Human CDT1 Associates with CDC7 and Recruits CDC45 to Chromatin during S Phase

Ballabeni, Andrea; Zamponi, Raffaella; Caprara, Greta; Melixetian, Marina; Bossi, Sergio; Masiero, Laura; Helin, Kristian

doi:10.1074/jbc.m803609200

Cited by 21 publications

(22 citation statements)

References 64 publications

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“…It is the high Cdk activity before cell division that allows for the accumulation of high levels of Cdt1 protein during mitosis, thus ensuring efficient formation of pre-RC during the next cell cycle. In addition to promoting chromatin licensing, Cdt1 and Cdc6 could also assist fast onset of DNA replication, as previously proposed (32,33). These results show that the basic APC/C Cdk oscillator operates in ES cells similar to somatic cells, and that the altered cell cycle of ES cells is because of altered regulation (Fig.…”

Section: Discussionsupporting

confidence: 57%

“…Briefly, mitotic synchronization of ES cells was obtained with 1.25 mM thymidine for 14 h followed by treatment with 50 ng/mL Nocodazole for 7 h. ES cells were differentiated by withdrawal of leukemia inhibitory factor (LIF) and treatment with 1 μM retinoic acid (RA) for 48 h. Chromatin fractionation was performed as previously reported (32). …”

Section: Methodsmentioning

confidence: 99%

“…4B). To evaluate if Cdt1 turnover is high in ES cells similar to somatic cells (13,32), we synchronized ES cells in mitosis and treated them either with protein synthesis inhibitor Cycloheximide or proteasome inhibitor MG132. Cdt1 levels rapidly decrease with Cycloheximide but increase when cells are exposed to MG132 (Fig.…”

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confidence: 99%

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Cell cycle adaptations of embryonic stem cells

Ballabeni¹,

Park

Zhao

et al. 2011

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

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ES cells proliferate with very short gap phases yet maintain their capacity to differentiate. It had been thought that the levels of cyclins and other substrates of ubiquitin ligase APC/C remain nearly constant and Cdk activity remains constitutively high in mouse ES cells. Here we demonstrate that APC/C (anaphase-promoting complex/cyclosome) enzyme is active in ES cells but attenuated by high levels of the Emi1 (early mitotic inhibitor-1) protein. Despite the presence of high Cdk activity during the G1 phase, chromatin can be effectively licensed for DNA replication and fast entry into the S phase can still occur. High Cdk activity during S-G2-M phases produces high levels of the DNA replication factor Cdt1, and this leads to efficient Mcm proteins loading on chromatin after mitotic exit. Although disturbing the usual balance between Cdk activity and APC/C activity found in somatic cells, a few key adaptations allow normal progression of a very rapid cell cycle.E mbryonic stem cells show unusual cell-cycle features: the duration of the S phase is comparable to somatic cells but they have remarkably short G1 and G2 phases (1-3). In somatic cells, the duration of G1 and G2 is determined by relative levels of Cdk kinase activity and other cell cycle-related proteins (4). Many of these proteins, including Cyclin A, Cyclin B, Cdt1, Cdc6, and Geminin fluctuate along the cell cycle because of degradation mediated by E3 ubiquitin ligase APC/C (anaphase-promoting complex/ cyclosome) together with E2 enzymes, such as UbcH10 and UBE2S (5-8). APC/C is activated at the end of mitosis by interaction with Cdc20 and Cdh1 proteins and inactivated just before the S phase by the pseudosubstrate inhibitor Emi1 (early mitotic inhibitor-1) and by the phosphorylation and degradation of Cdh1 (6, 9, 10). Cdk kinases are activated by Cyclins and phosphorylate a number of cell-cycle proteins important for mitotic and S phase progression. Cdk activity is inhibited during G1 in somatic cells because of degradation of Cyclins and presence of inhibitor proteins, like p21 (11). Inhibition of Cdk activity in the G1 phase allows the replication factors Cdt1 and Cdc6 to recruit Mcm proteins on chromatin, form prereplicative complexes (pre-RCs), and license DNA for replication (12)(13)(14). Geminin protein inhibits Cdt1 during the S phase and promotes its stabilization during mitosis (3,13,(15)(16)(17)(18)(19)(20). A puzzling feature of ES cells is that APC/C substrates were shown to be constant and Cdk activity to be high throughout the ES cell cycle (1,3,21), raising the question of whether the APC/C complex is functional and how ES cells regulate pre-RC assembly at G1. Remarkably, APC/C substrates and other positive cell-cycle regulators decrease after differentiation (1,3,22). We carefully reinvestigated cell-cycle dynamics in ES cells. Contrary to previous conclusions, APC/C substrate levels and Cdk activity both oscillate, although in a more muted manner compared with most studied somatic models. A few key adaptations promote an abbreviated...

