Mcm7p. Our data suggest that the loading of Mcm proteins onto origins is regulated by two mechanisms: first, by Cdc6p occupancy, and second, by S- and M-CDKs, whose activity during S, G2, and M phases prevents Mcm loading.

Binding Sites↗

Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase.

In S. cerevisiae, the chromatin structure of DNA replication origins changes as cells become competent for DNA replication, suggesting that G1 phase-specific association of replication factors with origin DNA regulates entry into S phase. We demonstrate that ORC, Cdc45p, and MCM proteins are components of prereplication complexes (pre-RC). The MCM-origin association is dependent upon ORC and Cdc6p. During S phase, MCM proteins and Cdc45p dissociate from origin DNA and associate with nonorigin DNA with similar kinetics as DNA Polymerase epsilon, which is present at DNA replication forks. Our results identify protein components of the pre-RC and a novel replication complex appearing at the G1/S transition (the RC), and suggest that after initiation MCM proteins and Cdc45p move with eukaryotic replication forks.

Carrier Proteins↗

Cloning and characterization of human MCM7 promoter.

MCM7 is a member of the MCM protein family which has been implicated in the regulatory machinery allowing DNA to replicate only once during S phase. In quiescent cells, human MCM7 (hMCM7) mRNA is almost undetectable. Stimulation of cells to enter the cell cycle results in induction of hMCM7 expression. Here, we report cloning and characterization of the hMCM7 promoter. We isolated and sequenced a 0.5 kb genomic fragment that contains putative transcription factor binding sites including three E2F sites, three GC boxes and an E box. Several transcription start sites, which were used upon growth stimulation, were identified. The minimal promoter region required for transcription of a luciferase reporter gene was delineated, and it contained an E box and one E2F site, which were important for promoter activity. Interestingly, the cloned sequence appears to act as a promoter for mu-adaptin-related protein 2 (mu-ARP2) gene in the opposite orientation.

Base Sequence↗

cDNA cloning and expression during development of Drosophila melanogaster MCM3, MCM6 and MCM7.

cDNAs encoding three Drosophila melanogaster MCM proteins, DmMCM3, DmMCM6 and DmMCM7, candidates of DNA replication-licensing factors, were cloned and sequenced. The deduced amino-acid sequences displayed 60, 59 and 68% identities with the respective Xenopus laevis homologues, XMCM3, XMCM6 and XMCM7. Six members of the D. melanogaster MCM family were found to share 31-36% identities in their amino-acid sequences, and to possess the five common domains carrying conserved amino-acid sequences as reported with X. laevis MCM proteins. DmMCM3, DmMCM6 and DmMCM7 genes were mapped to the 4F region on the X chromosome, the 6B region on the X chromosome and the 66E region on the third chromosome, respectively, by in situ hybridization. Contents of their mRNAs were proved to be high in unfertilized eggs and early embryos (0-4h after fertilization), then decrease gradually by the 12h time point, with only low levels detected at later stages of development except in adult females. This fluctuation pattern is similar to those of genes for proteins involved in DNA replication, such as DNA polymerase alpha and proliferating cell nuclear antigen, suggesting that expression of DmMCM genes is under the regulatory mechanism which regulates expression of other genes involved in DNA replication.

Amino Acid Sequence↗

The Mcm467 complex of Saccharomyces cerevisiae is preferentially activated by autonomously replicating DNA sequences.

We have analyzed the role of single-stranded DNA (ssDNA) in the modulation of the ATPase activity of Mcm467 helicase of the yeast Saccharomyces cerevisiae. The ATPase activity of the Mcm467 complex is modulated in a sequence-specific manner and that the ssDNA sequences derived from the origin of DNA replication of S. cerevisiae autonomously replicating sequence 1 (ARS1) are the most effective stimulators. Synthetic oligonucleotides, such as oligo(dA) and oligo(dT), also stimulated the ATPase activity of the Mcm467 complex, where oligo(dT) was more effective than oligo(dA). However, the preference of a thymidine stretch appeared unimportant, because with yeast ARS1 derived sequences, the A-rich strand was as effective in stimulating the ATPase activity, as was the T-rich strand. Both of these strands were more effective stimulators than either oligo(dA)( )()or oligo(dT). The DNA helicase activity of Mcm467 complex is also significantly stimulated by the ARS1-derived sequences. These results indicate that the ssDNA sequences containing A and B1 motifs of ARS1, activate the Mcm467 complex and stimulate its ATPase and DNA helicase activities. Our results also indicate that the yeast replication protein A stimulated the ATPase activity of the Mcm467 complex.

