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• Labib K, Diffley JF
• Is the MCM2-7 complex the eukaryotic DNA replication fork helicase?
• Curr Opin Genet Dev. 2001; 11: 64-70
• Display abstract

• The MCM2-7 complex is essential for both the initiation and elongation phases of eukaryotic chromosome replication. There is some evidence that MCM2-7 proteins may act as a DNA helicase; at the same time, a variety of other DNA helicases have also been implicated in the replication of eukaryotic chromosomes.

• Pasion SG, Forsburg SL
• Deconstructing a conserved protein family: the role of MCM proteins in eukaryotic DNA replication.
• Genet Eng (N Y). 2001; 23: 129-55

• Diffley JF
• DNA replication: building the perfect switch.
• Curr Biol. 2001; 11: 36770-36770
• Display abstract

• A sophisticated molecular switch ensures that replication origins are activated just once in each cell cycle. Recent work reveals how the proteolysis of a key replication inhibitor, geminin, by the anaphase promoting complex/cyclosome is an important component of this switch.

• Madine M, Laskey R
• Geminin bans replication licence.
• Nat Cell Biol. 2001; 3: 4950-4950

• Jares P, Donaldson A, Blow JJ
• The Cdc7/Dbf4 protein kinase: target of the S phase checkpoint?
• EMBO Rep. 2000; 1: 319-22
• Display abstract

• Cdc7/Dbf4 is a protein kinase that is required for the initiation of DNA replication in eukaryotes. Recent work has provided new clues to the role that Cdc7/Dbf4 plays in this process. A range of other observations suggest that Cdc7/Dbf4 also plays another, less well characterized, role in checkpoint function and in the maintenance of genomic integrity. In this review we attempt to bring together new information to explain how Cdc7/Dbf4 may perform these two distinct functions.

• Rouse J, Jackson SP
• LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae.
• EMBO J. 2000; 19: 5801-12
• Display abstract

• We identified YDR499W as a Saccharomyces cerevisiae open reading frame with homology to several checkpoint proteins, including S. cerevisiae Rfc5p and Schizosaccharomyces pombe Rad26. Disruption of YDR499W (termed LCD1) results in lethality that is rescued by increasing cellular deoxyribonucleotide levels. Cells lacking LCD1 are very sensitive to a range of DNA-damaging agents, including UV irradiation, and to the inhibition of DNA replication. LCD1 is necessary for the phosphorylation and activation of Rad53p in response to DNA damage or DNA replication blocks, and for Chk1p activation in response to DNA damage. LCD1 is also required for efficient DNA damage-induced phosphorylation of Rad9p and for the association of Rad9p with the FHA2 domain of Rad53p after DNA damage. In addition, cells lacking LCD1 are completely defective in the G(1)/S and G(2)/M DNA damage checkpoints. Finally, we reveal that endogenous Mec1p co-immunoprecipitates with Lcd1p both before and after treatment with DNA-damaging agents. These results indicate that Lcd1p is a pivotal checkpoint regulator, involved in both the essential and checkpoint functions of the Mec1p pathway.

• Lee DG, Bell SP
• ATPase switches controlling DNA replication initiation.
• Curr Opin Cell Biol. 2000; 12: 280-5
• Display abstract

• Proteins that bind and hydrolyze ATP are frequently involved in the early steps of DNA replication. Recent studies of Saccharomyces cerevisiae suggest that two members of the AAA+ ATPase family--the origin recognition complex and Cdc6p--have separable roles for ATP binding and ATP hydrolysis during eukaryotic DNA replication. Intriguingly, the proposed regulation of these eukaryotic replication proteins by ATP has functional similarities to the ATP-dependent control of the DnaA and DnaC initiation factors from Escherichia coli. Comparison of the ATP regulation of these factors suggests that ATP binding and hydrolysis acts as a molecular switch that couples key events during initiation of replication. This switch results in a significant change in protein function.

• Blow JJ, Tada S
• Cell cycle. A new check on issuing the licence.
• Nature. 2000; 404: 560-1

• Nishitani H, Lygerou Z, Nishimoto T, Nurse P
• The Cdt1 protein is required to license DNA for replication in fission yeast.
• Nature. 2000; 404: 625-8
• Display abstract

• 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.

• Leipe DD, Aravind L, Koonin EV
• Did DNA replication evolve twice independently?
• Nucleic Acids Res. 1999; 27: 3389-401
• Display abstract

• DNA replication is central to all extant cellular organisms. There are substantial functional similarities between the bacterial and the archaeal/eukaryotic replication machineries, including but not limited to defined origins, replication bidirectionality, RNA primers and leading and lagging strand synthesis. However, several core components of the bacterial replication machinery are unrelated or only distantly related to the functionally equivalent components of the archaeal/eukaryotic replication apparatus. This is in sharp contrast to the principal proteins involved in transcription and translation, which are highly conserved in all divisions of life. We performed detailed sequence comparisons of the proteins that fulfill indispensable functions in DNA replication and classified them into four main categories with respect to the conservation in bacteria and archaea/eukaryotes: (i) non-homologous, such as replicative polymerases and primases; (ii) containing homologous domains but apparently non-orthologous and conceivably independently recruited to function in replication, such as the principal replicative helicases or proofreading exonucleases; (iii) apparently orthologous but poorly conserved, such as the sliding clamp proteins or DNA ligases; (iv) orthologous and highly conserved, such as clamp-loader ATPases or 5'-->3' exonucleases (FLAP nucleases). The universal conservation of some components of the DNA replication machinery and enzymes for DNA precursor biosynthesis but not the principal DNA polymerases suggests that the last common ancestor (LCA) of all modern cellular life forms possessed DNA but did not replicate it the way extant cells do. We propose that the LCA had a genetic system that contained both RNA and DNA, with the latter being produced by reverse transcription. Consequently, the modern-type system for double-stranded DNA replication likely evolved independently in the bacterial and archaeal/eukaryotic lineages.

• Pines J
• Cell cycle. Checkpoint on the nuclear frontier.
• Nature. 1999; 397: 104-5

• Tye BK
• MCM proteins in DNA replication.
• Annu Rev Biochem. 1999; 68: 649-86
• Display abstract

• The MCM proteins are essential replication initiation factors originally identified as proteins required for minichromosome maintenance in Saccharomyces cerevisiae. The best known among them are a family of six structurally related proteins, MCM2-7, which are evolutionally conserved in all eukaryotes. The MCM2-7 proteins form a hexameric complex. This complex is a key component of the prereplication complex that assembles at replication origins during early G1 phase. New evidence suggests that the MCM2-7 proteins may be involved not only in the initiation but also in the elongation of DNA replication. Orchestration of the functional interactions between the MCM2-7 proteins and other components of the prereplication complex by cell cycle-dependent protein kinases results in initiation of DNA synthesis once every cell cycle.

