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. 2022 Apr 19;11:e77393. doi: 10.7554/eLife.77393

MCMBP promotes the assembly of the MCM2–7 hetero-hexamer to ensure robust DNA replication in human cells

Yuichiro Saito 1, Venny Santosa 1, Kei-ichiro Ishiguro 2, Masato T Kanemaki 1,3,✉
Editors: Michael R Botchan4, Kevin Struhl5
PMCID: PMC9018068  PMID: 35438632

Abstract

The MCM2–7 hetero-hexamer is the replicative DNA helicase that plays a central role in eukaryotic DNA replication. In proliferating cells, the expression level of the MCM2–7 hexamer is kept high, which safeguards the integrity of the genome. However, how the MCM2–7 hexamer is assembled in living cells remains unknown. Here, we revealed that the MCM-binding protein (MCMBP) plays a critical role in the assembly of this hexamer in human cells. MCMBP associates with MCM3 which is essential for maintaining the level of the MCM2–7 hexamer. Acute depletion of MCMBP demonstrated that it contributes to MCM2–7 assembly using nascent MCM3. Cells depleted of MCMBP gradually ceased to proliferate because of reduced replication licensing. Under this condition, p53-positive cells exhibited arrest in the G1 phase, whereas p53-null cells entered the S phase and lost their viability because of the accumulation of DNA damage, suggesting that MCMBP is a potential target for killing p53-deficient cancers.

Research organism: Human

Introduction

The minichromosome maintenance 2–7 (MCM2–7) hexamer is a replicative helicase that plays a central role in eukaryotic DNA replication (Bochman and Schwacha, 2009; Masai et al., 2010). Its six subunits are evolutionally related to each other and form a hetero-hexamer. In the late M to G1 phase, the MCM2–7 hexamer is loaded onto replication origins bound with ORC1–6, to form pre-replicative complexes (pre-RCs) in which the loaded MCM2–7 makes a head-to-head double-hexamer complex (Bleichert et al., 2017; Evrin et al., 2009; Remus et al., 2009). This reaction, which is also known as licensing, is aided by CDC6 and CDT1. Importantly, the MCM2–7 complex in pre-RCs is inactive as a helicase. Subsequently, upon the activation of two kinases, that is S-CDK and CDC7, MCM2–7 in pre-RCs is converted to the active CDC45-GINS-MCM (CMG) helicase, which becomes the core of the replisome and drives the replication forks in the S phase (Bleichert et al., 2017). The replication forks sometimes stall or collapse when they encounter obstacles of both intra- and extracellular origins, leading to the accumulation of ‘replication stress’ (Hills and Diffley, 2014; Zeman and Cimprich, 2014). Failure or incompletion of DNA replication causes genome instability, potentially resulting in cell death, cancer development, or genetic disorder. Moreover, cancer cells experience replication stress intrinsically, which is thought to drive cancer evolution (Hills and Diffley, 2014). After DNA replication, unused MCM2–7 hexamers at dormant origins are removed (Chong et al., 1995; Kubota et al., 1995; Todorov et al., 1995). The meeting of two converging forks during replication termination leads to the ubiquitylation of MCM7 in the CMG helicase, which is recognized and extracted by the p97/CDC48 segregase (Maric et al., 2014; Moreno et al., 2014). Finally, the MCM subunits released from chromatin are recycled to form a functional MCM2–7 hexamer, for the next round of replication (Sedlackova et al., 2020).

Proliferating cells express high levels of the MCM2–7 hexamer, which is loaded onto origins in 3- to 10-fold excess over the level used for normal origin firing (Edwards et al., 2002). Only a subset of loaded MCM2–7 hexamers at origins are used for DNA replication, with the remaining complexes creating dormant origins, which are used as a backup when cells are challenged by replication stress. It has also been proposed that MCM2–7 in dormant origins works as a roadblock to slow the replication forks, thus safeguarding DNA replication (Sedlackova et al., 2020). In fact, metazoan cells with reduced licensed origins are sensitive to replication stress and DNA damage (Ge et al., 2007; Ibarra et al., 2008; Woodward et al., 2006). Moreover, mice expressing a reduced level of MCM2–7 are prone to developing cancers (Kawabata et al., 2011; Kunnev et al., 2010; Pruitt et al., 2007; Shima et al., 2007). The maintenance of a high level of MCM2–7 is important for genome maintenance in proliferating cells. The MCM genes are transcriptionally upregulated by E2F following growth stimulation (Leone et al., 1998; Ohtani et al., 1999). However, other than transcriptional activation for the de novo synthesis of the MCM subunits, little is known about how they are assembled into the MCM2–7 hexamer and how it is kept at high levels in proliferating cells.

