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Published in final edited form as: Mol Cell. 2011 Oct 7;44(1):85–96. doi: 10.1016/j.molcel.2011.08.028

CDC-48/p97 Coordinates CDT-1 Degradation with GINS Chromatin Dissociation to Ensure Faithful DNA Replication

André Franz 1, Michael Orth 2, Paul A Pirson 1, Remi Sonneville 3, J Julian Blow 3, Anton Gartner 3, Olaf Stemmann 2, Thorsten Hoppe 1,*
PMCID: PMC3428722  EMSID: UKMS49022  PMID: 21981920

SUMMARY

Faithful transmission of genomic information requires tight spatiotemporal regulation of DNA replication factors. In the licensing step of DNA replication CDT-1 is loaded onto chromatin to subsequently promote the recruitment of additional replication factors including CDC-45 and GINS. During the elongation step the CDC-45/GINS complex moves with the replication fork, however it is largely unknown how its chromatin association is regulated. Here, we show that the chaperone-like ATPase CDC-48/p97 coordinates degradation of CDT-1 with release of the CDC-45/GINS complex. C. elegans embryos lacking CDC-48 or its cofactors UFD-1/NPL-4 accumulate CDT-1 on mitotic chromatin, indicating a critical role of CDC-48 in CDT-1 turnover. Strikingly, CDC-48UFD-1/NPL-4 deficient embryos show persistent chromatin association of CDC-45/GINS, which is a consequence of CDT-1 stabilization. Moreover, our data confirmed a similar regulation in Xenopus egg extracts, emphasizing a conserved coordination of licensing and elongation events during eukaryotic DNA replication by CDC-48/p97.

Keywords: CDC-48/p97, GINS, CDT-1, C. elegans, DNA replication

INTRODUCTION

Accurate duplication of DNA is a central and challenging task in dividing cells that requires the hierarchical order of timely separated events (Bell and Dutta, 2002; Masai et al., 2010). In embryonic cell cycles DNA replication starts during early mitosis with the loading of the pre-replication complex (pre-RC) onto origins of replication (Budirahardja and Gonczy, 2009). The licensing factors CDT-1 and CDC-6 are central to this process because they bind to origins and subsequently recruit the replication helicase, the MCM2-7 complex, to complete pre-RC formation. For initiation of replication in late mitosis, pre-RCs are activated by phosphorylation for recruitment of essential replication factors, including CDC-45 and the Go-Ichi-Ni-San complex (GINS, representative for the subunits SLD-5, PSF-1, PSF-2, and PSF-3) (Ilves et al., 2010; Masuda et al., 2003; Mimura et al., 2000; Sheu and Stillman, 2006; Yabuuchi et al., 2006). As part of the active replisome, CDC-45 and GINS move with the replication fork during the elongation step of DNA synthesis (Aparicio et al., 2009; Gambus et al., 2006; Moyer et al., 2006; Pacek et al., 2006). To prevent reassembly of pre-RCs and thus re-initiation of DNA replication within the same cell cycle, CDT-1 is targeted for degradation at the start of the elongation phase by the CUL-4CDT-2 ubiquitin ligase in C. elegans (Havens and Walter, 2009; Zhong et al., 2003). This timely separation between DNA licensing and the elongation step of replication has been shown to be crucial for the maintenance of genome integrity (Takeda and Dutta, 2005). Remarkably, although the assembly steps have been studied in great detail, mechanisms that determine spatiotemporal dynamics of the replisome are largely unknown, especially once DNA replication has been completed.

Interestingly, our recent findings identified an essential role for the chaperone-like ATPase CDC-48 in DNA replication that is linked to cell cycle progression (Deichsel et al., 2009; Mouysset et al., 2008). C. elegans CDC-48 and its orthologs in other species (termed Cdc48p in yeast and p97 in vertebrates) mediate mobilization and targeting of ubiquitylated proteins to the 26S proteasome (Rape et al., 2001). The substrate specificity is determined by different substrate-recruiting cofactors (Schuberth and Buchberger, 2008). For example, CDC-48/p97 cooperates with the UFD-1/NPL-4 heterodimer in endoplasmic reticulum (ER)-associated protein degradation (ERAD) and cell cycle progression (Mouysset et al., 2008; Mouysset et al., 2006; Rabinovich et al., 2002). Depletion of the CDC-48UFD-1/NPL-4 complex in worms causes severe defects in S phase progression of dividing cells, which depends on the DNA damage checkpoint. Moreover, reminiscent of the loss of the licensing factors CDT-1 and CDC-6, embryos lacking CDC-48, UFD-1, or NPL-4 are strongly reduced in DNA content. So far, interaction of CDC-48/p97 with DNA replication or repair proteins has been identified, however, the underlying mechanistic details remained unclear.

Here, we show a central regulatory role of CDC-48/p97 in the coordination of licensing and elongation events during eukaryotic DNA replication. C. elegans embryos lacking CDC-48 or its cofactors UFD-1/NPL-4 accumulate CDT-1 on mitotic chromatin, implicating a yet unknown function of CDC-48 in CDT-1 degradation. Moreover, CDC-48UFD-1/NPL-4 deficient embryos show persistent chromatin association of CDC-45/GINS after S phase is completed. Mechanistically, dissociation of CDC-45/GINS is a consequence of ubiquitin dependent CDT-1 turnover regulated by CDC-48. Since we show similar results in Xenopus egg extracts, our findings demonstrate that the spatiotemporal coordination of CDT-1 degradation and GINS release is evolutionarily conserved, which is important for eukaryotic DNA replication and cell cycle progression.

RESULTS

Chromatin Dissociation of CDC-45 and GINS Subunits Requires CDC-48UFD-1/NPL-4

In early C. elegans embryonic cell cycles S and M phases rapidly alternate without apparent gap phases, which can be monitored by time-lapse microscopy (Budirahardja and Gonczy, 2009). To characterize the mechanistic role of CDC-48 in DNA replication, we systematically analyzed the subcellular localization and dynamics of several conserved replication factors fused to green fluorescent protein (GFP) in C. elegans embryos that were depleted for the functional CDC-48UFD-1/NPL-4 complex. During these experiments chromatin was visualized by co-expression of mCherry-tagged histone H2B (mCherry::H2B). In wild-type embryos, CDC-45 and the GINS subunit SLD-5 similarly accumulate in the nucleus in S phase but never colocalize with H2B on mitotic chromosomes. In contrast, codepletion of CDC-48.1 and CDC-48.2 by RNA interference (RNAi) (hereafter referred to as CDC-48 depletion or cdc-48(RNAi)) or depletion of its cofactors UFD-1/NPL-4 specifically affects the localization of GFP::CDC-45 and GFP::SLD-5. Time-lapse microscopy identified that CDC-45 and SLD-5 remain associated with chromatin throughout cell cycle progression in cdc-48, ufd-1, and npl-4 RNAi embryos (Figure 1A, 1B and Movies S1, S2). In contrast, downregulation of ufd-1 does not change the cellular distribution of the pre-RC proteins ORC-2 and CDC-6 or the DNA helicase subunit MCM-2 (Figure S1A). As CDC-48 is involved in additional biological processes cooperating with other cofactors than UFD-1/NPL-4, complete downregulation of CDC-48 blocks embryonic cell division (Mouysset et al., 2008; Sasagawa et al., 2007; Yamanaka et al., 2004). To specifically address the replication related phenotype, we depleted ufd-1 and/ or npl-4 in further experiments.