• Faul T, Staib C, Nanda I, Schmid M, Grummt F
• Identification and characterization of mouse homologue to yeast Cdc7 protein and chromosomal localization of the cognate mouse gene Cdc7l.
• Chromosoma. 1999; 108: 26-31
• Display abstract

• The Cdc7 kinase is required for the G1/S-phase transition during the cell cycle and plays a direct role in the activation of individual origins of replication in Saccharomyces cerevisiae. Here, we report the identification of a mouse cDNA, MmCdc7, whose product is closely related in sequence to Saccharomyces cerevisiae Cdc7 as well as their human, Xenopus and Schizosaccharomyces pombe homologues. The MmCdc7p contains the conserved subdomains common to all protein-serine/threonine kinases and three kinase inserts that are characteristic of members of the Cdc7 protein family. We have mapped the locus of the MmCdc7 gene to chromosome 5, band 5E. Conservation of structures among members of the Cdc7-related proteins suggests that these proteins play a key role in the regulation of DNA replication during the cell cycle in all eukaryotes.

• Toth A, Ciosk R, Uhlmann F, Galova M, Schleiffer A, Nasmyth K
• Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication.
• Genes Dev. 1999; 13: 320-33
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• Sister chromatid cohesion is crucial for chromosome segregation during mitosis. Loss of cohesion very possibly triggers sister separation at the metaphase --> anaphase transition. This process depends on the destruction of anaphase inhibitory proteins like Pds1p (Cut2p), which is thought to liberate a sister-separating protein Esp1p (Cut1p). By looking for mutants that separate sister centromeres in the presence of Pds1p, this and a previous study have identified six proteins essential for establishing or maintaining sister chromatid cohesion. Four of these proteins, Scc1p, Scc3p, Smc1p, and Smc3p, are subunits of a 'Cohesin' complex that binds chromosomes from late G1 until the onset of anaphase. The fifth protein, Scc2p, is not a stoichiometric Cohesin subunit but it is required for Cohesin's association with chromosomes. The sixth protein, Eco1p(Ctf7p), is not a Cohesin subunit. It is necessary for the establishment of cohesion during DNA replication but not for its maintenance during G2 and M phases.

• Blow JJ, Prokhorova TA
• Saying a firm 'no' to DNA re-replication.
• Nat Cell Biol. 1999; 1: 1757-1757

• Tyler JK, Adams CR, Chen SR, Kobayashi R, Kamakaka RT, Kadonaga JT
• The RCAF complex mediates chromatin assembly during DNA replication and repair.
• Nature. 1999; 402: 555-60
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• Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.

• Roberts BT, Ying CY, Gautier J, Maller JL
• DNA replication in vertebrates requires a homolog of the Cdc7 protein kinase.
• Proc Natl Acad Sci U S A. 1999; 96: 2800-4
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• CDC7 is an essential gene required for DNA replication in Saccharomyces cerevisiae. Cdc7p homologs have recently been identified in vertebrates, but their role in DNA replication has not yet been addressed. Here we show that antibodies to the Xenopus laevis homolog, xCdc7, interfere with DNA replication in vivo in developing embryos and in vitro in cycling egg extracts. We also demonstrate cell cycle-dependent association of xCdc7 with the Mcm complex, which binds to replication origins and also is required for DNA synthesis. Taken together, these data indicate that the function of xCdc7 is conserved from fungi to vertebrates. xCdc7 protein accumulates after stimulation of resting oocytes with progesterone, suggesting a molecular explanation for previous observations that the development of the capacity for DNA replication requires protein synthesis late in meiosis I.

• Takei Y, Yamamoto K, Tsujimoto G
• Identification of the sequence responsible for the nuclear localization of human Cdc6.
• FEBS Lett. 1999; 447: 292-6
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• The Cdc6 is the essential protein for the initiation of DNA replication. Cdc6 is localized in the G1 nucleus, and abnormal nuclear localization of this protein induces irregular initiation of DNA replication. We identified here that amino acids K57 and R58 in the human Cdc6 protein play an important role in the nuclear localization of the protein. The fundamental features of the mechanism regulating the localization of Cdc6 seem to be maintained in yeast, Xenopus, and human, since the amino acid sequence surrounding K57 and R58, (S/T)PXKR(L/I), is conserved in these species. Substitution of amino acid residue S54 with E and not Q blocked partially the nuclear localization of the protein, implying that the phosphorylation at S54 is involved in the regulating mechanism of the cell cycle-dependent localization of Cdc6.

• Wang H, Elledge SJ
• DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae.
• Proc Natl Acad Sci U S A. 1999; 96: 3824-9
• Display abstract

• In addition to DNA polymerase complexes, DNA replication requires the coordinate action of a series of proteins, including regulators Cdc28/Clb and Dbf4/Cdc7 kinases, Orcs, Mcms, Cdc6, Cdc45, and Dpb11. Of these, Dpb11, an essential BRCT repeat protein, has remained particularly enigmatic. The Schizosaccharomyces pombe homolog of DPB11, cut5, has been implicated in the DNA replication checkpoint as has the POL2 gene with which DPB11 genetically interacts. Here we describe a gene, DRC1, isolated as a dosage suppressor of dpb11-1. DRC1 is an essential cell cycle-regulated gene required for DNA replication. We show that both Dpb11 and Drc1 are required for the S-phase checkpoint, including the proper activation of the Rad53 kinase in response to DNA damage and replication blocks. Dpb11 is the second BRCT-repeat protein shown to control Rad53 function, possibly indicating a general function for this class of proteins. DRC1 and DPB11 show synthetic lethality and reciprocal dosage suppression. The Drc1 and Dpb11 proteins physically associate and function together to coordinate DNA replication and the cell cycle.

• Fujita M
• Cell cycle regulation of DNA replication initiation proteins in mammalian cells.
• Front Biosci. 1999; 4: 81623-81623
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• Genomic DNA has to be replicated completely and only once during a single cell cycle in order to maintain integrity. Eukaryotes have developed highly regulated machinery for precisely replicating genomic DNA that is fragmented into multiple chromosomes. Our knowledge of such mechanisms largely depends on findings with budding yeast, since identification of specific DNA sequences acting as replication origins, autonomously replicating sequences, has allowed extensive analyses of the initiation of DNA replication. Several factors essential for regulation of initiation have been identified, including ORC, CDC6 and MCM. Subsequent work has suggested that the fundamental machinery for DNA replication may be conserved in metazoan embryonic cells in which replication occurs sequence-independently, and also in mammalian nonembryonic cells, where replication origins are more specific. However, there are specific differences. In this review, information on function and regulation of mammalian initiation factors, ORC, CDC6 and MCM, is summarized, and yeast and embryonic systems are compared. A hypothetical model for the state of prereplication chromatin in mammalian cell nuclei and regulation during the cell cycle is also proposed.

• Takahashi T, Masukata H
• [Initiation-protein complex for eukaryotic DNA replication]
• Tanpakushitsu Kakusan Koso. 1999; 44: 1804-12

• Miyake S, Yamashita S
• Identification of sna41 gene, which is the suppressor of nda4 mutation and is involved in DNA replication in Schizosaccharomyces pombe.
• Genes Cells. 1998; 3: 157-66
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• BACKGROUND: The replication licensing factor limits DNA replication to once in a cell cycle and is thought to contain MCM proteins as its component parts. Six MCM subtypes have been identified in various species. These MCM proteins are thought to bind each other to make a heteromeric complex. The Nda4 protein of Schizosaccharomyces pombe is one of the MCM proteins and is involved in DNA replication. RESULTS: The suppressor mutant of nda4 was isolated and the mutant gene was named sna41. The sna41-912 mutant demonstrated the ts phenotype, with an elongated cell shape at the restrictive temperature. Cells with 1C DNA content accumulated 2 h after shifting up to the restrictive temperature. This result suggests that sna41 is also involved in DNA replication. The sna41 genomic clone was isolated by a complementation of the ts phenotype of the mutant strain and was sequenced. The sna41 gene encodes a protein of 638 amino acids, which has low homology with CDC45 in S. cerevisiae. The gene disruption analysis showed that sna41 gene is essential for viability. CONCLUSIONS: The S. pombe sna41 mutation suppresses the nda4-108 mutation. Sna41 is involved in DNA replication and may play some roles in the regulation of DNA replication by the MCM proteins.