The MCM-binding protein (MCMBP) was identified as a protein that associates with the MCM subunits, with the exception of MCM2, and was proposed to form an alternative MCM–MCMBP complex via the replacement of MCM2 (Sakwe et al., 2007). However, overexpressed MCMBP can interact with all MCM subunits with different affinities (Nguyen et al., 2012). MCMBP is highly conserved in eukaryotes, with the exception of its absence in budding yeast, and has an MCM-like domain with no Walker A motifs, suggesting that it shares a common ancestor with the MCM proteins. The reported phenotypes stemming from the loss of MCMBP vary among species. Sister-chromatid cohesion was defective in an ETG1/MCMBP mutant of Arabidopsis thaliana (Takahashi et al., 2010). Defects in the cell cycle and licensing were found in fission yeast after the inactivation of Mcb1/MCMBP (Ding and Forsburg, 2011; Li et al., 2011; Santosa et al., 2013). Moreover, MCMBP depletion in Xenopus egg extracts yielded a defect in the unloading of MCM2–7 from chromatin in the late S phase (Nishiyama et al., 2011), whereas its depletion in Trypanosoma brucei caused defects in gene silencing and DNA replication (Kim, 2019; Kim et al., 2013). In turn, MCMBP depletion in human cells caused nuclear deformation (Jagannathan et al., 2012). The recent literature reported that MCMBP protects the newly synthesized MCM subunits from degradation and promotes their nuclear transport using a nuclear localization signal (NLS) within MCMBP (Sedlackova et al., 2020).

Here, we report that MCMBP mainly associated with de novo synthesized MCM3 (namely, nascent MCM3) under physiological conditions and was essential for the formation of the MCM2–7 hexamer using nascent MCM3. Although the association of MCMBP with MCM3 is required for maintaining the high levels of the MCM2–7 hexamer and, thus, for supporting normal proliferation, the NLS within MCMBP was dispensable, suggesting that the main function of MCMBP lies in the promotion of the assembly of the MCM2–7 hexamer.

Results

MCM3 associates with MCMBP to form a subcomplex

We initially examined the MCM2–7 complexes in human cells. For this purpose, we prepared soluble and chromatin extracts from human colorectal cancer HCT116 cells (Figure 1—figure supplement 1a) and subsequently size fractionated the extracts using a gel filtration column (Figure 1a). In the chromatin extract, all MCM subunits were detected at around 600 kDa (Figure 1a, right), suggesting that they form a hetero-hexamer (Prokhorova and Blow, 2000). In the soluble extract, the MCM2–7 subunits were detected in the similar 600 kDa fractions, but MCM3 was also detected in the fractions of smaller molecules (Figure 1a, left). Moreover, MCMBP was also observed in smaller fractions only in the soluble extracts, suggesting that it does not bind to chromatin and may associate with MCM3 in the soluble extract. We further examined the MCM2–7 hexamer in the soluble extract using native PAGE and detected the MCM2–7 hexamer and a subcomplex composed of MCM2/4/6/7, as reported previously (Figure 1—figure supplement 1b; Prokhorova and Blow, 2000). Interestingly, 68% of MCM3 was incorporated into the MCM2–7 hexamer, with the remainder existing as a monomer or in smaller complexes (Figure 1b, c and Figure 1—figure supplement 1c; Tamberg et al., 2018). To investigate whether MCM3 associates with MCMBP, a FLAG tag was fused to the endogenous MCM3 protein (Figure 1—figure supplement 1d). We carried out FLAG immunoprecipitation from the soluble extract after fractionation for large and small proteins, and found that MCM3 is associated with MCMBP, as well as MCM5, in the smaller fractions, as reported previously (Figure 1d; Prokhorova and Blow, 2000). These results suggest that MCM3 forms a subcomplex with MCMBP. To investigate MCM proteins interacting with MCMBP further, we used a cell line expressing 2HA-MCMBP and carried out HA immunoprecipitation after fractionation (Figure 1—figure supplement 1e). In the smaller fractions, MCMBP interacted with MCM3, 4, 5, 6, and 7 as well as weakly with MCM2, suggesting that MCMBP interacts with MCM monomers with different affinities as previously reported (Kusunoki and Ishimi, 2014; Nguyen et al., 2012; Sakwe et al., 2007). In the larger fractions, MCM4, 6, and 7 were mainly detected with MCMBP, suggesting that MCMBP also interacts with the MCM4/6/7 subcomplex (Kimura et al., 1996; Prokhorova and Blow, 2000). This immunoprecipitation data also indicated that MCMBP does not stably associate with the MCM2–7 hexamer.

