In Vivo Interaction of Human MCM Heterohexameric Complexes with Chromatin

Fujita, Masatoshi; Kiyono, Tohru; Hayashi, Yasuyuki; Ishibashi, Masayoshi

doi:10.1074/jbc.272.16.10928

Cited by 79 publications

(105 citation statements)

References 49 publications

(71 reference statements)

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“…3A). The remaining 90% of the cells were used for subcellular fractionation (31). Proteins in the soluble and nuclear͞chromatin fractions were resolved by SDS͞PAGE and then immunoblotted with 9E10 mAb or the antibody against chromatin-associated protein histone H1.…”

Section: P-labeledmentioning

confidence: 99%

“…Standard protocols for protein extraction, immunoblot analysis, and protein kinase assay were used as described (27,29). The chromatin͞nuclear matrix fractionation assay was performed as described (31). Briefly, U2OS cells were washed three times with ice-cold PBS and then lysed with 1 ml modified CSK buffer (10 mM Pipes, pH 6.8͞100 mM NaCl͞300 mM sucrose͞1 mM MgCl 2 ͞1 mM EGTA͞1 mM DTT͞1 mM PMSF͞10 units/ml aprotinin͞20 g/ml leupeptin͞50 M Na 3 VO 4 ͞5 mM ␤-glycerophosphate͞5 mM NaF͞2 mM ATP͞0.5% Triton X-100) for 10 min.…”

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confidence: 99%

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Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6

Jiang¹,

Wells²,

Hunter³

1999

Proc. Natl. Acad. Sci. U.S.A.

209

232

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We have characterized HsCdc6, a human protein homologous to the budding yeast Cdc6p that is essential for DNA replication. We show that, unlike Cdc6p, the levels of HsCdc6 protein remain constant throughout the cell cycle in human cells. However, phosphorylation of HsCdc6 is regulated during the cell cycle. HsCdc6 is an excellent substrate for Cdk2 in vitro and is phosphorylated in vivo at three sites (Ser-54, Ser-74, and Ser-106) that are phosphorylated by Cdk2 in vitro, strongly suggesting that HsCdc6 is an in vivo Cdk substrate. HsCdc6 is nuclear in G 1 , but translocates to the cytoplasm at the start of S phase via Crm1-dependent export. An HsCdc6A1A2A3 mutant, which mimics unphosphorylated HsCdc6, is exclusively nuclear, and its expression inhibits initiation of DNA replication. An HsCdc6E1E2E3 mutant, which mimics phosphorylated HsCdc6, is exclusively cytoplasmic and is not associated with the chromatin͞nuclear matrix fraction. Based on these results, we propose that phosphorylation of HsCdc6 by Cdks regulates DNA replication of at least two steps: first, by promoting initiation of DNA replication and, second, through nuclear exclusion preventing DNA rereplication.In all eukaryotic cells, DNA replication is a tightly regulated process that is strictly coupled to the progression of the cell cycle. It occurs only during S phase, and initiation of DNA replication occurs at discrete chromosomal locations (replication origins). When DNA replication is initiated, the cell must ensure that all its genome is replicated and that the replication of every DNA section occurs once and only once during the cell cycle. In the budding yeast, Cdc6p plays a unique role in regulating DNA replication. It is essential for initiation of DNA replication and required for assembly and maintenance of the prereplication complexes (pre-RCs) at replication origins (1, 2). In contrast to the origin recognition complex (ORC) and minichromosome maintenance (Mcm) proteins, Cdc6p is expressed only in G 1 phase of the Saccharomyces cerevisiae cell cycle (3, 4). Cdc6p and its Schizosaccharomyces pombe homologue Cdc18 physically interact with ORC, and Cdc6p is required for the loading of Mcm proteins at the replication origins in G 1 (5, 6). Cdc6p is a nucleotidedependent loading factor related to the eukaryotic and prokaryotic clamp loaders, such as PCNA (7,8). Both Cdc6p and Cdc18 also are associated with Cdks in vivo and can be phosphorylated by Cdks in vitro (9, 10). It is thought that Cdk phosphorylation of Cdc6p and Cdc18 triggers their degradation through a ubiquitination-mediated protein-degradation pathway (11-13). A gain-of-function Cdc6p mutant displays promiscuous initiation of DNA replication and promotes constant Mcm proteins association with chromatin throughout the cell cycle (14). Overexpression of a Cdc18 mutant that cannot be phosphorylated by Cdks in vitro causes greater DNA overreplication in Schizosaccharomyces pombe than expression of wild-type Cdc18 (13, 15). Taken together, these findings indicate that Cdc6p͞C...

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Section: P-labeledmentioning

confidence: 99%

mentioning

confidence: 99%

Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6

Jiang¹,

Wells²,

Hunter³

1999

Proc. Natl. Acad. Sci. U.S.A.

