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Yukio Ishimi

Publications and source records attributed to Yukio Ishimi.

13 recordsLinked to original sources
PubMed2006

Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells.

MCM4, a subunit of a putative replicative helicase, is phosphorylated during the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication. However, detailed characterization of the phosphorylation of MCM4 remains to be performed. We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera. Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phosphorylation that is greatly enhanced in the G2 and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3). We present data indicating that phosphorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2. Based on these results and those from in vitro phosphorylation of MCM4 with CDK2/cyclin A, we discuss the kinases responsible for MCM4 phosphorylation. Phosphorylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin. Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not generally colocalized with replicating DNA. Unexpectedly, MCM4 phosphorylated at site 32 was enriched in the nucleolus through the cell cycle. These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell cycle.

Amino Acid Sequence↗
PubMed2006

Phosphorylation of MCM4 at sites inactivating DNA helicase activity of the MCM4-MCM6-MCM7 complex during Epstein-Barr virus productive replication.

Induction of Epstein-Barr virus (EBV) lytic replication blocks chromosomal DNA replication notwithstanding an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. We report here that the phosphorylated form of MCM4, a subunit of the MCM complex essential for chromosomal DNA replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2/CDK1 targets whose phosphorylation inactivates MCM4-MCM6-MCM7 (MCM4-6-7) complex-associated DNA helicase. Expression of EBV-encoded protein kinase (EBV-PK) in HeLa cells caused phosphorylation of these sites on MCM4, leading to cell growth arrest. In vitro, the sites of MCM4 of the MCM4-6-7 hexamer were confirmed to be phosphorylated with EBV-PK, with the same loss of helicase activity as with CDK2/cyclin A. Introducing mutations in the N-terminal six Ser and Thr residues of MCM4 reduced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activities of both wild-type and mutant MCM4-6-7 hexamers, probably since EBV-PK can phosphorylate MCM6 and another site(s) of MCM4 in addition to the N-terminal residues. Therefore, phosphorylation of the MCM complex by redundant actions of CDK and EBV-PK during lytic replication might provide one mechanism to block chromosomal DNA replication in the infected cells through inactivation of DNA unwinding by the MCM4-6-7 complex.

Amino Acid Substitution↗
PubMed2005

Human CDK2 inhibition modifies the dynamics of chromatin-bound minichromosome maintenance complex and replication protein A.

Minichromosome maintenance (MCM) proteins form a complex and possess helicase activity to unwind the DNA duplex and establish a replication fork. To assure that origins only fire once per cell cycle, the MCM complex is removed from chromatin and inactivated as cells exit S phase. In this report, we demonstrate that CDK2 depletion in human cells leads to an overall phosphorylation defect at mitosis with increased rereplication, correlated with the accumulation of chromatin-bound MCM proteins. We show that CDK2 suppression results in decreased MCM4 phosphorylation at multiple serine and threonine sites. In addition, CDK2 inhibition induces an increase in chromatin-bound replication protein A (RPA) which should bind to single-stranded DNA regions, possibly establishing a replication intermediate that activates the ATR cascade. Finally, we observe that loss of CDK2 function in G1 delays replication initiation while it promotes rereplication in G2/M. Thus, by modulating the phospho-status of MCM4 and regulating origin firing, S phase CDK2 appears to be an integrated component of cellular machinery required for temporally controlling replication activity and maintaining genomic stability.

Blotting, Western↗
PubMed2004

Structural basis for inhibition of the replication licensing factor Cdt1 by geminin.

To maintain chromosome stability in eukaryotic cells, replication origins must be licensed by loading mini-chromosome maintenance (MCM2-7) complexes once and only once per cell cycle. This licensing control is achieved through the activities of geminin and cyclin-dependent kinases. Geminin binds tightly to Cdt1, an essential component of the replication licensing system, and prevents the inappropriate reinitiation of replication on an already fired origin. The inhibitory effect of geminin is thought to prevent the interaction between Cdt1 and the MCM helicase. Here we describe the crystal structure of the mouse geminin-Cdt1 complex using tGeminin (residues 79-157, truncated geminin) and tCdt1 (residues 172-368, truncated Cdt1). The amino-terminal region of a coiled-coil dimer of tGeminin interacts with both N-terminal and carboxy-terminal parts of tCdt1. The primary interface relies on the steric complementarity between the tGeminin dimer and the hydrophobic face of the two short N-terminal helices of tCdt1 and, in particular, Pro 181, Ala 182, Tyr 183, Phe 186 and Leu 189. The crystal structure, in conjunction with our biochemical data, indicates that the N-terminal region of tGeminin might be required to anchor tCdt1, and the C-terminal region of tGeminin prevents access of the MCM complex to tCdt1 through steric hindrance.