Adenosine Triphosphate↗

Identification of functional PDZ domain binding sites in several human proteins.

TIP-15 was previously identified as a cellular protein that can bind to the C-terminal end of the HTLV-1 Tax protein via its two PDZ domains. The sequence of the N-terminal part of TIP-15 is identical to that of the synaptic protein PSD-95. Both proteins are likely to be produced from the same gene by alternative splicing. Whereas expression of the PSD-95 mRNA was detected only with brain RNAs, that of TIP-15 was detected with RNAs from thymus, brain, skeletal muscle and Jurkat cells. The TIP-15 protein exhibits an apparent molecular weight of 40 kD and is weakly expressed in T cell lines. A two-hybrid screen performed with TIP-15 as bait revealed the presence of a PDZ binding site (PDZ-BS) in the following proteins: Lysyl tRNA synthetase, 6-phosphogluconolactonase (6-GPL), Stress-activated protein kinase 3 (SAPK3), NET-1, Diacylglycerol kinase zeta, MTMR1, MCM7, and hSec8. The sequence at the C-terminal ends of these proteins matches the X-S/T-X-V-COOH consensus previously defined for PDZ-BSs, with the exception of 6-GPL and SAPK3 which include a leucine as the C-terminal residue. For Lysyl tRNA synthetase, NET1, MTMR1 and hSec8, binding to TIP-15 was confirmed by co-immunoprecipitation experiments performed with the extracts of transfected COS7 cells. These results show the existence of functional PDZ-BSs in these proteins, but future studies will be necessary to establish whether or not TIP-15 represents a physiological partner. The significance of the presence of a PDZ-BS in these various proteins is discussed with respect to their function.

Amino Acid Sequence↗

Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin.

Eukaryotic replication origins are 'licensed' for replication early in the cell cycle by loading Mcm(2-7) proteins. As chromatin replicates, Mcm(2-7) are removed, thus preventing the origin from firing again. Here we report the purification of the RLF-B component of the licensing system and show that it corresponds to Cdt1. RLF-B/Cdt1 was inhibited by geminin, a protein that is degraded during late mitosis. Immunodepletion of geminin from metaphase extracts allowed them to assemble licensed replication origins. Inhibition of CDKs in metaphase stimulated origin assembly only after the depletion of geminin. These experiments suggest that geminin-mediated inhibition of RLF-B/Cdt1 is essential for repressing origin assembly late in the cell cycle of higher eukaryotes.

Animals↗

Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms.

The stable propagation of genetic information requires that the entire genome of an organism be faithfully replicated once and only once each cell cycle. In eukaryotes, this replication is initiated at hundreds to thousands of replication origins distributed over the genome, each of which must be prohibited from re-initiating DNA replication within every cell cycle. How cells prevent re-initiation has been a long-standing question in cell biology. In several eukaryotes, cyclin-dependent kinases (CDKs) have been implicated in promoting the block to re-initiation, but exactly how they perform this function is unclear. Here we show that B-type CDKs in Saccharomyces cerevisiae prevent re-initiation through multiple overlapping mechanisms, including phosphorylation of the origin recognition complex (ORC), downregulation of Cdc6 activity, and nuclear exclusion of the Mcm2-7 complex. Only when all three inhibitory pathways are disrupted do origins re-initiate DNA replication in G2/M cells. These studies show that each of these three independent mechanisms of regulation is functionally important.

CDC28 Protein Kinase, S cerevisiae↗