• Kamimura Y, Masumoto H, Sugino A, Araki H
• Sld2, which interacts with Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication.
• Mol Cell Biol. 1998; 18: 6102-9
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• The DPB11 gene, which genetically interacts with DNA polymerase II (epsilon), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint in Saccharomyces cerevisiae. To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal with dpb11-1) mutations which fall into six complementation groups (sld1 to -6). In this study, we characterized SLD2, encoding an essential 52-kDa protein. High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1. Conversely, high-copy DPB11 suppressed the temperature-sensitive growth defect caused by sld2-6. The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay. This interaction was evident at 25 degreesC but not at 34 degreesC when Sld2-6 or Dpb11-1 replaced its wild-type protein. No interaction between Sld2-6 and Dpb11-1 could be detected even at 25 degreesC. Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2. sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1 cells. Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift. These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication.

• Ronchetto F
• [Apparatus of control of the cell cycle]
• Recenti Prog Med. 1998; 89: 132-4

• Klann AG, Belanger AE, Abanes-De Mello A, Lee JY, Hatfull GF
• Characterization of the dnaG locus in Mycobacterium smegmatis reveals linkage of DNA replication and cell division.
• J Bacteriol. 1998; 180: 65-72
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• We have isolated a UV-induced temperature-sensitive mutant of Mycobacterium smegmatis that fails to grow at 42 degrees C and exhibits a filamentous phenotype following incubation at the nonpermissive temperature, reminiscent of a defect in cell division. Complementation of this mutant with an M. smegmatis genomic library and subsequent subcloning reveal that the defect lies within the M. smegmatis dnaG gene encoding DNA primase. Sequence analysis of the mutant dnaG allele reveals a substitution of proline for alanine at position 496. Thus, dnaG is an essential gene in M. smegmatis, and DNA replication and cell division are coupled processes in this species. Characterization of the sequences flanking the M. smegmatis dnaG gene shows that it is not part of the highly conserved macromolecular synthesis operon present in other eubacterial species but is part of an operon with a dgt gene encoding dGTPase. The organization of this operon is conserved in Mycobacterium tuberculosis and Mycobacterium leprae, suggesting that regulation of DNA replication, transcription, and translation may be coordinated differently in the mycobacteria than in other bacteria.

• Sherman DA, Pasion SG, Forsburg SL
• Multiple domains of fission yeast Cdc19p (MCM2) are required for its association with the core MCM complex.
• Mol Biol Cell. 1998; 9: 1833-45
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• 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.

• Dalton S
• Cell cycle control of chromosomal DNA replication.
• Immunol Cell Biol. 1998; 76: 467-72
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• Accurate replication and segregation of chromosomal DNA is essential for high-fidelity transmission of genetic information from generation to generation. Eukaryotic cells typically replicate by first duplicating their chromosomes during the S phase followed by their segregation between two daughter cells during the M phase. Over recent years, advances in our understanding of this process at the molecular level have been incredibly rapid. The present review will focus on molecular control of DNA replication and the mechanisms which operate to ensure that once replicated, chromosomes are not rereplicated in the same cell cycle.

• Fitzpatrick PJ, Toyn JH, Millar JB, Johnston LH
• DNA replication is completed in Saccharomyces cerevisiae cells that lack functional Cdc14, a dual-specificity protein phosphatase.
• Mol Gen Genet. 1998; 258: 437-41
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• The Cdc14 protein encodes a dual-specificity protein phosphatase which functions in late mitosis, and considerable genetic evidence suggests a role in DNA replication. We find that cdc14 mutants arrested in late mitosis maintain persistent levels of mitotic kinase activity, suggesting that Cdc14 controls inactivation of this kinase. Overexpression of Sicl, a cyclin-dependent protein kinase inhibitor, is able to suppress telophase mutants such as dbf2, cdc5 and cdc15, but not cdc14. It does, however, force cdc14-arrested cells into the next cell cycle, in which an apparently normal S phase occurs as judged by FACS and pulsed-field gel electrophoretic analysis. Furthermore, in a promoter shut-off experiment, cells lacking Cdc14 appear to carry out a normal S phase. Thus Cdc14 functions mainly in late mitosis and it has no essential role in S phase.

• Aves SJ, Tongue N, Foster AJ, Hart EA
• The essential schizosaccharomyces pombe cdc23 DNA replication gene shares structural and functional homology with the Saccharomyces cerevisiae DNA43 (MCM10) gene.
• Curr Genet. 1998; 34: 164-71
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• The fission yeast cdc23 gene is required for correct DNA replication: cdc23 mutants show reduced rates of DNA synthesis and become elongated after cell-cycle arrest. We have cloned the Schizosaccharomyces pombe cdc23 gene by complementation of the temperature-sensitive phenotype of cdc23-M36 and confirmed the identity of the gene by integrative mapping. Analysis of the DNA sequence reveals that cdc23 can encode a protein of 593 amino acids (Mr=67 kDa) with 22% overall identity and many structural homologies with the product of the Saccharomyces cerevisiae DNA43 (MCM10) gene which is required for correct initiation of DNA synthesis at chromosomal origins of replication. Construction of a cdc23 null allele has established that the cdc23 gene is essential for viability, with cdc23 deletion mutant spores germinating but undergoing arrest with undivided nuclei in the first or second cell cycle. The S. pombe cdc23 gene on an expression plasmid is able to complement the S. cerevisiae dna43-1 mutant. These structural and functional homologies between two distantly related species suggest that cdc23 and DNA43 may represent genes for a conserved essential eukaryotic DNA replication function.

• Sherman DA, Forsburg SL
• Schizosaccharomyces pombe Mcm3p, an essential nuclear protein, associates tightly with Nda4p (Mcm5p).
• Nucleic Acids Res. 1998; 26: 3955-60
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• MCM proteins are required for the proper regulation of DNA replication. There are six MCM proteins in all eukaryotes which interact to form a large complex. We report the cloning of fission yeast mcm3 +. mcm3 + is essential and spores carrying a Delta mcm3 disruption arrest with an apparently replicated DNA content. The protein is found constitutively in the nucleus and levels remain constant throughout the cell cycle. Mcm3p binds particularly tightly to Nda4p (Mcm5p), but is loosely associated with the other Schizosaccharomyces pombe MCM proteins. Thus, Mcm3p is a peripheral MCM subunit.