Figure 1. CDC-45 and subunits of the GINS complex persist on mitotic chromatin in embryos depleted for the CDC-48UFD-1/NPL-4 complex.

Figure 1

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(A, B) Selected pictures of time-lapse recordings of embryos expressing GFP::CDC-45 or GFP::SLD-5 (green) and mCherry::H2B (red) that are depleted for empty control, cdc-48 and/or ufd-1, npl-4, div-1, or codepleted for ufd-1/atl-1 by RNAi. Each image series shows representative cell-cycle phases (Mitosis or S phase) at distinct times of embryonic development (2 to 4 cell stage) of one single C. elegans embryo. Empty arrows indicate wild-type like mitotic localization, filled arrows indicate persistent association of the indicated proteins with mitotic chromatin. Anterior is to the left. Scale bars represent 5 μm.

We have previously shown that CDC-48UFD-1/NPL-4 depleted embryos exhibit a pronounced delay in S phase progression caused by activation of the DNA replication checkpoint kinases ATL-1/ATR and CHK-1/Chk1 (Abraham, 2001; Brauchle et al., 2003; Mouysset et al., 2008). Therefore, we tested whether activation of the replication checkpoint is responsible for the altered localization of CDC-45/GINS associated with the depletion of a functional CDC-48UFD-1/NPL-4 complex. However, checkpoint activation caused by depletion of the DNA polymerase α-subunit DIV-1 does not generate a comparable effect (Encalada et al., 2000) (Figure 1B). Whereas downregulation of atl-1 suppresses the cell division delay of ufd-1(RNAi) embryos (Mouysset et al., 2008), it does not reverse the GFP::SLD-5 mislocalization (Figure 1B). Together, these data suggest that misregulation of the CDC-45/GINS complex is caused by CDC-48UFD-1/NPL-4 depletion and is not a secondary effect of checkpoint activation.

Cell Cycle Progression Defects of Embryos Lacking UFD-1 or NPL-4 Depend on CDC-45/GINS

We have shown that the CDC-48UFD-1/NPL-4 complex is required for S phase progression of dividing cells of C. elegans embryos (Mouysset et al., 2008). The P0 zygote undergoes asymmetric division and generates an anterior AB cell, and a smaller posterior P1 cell. These cells have different developmental fates and division timing, with AB dividing approximately 2 min before P1 (Encalada et al., 2000; Pellettieri and Seydoux, 2002). Downregulation of cdc-48, ufd-1, and npl-4 activates the DNA damage checkpoint and further increases the cell division delay of P1 in comparison to AB, leading to a prolonged three-cell stage (Mouysset et al., 2008) (Figure 2A). Considering the persistent chromatin association, we tested the importance of CDC-45/GINS for the delayed S phase progression of embryos lacking CDC-48UFD-1/NPL-4. To perform this experiment, we established an RNAi protocol to deplete UFD-1 or NPL-4 in the first place and subsequently deplete CDC-45 or different GINS subunits (Figure 2A). Indeed, depletion of psf-3 or codepletion of both cdc-45 and sld-5 together significantly suppressed the P1 cell division delay of ufd-1 and npl-4 RNAi embryos (Figure 2B, 2C). Given that RNAi depletion of CDC-45/GINS on its own affects DNA replication and delays S phase, the suppression does not restore the cell division defect completely to the wild-type level. In contrast, RNAi-mediated downregulation of the DNA polymerase subunits DIV-1 and PRI-1, or the DNA repair protein RAD-51 (Wicky et al., 2004) had no effect or even increased the defect in cell cycle progression of embryos lacking UFD-1 (Brauchle et al., 2003) (Figure S2A). To test whether depletion of CDC-45/SLD-5 is able to reduce S phase progression defects in general that are not linked to CDC-48, we used the div-1(or148) temperature sensitive mutant that shows activation of the replication checkpoint at the restrictive temperature (Encalada et al., 2000). Strikingly, cdc-45/sld-5 codepletion significantly enhanced rather than reduced the P1 delay phenotype of div-1(or148) (Figure S2B). Therefore, defective CDC-45/GINS regulation seems to contribute specifically to activation of the DNA replication checkpoint in embryos lacking a functional CDC-48UFD-1/NPL-4 complex.

Figure 2. Suppression of the cell-cycle progression delay of embryos lacking UFD-1 or NPL-4 by cdc-45, sld-5 or psf-3 (RNAi) depletion.

Figure 2

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(A) Schematic illustration of the RNAi feeding procedure to achieve a sequential depletion of empty (light grey), ufd-1 or npl-4 (dark grey) for the first 48 h and cdc-45, sld-5, psf-3 or cdc-45+sld-5 (2nd (RNAi)) for the last 24 h. Subsequent time-lapse analysis was performed to visualize the previously described delay of ufd-1 and npl-4 RNAi embryos in cell-cycle progression of the P1 cell (Mouysset et al., 2008).

(B, C) Quantification of the time between division of AB and P1 cell (P1 division delay) of embryos depleted first for empty (light grey) and ufd-1 or npl-4 (dark grey) and sequentially for empty, cdc-45, sld-5, psf-3 or codepleted for cdc-45+sld-5 (2nd (RNAi)).

(D) Two-hybrid assay for the interaction of C. elegans UFD-1 with CDC-45. Yeast cells expressing the indicated proteins were streaked out on medium plates lacking histidine to test for interaction dependent activation of the HIS3 gene.

(E) Western-blot analysis of GFP fusions of CDC-45 and SLD-5 in C. elegans embryonic extracts depleted for empty, ufd-1 and cdc-45, or sld-5. Time is shown in hours (A) or minutes (B and C). Data are mean values. Error bars show standard error of the mean (s.e.m.). Statistical significance between cell-division timings are indicated by asterisks in B and C. The single asterisk indicates P ≤ 0.05 and the double asterisk indicates P ≤ 0.001.