Figure 1. 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 indicated on the top. The intensity of each band was quantified using ImageJ and is indicated as a relative value (max = 1.0) in the graphs shown below. (b, c) Native PAGE for detecting MCM3 in the MCM2–7 hexamer. Proteins were extracted from chromatin and soluble fractions and subjected to native PAGE for immunoblotting with an anti-MCM3 antibody. The intensity of the MCM3 signal was measured using the Image Lab 6.0.1 software (BioRad). The means of the intensities of three independent experiments are shown as relative values (total = 100%). The error bar represents the SD. p values were determined using one-way analysis of variance (ANOVA) with Tukey’s test. (d) Immunoprecipitation of MCM3 for detecting associating proteins in the large and small fractions. After gel filtration analysis, the indicated fractions (#28–33 or #40–45) were pooled and subjected to immunoprecipitation with an anti-FLAG antibody. Precipitated proteins were analyzed by immunoblotting using the indicated antibodies.

Figure 1.

Figure 1—figure supplement 1. MCM-binding protein (MCMBP) associates with MCM3 and other MCM proteins.

Figure 1—figure supplement 1.

(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 protein (MCMBP) separated using native PAGE. Proteins in the soluble extracts were subjected to native PAGE, and the indicated proteins were detected by immunoblotting. (c) MCM3 in the soluble and chromatin extracts were separated by native PAGE and subjected to immunoblotting. Intensity of MCM3 signal was measured using Image Lab 6.0.1 software (BioRad). (d) The expression of the endogenous and FLAG-tagged MCM3 was confirmed by immunoblotting using the indicated antibodies. (e) MCMs interacting with MCMBP in the large and small fractions. Soluble extract prepared from cells expressing 2HA-MCMBP was fractionated as Figure 1a. Subsequently, the indicated fractions were pooled and used for HA immunoprecipitation. Precipitated proteins were analyzed by immunoblotting using the indicated antibodies.
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MCMBP depletion causes loss of MCM3 and MCM5 from the MCM2–7 hexamer

To investigate the effect of MCMBP depletion on the MCM2–7 hexamer, we generated a conditional mutant using the AID2 system (Yesbolatova et al., 2020). The MCMBP protein fused with the mini-AID (mAID) degron was rapidly degraded after the addition of an auxin analog, 5-Ph-IAA (Figure 2a, b). We confirmed that endogenous MCMBP fused with mAID-Clover (MCMBP-mAC) was mainly expressed in the nucleus and became undetectable after the addition of 5-Ph-IAA for 4 hr (Figure 2—figure supplement 1a). Even without MCMBP, we did not observe a drastic effect on the cell-cycle distribution for up to 3 days (Figure 2—figure supplement 1b). However, we found that the expression levels of MCM3 and MCM5 gradually decreased in the soluble extract (Figure 2c, soluble). Furthermore, the level of all chromatin-bound MCMs decreased, suggesting a reduced level of the MCM2–7 hexamer in the absence of MCMBP (Figure 2c, chromatin). We prepared a soluble extract after MCMBP depletion for 2 days and fractionated it, as shown in Figure 1a. We found that MCM3 was lost from the 600 kDa fractions containing the MCM2–7 hexamer and migrated only in smaller fractions (Figure 2d). Interestingly, MCM5 was also lost from the 600 kDa fractions. The loss of MCM3 from the MCM2–7 hexamer was confirmed by native PAGE (Figure 2—figure supplement 1c), and the level of the MCM2–7 hexamer became half and one-quarter on Days 1 and 2, respectively (Figure 2—figure supplement 1d). Considering that the doubling time of HCT116 is about 24 hr, the MCMBP-depleted cells might have grown using the MCM2–7 hexamer formed before MCMBP depletion. These results suggest that MCMBP is required for the incorporation of MCM3 and MCM5 into the MCM2–7 hexamer.

