The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.01-08-0400 ◽

2002 ◽

Vol 13 (2) ◽

pp. 607-620 ◽

Cited By ~ 48

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.

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Regulation of Replication Licensing by Acetyltransferase Hbo1

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.1098-1108.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 1098-1108 ◽

Cited By ~ 132

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.

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PriC-mediated DNA Replication Restart Requires PriC Complex Formation with the Single-stranded DNA-binding Protein

Journal of Biological Chemistry ◽

10.1074/jbc.m113.478156 ◽

2013 ◽

Vol 288 (24) ◽

pp. 17569-17578 ◽

Cited By ~ 33

Author(s):

Sarah R. Wessel ◽

Aimee H. Marceau ◽

Shawn C. Massoni ◽

Ruobo Zhou ◽

Taekjip Ha ◽

...

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Complex Formation ◽

Binding Protein ◽

Dna Binding Protein ◽

Single Stranded Dna ◽

Replication Restart ◽

Dna Replication Restart

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

Molecular Biology of the Cell ◽

10.1091/mbc.02-01-0006 ◽

2002 ◽

Vol 13 (5) ◽

pp. 1462-1472 ◽

Cited By ~ 43

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.

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