Recent data on the regulation of the RNA polymerase II subunit Rpb1 supports the idea that CDC-48/p97 is frequently required to support degradation of chromatin bound proteins (Verma et al., 2011). By yeast two-hybrid analysis we identified a direct interaction between UFD-1 and CDC-45, suggesting a similar role in regulating the chromatin association of CDC-45/GINS (Figure 2D). Thus, we tested whether CDC-48UFD-1/NPL-4 dependent regulation of CDC-45 and GINS subunits also determines their stability. However, the protein levels of CDC-45 and SLD-5 are not changed in ufd-1(RNAi) embryos (Figure 2E).

CDC-48UFD-1/NPL-4 Is Required for CDT-1 Turnover at Mitotic Chromatin

Similar to cdc-45, sld-5, and psf-3 depletion, downregulation of the licensing factor CDT-1 reduced the P1 cell division delay of ufd-1 RNAi embryos (Figure 3A). Once per cell cycle CDT-1 is involved in the initiation of the pre-RC at origins of replication. To maintain genome stability, CDT-1 is targeted for degradation by different E3 ligases subsequent to DNA licensing (Kim and Kipreos, 2007; Zhong et al., 2003). Therefore, we wondered whether the CDC-48UFD-1/NPL-4 complex is also involved in the degradation of CDT-1. To clarify this hypothesis, cdt-1 mRNA and CDT-1 protein levels were monitored in embryonic extracts depleted for cdc-48, ufd-1, npl-4, or cdt-1 as control. Whereas cdt-1 mRNA levels remained unchanged, quantification of the immunoblot and normalization with Tubulin identified stabilization of CDT-1 upon downregulation of CDC-48, UFD-1, and NPL-4 (Figure 3B, 3C, and Figure S3A, 3B). Given the high abundance of CDC-48 protein in cells (up to 1% of all cellular proteins (Peters et al., 1990)), RNAi treatment is not efficient enough to deplete CDC-48 completely in wild type (Heubes and Stemmann, 2007). To solve this problem, we used the cdc-48.1(tm544) deletion mutant lacking one of two C. elegans genes encoding CDC-48 for complete downregulation (Figure 3C). In contrast to stabilization in embryos, CDT-1 did not accumulate significantly in whole worm lysates of a similar experiment, suggesting that CDT-1 degradation by the CDC-48UFD-1/NPL-4 complex is especially important in actively dividing tissues (Mouysset et al., 2008).

Figure 3. CDC-48UFD-1/NPL-4 depleted embryos show elevated levels of CDT-1 protein.

Figure 3

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(A) Quantification of the cell division delay between AB and P1 cell (P1 division delay) of embryos depleted first for empty (light grey) or ufd-1(RNAi) (dark grey) and sequentially for empty or cdt-1(RNAi).

(B, C, D) Western-blot analysis of CDT-1 protein levels in embryonic extracts that are depleted for the indicated gene products by RNAi. In (C) embryos were depleted for empty or cdc-48(RNAi) in wild-type or cdc-48.1(tm544) mutant background. In (D) ufd-1 and rbx-1(RNAi) bacteria were equally mixed either with empty control bacteria or together. Quantification of the signal intensity was calculated relative to the Tubulin level and normalized to the protein levels of the empty(RNAi) control.

In C. elegans, the SCF ligase subunit RBX-1 is required for CDT-1 degradation in somatic and germline cells (Jia et al., 2010). As we showed a role for the CDC-48UFD-1/NPL-4 complex in CDT-1 turnover, we examined CDT-1 protein levels after downregulation of ufd-1 and/or rbx-1 to address epistatic effects. Surprisingly, codepletion of UFD-1 together with RBX-1 enhanced the stabilization of CDT-1 observed in single RNAi experiments, indicating that they might act in parallel degradation pathways (Figure 3D). Consistent with this observation, CDT-1 strongly accumulates on mitotic chromatin in ufd-1 and npl-4 RNAi embryos, however not in rbx-1(RNAi) or upon depletion of the Skp2 homolog SKPT-1, CUL-1/Cullin1, or the DNA replication/repair protein PCN-1/PCNA, which is required for CDT-1 degradation on chromatin (Havens and Walter, 2009). Stabilization of CDT-1 in non-synchronized embryonic lysates lacking CDC-48, UFD-1, and NPL-4 appears mediocre but obvious given the low proportion of embryos undergoing mitosis, particularly when the progression through S phase is delayed (Figure 3B, 3C). In contrast to CDC-48, UFD-1, and NPL-4 depletion, CDT-1 is stabilized in S phase nuclei in rbx-1 and pcn-1 RNAi embryos (Figure 4A, 4B).

Figure 4. CDT-1 accumulates on mitotic chromatin in cdc-48, ufd-1 and npl-4 RNAi embryos.

Figure 4

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(A, B) Immunostainings of early C. elegans embryos treated with empty, cdc-48, ufd-1, npl-4, rbx-1 or pcn-1 (RNAi). CDT-1 (green), Tubulin (red) and DAPI (blue) staining is shown as merge images and in separate channels. Distinct cell cycle phases are indicated as Mitosis or S phase. Empty arrowheads indicate wild-type CDT-1 levels, whereas filled arrowheads indicate enhanced signal intensity on mitotic chromatin. In B cdc-48 (RNAi) was performed on cdc-48.1(tm544) mutant background.

(C) Selected pictures of time-lapse recordings of C. elegans embryos expressing GFP::SLD-5 (green) and mCherry::H2B (red) that are depleted for empty control, rbx-1, and pcn-1. Each image series shows representative cell-cycle phases of the first mitotic division of one single embryo. Empty arrowheads point to wild-type like SLD-5 localization. Scale bar represents 5 μm.

We addressed the importance of CDT-1 degradation for the chromatin association of CDC-45/GINS following GFP::SLD-5 localization during embryonic cell division. In contrast to CDC-48UFD-1/NPL-4 downregulation, SLD-5 does not persist on mitotic chromatin upon depletion of RBX-1, PCN-1, SKPT-1, CUL-1 or the Cullin neddylation enzyme DCN-1 (Kurz et al., 2005) (Figure 1B, Figure 4C and Figure S4B). This observation suggests that stabilization of CDT-1 in S phase does not affect CDC-45/GINS regulation. Consequently, CDC-48UFD-1/NPL-4 defines CDT-1 degradation in mitosis, which is specifically linked to chromatin dissociation of the CDC-45/GINS complex.