• Gravesen A, von Wright A, Josephsen J, Vogensen FK
• Replication regions of two pairs of incompatible lactococcal theta-replicating plasmids.
• Plasmid. 1997; 38: 115-27
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• Incompatibility tests were performed employing 12 replicons belonging to a family of homologous lactococcal theta-replicating plasmids. Two pairs of incompatible plasmids were found, namely, pFV1001 and pFV1201, and pJW565 and pFW094. The replicons of plasmids pFV1001, pFV1201, pJW565, pJW566, and pFW094 were sequenced. Alignments were made of the replicational origins (repA) and putative replication proteins (RepB) of these and 11 related plasmid sequences. Comparison of the alignments with the incompatibility data indicated that the incompatibility determinant could be contained within the 22-bp tandem repeats DRII and/or the inverted repeat IR1 in repA. In support, the incompatibility determinant of pJW563 was localized to a 743-bp fragment encompassing repA. A stretch of 13 amino acids of RepB was proposed to be responsible for the plasmid-specific initiation of replication. This stretch is part of a domain containing features that are highly conserved within the proposed DNA binding regions of the initiation proteins from several well-characterized plasmids from Gram-negative bacteria, including pSC101, R6K, and mini-F.

• Quintana DG, Hou Z, Thome KC, Hendricks M, Saha P, Dutta A
• Identification of HsORC4, a member of the human origin of replication recognition complex.
• J Biol Chem. 1997; 272: 28247-51
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• A new member of human origin recognition complex (ORC) has been cloned and identified as the human homologue of Saccharomyces cerevisiae ORC4. HsORC4 is a 45-kDa protein encoded by a 2.2-kilobase mRNA whose amino acid sequence is 29% identical to ScORC4. HsORC4 has a putative nucleotide triphosphate binding motif that is not seen in ScORC4. HsORC4P also reveals an unsuspected homology to the ORC1-Cdc18 family of proteins. HsORC4 mRNA expression and protein levels remain constant through the cell cycle. HsORC4P is coimmunoprecipitated from cell extracts with another subunit of human ORC, HsORC2P, consistent with it being a part of the putative human origin recognition complex.

• Rowles A, Blow JJ
• Chromatin proteins involved in the initiation of DNA replication.
• Curr Opin Genet Dev. 1997; 7: 152-7
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• Eukaryotic DNA replication is regulated at least in part by the assembly of initiation proteins onto origins of replication. The origin recognition complex (ORC) is bound to origins throughout most of the cell cycle. Other initiation proteins, such as Cdc6 and the MCM/P1 proteins, are assembled onto ORC-containing chromatin during G1 to define a prereplicative complex. During S phase, these proteins are displaced from chromatin and their reassembly is inhibited by protein-dependent kinases.

• Thommes P, Kubota Y, Takisawa H, Blow JJ
• The RLF-M component of the replication licensing system forms complexes containing all six MCM/P1 polypeptides.
• EMBO J. 1997; 16: 3312-9
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• Replication licensing factor (RLF) is involved in preventing re-replication of chromosomal DNA in a single cell cycle, and previously has been separated into two components termed RLF-M and RLF-B. Here we show that Xenopus RLF-M consists of all six members of the MCM/P1 protein family, XMcm2-XMcm7. The six MCM/P1 polypeptides co-eluted on glycerol gradients and gel filtration as complexes with a mol. wt of approximately 400 kDa. In crude Xenopus extract, all six MCM/P1 polypeptides co-precipitated with anti-XMcm3 antibody, although only XMcm5 quantitatively co-precipitated from purified RLF-M. Further fractionation separated RLF-M into two sub-components, one consisting of XMcms 3 and 5, the other consisting of XMcms 2, 4, 6 and 7. Neither of the sub-components provided RLF-M activity. Finally, we show that all six MCM/P1 proteins bind synchronously to chromatin before the onset of S-phase and are displaced as S-phase proceeds. These results strongly suggest that complexes containing all six MCM/P1 proteins are necessary for replication licensing.

• Dalton S, Hopwood B
• Characterization of Cdc47p-minichromosome maintenance complexes in Saccharomyces cerevisiae: identification of Cdc45p as a subunit.
• Mol Cell Biol. 1997; 17: 5867-75
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• Cdc47p is a member of the minichromosome maintenance (MCM) family of polypeptides, which have a role in the early stages of chromosomal DNA replication. Here, we show that Cdc47p assembles into stable complexes with two other members of the MCM family, Cdc46p and Mcm3p. The assembly of Cdc47p into complexes with Cdc46p does not appear to be cell cycle regulated, making it unlikely that these interactions per se are a rate-limiting step in the control of S phase. Cdc45p is also shown to interact with Cdc47p in vivo and to be a component of high-molecular-weight MCM complexes in cell lysates. Like MCM polypeptides, Cdc45p is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae; however, Cdc45p remains in the nucleus throughout the cell cycle, whereas MCMs are nuclear only during G1. We characterize two mutations in CDC47 and CDC46 which arrest cells with unduplicated DNA as a result of single base substitutions. The corresponding amino acid substitutions in Cdc46p and Cdc47p severely reduce the ability of these polypeptides to assemble in a complex with each other in vivo and in vitro. This argues that assembly of Cdc47p into complexes with other MCM polypeptides is important for its role in the initiation of chromosomal DNA replication.

• Bork P, Hofmann K, Bucher P, Neuwald AF, Altschul SF, Koonin EV
• A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins.
• FASEB J. 1997; 11: 68-76
• Display abstract

• Computer analysis of a conserved domain, BRCT, first described at the carboxyl terminus of the breast cancer protein BRCA1, a p53 binding protein (53BP1), and the yeast cell cycle checkpoint protein RAD9 revealed a large superfamily of domains that occur predominantly in proteins involved in cell cycle checkpoint functions responsive to DNA damage. The BRCT domain consists of approximately 95 amino acid residues and occurs as a tandem repeat at the carboxyl terminus of numerous proteins, but has been observed also as a tandem repeat at the amino terminus or as a single copy. The BRCT superfamily presently includes approximately 40 nonorthologous proteins, namely, BRCA1, 53BP1, and RAD9; a protein family that consists of the fission yeast replication checkpoint protein Rad4, the oncoprotein ECT2, the DNA repair protein XRCC1, and yeast DNA polymerase subunit DPB11; DNA binding enzymes such as terminal deoxynucleotidyltransferases, deoxycytidyl transferase involved in DNA repair, and DNA-ligases III and IV; yeast multifunctional transcription factor RAP1; and several uncharacterized gene products. Another previously described domain that is shared by bacterial NAD-dependent DNA-ligases, the large subunits of eukaryotic replication factor C, and poly(ADP-ribose) polymerases appears to be a distinct version of the BRCT domain. The retinoblastoma protein (a universal tumor suppressor) and related proteins may contain a distant relative of the BRCT domain. Despite the functional diversity of all these proteins, participation in DNA damage-responsive checkpoints appears to be a unifying theme. Thus, the BRCT domain is likely to perform critical, yet uncharacterized, functions in the cell cycle control of organisms from bacteria to humans. The carboxyterminal BRCT domain of BRCA1 corresponds precisely to the recently identified minimal transcription activation domain of this protein, indicating one such function.

• Liang C, Stillman B
• Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants.
• Genes Dev. 1997; 11: 3375-86
• Display abstract

• Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication.

