MCMBP promotes the assembly of the MCM2–7 hetero-hexamer to ensure robust DNA replication in human cells
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Japan
- Department of Chromosome Biology, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University, Japan
- Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Japan
Figures
Figure 1 with 1 supplement
MCM3 associates with MCM-binding protein (MCMBP) in soluble extracts.
(a) Size distribution of the MCM proteins extracted from soluble and chromatin extracts. Estimated size (using thyroglobulin, 669 kDa; ferritin, 440 kDa; aldolase, 158 kDa; and ovalbumin, 44 kDa) is …
Figure 1—figure supplement 1
MCM-binding protein (MCMBP) associates with MCM3 and other MCM proteins.
(a) Extracts used for the size fractionation assays. HSP90 and Tubulin were used as soluble protein markers. Histone H2B was used for a chromatin protein marker. (b) All MCM proteins and MCM-binding …
Figure 2 with 1 supplement
Depletion of MCM-binding protein (MCMBP) causes a defect in the formation of the MCM2–7 hexamer in the soluble extracts.
(a) Schematic illustration of MCMBP depletion by the AID2 system. Upon 5-Ph-IAA addition, mAID-Clover (mAC)-tagged MCMBP was recognized by OsTIR1(F74G) for degradation by the proteasome. (b) …
Figure 2—figure supplement 1
MCM-binding protein (MCMBP) depletion reduces the level of the MCM2–7 hexamer containing MCM3.
(a) Degradation of MCM-binding protein (MCMBP)-mAC was monitored by microscopy at 4 hr after the addition of 1 μM 5-Ph-IAA. (b) Cell-cycle distribution on Days 1, 2, and 3 after the addition of 1 μM …
Figure 3 with 1 supplement
The association between MCM3 and MCM-binding protein (MCMBP) is required for maintaining the level of the MCM2–7 hexamer and supporting cell proliferation.
(a) Illustration of the MCMBP truncations used in this study. NLS, nuclear localization signal; WB, Walker B; AF, arginine finger. (b) Interaction of MCMBP truncations with MCM3. HCT116 cells were …
Figure 3—figure supplement 1
The association between MCM3 mutants and MCM-binding protein (MCMBP).
(a) Representation of the MCM3 truncates. (b) Interaction of the MCM3 truncates with MCM-binding protein (MCMBP). FLAG-tagged MCM3 truncates were expressed and subjected to immunoprecipitation using …
Figure 4 with 2 supplements
MCM-binding protein (MCMBP) promotes MCM2–7 assembly using nascent MCM3.
(a) Size distribution of DD-FLAG-MCM3 in the presence or absence of MCMBP. Proteins were extracted and analyzed by immunoblotting at 6 or 24 hr after the addition of 0.5 μM Shield-1 with or without …
Figure 4—figure supplement 1
MCM-binding protein (MCMBP) promotes the loading of MCM2–7 containing nascent MCM3.
(a) Schematic illustration of the DD-MCM3 system. Ectopic expression of DD-(FLAG or mScarletI)-MCM3 was stabilized in the presence of a ligand, Shield-1. Without Shield-1, the fusion protein is …
Figure 4—figure supplement 2
The nuclear transport of nascent MCM4, but not that of nascent MCM3, depends on MCM-binding protein (MCMBP).
(a) The expression of the MCM4-DD was confirmed by immunoblotting using the indicated antibodies. Subcellular localization of MCM4-DD (b) and DD-MCM3 (c) with or without 1 μM of 5-Ph-IAA. In the …
Figure 5 with 1 supplement
p53-negative cells lose cell viability after MCMBP depletion.
(a) The growth of the indicated cell lines was measured every 2 days after the addition of 1 μM 5-Ph-IAA. Data represent the mean ± standard deviation (SD) of three independent experiments (Day 0 = …
Figure 5—figure supplement 1
The p53 checkpoint prevents DNA damage accumulation after the depletion of MCM-binding protein (MCMBP).
(a) Chromatin-bound MCM2 after MCM-binding protein (MCMBP) depletion to monitor the levels of replication licensing. Soluble proteins were extracted before staining with anti-MCM2 antibody. The …
Tables
Table 1
Cell lines used in this study.