Persistent Chromatin Binding of SLD-5 in ufd-1(RNAi) Embryos Depends on CDT-1

We tested the hypothesis whether CDT-1 stabilization might directly affect GINS chromatin binding by quantification of chromatin associated GFP::SLD-5 in embryos sequentially depleted for ufd-1 or ufd-1/cdt-1 (as described in Figure 2A). Importantly, depletion of cdt-1 alone does not affect the localization of SLD-5, whereas depletion of ufd-1 does. In contrast, sequential codepletion of ufd-1 and cdt-1 significantly reduced the amount of SLD-5::GFP that remained bound to chromatin after S phase (Figure 5A, 5B), indicating that GINS chromatin release and CDT-1 degradation are coordinated via CDC-48UFD-1/NPL-4. We speculate that cdt-1(RNAi) for 24 h affects the equilibrium between the free pool and chromatin bound CDT-1 rather than reducing the CDT-1 dependent recruitment of CDC-45/GINS during the licensing process, whereas simultaneous downregulation of UFD-1 and CDT-1 together might completely block GINS recruitment (Figure S5A). Moreover, downregulation of the licensing factors ORC-2 and CDC-6 neither suppressed the cell cycle progression phenotype of ufd-1(RNAi) nor reduced the amount of SLD-5::GFP accumulating on mitotic chromatin in UFD-1 depleted embryos which is in contrast to cdt-1(RNAi) (Figure 3A, 5A, B and Figure S5B, C).

Figure 5. Depletion of CDT-1 suppresses persistent SLD-5 chromatin association in ufd-1(RNAi) embryos.

Figure 5

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(A) Selected pictures of time-lapse recordings of embryos expressing GFP::SLD-5 (green) and mCherry::H2B (red) that are depleted first for empty or ufd-1 followed by empty or cdt-1 (seq(RNAi)). Representative pictures of indicated cell-cycle phases (Mitosis or S phase) at distinct time points of embryonic development (1 to 4 cell stage) of one single C. elegans embryo are shown. Empty arrows indicate wild-type like mitotic localization, filled arrows indicate persistent association with mitotic chromatin, shaded arrowheads indicate partial mislocalization. Percentage values represent the number of mitotic divisions where SLD-5 chromatin association was monitored under indicated experimental conditions.

(B) Quantification of the GFP signal intensity on mitotic chromatin in embryos treated with empty, cdt-1, ufd-1 or ufd-1/cdt-1 seq(RNAi) shown in (A). GFP::SLD-5 signal intensity is shown relative to the intensity for mCherry::H2B in the same area.

Anterior is to the left. Data are mean values. Error bars show standard error of the mean (s.e.m.). Statistical significance relative signal intensities are indicated by asterisks. The single asterisk indicates P ≤ 0.05. Scale bar represents 5 μm.

CDC-48/p97 Dependent Regulation of GINS/Cdt1 Is Conserved in Xenopus laevis

To investigate whether CDC-48/p97 together with Ufd1/Npl4 is also required to coordinate GINS and Cdt1 regulation in vertebrates, we took advantage of the Xenopus laevis egg extract system to re-isolate interphasic or mitotic chromatin and subsequently analyze associated proteins (Figure 6A). Remarkably, coimmunoprecipitation (co-IP) experiments identified that Ufd1 interacts with Cdt1 in egg extracts, whereas Npl4 or the alternative p97 cofactor p47 not. Since Npl4 did also not bind to p97 in this co-IP, interaction with Cdt1 is likely to require p97 (Figure 6B). Furthermore, sequential pulldown of Ufd1 and His-tagged Ubiquitin indicates interaction of ubiquitylated Cdt1 with Ufd1 (Figure 6C). Using affinity purified antibodies (Heubes and Stemmann, 2007), we specifically depleted extracts from either Ufd1/Npl4 or p47 without affecting the overall p97 level, respectively (Figure 6D). The depleted extracts were released from CSF arrest by addition of calcium and progression through interphase was then monitored via microscopy (Figure 6E). 85 minutes after addition of calcium, interphase nuclei were observed for all extracts (Figure 6E, middle panel). The nuclei were reisolated from one half of the extracts while the other half was driven back into mitosis for additional 90 minutes. When extracts re-entered mitosis (Figure 6E, bottom panel), sperm chromatin was reisolated and examined for the presence of Cdt1/GINS both by immunofluorescence experiments and western blot analysis. Strikingly, we observed persistent binding of Sld5 and Cdt1 on mitotic chromatin isolated from Ufd1/Npl4 depleted extracts which was not observed upon depletion of p47 (Figure 6F, 6G). As a component of the pre-RC, Cdt1 directly leaves chromatin after initiation of DNA replication (Maiorano et al., 2000). However, Cdt1 also accumulates on interphase chromatin specifically upon Ufd1/Npl4 depletion, which was isolated 85 minutes after calcium treatment when initiation has most likely occurred (Figure 6G, left part). In conclusion, these data suggest an evolutionarily conserved function of CDC-48/p97 in the degradation of Cdt1 linked to dissociation of GINS from chromatin, which seems to be important for accurate eukaryotic DNA replication.

Figure 6. Regulation of chromatin association of GINS and CDT-1 is conserved in Xenopus laevis egg extracts.

Figure 6

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(A) Schematic illustration of the experimental procedure to re-isolate sperm chromatin from S phase/interphase or mitotic Xenopus egg extracts.

(B) In vivo interaction between Ufd1 and Cdt1. Xenopus egg extracts were incubated with anti-Ufd1 antibodies to coupled to Protein A Dynabeads for immunoprecipitation experiments.

(C) Two sequential pulldowns against unspecific IgG/Ufd1 and His-tagged Ubiquitin were performed from Xenopus egg extracts arrested in Metaphase (M) or Interphase (I). Eluates were analyzed for the presence of indicated proteins by western blot. Cdt1 eluted from Ufd1 pulldowns can be precipitated by Ni-NTA (highlighted by <) indicating interaction of Ufd1 with ubiquitylated Cdt1. Asterisk indicates unspecific signal.

(D) Western blot analysis showing efficient immunodepletion of Ufd1, Npl4, and p47 after the first (1st IPΔ) and second (2nd IPΔ) round of incubation of egg extracts with the respective antibodies. Tubulin was used as loading control.

(E) Tubulin and DAPI staining of sperm chromatin incubated for 85 min or 175 min in Mock control, Ufd1, Npl4, and p47 immunodepleted extracts show successive cycling through S phase/Interphase and Mitosis.

(F) Immunostaining of mitotic sperm chromatin that was incubated for 175 min in Mock, Ufd1, Npl4, and p47 depleted egg extracts. SLD-5 (green) and DAPI (blue) staining is shown as merge images and in separated channels.

(G) Western blot analysis of reisolated S phase/interphasic or mitotic sperm chromatin from Mock, Ufd1, Npl4, and p47 depleted egg extracts. Cdt1 and Sld5 levels are shown. Phosphorylated Histone 3 was used as loading control.