• Tsuruga H, Yabuta N, Hashizume K, Ikeda M, Endo Y, Nojima H
• Expression, nuclear localization and interactions of human MCM/P1 proteins.
• Biochem Biophys Res Commun. 1997; 236: 118-25
• Display abstract

• We report here the comparative analysis of human Mcm/P1 proteins (HsMcm2, -3, -5 and -7), including a characterization of their mutual interactions, cell cycle dependent expression and nuclear localization during the cell cycle and the quiescent state. The mRNA levels of these genes, which undergo cell cycle dependent oscillations with a peak at G1/S phase, may be regulated by E2F motifs, two of which were detected in the 5' upstream region of the HsMCM5 gene. In contrast, the protein levels of these Mcm proteins were found to remain rather constant during the HeLa cell cycle. However, their levels gradually increased in a variable manner as KD cells progressed from GO into the G1/S phase. In the GO stage, the amounts of HsMcm2 and -5 proteins were much lower than those of HsMcm7 and -3 proteins, suggesting that they are not present in stoichiometric amounts, and that only a proportion of these molecules actively participate in cell cycle regulation as part of Mcm/P1 complexes.

• Kubota Y, Mimura S, Nishimoto S, Masuda T, Nojima H, Takisawa H
• Licensing of DNA replication by a multi-protein complex of MCM/P1 proteins in Xenopus eggs.
• EMBO J. 1997; 16: 3320-31
• Display abstract

• 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.

• Nishitani H, Nurse P
• The cdc18 protein initiates DNA replication in fission yeast.
• Prog Cell Cycle Res. 1997; 3: 135-42
• Display abstract

• Recent work has demonstrated that cdc18p plays a crucial role in regulating the onset of S phase in fission yeast. cdc18p is a major product of START specific transcription and associates with ORC and MCM proteins which are required for the initiation of DNA replication. High expression of cdc18p induces continuing DNA synthesis and is thought to drive the assembly of initiation complexes. In addition to its role in bringing about DNA replication, cdc18p participates in the cell cycle checkpoint control linking S phase to START and mitosis. We propose that cdc18p is central to the molecular mechanism co-ordinating S phase and M phase in concert with changes in activity of the master cell cycle regulator, the cdc2 protein kinase.

• Dutta A, Bell SP
• Initiation of DNA replication in eukaryotic cells.
• Annu Rev Cell Dev Biol. 1997; 13: 293-332
• Display abstract

• The recent identification of proteins that recognize origins of DNA replication and control the initiation of eukaryotic DNA replication has provided critical molecular tools to dissect this process. Dynamic changes in the assembly and disassembly of protein complexes at origins are important for the initiation of DNA replication and occur throughout the cell cycle. Herein, we review the key proteins required for the initiation of DNA replication, their involvement in the protein complex assembly at replication origins, and how the cell cycle machinery regulates this process.

• Forsburg SL, Sherman DA, Ottilie S, Yasuda JR, Hodson JA
• Mutational analysis of Cdc19p, a Schizosaccharomyces pombe MCM protein.
• Genetics. 1997; 147: 1025-41
• Display abstract

• The cdc19+ gene encodes an essential member of the MCM family of replication proteins in Schizosaccharomyces pombe. We have examined the structure and function of the Cdc19p protein using molecular and genetic approaches. We find that overproduction of wild-type Cdc19p in wild-type cells has no effect, but cdc19-P1 mutant cells do not tolerate elevated levels of other MCM proteins or overexpression of mutant forms of Cdc19p. We have found genetic interactions between cdc19+ and genes encoding subunits of DNA polymerase delta and the replication initiator cdc18+. We have constructed a series of point mutations and sequence deletions throughout Cdc19p, which allow us to distinguish essential from nonessential regions of the protein. Not surprisingly, conserved residues in the MCM homology domain are required for protein function, but some residues outside the core homology domain are dispensable.

• Hardy CF
• Identification of Cdc45p, an essential factor required for DNA replication.
• Gene. 1997; 187: 239-46
• Display abstract

• CDC45 is an essential gene required for initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. CDC45 interacts genetically with CDC46 and CDC47, both members of the MCM family of genes which have been implicated in the licensing of DNA replication. In this report, the isolation of CDC45 is described. The complementing gene is linked to an essential open reading frame on chromosome XII. CDC45 was found to be cell cycle regulated and steady-state mRNA levels are G1/S-specific. CDC45 encodes a protein structurally related to Tsd2p, a protein required for DNA replication in Ustilago maydis. CDC45 also interacts genetically with ORC2, the gene encoding the second subunit of the origin recognition complex, ORC, and MCM3, another member of the MCM family. The cdc45-1 mutant has a plasmid maintenance defect which is rescued by the addition of multiple potential origins to the plasmid.

• Thommes P, Blow JJ
• The DNA replication licensing system.
• Cancer Surv. 1997; 29: 75-90
• Display abstract

• The Xenopus cell free system has proved a good model system to study in vitro DNA replication and the mechanism preventing rereplication in a single cell cycle. Studies using this system resulted in the development of a model postulating the existence of a replication licensing factor (RLF), which binds to the chromatin before the G1-S transition of the cell cycle and is displaced during replication. The nuclear envelope prevents rebinding of RLF and hence relicensing. Nuclear envelope breakdown at mitosis is required to allow another round of replication. Protein kinase inhibitors block licensing factor activity and arrest Xenopus extracts in a G2 like state. These kinase inhibitors have allowed the development of an in vitro assay leading to the biochemical purification of RLF components. RLF can be separated into RLF-B and RLF-M, the latter consisting of several members of the MCM/P1 class of replication proteins. In Xenopus as well as in many other eukaryotes, the binding of MCM/P1 proteins to chromatin before S phase is essential for replication to occur. The proteins are then displaced as replication proceeds. These changes in subnuclear distribution are reflected by changes in the phosphorylation status. MCM/P1 proteins do not bind to the DNA on their own but need RLF-B to be loaded onto the chromatin. Their cycling behaviour is reminiscent of the existence of a prereplicative complex at the origins of replication in yeast, suggesting that the licensing mechanism is ubiquitous in eukaryotes.

• Tsuruga H, Yabuta N, Hosoya S, Tamura K, Endo Y, Nojima H
• HsMCM6: a new member of the human MCM/P1 family encodes a protein homologous to fission yeast Mis5.
• Genes Cells. 1997; 2: 381-99
• Display abstract

• BACKGROUND: The tight regulatory mechanism that prevents more than one round of chromosomal DNA replication per cell cycle is thought to require the function of Mcm/P1 proteins. We report here the structural and functional analyses of HsMcm6, a human homologue of the Mis5 of Schizosaccharomyces pombe. RESULTS: We demonstrate here that the transcription of the HsMCM6 gene was repressed in quiescent cells but was rapidly induced at the G1/S phase by growth factor stimulation. The 5' regulatory region of the HsMCM6 gene was found to harbour four putative E2F binding motifs, and these were responsible for the promoter activity. The HsMcm6 protein level oscillated during the cell cycle, with a peak at the G1/S phase. We also showed that the cell-cycle dependent change of subcellular localization of HsMcm6 resembles those of other Mcm/P1 proteins. HsMcm6 consists of two forms, a form extractable by Nonidet P-40 and the nucleus-bound form. A demonstration of the association of HsMcm6 with HsMcm2 and HsMcm7 in vivo supports the idea that they behave as a heteromeric complex. We mapped the HsMCM6 gene at 2q12-14. CONCLUSION: The results indicate that the behaviour of HsMcm6 is reminiscent of replication licensing factor like other Mcm/P1 family members.

