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.

scholarly journals Identification of a Preinitiation Step in DNA Replication That Is Independent of Origin Recognition Complex and cdc6, but Dependent on cdk2

1998 ◽  
Vol 140 (2) ◽  
pp. 271-281 ◽  
Author(s):  
Xuequn Helen Hua ◽  
John Newport
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Minichromosome Maintenance ◽  
Cdk2 Activity ◽  
Proteolytic Pathway ◽  
Egg Extracts ◽  
Dna Replication Origin ◽  
Initiation Of Dna Replication ◽  
Mcm Proteins ◽  
Origin Recognition

Before initiation of DNA replication, origin recognition complex (ORC) proteins, cdc6, and minichromosome maintenance (MCM) proteins bind to chromatin sequentially and form preinitiation complexes. Using Xenopus laevis egg extracts, we find that after the formation of these complexes and before initiation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic pathway. If this displacement is inhibited, DNA replication fails to initiate. We also find that after assembly of MCM proteins into preinitiation complexes, removal of the ORC from DNA does not block the subsequent initiation of replication. Importantly, under conditions in which both ORC and cdc6 protein are absent from preinitiation complexes, DNA replication is still dependent on cdk2 activity. Therefore, the final steps in the process leading to initiation of DNA replication during S phase of the cell cycle are independent of ORC and cdc6 proteins, but dependent on cdk2 activity.

scholarly journals Regulation of Replication Licensing by Acetyltransferase Hbo1

2006 ◽  
Vol 26 (3) ◽  
pp. 1098-1108 ◽  
Author(s):  
Masayoshi Iizuka ◽  
Tomoko Matsui ◽  
Haruhiko Takisawa ◽  
M. Mitchell Smith
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Cyclin Dependent Kinase ◽  
Chromatin Binding ◽  
Regulatory Factor ◽  
Minichromosome Maintenance ◽  
Egg Extracts ◽  
Sequential Binding ◽  
Initiation Of Dna Replication ◽  
Prereplicative Complex

ABSTRACT The initiation of DNA replication is tightly regulated in eukaryotic cells to ensure that the genome is precisely duplicated once and only once per cell cycle. This is accomplished by controlling the assembly of a prereplicative complex (pre-RC) which involves the sequential binding to replication origins of the origin recognition complex (ORC), Cdc6/Cdc18, Cdt1, and the minichromosome maintenance complex (Mcm2-Mcm7, or Mcm2-7). Several mechanisms of pre-RC regulation are known, including ATP utilization, cyclin-dependent kinase levels, protein turnover, and Cdt1 binding by geminin. Histone acetylation may also affect the initiation of DNA replication, but at present neither the enzymes nor the steps involved are known. Here, we show that Hbo1, a member of the MYST histone acetyltransferase family, is a previously unrecognized positive regulatory factor for pre-RC assembly. When Hbo1 expression was inhibited in human cells, Mcm2-7 failed to associate with chromatin even though ORC and Cdc6 loading was normal. When Xenopus egg extracts were immunodepleted of Xenopus Hbo1 (XHbo1), chromatin binding of Mcm2-7 was lost, and DNA replication was abolished. The binding of Mcm2-7 to chromatin in XHbo1-depleted extracts could be restored by the addition of recombinant Cdt1.

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.

scholarly journals CDC45, a novel yeast gene that functions with the origin recognition complex and Mcm proteins in initiation of DNA replication.

1997 ◽  
Vol 17 (2) ◽  
pp. 553-563 ◽  
Author(s):  
L Zou ◽  
J Mitchell ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Genetic Interaction ◽  
Sequence Similarity ◽  
Replication Initiation ◽  
Nonpermissive Temperature ◽  
Functional Connection ◽  
Initiation Of Dna Replication ◽  
Mcm Proteins ◽  
Origin Recognition

The CDC45 gene of Saccharomyces cerevisiae was isolated by complementation of the cold-sensitive cdc45-1 mutant and shown to be essential for cell viability. Although CDC45 genetically interacts with a group of MCM genes (CDC46, CDC47, and CDC54), the predicted sequence of its protein product reveals no significant sequence similarity to any known Mcm family member. Further genetic characterization of the cdc45-1 mutant demonstrated that it is synthetically lethal with orc2-1, mcm2-1, and mcm3-1. These results not only reveal a functional connection between the origin recognition complex (ORC) and Cdc45p but also extend the CDC45-MCM genetic interaction to all known MCM family members that were shown to be involved in replication initiation. Initiation of DNA replication in cdc45-1 cells was defective, causing a delayed entry into S phase at the nonpermissive temperature, as well as a high plasmid loss rate which could be suppressed by tandem copies of replication origins. Furthermore, two-dimensional gels directly showed that chromosomal origins fired less frequently in cdc45-1 cells at the nonpermissive temperature. These findings suggest that Cdc45p, ORC, and Mcm proteins act in concert for replication initiation throughout the genome.

