Association of Human Origin Recognition Complex 1 with Chromatin DNA and Nuclease-resistant Nuclear Structures

Tatsumi, Yasutoshi; Tsurimoto, Toshiki; Shirahige, Katsuhiko; Yoshikawa, Hiroshi; Obuse, Chikashi

doi:10.1074/jbc.275.8.5904

Cited by 46 publications

(63 citation statements)

References 51 publications

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“…Studies on Mcm10 modification revealed that phosphatase converted the slower migrating form in the metaphase extract to the faster migrating form in S phase extract. It seems that Mcm10 is phosphorylated by protein kinase(s) in G 2 /M phase, similar to Orc1 (40,41), Orc2 (42), Mcm2, and Mcm4 (21,43). One possible kinase candidate is cdc2/cyclin B1, since the mobility shift of Mcm10 to a slower migrating form occurred in conjunction with an increase in cyclin B1.…”

Section: Fig 3 Proteasome Inhibitors Stabilize Mcm10mentioning

confidence: 99%

Cell Cycle-dependent Proteolysis and Phosphorylation of Human Mcm10

Izumi

Yatagai

Hanaoka

2001

Journal of Biological Chemistry

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In eukaryotes, initiation of DNA replication is a strictly controlled process, so that chromosomal DNA is precisely duplicated once per cell cycle. Recent studies using different systems show that a number of proteins are involved in the initiation of DNA replication, a process that is largely conserved from yeast to human (1, 2). Current models indicate that initiation consists of two steps. In the first step, the origin recognition complex (ORC), 1 Cdc6, Cdt1, and Mcm2-7 proteins sequentially assemble on the replication origins to form the prereplicative complex, from late mitosis to early G 1 phase. The ORC, a complex of six proteins (Orc1-6), binds replication origins (3, 4) and recruits Cdc6. Cdc6 in turn coordinates with Cdt1 to load the Mcm2-7 complex (5-10), a presumed replicative helicase (11, 12), on the chromatin template. In the second step, Cdc7/Dbf4 kinase and S phase cyclin-dependent kinases (S-Cdks) activate the prereplicative complex and trigger DNA replication by loading Cdc45 onto each origin with programmed timing (13-16). Cdc45 facilitates assembly of the replication machinery by recruiting replication protein A and DNA polymerases (17, 18).Eukaryotes contain multiple parallel pathways to ensure that the prereplicative complex is not re-assembled until the segregation of chromosomes in mitosis. Cdc6 is either degraded via the ubiquitin-dependent pathway (19) or exported out of the nucleus (20). Phosphorylation of Mcm2-7 complex by cdc2 kinase initiates dissociation from chromatin during S phase (21). Cdt1 is regulated by protein expression as well as interactions with geminin to ensure that it is active only in the G 1 phase (22,23).Mcm10 (Dna43) was originally discovered in Saccharomyces cerevisiae while screening to identify other mcm mutants (24). Previous studies performed in S. cerevisiae suggest that Mcm10 has multiple roles in DNA replication. The mcm10 mutant is defective in initiation of DNA replication at the non-permissive temperature (24) and causes stalling of replication forks when the replication machinery passes through origins that do not fire (24). In addition, Mcm10 mediates the loading of the Mcm2-7 complex onto replication origins (25), and interacts genetically with Cdc45, DNA polymerase ␦ and ⑀, which are required for the elongation steps of DNA replication (26). Therefore, it appears that Mcm10 is involved in both origin activation and elongation, although the mechanisms by which the protein interacts with multiple replication factors at each step remain to be elucidated.Mcm10 homologs have additionally been discovered in Schizosaccharomyces pombe and Caenorhabditis elegans (25,27). Recently, we identified Drosophila and human homologs of Mcm10 and demonstrated that human Mcm10 interacts with the mammalian Orc2 and Mcm2-7 complex (28). We also confirmed that human Mcm10 binds chromatin during S phase and dissociates in G 2 phase (28), whereas yeast Mcm10 remains bound to chromatin throughout the cell cycle (25,26). To clarify the mechanism of regulation of Mcm10 funct...

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Section: Fig 3 Proteasome Inhibitors Stabilize Mcm10mentioning

confidence: 99%

Cell Cycle-dependent Proteolysis and Phosphorylation of Human Mcm10

Izumi

Yatagai

Hanaoka

2001

Journal of Biological Chemistry

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“…Briefly, cells were labeled with BrdUrd for 10 min and detected using a monoclonal antibody against BrdUrd and a fluorescein-conjugated second antibody, as specified by the manufacturer. Cells were also stained with 4,6-diamidino-2-phenylindole (DAPI) as described previously (25). The fluorescent signals were visualized with a Leica DMRBE and captured with Photometrics PXL.…”

