scholarly journals Association of Fission Yeast Orp1 and Mcm6 Proteins with Chromosomal Replication Origins

1999 ◽  
Vol 19 (10) ◽  
pp. 7228-7236 ◽  
Author(s):  
Yuya Ogawa ◽  
Tatsuro Takahashi ◽  
Hisao Masukata
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Chromatin Immunoprecipitation ◽  
Origin Recognition Complex ◽  
Replication Origins ◽  
Minichromosome Maintenance ◽  
Chromatin Immunoprecipitation Assay ◽  
Chromosomal Replication ◽  
Initiation Of Replication ◽  
Mcm Proteins

ABSTRACT We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by thenda1 +/cdc19+ andmis5+ genes, respectively, were associated with chromatin DNA only during the G1 and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G1 and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 andars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G1 and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication.

Essential role of human CDT1 in DNA replication and chromatin licensing

2002 ◽  
Vol 115 (7) ◽  
pp. 1435-1440 ◽  
Author(s):  
Mickael Rialland ◽  
Francesco Sola ◽  
Corrado Santocanale
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Complex Formation ◽  
Fission Yeast ◽  
Origin Recognition Complex ◽  
Essential Role ◽  
Large Body ◽  
Transformed Cells ◽  
Minichromosome Maintenance

Formation of pre-replicative complexes at origins is an early cell cycle event essential for DNA duplication. A large body of evidence supports the notion that Cdc6 protein, through its interaction with the origin recognition complex, is required for pre-replicative complex assembly by loading minichromosome maintenance proteins onto DNA. In fission yeast and Xenopus, this reaction known as the licensing of chromatin for DNA replication also requires the newly identified Cdt1 protein. We studied the role of hCdt1 protein in the duplication of the human genome by antibody microinjection experiments and analyzed its expression during the cell cycle in human non-transformed cells. We show that hCdt1 is essential for DNA replication in intact human cells, that it executes its function in a window of the cell cycle overlapping with pre-replicative complex formation and that it is necessary for the loading of minichromosome maintenance proteins onto chromatin. Intriguingly, we observed that hCdt1 protein, in contrast to other licensing factors, is already present in serum-deprived G0 arrested cells and its levels increase only marginally upon re-entry in the cell cycle.

scholarly journals Chromatin Association of Human Origin Recognition Complex, Cdc6, and Minichromosome Maintenance Proteins during the Cell Cycle: Assembly of Prereplication Complexes in Late Mitosis

2000 ◽  
Vol 20 (22) ◽  
pp. 8602-8612 ◽  
Author(s):  
Juan Méndez ◽  
Bruce Stillman
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Origin Recognition Complex ◽  
Proteasome Inhibitors ◽  
Minichromosome Maintenance ◽  
Mitotic Kinase ◽  
Late Mitosis ◽  
Mcm Proteins ◽  
Origin Recognition

ABSTRACT Evidence obtained from studies with yeast and Xenopusindicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G1 and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.

scholarly journals Drosophila Minichromosome Maintenance 6 Is Required for Chorion Gene Amplification and Genomic Replication

2002 ◽  
Vol 13 (2) ◽  
pp. 607-620 ◽  
Author(s):  
Gina Schwed ◽  
Noah May ◽  
Yana Pechersky ◽  
Brian R. Calvi
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Genetic Dissection ◽  
Minichromosome Maintenance ◽  
Multiple Origins ◽  
Origin Licensing ◽  
Mcm Complex ◽  
Mcm Proteins ◽  
Prereplicative Complex ◽  
Cycle Regulation

Duplication of the eukaryotic genome initiates from multiple origins of DNA replication whose activity is coordinated with the cell cycle. We have been studying the origins of DNA replication that control amplification of eggshell (chorion) genes duringDrosophila oogenesis. Mutation of genes required for amplification results in a thin eggshell phenotype, allowing a genetic dissection of origin regulation. Herein, we show that one mutation corresponds to a subunit of the minichromosome maintenance (MCM) complex of proteins, MCM6. The binding of the MCM complex to origins in G1 as part of a prereplicative complex is critical for the cell cycle regulation of origin licensing. We find that MCM6 associates with other MCM subunits during amplification. These results suggest that chorion origins are bound by an amplification complex that contains MCM proteins and therefore resembles the prereplicative complex. Lethal alleles of MCM6 reveal it is essential for mitotic cycles and endocycles, and suggest that its function is mediated by ATP. We discuss the implications of these findings for the role of MCMs in the coordination of DNA replication during the cell cycle.

scholarly journals DNA Replication Origins Fire Stochastically in Fission Yeast

2006 ◽  
Vol 17 (1) ◽  
pp. 308-316 ◽  
Author(s):  
Prasanta K. Patel ◽  
Benoit Arcangioli ◽  
Stephen P. Baker ◽  
Aaron Bensimon ◽  
Nicholas Rhind
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Replication Initiation ◽  
Epigenetic Mechanism ◽  
Replication Origins ◽  
Origin Firing ◽  
Cell Cycles ◽  
Initiation Of Replication ◽  
Dna Combing ◽  
Dna Replication Origins

