Mcm10 proteolysis initiates before the onset of M-phase
© Kaur et al; licensee BioMed Central Ltd. 2010
Received: 8 June 2010
Accepted: 28 October 2010
Published: 28 October 2010
Mcm10 protein is essential for initiation and elongation phases of replication. Human cells proteolyze Mcm10 during mitosis, presumably to ensure a single round of replication. It has been proposed that anaphase promoting complex ubiquitinates Mcm10 in late M and early G1 phases.
In contrast to the previous work, we report that the degradation of Mcm10 is initiated at the onset of mitosis. Immunoblotting and immunofluorescence assays display that Mcm10 levels are low in all phases of mitosis. We report that Mcm10 degradation is not dependent on anaphase promoting complex. Further, the proteolysis in M-phase can be independently mediated by non-overlapping regions of Mcm10, apparently employing a redundant mechanism to ensure downregulation.
It is believed that the proteolysis of Mcm10 during mitosis is a vital mechanism to prevent aberrant initiation of replication and the present study describes the regulation of Mcm10 during this phase of the cell-cycle.
DNA replication in eukaryotes begins with the assembly of the pre-replicative complex comprising of replication initiators, the origin recognition complex, Cdc6, Cdt1, and the replicative helicase, Mcm2-7 complex . Increase in the activity of cyclin dependent kinases and loading of replication factor, Mcm10 marks the transition from G1 to S phase. These events promote the loading of Cdc45, RPA and DNA polymerases at the replication origins to initiate DNA synthesis. The essential requirement of Mcm10 in DNA replication initiation and elongation has been exhibited across species [2–7]. Mutations in Mcm10 cause a decrease in initiation of replication, slow progression of DNA synthesis and stalling of replication forks during elongation . Since Mcm10 is essential for replication initiation and elongation, its activity is regulated in a cell-cycle manner to ensure a single round of replication. S. cerevisiae Mcm10 protein is present in all phases of the cell cycle though its association with chromatin is regulated to ensure replication licensing .
Human Mcm10 protein is known to decrease in the early G1 phase . The levels of MCM10 mRNA have been evaluated as the cells pass from M-phase into G1 phase. Though the Mcm10 protein decreased, the levels of MCM10 mRNA increased within the same time period. These results demonstrate that the decrease in the Mcm10 activity during the G1 phase is not due to decrease in transcription but because of protein turnover. In this communication, we report that proteolysis of human Mcm10 protein initiates before the onset of mitosis. In an immunoblot with anti-Mcm10 antibody, we observed that the M-phase blocked cells have downregulated the Mcm10 protein. On the basis of single cell immunofluorescence, we show that asynchronously growing cells in different phases of mitosis have reduced levels of Mcm10. Also, Mcm10 downregulation in M-phase is independent of the APC recognition motifs: the destruction and the KEN box. We demonstrate that Mcm10 degradation is not dependent on anaphase promoting complex or on its recognition motifs but rather mediated by non-overlapping regions, apparently employing a redundant mechanism to ensure downregulation.
Cell-cycle proteolysis of Mcm10 initiates before the onset of mitosis
Single-cell imaging displays reduced levels of Mcm10 during mitosis
Live-cell imaging demonstrates that Mcm10 is absent during the M-phase
We have reported that the 61 aa ZF motif (783-843aa) of Mcm10 is sufficient for M phase proteolysis and therefore we expressed EGFP-tagged ZF motif of Mcm10 and tracked the EGFP signal and cell division of HeLa cells by fluorescence and phase-contrast microscopy respectively as the cells progressed through mitosis. As shown in Figure S4C (Additional file 5), we located a mitotic cell that undergoes cytokines after 1 h 20 min (the two daughter cells have been marked by arrows). We observed that the cell was negative for Mcm10 during mitosis but the daughter cells accumulated Mcm10 after around 5 h. Therefore, utilizing a different cell-line, we establish that the full-length Mcm10 is low during mitosis. Summing up, using this antibody-independent approach, we demonstrated that the Mcm10 levels are low during mitosis.