Animals↗
PubMed2003

Thymine-rich single-stranded DNA activates Mcm4/6/7 helicase on Y-fork and bubble-like substrates.

The presence of multiple clusters of runs of asymmetric adenine or thymine is a feature commonly found in eukaryotic replication origins. Here we report that the helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine stretches. The Mcm helicase is specifically activated by a synthetic bubble structure which mimics an activated replication origin, as well as by a Y-fork structure, provided that a single-stranded DNA region of sufficient length is present in the unwound segment or 3' tail, respectively, and that it carries clusters of thymines. Sequences derived from the human lamin B2 origin can serve as a potent activator for the Mcm helicase, and substitution of its thymine clusters with guanine leads to loss of this activation. At the fork, Mcm displays marked processivity, expected for a replicative helicase. These findings lead us to propose that selective activation by stretches of thymine sequences of a fraction of Mcm helicases loaded onto chromatin may be the determinant for selection of initiation sites on mammalian genomes.

Adenosine Triphosphatases↗
PubMed2003

The chromatin-remodeling complex WINAC targets a nuclear receptor to promoters and is impaired in Williams syndrome.

We identified a human multiprotein complex (WINAC) that directly interacts with the vitamin D receptor (VDR) through the Williams syndrome transcription factor (WSTF). WINAC has ATP-dependent chromatin-remodeling activity and contains both SWI/SNF components and DNA replication-related factors. The latter might explain a WINAC requirement for normal S phase progression. WINAC mediates the recruitment of unliganded VDR to VDR target sites in promoters, while subsequent binding of coregulators requires ligand binding. This recruitment order exemplifies that an interaction of a sequence-specific regulator with a chromatin-remodeling complex can organize nucleosomal arrays at specific local sites in order to make promoters accessible for coregulators. Furthermore, overexpression of WSTF could restore the impaired recruitment of VDR to vitamin D regulated promoters in fibroblasts from Williams syndrome patients. This suggests that WINAC dysfunction contributes to Williams syndrome, which could therefore be considered, at least in part, a chromatin-remodeling factor disease.

Active Transport, Cell Nucleus↗
PubMed2003

Identification of MCM4 as a target of the DNA replication block checkpoint system.

Inhibition of the progression of DNA replication prevents further initiation of DNA replication and allows cells to maintain arrested replication forks, but the proteins that are targets of the replication checkpoint system remain to be identified. We report here that human MCM4, a subunit of the putative DNA replicative helicase, is extensively phosphorylated in HeLa cells when they are incubated in the presence of inhibitors of DNA synthesis or are exposed to UV irradiation. The data presented here indicate that the consecutive actions of ATR-CHK1 and CDK2 kinases are involved in this phosphorylation in the presence of hydroxyurea. The phosphorylation sites in MCM4 were identified using specific anti-phosphoantibodies. Based on results that showed that the DNA helicase activity of the MCM4-6-7 complex is negatively regulated by CDK2 phosphorylation, we suggest that the phosphorylation of MCM4 in the checkpoint control inhibits DNA replication, which includes blockage of DNA fork progression, through inactivation of the MCM complex.

Cell Cycle Proteins↗
PubMed2003

Mammalian Mcm2/4/6/7 complex forms a toroidal structure.

BACKGROUND: The Mcm proteins are a family of six homologous proteins (Mcm2-7) that play an important role in DNA replication. They form Mcm4/6/7 and Mcm2/4/6/7 complexes, but their structures are not known. RESULTS: We found that the human Mcm2/4/6/7 tetramer forms a toroidal structure, with a central cavity about 3-4 nm in diameter. Observations were made using electron microscopy, employing the image analysis of single particles. The most predominant averaged image displayed a toroid harbouring four bulges forming corners, one of which was larger than the others. This structure was very similar to the mouse Mcm2/4/6/7 tetramer that was independently prepared and analysed by electron microscopy. These toroidal structures are distinct from that of the Mcm4/6/7 hexamer, which was also examined by electron microscopy. GST(glutathione S-transferase)-pull down and two hybrid experiments suggest that a putative Mcm6-Mcm6 hinge contributes to the formation of the Mcm7/4/6/6/4/7 heterohexamer. CONCLUSIONS: The Mcm2/4/6/7 tetramer forms a toroidal structure that is distinct from that of the Mcm4/6/7 hexamer in size and shape.