Figure 2. 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) MCMBP-mAC was induced to degrade by the addition of 1 μM 5-Ph-IAA for 4 hr. MCMBP expression was analyzed by immunoblotting. (c) Protein levels of soluble- and chromatin-bound MCM2–7 after MCMBP depletion. Proteins were extracted from soluble and chromatin fractions on Days 1, 2, and 3 after MCMBP depletion. The intensity of each band was measured using ImageJ and is indicated as a relative value (Day 0 = 1.0) in the graphs. Data represent the mean ± standard deviation (SD) of three independent experiments. (d) Size distribution of the MCM proteins in soluble extracts after MCMBP depletion. Soluble extracts were prepared 2 days after MCMBP depletion and analyzed as in Figure 1a. The intensity of each band was quantified using ImageJ and is indicated as a relative value (max in control = 1.0) in the graphs.

Figure 2.

Figure 2—figure supplement 1. MCM-binding protein (MCMBP) depletion reduces the level of the MCM2–7 hexamer containing MCM3.

Figure 2—figure supplement 1.

(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 5-Ph-IAA. (c, d) MCM3 in the soluble extract was separated by native PAGE on Days 1, 2, and 3 after MCMBP depletion. Intensity of the MCM3 band at the hexamer size was measured using Image Lab 6.0.1 software (BioRad). The mean of intensities of three independent experiments was indicated as relative value (hexamer at 0 d = 1.0). Error bar represents SD.
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Next, we wished to determine whether the association of MCMBP with MCM3 is essential for maintaining the high levels of the MCM2–7 hexamer. For this purpose, we initially identified the interaction domain within MCMBP. The full-length MCMBP (FL) and a mutant lacking the nuclear localization signal (ΔNLS) bound to MCM3 (Figure 3a and b). Conversely, a mutant lacking the N terminus of MCMBP (ΔN) lost its association with MCM3. We also found that the middle and C-terminal domains of the protein were necessary for the association, although the expression levels of these truncation mutants were lower than those of the other mutants. Subsequently, we found that the N terminus of MCM3 was bound to MCMBP (Figure 3—figure supplement 1a, b).

Figure 3. 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 transiently transfected with the HA-tagged MCMBP mutants and subjected to immunoprecipitation using an anti-HA antibody. Coprecipitated MCM3 was analyzed by immunoblotting. (c) Levels of the MCM2–7 hexamer in cells expressing the MCMBP mutants. The indicated MCMBP truncations were expressed in MCMBP-mAC cells; subsequently, MCMBP-mAC was depleted before preparation of the soluble extracts. Proteins were separated using native PAGE and detected using the indicated antibodies. Tubulin was used as a loading control. (d) The MCM2–7 hexamer in the cells expressing the indicated MCMBP truncation. The intensity of the MCM3 signal within the hexamer was quantified using the Image Lab 6.0.1 software (BioRad). The mean of the intensities of three independent experiments is indicated as a relative value (hexamer in control = 1.0). The error bar represents the SD. p values were determined using one-way analysis of variance (ANOVA) with Tukey’s test. (e) Growth curves were obtained 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 = 100).

Figure 3.

Figure 3—figure supplement 1. The association between MCM3 mutants and MCM-binding protein (MCMBP).

Figure 3—figure supplement 1.

(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 FLAG antibody. Coprecipitated MCMBP and MCM2 (as a control) were detected by immunoblotting. (c) Subcellular distribution of the MCMBP mutants were captured using a Delta Vision microscope.
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Next, we introduced an MCMBP transgene encoding FL, ΔN, and ΔNLS into MCMBP-mAC cells. We depleted endogenous MCMBP-mAC and assessed whether those MCMBP mutants support the retention of MCM3 in the MCM2–7 hexamer. For this purpose, we treated the cells with 5-Ph-IAA for 2 days, prepared soluble extracts and ran a native PAGE. We found that, in cells expressing MCMBP FL and ΔNLS, the level of MCM3 in the MCM2–7 hexamer was maintained (Figure 3c, d). Conversely, in the cells expressing MCMBP ΔN, the level of MCM3 was reduced. Subsequently, we analyzed cell proliferation after depletion of MCMBP-mAC. Cells with MCMBP-mAC depletion exhibited a gradual slowing of proliferation, which was the same even if MCMBP ΔN was expressed (Figure 3e). Conversely, the cells expressing MCMBP FL and ΔNLS proliferated in a manner similar to the controls after MCMBP-mAC depletion. These results indicate that the interaction of MCMBP with MCM3 is essential for maintaining MCM3 in the MCM2–7 hexamer, which is crucial for normal cell proliferation.