DISCUSSION

Since CDC-48/p97 was identified as a crucial factor for cell cycle progression in yeast, the precise role during cell division has remained unclear (Moir et al., 1982). Direct interaction with several factors involved in DNA metabolism has been reported in diverse organisms, which supports the recently described role of CDC-48 in DNA replication (Deichsel et al., 2009; Mouysset et al., 2008). Here, we discovered a conserved regulatory function of CDC-48/p97 in the coordination of licensing and elongation events during eukaryotic DNA replication both in C. elegans and Xenopus laevis. CDC-48UFD-1/NPL-4 deficient embryos stabilize the licensing factor CDT-1 exclusively on mitotic chromatin (Figure 3B, 3C, and 4A, 4B). Furthermore, worm embryos lacking cdc-48, ufd-1, and npl-4, show persistent CDC-45 and SLD-5 on chromatin, becoming visible on condensing chromosomes after S phase is completed (Figure 1A, 1B). Thus, our findings suggest that CDC-48/p97 orchestrates both CDT-1 degradation and chromatin dissociation of the CDC-45/GINS complex during eukaryotic DNA replication directly at the DNA. We show that downregulation of the licensing factors ORC-2 and CDC-6 neither suppress the P1 division delay phenotype of ufd-1(RNAi) nor reduced the amount of SLD-5::GFP, which accumulates on mitotic chromatin in UFD-1 depleted embryos which is in contrast to cdt-1(RNAi) (Figure. 3A, 3B, 5A, 5B, and S5B, C). Consequently, the reduction of chromatin associated CDC-45/GINS in UFD-1 depleted embryos observed after cdt-1(RNAi) is not secondary to reduced recruitment of CDC-45/GINS during the licensing process. Therefore, the persistent association of CDC-45/GINS in embryos lacking UFD-1 and NPL-4 is specifically dependent on CDT-1 stabilization in late mitosis.

In contrast to CDC-48, RBX-1 and PCN-1 support CDT-1 degradation during S phase to prevent enhanced chromatin loading and re-replication (Figure 3D, 4A, 4B) (Havens and Walter, 2009; Zhong et al., 2003). The fact that the GINS subunit SLD-5 does not persist on mitotic chromatin in the absence of rbx-1 and pcn-1 further supports the idea that stabilization of CDT-1 in S phase does not enhance CDC-45/GINS recruitment (Figure 1B and 4C). Unlike CDC-45/GINS, other replication factors known to be recruited by CDT-1 do not persist on chromatin in embryos lacking UFD-1 (Figure S1A). Therefore, we propose a highly specific role of CDC-48/p97 in the regulation of CDT-1 degradation in mitosis, which is linked to the dissociation of CDC-45/GINS. The misregulation of CDC-45/GINS seems to contribute to DNA replication defects in cdc-48, ufd-1, or npl-4 RNAi embryos because depletion of CDC-45/SLD-5 and CDT-1 is able to suppress the P1 cell division delay of CDC-48UFD-1/NPL-4 deficient embryos (Figure 2B, 2C and 3A). However, given the significant delay in S phase progression still detectable in codepleted embryos, suppression of cell cycle progression does not reflect restoration of DNA replication (Figure 2B).

Based on its function as a ubiquitin-selective chaperone, CDC-48/p97 is thought to provide segregase activity that separates ubiquitylated proteins from tightly bound partners (Ye, 2006). The best studied segregase-like function is described for the ERAD pathway where CDC-48UFD-1/NPL-4 mediates re-translocation of damaged proteins from the ER lumen to the cytosol for proteasomal degradation (Jarosch et al., 2002; Ye et al., 2001). Considering recent findings, it is intriguing to speculate that CDC-48/p97 especially extracts proteins from chromatin. For example, yeast Cdc48 is required for the turnover of the RNA Polymerase II subunit Rpb1 upon DNA damage induction (Verma et al., 2011). In light of this observation, CDC-48/p97 probably facilitates extraction of ubiquitylated CDT-1 from mitotic chromatin, resulting in degradation of CDT-1 and dissociation of bound CDC-45/GINS subunits. Consequently, CDT-1/Cdt1 accumulates on mitotic chromatin of C. elegans embryos and Xenopus egg extract depleted for CDC-48, UFD-1/Ufd1, or NPL-4/Npl4 (Figure 3C, 4A, 6B, and 6G). In line with our observation Ballabeni et al. observed Cdt1 accumulation in mitosis depending its ubiquitylation supporting the existence of a CDT-1/Cdt1 degradation pathway besides its known turnover during S phase also in human cells (Ballabeni et al., 2004). Stabilization of CDT-1 in mitosis might keep CDC-45/GINS tightly associated with chromatin, which may interfere with dynamic progression of the replication fork in S phase. Defects in GINS dissociation and DNA replication of ufd-1(RNAi) embryos are independent of ATL-1/CHK-1 and therefore not secondary to checkpoint activation (Figure 1B) (Mouysset et al., 2008). We propose that defects in CDC-45/GINS dissociation already take place in late mitosis/beginning of S phase but are first detectable with condensing chromosomes at the end of S phase (Figure 7). The CDC-48 complex seems to coordinate both events since UFD-1/Ufd1 binds CDC-45 and Cdt1 (Figure 2D and 6B).

Figure 7. Hypothetical model for the coordination of CDT-1 turnover and GINS chromatin extraction.

Figure 7

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CDC-48/p97 together with the cofactors UFD-1/NPL-4 coordinates the turnover of ubiquitylated CDT-1 during the licensing phase with chromatin dissociation of CDC-45/GINS. Failure in CDT-1 turnover keeps CDC-45/GINS tightly associated with chromatin, becoming visible with condensing chromosomes at the end of S phase. CDC-45/GINS misregulation may interfere with dynamic progression of the replication fork in S phase. The CDC-48/p97 complex seems to coordinate both events since UFD-1/Ufd1 binds both CDC-45 and Cdt1.

Taken together, our findings reveal that the spatial and temporal regulation between dynamic protein complexes at the replication fork is governed by CDC-48/p97. In conclusion, CDC-48/p97 coordinates CDT-1 turnover and dissociation of the CDT-1 bound CDC-45/GINS complex, which is important for adjusting DNA replication and cell cycle progression.