Animals↗
PubMed2003

Enhanced expression of Mcm proteins in cancer cells derived from uterine cervix.

Minichromosome maintenance proteins (Mcm) 2-7 play essential roles in eukaryotic DNA replication. Several reports have indicated the usefulness of Mcm proteins as markers of cancer cells in histopathological diagnosis. However, their mode of expression and pathophysiological significance in cancer cells remain to be clarified. We compared the level of expression of Mcm proteins among human HeLa uterine cervical carcinoma cells, SV40-transformed human fibroblast GM00637 cells and normal human fibroblast WI-38 cells. All the proteins examined were detected in HeLa and GM cells at 6-10 times the level found in WI-38 cells on average. This increase was observed both in total cellular proteins and in the chromatin-bound fraction. Consistently, Mcm2 mRNA was enriched in HeLa cells to approximately four times the level in WI-38 cells, and the synthesis of Mcm4, 6 and 7 proteins was accelerated in HeLa cells. Immunohistochemical studies of surgical materials from human uterine cervix showed that Mcm3 and 4 are ubiquitously expressed in cancer cells. Further, the positive rate and level of Mcm3 and 4 expression appeared to be higher in cancer cells than in normal proliferating cells of the uterine cervix and dysplastic cells, suggesting that they can be useful markers to distinguish these cells.

Animals↗
PubMed2002

Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex.

Mcm, which is composed of six structurally related subunits (Mcm2-7), is essential for eukaryotic DNA replication. A subassembly of Mcm, the Mcm4/6/7 double-trimeric complex, possesses DNA helicase activity, and it has been proposed that Mcm may function as a replicative helicase at replication forks. We show here that conserved ATPase motifs of Mcm7 are essential for ATPase and DNA helicase activities of the Mcm4/6/7 complex. Because uncomplexed Mcm7 displayed neither ATPase nor DNA helicase activity, Mcm7 contributes to the DNA helicase activity of the Mcm complex through interaction with other subunits. In contrast, the Mcm4/6/7 complex containing a zinc finger mutant of Mcm4 with partially impaired DNA binding activity exhibited elevated DNA helicase activity. The Mcm4/6/7 complex containing this Mcm4 mutant tended to dissociate into trimeric complexes, suggesting that the zinc finger of Mcm4 is involved in subunit interactions of trimers. The Mcm4 mutants lacking the N-terminal 35 or 112 amino acids could form hexameric Mcm4/6/7 complexes, but displayed very little DNA helicase activity. In conjunction with the previously reported essential role of Mcm6 in ATP binding (You, Z., Komamura, Y., and Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003-8015), our data indicate distinct roles of Mcm4, Mcm6, and Mcm7 subunits in activation of the DNA helicase activity of the Mcm4/6/7 complex.

Adenosine Triphosphatases↗
PubMed2002

Nuclear organization of DNA replication initiation proteins in mammalian cells.

Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Here we show that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells using an in vivo chemical cross-linking method. CDC6 proteins were also found to assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC1 has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. We also found that some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder binds to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. On the other hand, our results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed.

Cell Cycle Proteins↗
PubMedDate not supplied

Levels of MCM4 phosphorylation and DNA synthesis in DNA replication block checkpoint control.

Blockage of a DNA replication fork movement not only stabilizes the fork structure but also prevents initiation of DNA replication. We reported that MCM4, a subunit of a putative replicative DNA helicase, is extensively phosphorylated in the presence of hydroxyurea (HU) or after exposure to UV irradiation. Here we examined the relationship between levels of MCM4 phosphorylation and DNA synthesis during DNA replication checkpoint control and after release of the control. The results suggest that there is roughly inverse correlation between these two levels; namely the higher the level of MCM4 phosphorylation, the lower the level of DNA synthesis. The presence of HU or UV irradiation can stimulate phosphorylation at several cyclin-dependent kinase (CDK) sites in MCM4, which can lead to inhibition of MCM4/6/7 helicase activity. These results are consistent with the notion that the phosphorylation of MCM4 is involved in regulation of DNA synthesis in the checkpoint control.

Binding Sites↗