• Ariga H, Iguchi-Ariga SM
• [Structure of origin of replication]
• Tanpakushitsu Kakusan Koso. 1996; 41: 2254-8

• Sabelli PA, Burgess SR, Kush AK, Young MR, Shewry PR
• cDNA cloning and characterisation of a maize homologue of the MCM proteins required for the initiation of DNA replication.
• Mol Gen Genet. 1996; 252: 125-36
• Display abstract

• A central question in cell cycle regulation is how DNA replication is initiated and executed only once in each cell cycle. The cell cycle-regulated assembly of specific initiation protein complexes at chromosomal origins appears to specify the initial sites and timing of DNA replication, and to restrict this process to only one round in the somatic cell cycle. Among the enzymes involved in origin activation, the MCM proteins play a conserved key role. In particular, MCM3 homologues have been shown to be components of the DNA replication licensing activity in yeast and vertebrates. In spite of our detailed knowledge of the regulation of the initiation of DNA synthesis in yeast, there is virtually no information available on the molecules involved in origin activation in higher plants. We have isolated a cDNA from maize root apices, termed ROA (Replication Origin Activator), encoding a protein which shares a high degree of homology with the MCM3 subfamily of MCM proteins. Analysis of gene organisation by Southern blotting shows 2-4 copies per haploid genome of closely related ROA sequences and the presence of further less related sequences in a multigene family. The steady-state levels of ROA mRNA are under developmental control, being relatively high in proliferative tissues such as the root apex, the developing cob and the coleoptile, and are strongly correlated with that of the histone H4 transcript. In situ hybridisation analysis in the root apex reveals that ROA mRNA expression is limited to specific subpopulations of cycling cells, which is typical of cell cycle-regulated expression. The isolation of nearly identical sequences from barley and Arabidopsis by the polymerase chain reaction indicates that MCM-related proteins are conserved in higher plants.

• Muzi Falconi M, Brown GW, Kelly TJ
• cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe.
• Proc Natl Acad Sci U S A. 1996; 93: 1566-70
• Display abstract

• In the fission yeast Schizosaccharomyces pombe the cdc18'+gene is required both for initiation of DNA replication and for coupling mitosis to the completion of S phase. Cells lacking Cdc18 fail to enter S phase but still undergo nuclear division. Expression of cdc18+ is sufficient to drive a G1-arrested cdc10ts mutant into the S phase of the cell cycle, indicating that cdc18+ represents a critical link between passage through START and the initiation of DNA replication. Here we show that Cdcl8 is a highly unstable protein that is expressed only once per cell cycle at the boundary between GI and S phase. De novo synthesis of Cdc18 is required before, but not after, the initiation of DNA replication, indicating that Cdc18 function is not necessary once the initiation event has occurred. Overproduction of the protein results in an accumulation of cells with DNA content of greater than 2C and delays mitosis, suggesting that Cdc18 is sufficient to cause reinitiation of DNA replication within a given cell cycle. Our data indicate that the synthesis of Cdc18 protein is a critical rate-limiting step in the initiation of DNA replication during each cell cycle. The extreme lability of the protein may contribute to the prevention of reinitiation.

• Takisawa H
• [Licensing of chromosomal DNA replication and cell cycle control]
• Tanpakushitsu Kakusan Koso. 1996; 41: 1769-76

• Takisawa H, Kubota Y
• [DNA replication licensing factor: MCM protein family]
• Tanpakushitsu Kakusan Koso. 1996; 41: 356-61

• Romanowski P, Madine MA, Laskey RA
• XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication.
• Proc Natl Acad Sci U S A. 1996; 93: 10189-94
• Display abstract

• A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.

• Holthoff HP, Hameister H, Knippers R
• A novel human Mcm protein: homology to the yeast replication protein Mis5 and chromosomal location.
• Genomics. 1996; 37: 131-4
• Display abstract

• Mcm proteins perform functions related to the regulation of eukaryotic genome replication. Previous work has shown that human cells contain at least five different Mcm proteins. We report now the amino acid sequence of an additional human Mcm protein, p105Mcm, and show that it is homologous to the Schizosaccharomyces pombe protein Mis5. We demonstrate that the gene for protein p105Mcm (HGMW-approved symbol, MCM6) is located on human chromosome 2q14-q21. All six known human Mcm proteins have now been sequenced and compared to their yeast counterparts.

• Hopwood B, Dalton S
• Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication.
• Proc Natl Acad Sci U S A. 1996; 93: 12309-14
• Display abstract

• We report the isolation and characterization of CDC45, which encodes a polypeptide of 650 amino acids that is essential for the initiation of chromosomal DNA replication in the budding yeast, Saccharomyces cerevisiae. CDC45 genetically interacts with at least two members of the MCM (minichromosome maintenance) family of replication genes, CDC46 and CDC47, which are proposed to perform a role in restricting initiation of DNA replication to once per cell cycle. Like mutants in several MCM genes, alleles of CDC45 also show a severe minichromosome maintenance defect. Together, these observations imply that Cdc45p performs a role in the control of initiation events at chromosomal replication origins. We investigated this possibility further and present evidence demonstrating that Cdc45p is assembled into complexes with one MCM family member, Cdc46p/Mcm5p. These observations point to a role for Cdc45p in controlling the early steps of chromosomal DNA replication in conjunction with MCM polypeptide complexes. Unlike the MCMs, however, the subcellular localization of Cdc45p does not vary with the cell cycle, making it likely that Cdc45p interacts with MCMs only during the nuclear phase of MCM localization in G1.

• Chong JP, Blow JJ
• DNA replication licensing factor.
• Prog Cell Cycle Res. 1996; 2: 83-90
• Display abstract

• DNA Replication Licensing Factor (RLF) is an essential activity required to restrict the duplication of genomic DNA to precisely once per cell cycle. Recent fractionation of RLF activity from Xenopus egg extracts has resulted in the identification of two essential components, RLF-B and RLF-M. RLF-M has been purified to homogeneity and has been shown to consist of a complex of proteins in the MCM/P1 family. RLF-B is still unidentified, but possible candidates for this activity have been identified in yeast. Elucidation of the RLF mechanism will provide important insights into the way that chromosome replication is controlled.

• Setkov NA
• [Negative control of proliferation depends on protein biosynthesis in resting cells]
• Dokl Akad Nauk. 1996; 346: 708-11

• Cooper JA, Kiehart DP
• Septins may form a ubiquitous family of cytoskeletal filaments.
• J Cell Biol. 1996; 134: 1345-8

• Donovan S, Diffley JF
• Replication origins in eukaroytes.
• Curr Opin Genet Dev. 1996; 6: 203-7
• Display abstract

• Recent experiments in budding yeast and Xenopus have 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.

• Kearsey SE, Labib K, Maiorano D
• Cell cycle control of eukaryotic DNA replication.
• Curr Opin Genet Dev. 1996; 6: 208-14
• Display abstract

• A clearer picture of replication control is emerging through the characterization of proteins, such as cdc18/Cdc6 and members of the mini-chromosome maintenance (MCM) protein family, that are involved in the initiation step. Cyclin B dependent kinases have conserved roles in both Saccharomyces cerevisiae and Schizosaccharomyces pombe, switching on DNA replication in G1 and preventing re-replication in G2. A model is suggested where MCMs and CDKs play complementary roles to ensure 'once-per-cell-cycle' replication, with CDKs maintaining a G1 or G2 state, whereas MCMs provide a cis-acting control on chromatin to prevent reinitiation during a single S phase.