EXPERIMENTAL PROCEDURES

Strains

Worms were grown according to the standard protocols at 20°C, unless otherwise stated (Brenner, 1974). The C. elegans Bristol strain N2 was used as wild-type strain. Mutations and transgenes used in this study are listed as follows: div-1(or148)III, cdc-48.1(tm544)II, unc-119(ed3)III; gtIs[unc-119(+), Ppie-1::GFP::mcm-2::pie-1-3′UTR], unc-119(ed3)III; gtIs[unc-119(+), Ppie-1::GFP::orc-2::pie-1-3′UTR], du[unc-119(+), Ppie-1::mCherry::H2B::pie-1-3′UTR], unc-119(ed3)III; gtIs[unc-119(+), Ppie-1::GFP::cdc-6::pie-1-3′UTR], du[unc-119(+), Ppie-1::mCherry::H2B::pie-1-3′UTR], unc-119(ed3)III; gtIs[unc-119(+), Ppie-1::GFP::cdc-45::pie-1-3′UTR], unc-119(ed3)III; gtIs[unc-119(+), Ppie-1::mCherry::H2B::pie-1-3′UTR], unc-119(ed3)III; gtIs[unc-119(+), Ppie-1::GFP::sld-5::pie-1-3′UTR], du[unc-119(+), Ppie-1::mCherry::H2B::pie-1-3′UTR], unc-119(ed3)III; [unc-119(+), Ppie-1::GFP::psf-3]. The generation of orc-2, cdc-6, mcm-2, cdc-45, and sld-5 transgenes fused to gfp and controlled by the pie-1 promoter, as well as the detailed dynamic behavior of the respective fusion proteins and their biological validation will be described elsewere (R. Sonneville, J.J. Blow, A. Gartner in preparation).

RNAi

RNAi-mediated depletion was achieved using the feeding method (Kamath et al., 2001). RNAi was fed to L3/L4 larvae at 15°C for 72 hours. To induce stronger expression of the fluorescent reporter constructs, worms were shifted to 20°C or 25°C over night the day before time-lapse analysis. For sequential RNAi (seq(RNAi)) depletion worms were fed with the dsRNA-containing bacteria against the first target gene for 48 h and then switched to bacteria containing dsRNA against the second target. For the simultaneous depletion of two genes the respective bacteria were mixed 1:1 in cell density. For the preparation of embryonic lysates worms were kept at 20°C during the entire experimental procedure. In RNAi control experiments, bacteria only contained the empty vector pPD129.36.

Yeast two-hybrid analysis

The full-length protein UFD-1 was fused to the GAL4 DNA binding domain using the vector pGBKT7 and co-expressed with CDC-45 fused to the GAL4 DNA activation domain using the vector pGADT7 in the yeast host strain AH109 (Clontech, Palo Alto, C.A.). Protein interaction studies were carried out according to the manufacturer’s instructions.

Preparation of embryonic lysates

To produce embryonic lysates, L1 larvae were kept on control RNAi plates at 20°C until they reached the L3/L4 larval stage and then spread onto respective target RNAi plates. Embryos of gravid worms were harvested and resuspended in 2x Laemmli buffer. Embryos were sonicated twice for 15 seconds (Bandelin, Microtip MS 1,5) and incubated at 95°C for 5 min and centrifugated at 14.000 g for 5 min before SDS-PAGE analysis.

Microscopy and image aquisition

For time-lapse microscopy, embryos were mounted on agar pads essentially as described before (Mouysset et al., 2008). An Axio-Imager.M1 microscope equipped with a AxioCam MRm camera (Carl Zeiss) was used for image acquisition. Time-lapse recordings in 90 s intervals were aquired using 2×2 mono binning to avoid photobleaching and –toxicity. To allow direct comparison of signal intensities, images were recorded under identical conditions. Analysis of time-lapse recordings was done in the AxioVision 4.7 software. Timing of cell division was estimated as described previously (Mouysset et al., 2008). Processing of selected pictures was done in Adobe Photoshop CS4. Images of immunostainings were also acquired with AxioImager.M1 and AxioCam MRm but using full resolution of the camera.

Immunotechniques

Immunostaining of early embryos was done essentially according to the “freeze-crack” protocol (Kemphues et al., 1986). Gravid worms were dissected onto polylysine coated slides (Thermo Scientific) and frozen in liquid Nitrogen followed by incubation in -20°C methanol for 20 minutes and -20°C acetone for 5 minutes. After rehydration in PBS and blocking in 5 % BSA, embryos were incubated with the primary antibody over night at 4°C (anti-CDT-1 1:300, anti-αTubulin 1:200 (Sigma, clone DM1A)). Incubation with the secondary antibodies (Alexa488 or Alexa 594 conjugated, Invitrogen) was done at room temperature for one hour (1:400). Embryos were then mounted in Dapi Fluoromount G medium (SouthernBiotech). Quantification of signal intensities was done using ImageJ (National Institutes of Health). Background signal in the surrounding area was substracted from values in the area of interest. For western-blotting, proteins were separated by SDS-PAGE and transferred to Nitrocellulose membranes (Whatman, Protran). Membranes were blocked in 3 % milk solution and incubated with the primary antibodies over night at 4°C in RotiBlock (Roth) (anti-CDT-1 1:300, anti-GFP 1:5.000 (Clonetech), anti-Tubulin 1:5.000 (Sigma, clone DM1A), anti-UFD-1 1:50.000, anti-CDC-48 1:50.000). Incubation with fluorescently labeled secondary antibodies (1:10.000) was done at room temperature, before detection of signals using the Li-cor Odyssey scanner. Quantification of signal intensities was done using the Odyssey V3.0 software (Li-cor). Backround signal in the surrounding area was substracted from values in the area of interest.

Xenopus laevis egg extract preparation and reisolation of sperm chromatin

CSF extracts were prepared as described previously (Murray, 1991). For immunodepletion, antibodies were coupled to Protein A Dynabeads™ (Invitrogen) overnight at 4°C. Extracts were depleted at 12°C in two rounds each 30′ as previously described (Heubes and Stemmann, 2007). After depletion, extracts were supplemented with Rhodamine-labeled tubulin and sperm to a final concentration of 6.000 nuclei/μl. Extracts were released from metaphase II arrest at 20°C by addition of Ca2+ and cell cycle stages were followed by fluorescence microscopy of fixed, Hoechst33342 stained aliquots. Interphase extracts were split into halves. Sperm chromatin of one half was reisolated (see below) while the other half was driven back into mitosis by addition of an equal volume of depleted CSF extract. Re-isolation of sperm chromatin was carried out as described previously (Stemmann et al., 2001).

Interaction analysis of ubiquitylated Cdt1 with Ufd1

CSF extract was prepared as described (Murray et al., 1991) and supplemented with sperm nuclei (4.000 μl−1) and His6-tagged ubiquitin (2,5 μg/μl). To obtain interphase extract, half of the sample was released by addition of CaCl2 to 0,6 mM and incubated for 20 minutes at 22°C. Then, CSF- and interphase extracts were supplemented with Cycloheximide (0,1 mg/ml), the proteasome inhibitor Bortezomib (200 μM) and N-Ethylmaleimide (10 mM). Antibodies (80 μg each) were coupled to magnetic protein A beads and pulldowns from 1ml of extract each were performed for 60 minutes at 12°C. Beads were washed three times with XB buffer supplemented with 300 mM NaCl and 0,1% TritonX100. Precipitated proteins were eluted in 6M Guanidiniumhydrochloride, 100 mM NaH2PO4, 10 mM Tris/HCl pH8,0, 0,1% TritonX100 and subjected to Ni-NTA pulldown for 3h at room temperature. Beads were washed five times in 8M Urea, 100 mM NaH2PO4, 10 mM Tris/HCl pH8,0, 0,1% TritonX100 and two times in PBS according to the protocol described by Siepe and Jentsch (Siepe and Jentsch, 2009). Proteins were eluted with SDS-sample buffer at 95°C and analyzed by Western.