| Figure | Cell line |
|---|---|
| Figure 1d | HCT116 CMV-OsTIR1F74G Stag-3FLAG-MCM3 |
| Figure 2b–d | HCT116 CMV-OsTIR1F74G MCMBP-mAC |
| Figure 3c–e, Figure 1—figure supplement 1e | HCT116 CMV-OsTIR1F74G MCMBP-mAC 2HA-MCMBP FL |
| Figure 3c–e | HCT116 CMV-OsTIR1F74G MCMBP-mAC 2HA-MCMBPΔN |
| Figure 3c–e | HCT116 CMV-OsTIR1F74G MCMBP-mAC 2HA-MCMBPΔNLS |
| Figure 4a and f | HCT116 CMV-OsTIR1F74G MCMBP-mAC PiggyBac-EF1-DD-3FLAG-MCM3 |
| Figure 4b–e | HCT116 CMV-OsTIR1F74G MCMBP-mAC PiggyBac-EF1-DD-mScarletI-MCM3 |
| Figure 5a–e | HCT116 CMV-OsTIR1F74G MCMBP-mAC p53−/− |
| Figure 4—figure supplement 2a, d | HCT116 CMV-OsTIR1F74G MCMBP-mAC PiggyBac-EF1-MCM4-3FLAG-DD |
| Figure 4—figure supplement 2b | HCT116 CMV-OsTIR1F74G MCMBP-mAC PiggyBac-EF1-MCM4-mScarletI-DD |
Table 2
Plasmids used in this study.
| Figure | Plasmid |
|---|---|
| Figure 1d | MCM3-N-tagging CRISPR in pX330 |
| Figure 1d | Stag-3FLAG-N-MCM3 donor |
| Figure 2b | MCMBP-C-tagging CRISPR in pX330 |
| Figure 2b | MCMBP-C-tagging CRISPR in pX330 |
| Figure 3a, b | CMV-2HA-MCMBP FL |
| Figure 3a, b | CMV-2HA-MCMBPΔN |
| Figure 3a, b | CMV-2HA-MCMBPΔNLS |
| Figure 3a, b | CMV-2HA-MCMBPΔmid |
| Figure 3a, b | CMV-2HA-MCMBPΔC |
| Figure 3c–e, Figure 1—figure supplement 1e | Piggy–Bac-EF1-2HA-MCMBP FL |
| Figure 3c–e | PiggyBac-EF1-2HA-MCMBPΔN |
| Figure 3c–e | PiggyBac-EF1-2HA-MCMBPΔNLS |
| Figure 4a, f | PiggyBac-EF1-DD-3FLAG-MCM3 |
| Figure 4b–e | PiggyBac-EF1-DD-mScarletI-MCM3 |
| Figure 5a–e | TP53-KO CRISPR in pX330 |
| Figure 3—figure supplement 1a, b | TP53-KO CRISPR in pX330 |
| Figure 3—figure supplement 1a, b | PiggyBac-EF1-3FLAG-MCM3 FL |
| Figure 3—figure supplement 1a, b | PiggyBac-EF1-3FLAG-MCM3ΔN |
| Figure 3—figure supplement 1a, b | PiggyBac-EF1-3FLAG-MCM3ΔMCM |
| Figure 4—figure supplement 2a, d | PiggyBac-EF1-3FLAG-MCM3ΔC |
| Figure 4—figure supplement 2b | PiggyBac-EF1-MCM4-3FLAG-DD |
Table 3
Antibodies used in this study.
| Antigen | Manufacturer | Code | Method(s) | Dilution(s) |
|---|---|---|---|---|
| MCM2 | CST | 3619 | IB and IF | 1:10,000 for IB; 1:1000 for IF |
| MCM3 | Proteintech | 15597-1-AP | IB | 1:5000 |
| MCM4 | Abcam | ab4459 | IB | 1:5000 |
| MCM5 | Proteintech | 11703-1-AP | IB | 1:3000 |
| MCM6 | Proteintech | 13347-2-AP | IB | 1:5000 |
| MCM7 | Proteintech | 11225-1-AP | IB | 1:5000 |
| MCMBP | Proteintech | 19573-1-AP | IB | 1:5000 |
| FLAG | SIGMA | F1804 | IB | 1:10,000 |
| HA | MBL | M132-3 | IB | 1:5000 |
| HSP90 | Proteintech | 60,318 | IB | 1:5000 |
| Tubulin | BioRad | 12004165 | IB | 1:10,000 |
| histone H2B | Abcam | ab1790 | IB | 1:10,000 |
| p53 | MBL | K0181-3 | IB | 1:5000 |
| p21 | CST | 2946 | IB | 1:3000 |
| gH2AX | Millipore | 05-636 | IB | 1:3000 |
| anti-rabbit IgG | Abcam | ab216773 | IB | 1:3000 |
| anti-mouse IgG | Abcam | ab216772 | IB | 1:3000 |
| anti-mouse IgG | BioRad | 12004158 | IB | 1:3000 |
| anti-rabbit IgG | BioRad | 12004161 | IB | 1:3000 |
| anti-rabbit IgG | Life Technologies | A-11037 | IF | 1:500 |
| anti-mouse IgG | Life Technologies | A-21236 | IF | 1:500 |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/77393/elife-77393-transrepform1-v1.docx
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Source data 1
Underlying data for all graphs.
- https://cdn.elifesciences.org/articles/77393/elife-77393-data1-v1.xlsx
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Source data 2
All original immunoblot images.
- https://cdn.elifesciences.org/articles/77393/elife-77393-data2-v1.zip