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 The yeast Mcm1 protein is regulated posttranscriptionally by the flux of glycolysis.

1995 ◽  
Vol 15 (8) ◽  
pp. 4631-4639 ◽  
Author(s):  
Y Chen ◽  
B K Tye
Keyword(s):  
Glycolytic Enzyme ◽  
Yeast Cells ◽  
Phosphoglycerate Mutase ◽  
Protein Activity ◽  
Minichromosome Maintenance ◽  
Multifunctional Protein ◽  
Global Regulators ◽  
Genes Encoding ◽  
Initiation Of Dna Replication ◽  
Reduced Rate

Mcm1 is a multifunctional protein which plays a role both in the initiation of DNA replication and in the transcriptional regulation of diverse genes in Saccharomyces cerevisiae. The mcm1-1 mutation results in instability of minichromosomes and alpha-specific sterility. Second-site suppressors that restore minichromosome stability but not fertility to the mcm1-1 mutant were isolated. Two of the suppressors, pgm1-1 and pgm1-2, are mutant alleles of PGM1 which encodes a glycolytic enzyme, phosphoglycerate mutase. We show that the pgm1-1 mutation suppresses the minichromosome maintenance (Mcm) defect by increasing the protein activity or level of Mcm1-1 posttranscriptionally. This increase in the intracellular Mcm1-1 activity is sufficient to suppress the Mcm defect but only minimally suppresses the mating defect. Mutations in genes encoding other glycolytic enzymes, such as eno2::URA3, can also suppress the Mcm phenotype of mcm1-1. Suppression by these glycolytic enzyme mutations correlates with a reduced rate of glycolysis rather than a reduced rate of cell growth. This study suggests that in response to changes in their nutritional states yeast cells may attain homeostasis by modulating the activity of global regulators like Mcm1, which plays a central role in the regulation of energy-expensive anabolic processes.

scholarly journals Essential Role of MCM Proteins in Premeiotic DNA Replication

2002 ◽  
Vol 13 (2) ◽  
pp. 435-444 ◽  
Author(s):  
Karola Lindner ◽  
Juraj Gregán ◽  
Stuart Montgomery ◽  
Stephen E. Kearsey
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Yeast Cells ◽  
Critical Event ◽  
Minichromosome Maintenance ◽  
Cell Cycles ◽  
Eukaryotic Dna ◽  
Mcm Proteins ◽  
Meiotic Cycle

A critical event in eukaryotic DNA replication involves association of minichromosome maintenance (MCM2–7) proteins with origins, to form prereplicative complexes (pre-RCs) that are competent for initiation. The ability of mutants defective in MCM2–7 function to complete meiosis had suggested that pre-RC components could be irrelevant to premeiotic S phase. We show here that MCM2–7 proteins bind to chromatin in fission yeast cells preparing for meiosis and during premeiotic S phase in a manner suggesting they in fact are required for DNA replication in the meiotic cycle. This is confirmed by analysis of a degron mcm4 mutant, which cannot carry out premeiotic DNA replication. Later in meiosis, Mcm4 chromatin association is blocked between meiotic nuclear divisions, presumably accounting for the absence of a second round of DNA replication. Together, these results emphasize similarity between replication mechanisms in mitotic and meiotic cell cycles.

scholarly journals The Croonian Lecture 2001 Hunting the antisocial cancer cell: MCM proteins and their exploitation

2005 ◽  
Vol 360 (1458) ◽  
pp. 1119-1132 ◽  
Author(s):  
Ronald Laskey
Keyword(s):  
Cell Cycle ◽  
Screening Tests ◽  
Eukaryotic Cells ◽  
Replication Forks ◽  
Minichromosome Maintenance ◽  
A Cell ◽  
The Common ◽  
Initiation Of Dna Replication ◽  
Mcm Proteins ◽  
Eukaryotic Genomes

Replicating large eukaryotic genomes presents the challenge of distinguishing replicated regions of DNA from unreplicated DNA. A heterohexamer of minichromosome maintenance (MCM) proteins is essential for the initiation of DNA replication. MCM proteins are loaded on to unreplicated DNA before replication begins and displaced progressively during replication. Thus, bound MCM proteins license DNA for one, and only one, round of replication and this licence is reissued each time a cell divides. MCM proteins are also the best candidates for the replicative helicases that unwind DNA during replication, but interesting questions arise about how they can perform this role, particularly as they are present on only unreplicated DNA, rather than clustered at replication forks. Although MCM proteins are bound and released cyclically from DNA during the cell cycle, higher eukaryotic cells retain them in the nucleus throughout the cell cycle. In contrast, MCMs are broken down when cells exit the cycle by quiescence or differentiation. We have exploited these observations to develop screening tests for the common carcinomas, starting with an attempt to improve the sensitivity of the smear test for cervical cancer. MCM proteins emerge as exceptionally promising markers for cancer screening and early diagnosis.

