Control of the Cdc6 replication licensing factor in metazoa: The role of nuclear export and the CUL4 ubiquitin ligase

Kim, Jihyun; Kipreos, Edward T.

doi:10.4161/cc.7.2.5282

Cited by 20 publications

(24 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This debate obviously stems from different experimental systems for the detection of endogenous versus 'ectopically' expressed, tagged Cdc6. 7,27,36 Here, we demonstrate that cytoplasmic YFP-tagged Cdc6 binds to chromatin preparations of S phase cells in the same way as endogenous Cdc6, and we provide evidence that this binding takes place during the preparation procedure when the nuclear membrane is lysed by detergents. The fact that the protein remains stably bound to chromatin during subsequent extraction procedures further implies that cytoplasmic Cdc6 retains a high affinity for chromatin.…”

Section: Discussionmentioning

confidence: 80%

“…In synopsis with the current knowledge about replication licensing and Cdc6 regulation 7,27,36 we suggest the following chronology of regulatory events affecting Cdc6 (Fig. 6): Upon breakdown of the nuclear envelope in prophase, Cdc6 gains access to condensing chromosomes.…”

Section: Discussionmentioning

confidence: 99%

“…42 As cells proceed through S phase, Cyclin A/Cdk2-dependent phosphorylation of newly synthesized Cdc6 then results in its protection from proteasomal degradation and export to the cytoplasm by Crm1. 9,10,[17][18][19]36 Finally, exported Cdc6 binds to centrosomes in late G2 and throughout mitosis, where it may carry out a functional role in regulating proper chromosomal alignment. 21 …”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

et al. 2015

View full text Add to dashboard Cite

To maintain genome stability, the thousands of replication origins of mammalian genomes must only initiate replication once per cell cycle. This is achieved by a strict temporal separation of ongoing replication in S phase, and the formation of pre-replicative complexes in the preceding G1 phase, which "licenses" each origin competent for replication. The contribution of the loading factor Cdc6 to the timing of the licensing process remained however elusive due to seemingly contradictory findings concerning stabilization, degradation and nuclear export of Cdc6. Using fluorescently tagged Cdc6 (Cdc6-YFP) expressed in living cycling cells, we demonstrate here that Cdc6-YFP is stable and chromatin-associated during mitosis and G1 phase. It undergoes rapid proteasomal degradation during S phase initiation followed by active export to the cytosol during S and G2 phases. Biochemical fractionation abolishes this nuclear exclusion, causing aberrant chromatin association of Cdc6-YFP and, likely, endogenous Cdc6, too. In addition, we demonstrate association of Cdc6 with centrosomes in late G2 and during mitosis. These results show that multiple Cdc6-regulatory mechanisms coexist but are tightly controlled in a cell cycle-specific manner.

show abstract

Section: Discussionmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

et al. 2015

View full text Add to dashboard Cite

show abstract

“…In Schizosaccharomyces pombe, both Cdc6 and Cdt1 are subject to proteolytic degradation in post-G 1 cells (17,19,21,22,43). In mammalian cells, multiple parallel mechanisms operate to prevent Cdc6 and Cdt1 activity in post-G 1 phase: nuclear export of Cdc6 (25), cleavage of Cdc6 (41), SCF Skp2 -and Cul4/Ddb1-mediated degradation of Cdt1 (39,65), and the presence of the Cdt1 inhibitor geminin (34). Geminin is predominantly present in post-G 1 cells and is degraded by APC Cdh1 during late mitosis to enable Cdt1 to assemble on chromatin (5,27,28,50,61).…”

mentioning

confidence: 99%

Dynamic Association of ORCA with Prereplicative Complex Components Regulates DNA Replication Initiation

Shen

Chakraborty

Jain

et al. 2012

Molecular and Cellular Biology

View full text Add to dashboard Cite

In eukaryotes, initiation of DNA replication requires the assembly of a multiprotein prereplicative complex (pre-RC) at the origins. We recently reported that a WD repeat-containing protein, origin recognition complex (ORC)-associated (ORCA/LRWD1), plays a crucial role in stabilizing ORC to chromatin. Here, we find that ORCA is required for the G 1 -to-S-phase transition in human cells. In addition to binding to ORC, ORCA associates with Cdt1 and its inhibitor, geminin. Single-molecule pulldown experiments demonstrate that each molecule of ORCA can bind to one molecule of ORC, one molecule of Cdt1, and two molecules of geminin. Further, ORCA directly interacts with the N terminus of Orc2, and the stability of ORCA is dependent on its association with Orc2. ORCA associates with Orc2 throughout the cell cycle, with Cdt1 during mitosis and G 1 , and with geminin in post-G 1 cells. Overexpression of geminin results in the loss of interaction between ORCA and Cdt1, suggesting that increased levels of geminin in post-G 1 cells titrate Cdt1 away from ORCA. We propose that the dynamic association of ORCA with pre-RC components modulates the assembly of its interacting partners on chromatin and facilitates DNA replication initiation.