Statistical Analysis

Statistical analysis was performed in Microsoft Excel. Statistical significance was calculated with two-tailed paired student’s T-test. P-values of P ≤ 0.05 are indicated with a single asterisk and double asterisks indicate P ≤ 0.001. Comparison of cell division timings was done for experiments that were done on one single day, whereas Figure 2B shows summarized values for the controls of all experiments for better visualization.

Supplementary Material

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ACKNOWLEDGMENTS

We thank A. Fire, E.T. Kipreos, M. Mechali, L. Pintard, H. Takisawa, the Caenorhabditis Genetics Center (funded by the NIH Center for Research Resources), and the Dana-Farber Cancer Institute and Geneservice Ltd for antibodies, plasmids, cDNAs, and strains. We particularly thank J.W. Harper for exchange of unpublished results. We also thank A. Segref for critical reading of the manuscript. This work is supported by grants of the Deutsche Forschungsgemeinschaft (especially the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, FOR885, and SFB635 to T.H. and SPP1384 to O.S.), and the Rubicon European Union Network of Excellence to T.H., a Cancer Research U.K. programme grant to J.J.Blow., a Cancer Research U.K. CDA grant and a Wellcome Trust Senior Research Fellowship to A.G., and a postdoctoral EMBO fellowship to R.S.. T.H. is an EMBO Young Investigator.

Footnotes

COMPETING INTEREST The authors declare that they have no competing financial interests.