scholarly journals SpSld3 Is Required for Loading and Maintenance of SpCdc45 on Chromatin in DNA Replication in Fission Yeast

2002 ◽  
Vol 13 (5) ◽  
pp. 1462-1472 ◽  
Author(s):  
Reiko Nakajima ◽  
Hisao Masukata
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Temperature Sensitive ◽  
Minichromosome Maintenance ◽  
Origin Firing ◽  
Temporal Regulation ◽  
Ordered Assembly ◽  
Initiation Of Dna Replication ◽  
Mcm Proteins ◽  
Stable Association

Initiation of DNA replication in eukaryotic cells is regulated through the ordered assembly of replication complexes at origins of replication. Association of Cdc45 with the origins is a crucial step in assembly of the replication machinery, hence can be considered a target for the regulation of origin activation. To examine the process required for SpCdc45 loading, we isolated fission yeast SpSld3, a counterpart of budding yeast Sld3 that interacts with Cdc45. SpSld3 associates with the replication origin during G1–S phases and this association depends on Dbf4-dependent (DDK) kinase activity. In the corresponding period, SpSld3 interacts with minichromosome maintenance (MCM) proteins and then with SpCdc45. A temperature-sensitive sld3-10 mutation suppressed by the multicopy of the sna41+encoding SpCdc45 impairs loading of SpCdc45 onto chromatin. In addition, this mutation leads to dissociation of preloaded Cdc45 from chromatin in the hydroxyurea-arrested S phase, and DNA replication upon removal of hydroxyurea is retarded. Thus, we conclude that SpSld3 is required for stable association of Cdc45 with chromatin both in initiation and elongation of DNA replication. The DDK-dependent origin association suggests that SpSld3 is involved in temporal regulation of origin firing.

scholarly journals Laminar shear stress upregulates endothelial Ca2+-activated K+ channels KCa2.3 and KCa3.1 via a Ca2+/calmodulin-dependent protein kinase kinase/Akt/p300 cascade

2013 ◽  
Vol 305 (4) ◽  
pp. H484-H493 ◽  
Author(s):  
Jun Takai ◽  
Alexandra Santu ◽  
Haifeng Zheng ◽  
Sang Don Koh ◽  
Masanori Ohta ◽  
...  
Keyword(s):  
Shear Stress ◽  
Transcriptional Activation ◽  
Static Condition ◽  
Fold Increase ◽  
Laminar Shear Stress ◽  
Human Coronary Artery ◽  
Intracellular Ca ◽  
Kinase Cascade ◽  
Dependent Protein ◽  
Laminar Shear

In endothelial cells (ECs), Ca2+-activated K+ channels KCa2.3 and KCa3.1 play a crucial role in the regulation of arterial tone via producing NO and endothelium-derived hyperpolarizing factors. Since a rise in intracellular Ca2+ levels and activation of p300 histone acetyltransferase are early EC responses to laminar shear stress (LS) for the transcriptional activation of genes, we examined the role of Ca2+/calmodulin-dependent kinase kinase (CaMKK), the most upstream element of a Ca2+/calmodulin-kinase cascade, and p300 in LS-dependent regulation of KCa2.3 and KCa3.1 in ECs. Exposure to LS (15 dyn/cm2) for 24 h markedly increased KCa2.3 and KCa3.1 mRNA expression in cultured human coronary artery ECs (3.2 ± 0.4 and 45 ± 10 fold increase, respectively; P < 0.05 vs. static condition; n = 8–30), whereas oscillatory shear (OS; ± 5 dyn/cm2 × 1 Hz) moderately increased KCa3.1 but did not affect KCa2.3. Expression of KCa2.1 and KCa2.2 was suppressed under both LS and OS conditions, whereas KCa1.1 was slightly elevated in LS and unchanged in OS. Inhibition of CaMKK attenuated LS-induced increases in the expression and channel activity of KCa2.3 and KCa3.1, and in phosphorylation of Akt (Ser473) and p300 (Ser1834). Inhibition of Akt abolished the upregulation of these channels by diminishing p300 phosphorylation. Consistently, disruption of the interaction of p300 with transcription factors eliminated the induction of these channels. Thus a CaMKK/Akt/p300 cascade plays an important role in LS-dependent induction of KCa2.3 and KCa3.1 expression, thereby regulating EC function and adaptation to hemodynamic changes.

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