show abstract

“…However, even though there is an abundance of MCM2-7 protein on the chromatin throughout the cell cycle, most of the MCM2-7 is kept inactive, and only a small population is recruited and associated with active replicons to participate in DNA synthesis (1)(2)(3). During early G 1 phase, the MCM2-7 protein is recruited to the active replication origins, where it forms part of the pre-replication complex, and its recruitment requires the CDC6 and CDT1 licensing factors (4,5). To ensure the accurate timing of replication initiation, the activity of the chromatin-bound MCM2-7 protein is tightly regulated both by post-translational modifications and protein-protein interactions.…”

mentioning

confidence: 99%

TIMELESS Suppresses the Accumulation of Aberrant CDC45·MCM2-7·GINS Replicative Helicase Complexes on Human Chromatin

Wang

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

The replication licensing factor CDC6 recruits the MCM2-7 replicative helicase to the replication origin, where MCM2-7 is activated to initiate DNA replication. MCM2-7 is activated by both the CDC7-Dbf4 kinase and cyclin-dependent kinase and via interactions with CDC45 and go-ichi-ni-san complex (GINS) to form the CDC45⅐MCM2-7⅐GINS (CMG) helicase complex. TIMELESS (TIM) is important for the subsequent coupling of CMG activity to DNA polymerases for efficient DNA synthesis. However, the mechanism by which TIM regulates CMG activity for proper replication fork progression remains unclear. Here we show that TIM interacts with MCM2-7 prior to the initiation of DNA replication. TIM depletion in various human cell lines results in the accumulation of aberrant CMG helicase complexes on chromatin. Importantly, the presence of these abnormal CMG helicase complexes is not restricted to cells undergoing DNA synthesis. Furthermore, even though these aberrant CMG complexes interact with the DNA polymerases on human chromatin, these complexes are not phosphorylated properly by cyclin-dependent kinase/CDC7-Dbf4 kinase and exhibit reduced DNA unwinding activity. This phenomenon coincides with a significant accumulation of the p27 and p21 replication inhibitors, reduced chromatin association of CDC6 and cyclin E, and a delay in S phase entry. Our results provide the first evidence that TIM is required for the correct chromatin association of the CMG complex to allow efficient DNA replication.During cell growth, faithful DNA duplication is essential for correctly transmitting genetic material to daughter cells during cell division, and defects in this process can result in genome instability, tumorigenesis, or cell death. During DNA replication, double-stranded DNA must be unwound by the MCM2-7 a hexameric helicase to generate ssDNA, 2 which is then used as the template for DNA synthesis by DNA polymerase. However, even though there is an abundance of MCM2-7 protein on the chromatin throughout the cell cycle, most of the MCM2-7 is kept inactive, and only a small population is recruited and associated with active replicons to participate in DNA synthesis (1-3). During early G 1 phase, the MCM2-7 protein is recruited to the active replication origins, where it forms part of the pre-replication complex, and its recruitment requires the CDC6 and CDT1 licensing factors (4, 5). To ensure the accurate timing of replication initiation, the activity of the chromatin-bound MCM2-7 protein is tightly regulated both by post-translational modifications and protein-protein interactions. Specifically, the catalytic activity of the MCM2-7 helicase is suppressed during G 1 phase and can only be activated both via the interaction with GINS and CDC45, which forms the CMG complex, and via phosphorylation by DDK and CDK during replication initiation (6, 7). Mammalian TIM is an essential protein for embryonic development (8). TIM and its interacting protein, TIPIN, are highly conserved replication factors that directly interact with the MCM2-7 helicase...

show abstract

Control of the Cdc6 replication licensing factor in metazoa: The role of nuclear export and the CUL4 ubiquitin ligase

Cited by 20 publications

References 56 publications

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

Dynamic Association of ORCA with Prereplicative Complex Components Regulates DNA Replication Initiation

TIMELESS Suppresses the Accumulation of Aberrant CDC45·MCM2-7·GINS Replicative Helicase Complexes on Human Chromatin

Contact Info

Product

Resources

About