REFERENCES

  1. Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 2001;15:2177–2196. doi: 10.1101/gad.914401. [DOI] [PubMed] [Google Scholar]
  2. Aparicio T, Guillou E, Coloma J, Montoya G, Mendez J. The human GINS complex associates with Cdc45 and MCM and is essential for DNA replication. Nucleic Acids Res. 2009;37:2087–2095. doi: 10.1093/nar/gkp065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ballabeni A, Melixetian M, Zamponi R, Masiero L, Marinoni F, Helin K. Human geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis. EMBO J. 2004;23:3122–3132. doi: 10.1038/sj.emboj.7600314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bell SP, Dutta A. DNA replication in eukaryotic cells. Annu Rev Biochem. 2002;71:333–374. doi: 10.1146/annurev.biochem.71.110601.135425. [DOI] [PubMed] [Google Scholar]
  5. Brauchle M, Baumer K, Gonczy P. Differential activation of the DNA replication checkpoint contributes to asynchrony of cell division in C. elegans embryos. Curr Biol. 2003;13:819–827. doi: 10.1016/s0960-9822(03)00295-1. [DOI] [PubMed] [Google Scholar]
  6. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. doi: 10.1093/genetics/77.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Budirahardja Y, Gonczy P. Coupling the cell cycle to development. Development. 2009;136:2861–2872. doi: 10.1242/dev.021931. [DOI] [PubMed] [Google Scholar]
  8. Deichsel A, Mouysset J, Hoppe T. The ubiquitin-selective chaperone CDC-48/p97, a new player in DNA replication. Cell Cycle. 2009;8:185–190. doi: 10.4161/cc.8.2.7356. [DOI] [PubMed] [Google Scholar]
  9. Encalada SE, Martin PR, Phillips JB, Lyczak R, Hamill DR, Swan KA, Bowerman B. DNA replication defects delay cell division and disrupt cell polarity in early Caenorhabditis elegans embryos. Dev Biol. 2000;228:225–238. doi: 10.1006/dbio.2000.9965. [DOI] [PubMed] [Google Scholar]
  10. Gambus A, Jones RC, Sanchez-Diaz A, Kanemaki M, van Deursen F, Edmondson RD, Labib K. GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat Cell Biol. 2006;8:358–366. doi: 10.1038/ncb1382. [DOI] [PubMed] [Google Scholar]
  11. Havens CG, Walter JC. Docking of a specialized PIP Box onto chromatin-bound PCNA creates a degron for the ubiquitin ligase CRL4Cdt2. Mol Cell. 2009;35:93–104. doi: 10.1016/j.molcel.2009.05.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heubes S, Stemmann O. The AAA-ATPase p97-Ufd1-Npl4 is required for ERAD but not for spindle disassembly in Xenopus egg extracts. J Cell Sci. 2007;120:1325–1329. doi: 10.1242/jcs.006924. [DOI] [PubMed] [Google Scholar]
  13. Ilves I, Petojevic T, Pesavento JJ, Botchan MR. Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. Mol Cell. 2010;37:247–258. doi: 10.1016/j.molcel.2009.12.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jarosch E, Taxis C, Volkwein C, Bordallo J, Finley D, Wolf DH, Sommer T. Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nat Cell Biol. 2002;4:134–139. doi: 10.1038/ncb746. [DOI] [PubMed] [Google Scholar]
  15. Jia L, Bickel JS, Wu J, Morgan MA, Li H, Yang J, Yu X, Chan RC, Sun Y. RBX1 (RING box protein 1) E3 ubiquitin ligase is required for genomic integrity by modulating DNA replication licensing proteins. J Biol Chem. 2010;286:3379–3386. doi: 10.1074/jbc.M110.188425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, Ahringer J. Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2001;2 doi: 10.1186/gb-2000-2-1-research0002. RESEARCH0002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kemphues KJ, Wolf N, Wood WB, Hirsh D. Two loci required for cytoplasmic organization in early embryos of Caenorhabditis elegans. Dev Biol. 1986;113:449–460. doi: 10.1016/0012-1606(86)90180-6. [DOI] [PubMed] [Google Scholar]
  18. Kim Y, Kipreos ET. Cdt1 degradation to prevent DNA re-replication: conserved and non-conserved pathways. Cell Div. 2007;2:18. doi: 10.1186/1747-1028-2-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kurz T, Ozlu N, Rudolf F, O’Rourke SM, Luke B, Hofmann K, Hyman AA, Bowerman B, Peter M. The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae. Nature. 2005;435:1257–1261. doi: 10.1038/nature03662. [DOI] [PubMed] [Google Scholar]
  20. Maiorano D, Moreau J, Mechali M. XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis. Nature. 2000;404:622–625. doi: 10.1038/35007104. [DOI] [PubMed] [Google Scholar]
  21. Masai H, Matsumoto S, You Z, Yoshizawa-Sugata N, Oda M. Eukaryotic chromosome DNA replication: where, when, and how? Annu Rev Biochem. 2010;79:89–130. doi: 10.1146/annurev.biochem.052308.103205. [DOI] [PubMed] [Google Scholar]
  22. Masuda T, Mimura S, Takisawa H. CDK- and Cdc45-dependent priming of the MCM complex on chromatin during S-phase in Xenopus egg extracts: possible activation of MCM helicase by association with Cdc45. Genes Cells. 2003;8:145–161. doi: 10.1046/j.1365-2443.2003.00621.x. [DOI] [PubMed] [Google Scholar]
  23. Mimura S, Masuda T, Matsui T, Takisawa H. Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts. Genes Cells. 2000;5:439–452. doi: 10.1046/j.1365-2443.2000.00340.x. [DOI] [PubMed] [Google Scholar]
  24. Moir D, Stewart SE, Osmond BC, Botstein D. Cold-sensitive cell-division-cycle mutants of yeast: isolation, properties, and pseudoreversion studies. Genetics. 1982;100:547–563. doi: 10.1093/genetics/100.4.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mouysset J, Deichsel A, Moser S, Hoege C, Hyman AA, Gartner A, Hoppe T. Cell cycle progression requires the CDC-48UFD-1/NPL-4 complex for efficient DNA replication. Proc Natl Acad Sci U S A. 2008;105:12879–12884. doi: 10.1073/pnas.0805944105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mouysset J, Kahler C, Hoppe T. A conserved role of Caenorhabditis elegans CDC-48 in ER-associated protein degradation. J Struct Biol. 2006;156:41–49. doi: 10.1016/j.jsb.2006.02.015. [DOI] [PubMed] [Google Scholar]
  27. Moyer SE, Lewis PW, Botchan MR. Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc Natl Acad Sci U S A. 2006;103:10236–10241. doi: 10.1073/pnas.0602400103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Murray AW. Cell cycle extracts. Methods Cell Biol. 1991;36:581–605. [PubMed] [Google Scholar]
  29. Murray MT, Krohne G, Franke WW. Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos. J Cell Biol. 1991;112:1–11. doi: 10.1083/jcb.112.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pacek M, Tutter AV, Kubota Y, Takisawa H, Walter JC. Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. Mol Cell. 2006;21:581–587. doi: 10.1016/j.molcel.2006.01.030. [DOI] [PubMed] [Google Scholar]
  31. Pellettieri J, Seydoux G. Anterior-posterior polarity in C. elegans and Drosophila--PARallels and differences. Science. 2002;298:1946–1950. doi: 10.1126/science.1072162. [DOI] [PubMed] [Google Scholar]
  32. Peters JM, Walsh MJ, Franke WW. An abundant and ubiquitous homo-oligomeric ring-shaped ATPase particle related to the putative vesicle fusion proteins Sec18p and NSF. EMBO J. 1990;9:1757–1767. doi: 10.1002/j.1460-2075.1990.tb08300.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rabinovich E, Kerem A, Frohlich KU, Diamant N, Bar-Nun S. AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol Cell Biol. 2002;22:626–634. doi: 10.1128/MCB.22.2.626-634.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rape M, Hoppe T, Gorr I, Kalocay M, Richly H, Jentsch S. Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone. Cell. 2001;107:667–677. doi: 10.1016/s0092-8674(01)00595-5. [DOI] [PubMed] [Google Scholar]
  35. Sasagawa Y, Yamanaka K, Nishikori S, Ogura T. Caenorhabditis elegans p97/CDC-48 is crucial for progression of meiosis I. Biochem Biophys Res Commun. 2007;358:920–924. doi: 10.1016/j.bbrc.2007.05.022. [DOI] [PubMed] [Google Scholar]
  36. Schuberth C, Buchberger A. UBX domain proteins: major regulators of the AAA ATPase Cdc48/p97. Cell Mol Life Sci. 2008;65:2360–2371. doi: 10.1007/s00018-008-8072-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sheu YJ, Stillman B. Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression. Mol Cell. 2006;24:101–113. doi: 10.1016/j.molcel.2006.07.033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Siepe D, Jentsch S. Prolyl isomerase Pin1 acts as a switch to control the degree of substrate ubiquitylation. Nat Cell Biol. 2009;11:967–972. doi: 10.1038/ncb1908. [DOI] [PubMed] [Google Scholar]
  39. Stemmann O, Zou H, Gerber SA, Gygi SP, Kirschner MW. Dual inhibition of sister chromatid separation at metaphase. Cell. 2001;107:715–726. doi: 10.1016/s0092-8674(01)00603-1. [DOI] [PubMed] [Google Scholar]
  40. Takeda DY, Dutta A. DNA replication and progression through S phase. Oncogene. 2005;24:2827–2843. doi: 10.1038/sj.onc.1208616. [DOI] [PubMed] [Google Scholar]
  41. Verma R, Oania R, Fang R, Smith GT, Deshaies RJ. Cdc48/p97 mediates UV-dependent turnover of RNA Pol II. Mol Cell. 2011;41:82–92. doi: 10.1016/j.molcel.2010.12.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wicky C, Alpi A, Passannante M, Rose A, Gartner A, Muller F. Multiple genetic pathways involving the Caenorhabditis elegans Bloom’s syndrome genes him-6, rad-51, and top-3 are needed to maintain genome stability in the germ line. Mol Cell Biol. 2004;24:5016–5027. doi: 10.1128/MCB.24.11.5016-5027.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yabuuchi H, Yamada Y, Uchida T, Sunathvanichkul T, Nakagawa T, Masukata H. Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins. EMBO J. 2006;25:4663–4674. doi: 10.1038/sj.emboj.7601347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yamanaka K, Okubo Y, Suzaki T, Ogura T. Analysis of the two p97/VCP/Cdc48p proteins of Caenorhabditis elegans and their suppression of polyglutamine-induced protein aggregation. J Struct Biol. 2004;146:242–250. doi: 10.1016/j.jsb.2003.11.017. [DOI] [PubMed] [Google Scholar]
  45. Ye Y. Diverse functions with a common regulator: ubiquitin takes command of an AAA ATPase. J Struct Biol. 2006;156:29–40. doi: 10.1016/j.jsb.2006.01.005. [DOI] [PubMed] [Google Scholar]
  46. Ye Y, Meyer HH, Rapoport TA. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature. 2001;414:652–656. doi: 10.1038/414652a. [DOI] [PubMed] [Google Scholar]
  47. Zhong W, Feng H, Santiago FE, Kipreos ET. CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing. Nature. 2003;423:885–889. doi: 10.1038/nature01747. [DOI] [PubMed] [Google Scholar]

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