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.

scholarly journals Identification of New Human Origins of DNA Replication by an Origin-Trapping Assay

2006 ◽  
Vol 26 (20) ◽  
pp. 7731-7746 ◽  
Author(s):  
Jeannine Gerhardt ◽  
Samira Jafar ◽  
Mark-Peter Spindler ◽  
Elisabeth Ott ◽  
Aloys Schepers
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Origin Recognition Complex ◽  
Human Origins ◽  
Replication Origins ◽  
Barr Virus ◽  
Human Dna ◽  
The Family ◽  
Epstein Barr ◽  
Dyad Symmetry

ABSTRACT Metazoan genomes contain thousands of replication origins, but only a limited number have been characterized so far. We developed a two-step origin-trapping assay in which human chromatin fragments associated with origin recognition complex (ORC) in vivo were first enriched by chromatin immunoprecipitation. In a second step, these fragments were screened for transient replication competence in a plasmid-based assay utilizing the Epstein-Barr virus latent origin oriP. oriP contains two elements, an origin (dyad symmetry element [DS]) and the family of repeats, that when associated with the viral protein EBNA1 facilitate extrachromosomal stability. Insertion of the ORC-binding human DNA fragments in oriP plasmids in place of DS enabled us to screen functionally for their abilities to restore replication. Using the origin-trapping assay, we isolated and characterized five previously unknown human origins. The assay was validated with nascent strand abundance assays that confirm these origins as active initiation sites in their native chromosomal contexts. Furthermore, ORC and MCM2-7 components localized at these origins during G1 phase of the cell cycle but were not detected during mitosis. This finding extends the current understanding of origin-ORC dynamics by suggesting that replication origins must be reestablished during the early stages of each cell division cycle and that ORC itself participates in this process.

scholarly journals Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Mitotic Cycles

1997 ◽  
Vol 139 (1) ◽  
pp. 13-21 ◽  
Author(s):  
Tin Tin Su ◽  
Patrick H. O'Farrell
Keyword(s):  
Cell Cycle ◽  
Cyclin E ◽  
Embryonic Cell ◽  
Cyclin B ◽  
Chromatin Interaction ◽  
G1 Phase ◽  
Minichromosome Maintenance ◽  
Cell Cycles ◽  
Mcm Proteins ◽  
Replication Factors

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM–chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM–chromatin association.

scholarly journals Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins

2012 ◽  
Vol 287 (15) ◽  
pp. 11891-11898 ◽  
Author(s):  
Kyung Yong Lee ◽  
Sung Woong Bang ◽  
Sang Wook Yoon ◽  
Seung-Hoon Lee ◽  
Jong-Bok Yoon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Replication Origins ◽  
Eukaryotic Chromosome ◽  
M Phase ◽  
Cycle Dependent ◽  
Prereplicative Complex ◽  
Origin Recognition

During the late M to the G1 phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2–5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2–5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.

Identification of Cdc6 protein domains involved in interaction with Mcm2 protein and Cdc4 protein in budding yeast cells

2001 ◽  
Vol 354 (3) ◽  
pp. 655-661 ◽  
Author(s):  
Sung-Wuk JANG ◽  
Suzanne ELSASSER ◽  
Judith L. CAMPBELL ◽  
Jiyoung KIM
Keyword(s):  
Origin Recognition Complex ◽  
Fold Increase ◽  
Yeast Cells ◽  
Amino Acid Residues ◽  
Minichromosome Maintenance ◽  
Interaction Domain ◽  
Dependent Protein ◽  
Initiation Of Dna Replication ◽  
Mcm Proteins ◽  
Two Hybrid

The Cdc6 protein (Cdc6p) has essential roles in regulating initiation of DNA replication. Cdc6p is recruited to origins of replication by the origin recognition complex (ORC) late in mitosis; Cdc6p in turn recruits minichromosome maintenance (Mcm) proteins to form the pre-replicative complex. Cdc6p is thought to interact with one or more Mcm proteins but this point has not yet been demonstrated. In the present study we observed that Cdc6p interacted significantly only with Mcm2p out of six Mcm proteins in yeast two-hybrid cells. Our results indicate that the interaction of Cdc6p with Mcm2p is specific, although we cannot exclude the possibility that the interaction might not be direct. In attempts to identify domains of Cdc6p important for interaction with Mcm2p, we tested interactions of various deleted versions of Cdc6p with Mcm2p and also with Cdc4p, which was previously known to interact with Cdc6p. The portion of Cdc6p from amino acid residues 51 to 394 was able to interact with Mcm2p. During the course of the studies we also discovered a previously undetected Cdc4p interaction domain between residues 51 and 394. Interestingly, when all six putative Cdc28 phosphorylation sites in Cdc6p were changed to alanine, a 6–7-fold increase in binding to Mcm2p was observed. This result suggests that unphosphorylated Cdc6p has higher affinity than phosphorylated Cdc6p for Mcm2p; this might partly explain the previous observation that Cdc6p failed to load Mcm proteins on replication origins during S phase when the cyclin-dependent protein kinase was active, thus helping to prevent the reinitiation of activated replicons.

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