209

232

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“…Minichromosome maintenance (MCM) proteins play an important role in the replication of eukaryotic DNA by binding to chromatin before the initiation of DNA replication (Fujita et al, 1997;Ogawa et al, 1999). Minichromosome maintenance protein 6 (MCM6) is one of six members (MCM2 -7) of the MCM family (Lindner et al, 2002) and consists of 821 amino acids with a molecular mass of 105 kDa (Heidebrecht et al, 2001).…”

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confidence: 99%

Minichromosome maintenance protein 6, a proliferation marker superior to Ki-67 and independent predictor of survival in patients with mantle cell lymphoma

et al. 2005

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Minichromosome maintenance protein 6 (MCM6) is one of six proteins of the MCM family which are involved in the initiation of DNA replication and thus represent a marker of proliferating cells. Since the level of cell proliferation is the most valuable predictor of survival in mantle cell lymphoma (MCL), we investigated lymph node biopsy specimens from 70 patients immunohistochemically with a monoclonal antibody against MCM6. The percentage of MCM6 expressing lymphoma cells ranged from 12.0 to 95.6%, with a mean of 61.0%, and was significantly higher than the percentage of Ki-67-positive cells (Po0.0001). Surprisingly, the ratio of MCM6-positive cells to Ki-67-positive cells was higher than in normal stimulated peripheral blood mononuclear cells, indicating a cell early G1-phase arrest in MCL. A high MCM6 expression level of more than 75% positive cells was associated with a significantly shorter overall survival time (16 months) compared to MCL with a low MCM6 expression level of less than 25% (no median reached, Po0.0001). Multivariate analysis revealed MCM6 to be an independent predictor of survival that is superior to the international prognostic factor and the Ki-67 index. Therefore, aside from gene expression profiling, immunohistochemical detection of MCM6 seems to be the most promising marker for predicting the outcome in MCL.

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“…MCM4, -6, and -7 have been shown to unwind DNA helices in a ATP-dependent manner, whereas the heterohexameric MCM2-7 does not, suggesting that MCM4, -6, and -7 may function as the catalytic core and the replicative helicase during DNA replication (Fujita et al, 1997;Kubota et al, 1997;Labib et al, 2000). Association of the MCM complex with the chromatin is cell cycle regulated: MCM2-7 binds to chromatin only during the G1/S phase and dissociates from as replication proceeds (Kearsey and Labib, 1998).…”

Section: Introductionmentioning

confidence: 99%

Udu Deficiency Activates DNA Damage Checkpoint

Lim

Chong

Jiang

2009

MBoC

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Udu has been shown to play an essential role during blood cell development; however, its roles in other cellular processes remain largely unexplored. In addition, ugly duckling (udu) mutants exhibited somite and myotome boundary defects. Our fluorescence-activated cell sorting analysis also showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased DNA damage in udu tu24 mutants. We further showed that the extensive p53-dependent apoptosis in udu tu24 mutants is a consequence of activation in the Atm-Chk2 pathway. Udu seems not to be required for DNA repair, because both wild-type and udu embryos similarly respond to and recover from UV treatment. Yeast two-hybrid and coimmunoprecipitation data demonstrated that PAH-L repeats and SANT-L domain of Udu interacts with MCM3 and MCM4. Furthermore, Udu is colocalized with 5-bromo-2 -deoxyuridine and heterochromatin during DNA replication, suggesting a role in maintaining genome integrity. INTRODUCTIONugly duckling (udu tu24 ) mutant was first isolated from the 1996 Tü bingen screen and has been shown to exhibit a short body-axis with massive cell death (Hammerschmidt et al., 1996). Another udu allele, udu sq1 , was isolated in a genetic screen aiming at mutants with defects in hematopoiesis (Liu et al., 2007). Positional cloning revealed that Udu protein encodes a novel nuclear factor consisting of three conserved regions (CR-1, CR-2, and CR-3) that do not share similarity with any known domains, two paired amphipathic ␣-helix like (PAH-L) repeats, and one putative SW13, ADA2, N-Cor and TFIIIB like (SANT-L) domain. The C-terminal PAH-L and SANT-L domains have been shown to be essential in primitive erythroid cell development (Liu et al., 2007). The PAH domain, first identified in yeast SIN3 (Wang et al., 1990), has been demonstrated to mediate protein-protein interactions (Spronk et al., 2000). SIN3 has no intrinsic DNA binding ability and has to be targeted to gene promoters by interacting with DNA binding proteins, thereafter positively and negatively regulating genes involved in diverse cellular functions (Silverstein and Ekwall, 2005). The SANT domain is a highly conserved motif with similarity to Myb DNA binding domain (Aasland et al., 1996), which has been shown to be critical in regulating chromatin accessibility (Boyer et al., 2002(Boyer et al., , 2004.The DNA damage response pathway is a cellular surveillance system that senses the presence of damaged DNA and elicits checkpoint activation and subsequent lesion repair in preventing amplification or loss of genes or chromosomes (Zhou and Elledge, 2000). The critical components of DNA damage checkpoint are two phosphatidyl inositol 3-kinaselike kinase family proteins: Ataxia telangiectasia mutated (ATM) and ATM-and Rad3-related (ATR) (Abraham, 2003;Shiloh, 2003). ATR functions as a sensor and transducer in response to UV light and presumably to genotoxic agents that give rise to stalled replication forks, and subsequently activates Checkp...

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In Vivo Interaction of Human MCM Heterohexameric Complexes with Chromatin

Cited by 79 publications

References 49 publications

Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6

Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6

Minichromosome maintenance protein 6, a proliferation marker superior to Ki-67 and independent predictor of survival in patients with mantle cell lymphoma

Udu Deficiency Activates DNA Damage Checkpoint

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