• Fujita M, Kiyono T, Hayashi Y, Ishibashi M
• hCDC47, a human member of the MCM family. Dissociation of the nucleus-bound form during S phase.
• J Biol Chem. 1996; 271: 4349-54
• Display abstract

• hCDC47 is a putative human homologue of yeast CDC47 and a member of the MCM protein family, which has been implicated in the regulatory machinery causing DNA to replicate only once in the S phase. In the present study, we performed an initial characterization of hCDC47. We found that hCDC47 protein was present in the nucleus of cultured human cells in two different forms; one extractable by a non-ionic detergent and the other resistant to such extraction and tightly associated with the nucleus. The levels of the nucleus-bound form gradually diminished during S phase progression, although the total amount of nuclear hCDC47 protein remained relatively constant, suggesting that the nucleus-bound form becomes dissociated from the nuclear structure during DNA replication. This behavior of hCDC47 protein is very similar to that of other mammalian MCM proteins reported recently. We also found that expression of hCDC47 mRNA was repressed in quiescent cells but was induced at the late G1 to S phase by growth factor stimulation. Together, these findings indicate that hCDC47 protein together with other MCM proteins participates in the regulation of mammalian DNA replication.

• Miyake S, Saito I, Kobayashi H, Yamashita S
• Identification of two Xenopus laevis genes, xMCM2 and xCDC46, with sequence homology to MCM genes involved in DNA replication.
• Gene. 1996; 175: 71-5
• Display abstract

• The Schizosaccharomyces pombe genes, nda1 and nda4, are essential for the normal regulation of DNA replication and belong to the MCM gene family. This gene family includes Saccharomyces cerevisiae MCM2, MCM3, MCM5/CDC46 and CDC47, S. pombe nda1, nda4, cdc21 and mis5, and genes encoding human BM28, P1MCM3 and P1.1MCM3 and mouse P1MCM3, most of which are considered to be required for the initiation of DNA replication. We isolated two homologues of the MCM genes, xMCM2 and xCDC46, from a Xenopus laevis cDNA library using the polymerase chain reaction (PCR) method. The predicted amino acid (aa) sequences of xMCM2 and xCDC46 are most similar to those of human BM28 (78% identity) and S. pombe Nda4 (48% identity), respectively. By Western blot analysis using anti-xMCM2 and anti-xCDC46 polyclonal antibodies (Ab) raised against glutathione S-transferase (GST)::xMCM2 or GST::xCDC46 fusion proteins, xMCM2 and xCDC46 were identified as 120- and 95-kDa proteins, respectively. When either xMCM2 or xCDC46 was immunoprecipitated with the specific Ab, the other was also co-precipitated. These results suggest that xMCM2 and xCDC46 physically interact with each other.

• Heichman KA
• Cdc6 and DNA replication: limited to humble origins.
• Bioessays. 1996; 18: 859-62
• Display abstract

• The budding yeast Cdc6 protein is important for regulating DNA replication initiation. Cdc6p acts at replication origins, and cdc6-1 mutants arrest with unreplicated DNA and show elevated minichromosome loss rates. Overexpression of the related Cdc18 protein in fission yeast results in DNA rereplication; however, Cdc6p overexpression does not cause this result. A recent paper further defines the role of Cdc6p in DNA replication. Cdc6p only promotes DNA replication between the end of mitosis and late G1, and although the Cdc6 protein is highly unstable, neither degradation nor nuclear localization is critical for limiting DNA replication to this interval.

• Maiorano D, Van Assendelft GB, Kearsey SE
• Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle.
• EMBO J. 1996; 15: 861-72
• Display abstract

• The fission yeast cdc21 protein belongs to the MCM family, implicated in the once per cell cycle regulation of chromosome replication. In budding yeast, proteins in this family are eliminated from the nucleus during S phase, which has led to the suggestion that they may serve to distinguish unreplicated from replicated DNA, as in the licensing factor model. We show here that, in contrast to the situation in budding yeast, cdc21 remains in the nucleus after S phase, as is found for related proteins in mammalian cells. We suggest that regulation of nuclear import of these proteins may not be an essential aspect of their function in chromosome replication. To determine the function of cdc21+, we have analysed the phenotype of a gene deletion. cdc21+ is required for entry into S phase and, unexpectedly, a proportion of cells depleted of the gene product are able to enter mitosis in the absence of DNA replication. These results are consistent with the view that individual proteins in the MCM family are required for all initiation events, and defective initiation may impair the coordination between mitosis and S phase.

• Kearsey SE, Maiorano D, Holmes EC, Todorov IT
• The role of MCM proteins in the cell cycle control of genome duplication.
• Bioessays. 1996; 18: 183-90
• Display abstract

• The regulatory mechanism which ensures that eukaryotic chromosomes replicate precisely once per cell cycle is a basic and essential cellular property of eukaryotes. This fundamental aspect of DNA replication is still poorly understood, but recent advances encourage the view that we may soon have a clearer picture of how this regulation is achieved. This review will discuss in particular the role of proteins in the minichromosome maintenance (MCM) family, which may hold the key to understanding how DNA is replicated once, and only once, per cell cycle.

• Adachi Y
• [MCM protein complex in fission yeast]
• Tanpakushitsu Kakusan Koso. 1996; 41: 1777-83

• Chant J
• Septin scaffolds and cleavage planes in Saccharomyces.
• Cell. 1996; 84: 187-90

• Matsukage A
• [Replication of chromosomal DNA: process for its initiation]
• Tanpakushitsu Kakusan Koso. 1996; 41: 2369-77

• Takizawa N, Kimura H, Sugimoto K
• Sequence of mouse CDC47, a member of the minichromosome maintenance (Mcm) family involved in the DNA replication licensing system.
• Gene. 1995; 167: 343-4
• Display abstract

• 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.

• Ford C, Chevalier S
• DNA replication. Almost licensed.
• Curr Biol. 1995; 5: 1009-12
• Display abstract

• Exact duplication of all the DNA in a cell occurs during each S phase, and only once in each cell cycle. Recent results show that conserved proteins of the MCM family contribute to these precisely regulated events.

• Huberman JA
• Cell cycle. A licence to replicate.
• Nature. 1995; 375: 360-1

• Chong JP, Mahbubani HM, Khoo CY, Blow JJ
• Purification of an MCM-containing complex as a component of the DNA replication licensing system.
• Nature. 1995; 375: 418-21
• Display abstract

• Replication licensing factor (RLF) ensures that eukaryotic chromosomal DNA is replicated exactly once in each cell cycle. On exit from metaphase, RLF is activated and binds to or modifies chromatin. This modification (the 'licence') is required for subsequent DNA replication; the licence is also inactivated in the process of replication. Active RLF is not imported into the nucleus, so further DNA replication cannot occur until the DNA is relicensed by passage throught mitosis. We have developed an assay to purify RLF from Xenopus eggs. Activity resolves into two components, RLF-M and RLF-B, both of which are required for licensing. RLF-M has been purified to apparent homogeneity: it consists of three polypeptides, one of which is a Xenopus homologue of the yeast MCM3 protein. Xenopus Mcm3 associates with chomatin in G1 and is removed during replication, consistent with its being a component of the RLF system.