APC is not required for cell cycle degradation of Mcm10
APC recognition motifs are not required for cell cycle degradation of Mcm10
We observed that the NTD+ID domain of Mcm10, which contains the KEN box, was resistant to cell cycle-regulated degradation. In the present study, we tested whether the WH motif, that contains the destruction box, is proteolyzed during the M-phase. Stable U2OS cells expressing WH motif of Mcm10 (707-770 aa) was blocked with nocodazole, which arrested the cells in M-phase. The obtained mitotic cells were released from the block and harvested at regular intervals in order to collect cells in different phases of the cell cycle. The flow cytometry profile of propidium iodide-stained DNA and the levels of cyclin B demonstrate a block in M-phase and subsequent progression through the cell cycle (Figure 2D). As noted previously, Mcm10 was absent in nocodazole-released cells, demonstrating the natural proteolysis of Mcm10 in M phase which began to increase after 8 h, displaying the natural cycling of Mcm10 levels (Figure 2C). Full-length Mcm10 expressed from the retroviral vector showed a degradation pattern similar to that of the endogenous protein, validating our assay for evaluating the cell cycle-regulated degradation of Mcm10. We observed that the WH motif fragment was resistant to cell cycle-regulated degradation. We confirmed that the nuclear localization signal expressed in fusion with the WH motif steered it to the nucleus and therefore change in cellular localization is not the reason for resistance to proteolysis (Figure 2E). We have previously reported that the LNK domain which does not have either the KEN box or destruction box is proteolyzed as cells pass through M-phase. Therefore, we conclude that the KEN and destruction box of Mcm10 are neither essential nor sufficient for its M-phase proteolysis.
Mcm10 proteolysis utilizes independent non-overlapping signals
We further divided the CTD (607-875 aa) into three fragments 607-707 aa, 707-770 aa (WH motif) and 770-875 aa. We observed that the 607-707 aa and 707-770 aa fragment were resistant to proteolysis (Figure 8A, panel 1 and results of WH motif in Figure 2C). However, the 770-875 aa fragment decreased in M-phase, demonstrating that the signal for degradation lies in this fragment. In order to identify the minimum sequence required for Mcm10 degradation, we further divided the 770-875 aa fragment into three parts: 770-783 aa, 783-843 aa and 843-875 aa. 770-783 aa fragment was partially stable indicating that there could be an incomplete degron in this fragment (Figure 8A, panel 3). The 783-843 aa (ZF motif) and 843-875 aa decreased in M-phase demonstrating that there are non-overlapping regions that are sufficient for Mcm10 proteolysis (Figure 8A, panel 4 and results of ZF motif in ). The ZF motif was divided into 3 segments each of 20 amino acids: 783-803 aa, 803-823 aa and 823-843 aa. We observed that 783-803 aa region was essential and sufficient for degradation while 803-823 aa and 823-843 aa were not proteolyzed (Figure 8C and 8E). Therefore, we have narrowed to two regions that contain the degron: a 20 aa region (783-803 aa) within the ZF motif and C-terminal end of Mcm10 (843-875 aa).
Another independent region that is sufficient for Mcm10 downregulation is the linker domain (440-607 aa). We divided the linker domain into three parts: 440-471 aa, 471-525 aa and 525-607 aa. We observed that though the linker domain is decreased during M-phase, none of the three constituent fragments were proteolyzed (Figure 9A). However, when we combined the 440-471 aa and 471-525 aa fragments, the degradation ability was restored (Figure 9C, panel 1). Combining 440-471 aa with 525-607 aa or combing 471-525 aa with 525-607 aa did not result in M-phase degradation (Figure 9C, panel 2 and 3). Therefore, the degron in the linker domain lies within the stretch of 440-525 aa. Hence, it seems that Mcm10 degradation is mediated by non-overlapping regions, apparently to ensure downregulation of Mcm10 even if proteolysis at any one region is somehow blocked. There is no apparent sequence similarity between the three minimal regions that are competent for M-phase proteolysis: 440-525 aa, 783-803 aa and 843-875 aa. This would suggest that the ubiquitination machinery is adaptable to recognize multiple sequences to ensure Mcm10 proteolysis. Recognition of many biological targets is defined by similar 2-D structures rather than primary sequence and that possibility cannot be ruled for recognition of different domains of Mcm10.