Section: Methodsmentioning

confidence: 99%

“…Hydroxyurea (HU) was included at 2.5 mM. Synchronized growth was monitored by flow cytometry (BD Biosciences) of propidium iodide-stained cells (25), by immunoblot analysis of cyclin E and cyclin A levels, and by 5-bromo-2Ј-deoxy-uridine (BrdUrd) incorporation. Incorporated BrdUrd was detected using the 5-Bromo-2Ј-deoxyuridine Labeling and Detection Kit I (Roche Applied Science, Germany).…”

Section: Methodsmentioning

confidence: 99%

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The ORC1 Cycle in Human Cells

Tatsumi

Ohta

Kimura

et al. 2003

Journal of Biological Chemistry

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101

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Components of ORC (the origin recognition complex) are highly conserved among eukaryotes and are thought to play an essential role in the initiation of DNA replication. The level of the largest subunit of human ORC (ORC1) during the cell cycle was studied in several human cell lines with a specific antibody. In all cell lines, ORC1 levels oscillate: ORC1 starts to accumulate in mid-G 1 phase, reaches a peak at the G 1 /S boundary, and decreases to a basal level in S phase. In contrast, the levels of other ORC subunits (ORCs 2-5) remain constant throughout the cell cycle. The oscillation of ORC1, or the ORC1 cycle, also occurs in cells expressing ORC1 ectopically from a constitutive promoter. Furthermore, the 26 S proteasome inhibitor MG132 blocks the decrease in ORC1, suggesting that the ORC1 cycle is mainly due to 26 S proteasome-dependent degradation. Arrest of the cell cycle in early S phase by hydroxyurea, aphidicolin, or thymidine treatment is associated with basal levels of ORC1, indicating that ORC1 proteolysis starts in early S phase and is independent of S phase progression. These observations indicate that the ORC1 cycle in human cells is highly linked with cell cycle progression, allowing the initiation of replication to be coordinated with the cell cycle and preventing origins from refiring.The replication of chromosomal DNA in eukaryotes is limited to once per cell division cycle. This control appears to be achieved mainly by the regulation of replication origins so that they fire only once per cell cycle. The origin recognition complex (ORC), 1 identified in budding yeast as a protein complex that binds origins, consists of six gene products (1-5). In addition to ORC, several factors highly conserved among eukaryotes are involved in initiation (6 -8). The sequential assembly of these factors on origin-ORC complexes precedes initiation, as has been shown in yeast and Xenopus systems. For example, minichromosome maintenance (MCM) proteins are loaded onto origins in the presence of ORC and CDC6, which establishes the pre-replicative complex (pre-RC) necessary for subsequent protein assembly (9 -13). After origin firing, the pre-RC changes to a post-replicative form by the dissociation of MCM from the complex (12, 14, 15). These associations provide an important mechanism that 1) ensures that replication origins fire at precise times and 2) prevents re-initiation. Budding yeast ORC is a static complex that is maintained at a constant level and remains bound to origins throughout the cell cycle (4, 5). Thus, MCM loading in yeast is mainly regulated by other factors such as cell cycle-regulated Cdc6 or CDK kinase activities (7,8,16). It is also known that the phosphorylation status of ORC subunits correlates with the timing of pre-RC formation, suggesting a role for ORC phosphorylation in MCM loading (16). Similar phosphorylation of ORC subunits was found in a Xenopus egg extract system, suggesting a conserved mechanism for the regulation of ORC functions (17, 18). Recent studies have elucidated possible...

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Comparative analysis of pre‐replication complex proteins in transformed and normal cells

Paola

Zannis‐Hadjopoulos

2012

J of Cellular Biochemistry

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This study examines the abundance of the major protein constituents of the pre-replication complex (pre-RC), both genome-wide and in association with specific replication origins, namely the lamin B2, c-myc, 20mer1, and 20mer2 origins. Several pre-RC protein components, namely ORC1-6, Cdc6, Cdt1, MCM4, MCM7, as well as additional replication proteins, such as Ku70/86, 14-3-3, Cdc45, and PCNA, were comparatively and quantitatively analyzed in both transformed and normal cells. The results show that these proteins are overexpressed and more abundantly bound to chromatin in the transformed compared to normal cells. Interestingly, the 20mer1, 20mer2, and c-myc origins exhibited a two- to threefold greater origin activity and a two- to threefold greater in vivo association of the pre-RC proteins with these origins in the transformed cells, whereas the origin associated with the housekeeping lamin B2 gene exhibited both similar levels of activity and in vivo association of these pre-RC proteins in both cell types. Overall, the results indicate that cellular transformation is associated with an overexpression and increased chromatin association of the pre-RC proteins. This study is significant, because it represents the most systematic comprehensive analysis done to date, using multiple replication proteins and different replication origins in both normal and transformed cell lines.

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Association of Human Origin Recognition Complex 1 with Chromatin DNA and Nuclease-resistant Nuclear Structures

Cited by 46 publications

References 51 publications

Cell Cycle-dependent Proteolysis and Phosphorylation of Human Mcm10

Cell Cycle-dependent Proteolysis and Phosphorylation of Human Mcm10

The ORC1 Cycle in Human Cells

Comparative analysis of pre‐replication complex proteins in transformed and normal cells

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