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Section: Discussionsupporting

confidence: 57%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Cell cycle adaptations of embryonic stem cells

Ballabeni¹,

Park

Zhao

et al. 2011

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

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“…At S phase onset, Cdt1 is degraded more efficiently by the activation of the ubiquitin ligase activity mediated by Cul4-DDB1-Cdt2 (32), and its level decreases dramatically; only low levels of Cdt1 remain. This residual Cdt1, possibly associated with chromatin, could have some role in the efficient execution of DNA replication through interactions with DDK complex and cell division cycle 45 (Cdc45) (28,30,33,34), but it no longer can form pre-RCs. It also is possible that the low S-phase levels of Cdt1 may be completely inactive by binding to Geminin, CDK and DDK.…”

Section: Discussionmentioning

confidence: 99%

Geminin deploys multiple mechanisms to regulate Cdt1 before cell division thus ensuring the proper execution of DNA replication

Ballabeni

Zamponi²,

Moore

et al. 2013

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

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Cdc10-dependent transcript 1 (Cdt1) is an essential DNA replication protein whose accumulation at the end of the cell cycle promotes the formation of pre-replicative complexes and replication in the next cell cycle. Geminin is thought to be involved in licensing replication by promoting the accumulation of Cdt1 in mitosis, because decreasing the Geminin levels prevents Cdt1 accumulation and impairs DNA replication. Geminin is known to inhibit Cdt1 function; its depletion during G2 leads to DNA rereplication and checkpoint activation. Here we show that, despite rapid Cdt1 protein turnover in G2 phase, Geminin promotes Cdt1 accumulation by increasing its RNA and protein levels in the unperturbed cell cycle. Therefore, Geminin is a master regulator of cell-cycle progression that ensures the timely onset of DNA replication and prevents its rereplication.

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“…Work in mammalian cells indicates that Cdc7/Dbf4-dependent recruitment of Cdc45 to the pre-IC is mediated through Cdt1 (Ballabeni et al 2009). Following the recruitment of Cdc45 and the GINS complex, the replicative DNA polymerases responsible for copying the leading and lagging strands, the DNA processivity clamp PCNA, as well as the rest of the proteins required to form a stable and functional replisome are recruited to the origin.…”

mentioning

confidence: 99%

Break-induced replication requires all essential DNA replication factors except those specific for pre-RC assembly

Lydeard¹,

Lipkin-Moore²,

Sheu³

et al. 2010

Genes Dev.

151

169

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Break-induced replication (BIR) is an efficient homologous recombination (HR) pathway employed to repair a DNA double-strand break (DSB) when homology is restricted to one end. All three major replicative DNA polymerases are required for BIR, including the otherwise nonessential Pol32 subunit. Here we show that BIR requires the replicative DNA helicase (Cdc45, the GINS, and Mcm2-7 proteins) as well as Cdt1. In contrast, both subunits of origin recognition complex (ORC) and Cdc6, which are required to create a prereplication complex (pre-RC), are dispensable. The Cdc7 kinase, required for both initiation of DNA replication and post-replication repair (PRR), is also required for BIR. Ubiquitination and sumoylation of the DNA processivity clamp PCNA play modest roles; in contrast, PCNA alleles that suppress pol32D's cold sensitivity fail to suppress its role in BIR, and are by themselves dominant inhibitors of BIR. These results suggest that origin-independent BIR involves cross-talk between normal DNA replication factors and PRR.[Keywords: Break-induced replication (BIR); DNA replication; recombination; Saccharomyces cerevisiae; double-strand break (DSB); post-replication repair] Supplemental material is available at http://www.genesdev.org.

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Human CDT1 Associates with CDC7 and Recruits CDC45 to Chromatin during S Phase

Cited by 21 publications

References 64 publications

Cell cycle adaptations of embryonic stem cells

Cell cycle adaptations of embryonic stem cells

Geminin deploys multiple mechanisms to regulate Cdt1 before cell division thus ensuring the proper execution of DNA replication

Break-induced replication requires all essential DNA replication factors except those specific for pre-RC assembly

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