Discussion and Conclusions
We have observed that the Mcm10 levels are significantly reduced in nocodazole blocked cells. This is in contrast to the previous report where on the basis of estimation of Mcm10 levels in asynchronous and nocodazole released cells by immunoblots, the authors inferred that Mcm10 degradation begins after metaphase . It is possible that the Mcm10 protein observed after nocodazole treatment was contributed by cells trapped at G2/M boundary. The stabilization of Mcm10 by MG132 in nocodazole released cells observed by them is likely due to non-degradation of Mcm10 in these cells. It has been suggested that a destruction box sequence (719-727 aa) could be recognized by the anaphase-promoting complex for ubiquitination. Additionally, Mcm10 also contains a KEN box sequence (71-73 aa) that is present in targets ubiquitinated by APC. However, the NTD domain, which contains the KEN box is not degraded during M-phase . Similarly, the WH motif which contains the destruction box is stable during mitosis (Figure 2C). However, the linker region and ZF motif, which do not have either destruction or KEN box, are proteolyzed during M-phase. In summation, the above data would suggest that the APC recognition motifs are not required for M-phase degradation of Mcm10. In this study, we have also demonstrated that inhibition of APC does not block cell-cycle degradation of Mcm10.
This study has discovered at least three independent regions of Mcm10 that are sufficient for proteolysis during M phase. Since the non-overlapping regions of Mcm10 sufficient for M-phase proteolysis did not display sequence similarity, it suggests of a limited degeneracy of the E3 ligase to allow for recognition of multiple substrates. CRL4 is known to mediate the ubiquitination of almost two dozen substrates, including Cdt1, PCNA and histones that do not share sequence similarity . It is widely believed that the proteolysis of Mcm10 during mitosis is a mechanism to prevent aberrant initiation of replication and therefore it is possible that utilization of independent regions is a means to ensure downregulation of Mcm10 even if proteolysis at any one region is somehow blocked. The cloning of stable mutants of full length Mcm10 would help us evaluate the effect of stable Mcm10 on cell-cycle progression and genomic stability. It is believed that the proteolysis of Mcm10 during mitosis is a vital mechanism to prevent aberrant initiation of replication and the present study describes the regulation of Mcm10 during this phase of the cell-cycle.
Cell culture, chemicals, antibodies, cell synchronization and FACS analysis
Cell lines were maintained in DMEM supplemented with fetal bovine serum and antibiotics. Specific chemicals and antibodies used in this study are mentioned in the supplementary methods (Additional file 6). HeLa cells were transfected with specific siRNA oligos consecutively for three days and were blocked with 40 ng/ml nocodazole for 15 h. Later, cells were released in drug-free medium and cells arrested in the M-phase were collected by 'mitotic-shake off'. The obtained mitotic cells were then re-plated in drug-free medium and harvested till 12 h to collect cells in different phases of the cell cycle. HeLa and U2OS cells were incubated with 40 ng/ml or 0.10 μg/ml nocodazole, 0.05 μg/ml or 0.2 μg/ml vincristine, 0.15 μg/ml or 0.5 μg/ml colchicine or 0.1 μg/ml or 0.3 μg/ml taxol for 15 h or 16 h respectively to obtain mitotic cells. For cell cycle analysis, the cells were fixed with 70% ethanol after washing with 1× PBS. Subsequently, the cell pellet was resuspended in 1× PBS with 0.1% Triton X-100, 20 μg/ml RNase and 70 μg/ml propidium iodide and the flow cytometry was performed.The flow cytometry data was acquired on Becton Dickinson FACS Calibur machine by Cell Quest Pro software. Cell cycle analysis was done by Dean/Jett/Fox method of FlowJo software.