• Madine MA, Khoo CY, Mills AD, Laskey RA
• MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells.
• Nature. 1995; 375: 421-4
• Display abstract

• An intact nuclear membrane restricts DNA replication to only one round in each cell cycle, apparently by excluding an essential replication-licensing factor throughout interphase. A family of related yeast replication proteins, MCM2, 3 and 5 (also called, after cell-division cycle, CDC46), resemble licensing factor, entering the nucleus only during mitosis. We have cloned a Xenopus homologue of MCM3 (XMCM3) and raised antibodies against expressed protein. Immunodepletion of Xenopus egg extracts removes a complex of MCM2, 3 and 5 homologues and inhibits replication of Xenopus sperm nuclei or permeable G2 HeLa nuclei. However, G1 HeLa nuclei still replicate efficiently. Mock-depleted extracts replicate all three templates. XMCM3 accumulates in nuclei before replication but anti-XMCM3 staining decreases during replication. These results can explain why replicated nuclei are unable to reinitiate replication in a single cell cycle.

• Todorov IT, Attaran A, Kearsey SE
• BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication.
• J Cell Biol. 1995; 129: 1433-45
• Display abstract

• We have recently cloned and characterized a human member (BM28) of the MCM2-3-5 family of putative relication factors (Todorov, I.T., R. Pepperkok, R.N. Philipova, S. Kearsey, W. Ansorge, and D. Werner. 1994. J. Cell Sci. 107:253-265). While this protein is located in the nucleus throughout interphase, we report here a dramatic alteration in its nuclear binding during the cell cycle. BM28 is retained in the nucleus after Triton X-100 extraction in G1 and early S phase cells, but is progressively lost as S phase proceeds, and little BM28 is retained in detergent-extracted G2 nuclei. BM28 that is resistant to extraction in G1 nuclei is removed by DNase I digestion, suggesting that the protein is chromatin associated. In addition, we present evidence for variations in the electrophoretic mobility of BM28 that may reflect posttranslational modifications of BM28 during the cell cycle. During mitosis, BM28 is present as a fast-migrating form, but on entry into G1, the protein is converted into a slow-migrating form. With the onset of S phase, the slow-migrating form is progressively converted into the fast form. BM28 is phosphorylated at all stages of the cell cycle, but during interphase the fast form is hyperphosphorylated compared with the slow form. These apparent changes in modification may reflect or effect changes in the nuclear binding of BM28. The behavior of BM28 is not dissimilar to related proteins in Saccharomyces cerevisiae, such as Mcm2p, which are excluded from the nucleus after DNA replication. We speculate that BM28 may be involved in the control that limits eukaryotic DNA replication to one round per cell cycle.

• Vaisman N, Tsouladze A, Robzyk K, Ben-Yehuda S, Kupiec M, Kassir Y
• The role of Saccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance.
• Mol Gen Genet. 1995; 247: 123-36
• Display abstract

• Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, in Saccharomyces cerevisiae, the CDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence of CDC40 (455 amino acids) contains four copies of a beta-transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in the cdc40-1 allele) results in normal vegetative growth at 23 degrees C, and cell cycle arrest at 36 degrees C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36 degrees C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.

• Nishitani H, Nurse P
• p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast.
• Cell. 1995; 83: 397-405
• Display abstract

• A key problem in the cell cycle is understanding what brings about the initiation of DNA replication and how this is linked with global cell cycle controls. The fission yeast gene cdc18 is required for DNA replication and is transcriptionally activated by the cdc10/res1/res2 control acting at START in late G1. We show here that overexpressing cdc18 is able to bring about repeated rounds of DNA synthesis in the absence of mitosis and of continuing protein synthesis. The level of the cdc18-encoded protein p65cdc18 is periodic in the cell cycle, peaking at the G1 to S phase transition, and p65cdc18 is located in the nucleus when cdc18 is overexpressed. We propose that p65cdc18 acts at the initiation of DNA replication and plays a major role in controlling the onset of S phase.

• Musahl C, Schulte D, Burkhart R, Knippers R
• A human homologue of the yeast replication protein Cdc21. Interactions with other Mcm proteins.
• Eur J Biochem. 1995; 230: 1096-101
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• We present the amino acid sequence of the human homologue of the yeast replication protein Cdc21, a member of the Mcm family of nuclear proteins. Specific antibodies, raised against protein hCdc21, were used to investigate the expression of the protein through the cell cycle. The protein is highly phosphorylated in mitotic cells. The phosphorylated form of protein hCdc21 appears to be less tightly bound to nuclear structures than the underphosphorylated form suggesting that phosphorylation/dephosphorylation reactions may determine the nuclear distribution of the protein. Protein hCdc21 forms a stable trimeric complex with two novel human Mcm proteins, p85Mcm and p105Mcm. Protein BM28/Mcm2 is more loosely associated with the trimeric hCdc21 complex.

• Kubota Y, Mimura S, Nishimoto S, Takisawa H, Nojima H
• Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor.
• Cell. 1995; 81: 601-9
• Display abstract

• Replication licensing factor is thought to be involved in the strict control of the initiation of DNA replication in eukaryotes. We identified a 100 kDa protein as a candidate for the licensing factor in Xenopus egg extracts. This protein was required for replication; it bound to sperm DNA before the formation of nuclei and apparently dissociated from the nuclear DNA during the progression of replication without being transported into the nuclei. An immunologically homologous protein in HeLa cells behaved similarly to the Xenopus protein during the cell cycle. Cloning and sequencing of the cDNAs encoding the Xenopus and human proteins revealed that they are homologs of yeast Mcm3, a putative yeast DNA replication licensing factor.

• Gasser SM
• Chromosome structure. Coiling up chromosomes.
• Curr Biol. 1995; 5: 357-60
• Display abstract

• The mechanism by which eukaryotic chromosomes condense as cells enter mitosis has long been inaccessible to molecular biologists. An important clue has now been provided by a ubiquitous protein family, the SMCs.

• Feger G, Vaessin H, Su TT, Wolff E, Jan LY, Jan YN
• dpa, a member of the MCM family, is required for mitotic DNA replication but not endoreplication in Drosophila.
• EMBO J. 1995; 14: 5387-98
• Display abstract

• We have isolated the Drosophila disc proliferation abnormal (dpa) gene, a member of the MCM family of DNA replication factors. Members of this family of proteins are required for DNA replication in yeast. A dpa null mutant dies during pupal stages because imaginal tissues necessary for the formation of the adult fly fail to proliferate normally. Beginning in late embryogenesis BrdU labeling reveals DNA replication defects in mitotically proliferating cells. In contrast, dpa is dispensable for endoreplication, a specialized cell cycle consisting of consecutive rounds of S phases without intervening mitosis. Our studies suggest an essential role for dpa in mitotic DNA replication but not in endoreplication. Thus, dpa is not a general replication factor but may play a specialized regulatory role in DNA replication.

• Kelly TJ, Nurse P, Forsburg SL
• Coupling DNA replication to the cell cycle.
• Cold Spring Harb Symp Quant Biol. 1993; 58: 637-44

• Mainwaring WI
• Hormones, growth, and DNA replication.
• Prog Clin Biol Res. 1981; 74: 413-26

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