PCNA Is a Cofactor for Cdt1 Degradation by CUL4/DDB1-mediated N-terminal Ubiquitination

Senga, Takeshi; Sivaprasad, Umasundari; Zhu, Wenge; Park, Jong Hoon; Arias, Emily E.; Walter, Johannes C.; Dutta, Anindya

doi:10.1074/jbc.m512705200

Cited by 231 publications

(315 citation statements)

References 33 publications

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“…We tested this by overexpressing Cdt1-(6 -546). As this fragment lacks part of the PCNA-interacting protein box motif, PCNA-mediated degradation is disrupted (25). This fragment was still able to induce re-replication, suggesting that titration of the PCNAmediated degradation apparatus alone is not required to induce re-replication (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, as Cdt1 is stabilized in S-phase only when both PCNA/Cul4 and cyclin A/Cul1-mediated pathways are inhibited (25), it seems surprising that titrating one alone could activate endogenous Cdt1. In fact, no increase in endogenous Cdt1 level is observed following overexpression of Cdt1-(1-370) (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Geminin antibody was described previously (16). hsCdt1 antibody was described previously (25). siRNA was performed according to common methods using RNAi MAX (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

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Human Cdt1 Lacking the Evolutionarily Conserved Region That Interacts with MCM2–7 Is Capable of Inducing Re-replication

Teer

Dutta

2008

Journal of Biological Chemistry

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Replication initiation must be a carefully regulated process to avoid genomic instability caused by aberrant replication. In eukaryotic cells, distinct steps of protein loading (origin licensing) and replication activation are choreographed such that a cell can replicate only once per cell cycle. The first proteins recruited to the origins form the pre-replication complex. Of these proteins, Cdt1 is of interest, as it is the focus of several pathways to control replication initiation. It is degraded by two different pathways, mediated by the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) or with cyclin-Cdk2 and inhibited by geminin once cells are in S-phase, presumably to prevent reloading of pre-replication complexes once S-phase has begun. Although the requirement of Cdt1 in loading MCM2-7 is known, the mechanism by which overexpressed Cdt1 stimulates re-replication is unclear. In this study we have designed various mutations in Cdt1 to determine which portion of Cdt1 is important for re-replication, providing insight into possible mechanisms. Surprisingly, we found that mutants of Cdt1 that do not interact with MCM2-7 are able to induce rereplication when overexpressed. The re-replication is not due to titration of geminin from endogenous Cdt1 and is not accompanied by stabilization of endogenous Cdt1. Additionally, the N-terminal one-third of Cdt1 is sufficient to induce re-replication. The N terminus contains the PCNA-and cyclin-interacting motifs, and deletion of both motifs simultaneously in the overexpressed Cdt1 prevents re-replication. These findings suggest that exogenous Cdt1 induces re-replication by de-repressing endogenous Cdt1 through the titration of PCNA and cyclin.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Human Cdt1 Lacking the Evolutionarily Conserved Region That Interacts with MCM2–7 Is Capable of Inducing Re-replication

Teer

Dutta

2008

Journal of Biological Chemistry

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“…8 Although by a still elusive mechanism, the rapid and dose-dependent degradation of c-MYC in cells after UV-irradiation that we report here is reminiscent of the UV-induced degradation of the replication licencing factor Cdt1 that requires the presence of L2DTL, PCNA and the DDB1/CUL4 E3 ligase complexes. [60][61][62] It has been shown that c-MYC overexpression antagonizes the p53-dependent cell cycle arrest following DNA damage. 63,64 Consequently, an excess of c-MYC promotes chromosomal instability probably by combined effects since it increases DNA damage and it overcomes cell cycle arrest.…”

Section: Discussionmentioning

confidence: 99%

“…The wild-type, ΔMBI (aa [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63] and ΔMBII (aa 129-143) N-terminally FLAG tagged c-MYC2 coding pcDNA3 plasmids were kindly provided by Dr B. Lüscher (Institut für Biochemie, Aachen, Germany). ΔMBIII (aa 129-143), ΔA (aa 1-63), ΔB (aa 64-126), ΔC (aa 127-189), ΔD (aa 190-252), ΔE (aa 253-315), ΔF (aa 316-378), ΔG (aa 379-439), ΔCT (aa 354-439), T58A S62A and T358A S373A T400A deletions and point mutations were done by PCR.…”

Section: Methodsmentioning

confidence: 99%

c-Myc protein is degraded in response to UV irradiation

Britton¹,

Salles²,

Calsou³

2008

Cell Cycle

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The c-MYC proto-oncogene encodes a transcription factor that is critical for cell growth and proliferation. It is one of the genes frequently altered in cancer cells in which it exhibits constitutive activity. The half-life of c-MYC is very short in quiescent cells due to ubiquitin-mediated proteolysis. We report here the rapid and dose-dependent decline of c-MYC protein level after UV-irradiation in various human and rodent cells. This decline is due to a proteasomal degradation of c-MYC protein and does not require the binding sites for the FBW7 and SKP2 ubiquitin ligases. Together, our data exclude a prominent role for the stress-responsive kinase PAK2, for the major phosphoinositide 3-kinase related protein kinases ATR, ATM, DNA-PK and mTOR and for ERK, JNK and p38 mitogen activated protein kinases in this UV-induced degradation process. We propose that c-MYC degradation is part of the global cell response to UV-damage, complementary to the accumulation and activation of the p53 transcription factor. By contributing to the replication arrest after infliction of lesions to the genome, the induced degradation of c-MYC may be part of the safeguard mechanisms maintaining genome stability.

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