Antibodies for immunoblotting and immunofluorescence
Anti-human Mcm10 antibody was produced using recombinant His-tagged Mcm10 (cloned in pET28a vector), purified on nickel-NTA column (Qiagen).Rabbits were injected with recombinant protein along with complete Freund's adjuvant to obtain Mcm10 Ab (N) and Ab (FL). Anti-Mcm10 sera was affinity-purified using Mcm10 conjugated sepharose column. Rabbit polyclonal anti-cyclin B1 (Cat. No. Sc-752) was purchased from Santa Cruz Biotechnology Mouse monoclonal anti-APC3 (Cat. No. ab10538) and anti-alpha tubulin-FITC (Cat. No. ab64503) were purchased from Abcam.Mouse monoclonal anti-cyclin A (Cat. No. CS-4656) was obtained from Cell Signaling Technology.Mouse monoclonal anti-HA (Cat.No. H3663) was obtained from Sigma. Polyclonal goat anti-rabbit HRP (Cat.No. P0448), polyclonal rabbit anti-mouse HRP (Cat.No. P0161) and polyclonal swine anti-rabbit TRITC (Cat No.R0156) were obtained from Dako. Alexa Fluor 488 goat anti-rabbit IgG (Cat.No. A11008) and Alexa Fluor 594 goat anti-mouse IgG (Cat.No. A110050) were obtained from Molecular Probes, Invitrogen. For western blotting, cells were harvested in 1× SDS sample buffer. Equal amount of protein was separated on SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes. Finally the results were assayed using the Enhanced Chemiluminescence method.
RNAi silencing and reverse-transcriptase PCR
Silencing of genes was done by transfecting specific 40-80 nM siRNA duplex on three consecutive days and cells were harvested 24 h post the last transfection.The levels of protein and mRNA were evaluated by immunoblotting and reverse-transcriptase PCR respectively. For reverse-transcriptase PCR, RNA was extracted using TRIzol method and 0.25-1 μg RNA was used for cDNA synthesis. The primers used for PCR are described in the supplementary methods (Additional file 6)
Plasmid construction, transfection, immunoblotting and immunofluorescence
Full-length Mcm10 was subcloned into BglII and SalI sites of pEGFP-C3 vector, which carries the GFP sequence at the N-terminal of the vector. Similarly, Mcm10 cDNA was digested with either BglII and EcoRI or BglII and MfeI and cloned in BamHI and EcoRI sites of pMX-puro-NLS-HA vector. The sequences of the cloning primers are provided in the supplementary methods (Additional file 6). Stable U2OS cells expressing Mcm10 and its fragments were generated as described previously . Cells of almost equal confluency were lysed in proportionate volumes of Laemmli buffer for immunoblotting. To demonstrate equal protein loading in each lane, immunoblotting was performed and a nonspecific protein band was displayed. For indirect immunofluorescence studies, HeLa cells grown on glass coverslips in DMEM supplemented with 10% fetal bovine serum (FBS) were fixed with 4% formaldehyde in PBS for 10 min and then permeabilized with 0.2% Triton X-100 in PBS for 5 min. Later, the cells were blocked with 10% FBS in PBS with 0.1% Tween-20. The cells were then stained with primary antibody (1:100 or 1:500 dilution) for an hour followed by either fluorescein isothiocyanate, Alexa-488 or Alexa-594-conjugated anti-rabbit or anti-mouse antibody (1:500 dilution) at room temperature. The coverslips were then mounted with a mounting reagent with DAPI and viewed under the Nikon TE2000-S inverted fluorescence microscope. Images were captured on Evolution VF (Media Cybernetics) 12-bit color digital camera using the 'Q capture Pro' software and contrast enhancements were identically done for all the images of a particular antibody/protein in an experiment. Some images were captured using the Zeiss LSM 510 confocal microscope and viewed using the Zeiss LSM Image Browser Version 4,2,0,121 software.
We thank Prof. Avadhesha Surolia, Director, National Institute of Immunology, for his help and support in our research projects. We thank Sourabh M. Ranade, Puneeta Bhatia and Kanika Jain for their help in immunoblotting and immunofluorescence assays. This work was supported by sanctioned projects from Department of Biotechnology & Science of Technology, Government of India and NET fellowship to M.K. and A.S. The authors declare that they have no competing financial interests.
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