MCMBP promotes the assembly of the MCM2–7 hexamer using nascent MCM3

In the absence of MCMBP, the level of the MCM2–7 hexamer decreased with time (Figure 2—figure supplement 1c, d), suggesting that the formation of the nascent MCM2–7 hexamer was defective. In support of this finding, Sedlackova et al., 2020 recently reported that the stabilization and nuclear translocation of the nascent MCM subunits require MCMBP. Therefore, we reasoned that MCMBP might associate with nascent MCM3 and promote the assembly of the MCM2–7 hexamer. To monitor nascent MCM3, we introduced a transgene encoding the MCM3 protein fused with a destabilizing domain (DD) into MCMBP-mAC cells (Figure 4—figure supplement 1a). The DD-MCM3 protein was degraded in the absence of a DD stabilizing ligand, Shield-1, but was expressed after its addition (Figure 4—figure supplement 1b). We added Shield-1 to the cells with or without MCMBP and prepared soluble extracts at 0, 6, and 24 hr after its addition. Subsequently, we size fractionated the extracts and looked at nascent DD-MCM3 (Figure 4a). In the cells expressing MCMBP, DD-MCM3 showed two peaks around 160 and 600 kDa, respectively, at 6 hr, and the peak at 600 kDa corresponds to MCM3 within the MCM2–7 hexamer. Subsequently, the 600 kDa peak increased at 24 hr, indicating that more nascent MCM3 was incorporated, to form the MCM2–7 hexamer, following time. Conversely, in cells with MCMBP depletion, DD-MCM3 was detected around 160 kDa, and the peak at higher molecular weights was never observed. These observations are consistent with the hypothesis that MCMBP promotes the incorporation of nascent MCM3 into the MCM2–7 hexamer.

Figure 4. 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 1 μM 5-Ph-IAA. The intensity of each band was measured using ImageJ and is indicated as a relative value (max in control = 1.0) in the graphs. (b, c) Total DD-mScarletI-MCM3 after the addition of 0.5 μM Shield-1 was detected using a Delta Vision microscope. Circles with a dotted line indicate the nucleus. In the graphs, the central lines are medians. The boxes represent 25th and 75th percentiles, and the whiskers represent 5th and 95th percentiles. n > 200 cells per condition. AU, arbitrary units. p values were determined using one-way analysis of variance (ANOVA) with Turkey’s test. (d, e) Chromatin-bound DD-mScarletI-MCM3 was visualized after the extraction of soluble proteins. Circles with a dotted line indicate the nucleus. The data were quantified and presented as in panel c. (f) Interaction of DD-FLAG-MCM3 with other MCM proteins. After immunoprecipitation using an anti-FLAG antibody, the indicated proteins were detected by immunoblotting.

Figure 4.

Figure 4—figure supplement 1. MCM-binding protein (MCMBP) promotes the loading of MCM2–7 containing nascent MCM3.

Figure 4—figure supplement 1.

(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 degraded. (b) Expression of the DD-MCM3 was confirmed by immunoblotting using anti-MCM3 and -FLAG antibodies. (c) Experimental scheme in Figure 4b–f. (d) Cell-cycle progression of the cell used in Figure 4b–f. After releasing from G1 synchronization using lovastatin, 0.5 μM Shield-1 was added at 14 hr for expressing DD-MCM3. (e) Expression and chromatin association of DD-MCM3. Synchronized cells were released into medium containing 1 μM 5-Ph-IAA for MCMBP-depletion. DD-MCM3 was expressed by adding Shield-1. Proteins were extracted from soluble and chromatin fractions at 17, 21, or 25 hr after release for S, G2, or next G1 phase, respectively, and then analyzed by immunoblotting using anti-FLAG antibodies. (f) Mass spectrometry analysis of DD-MCM3-binding proteins with or without MCMBP. The log2 change of PSM (peptide-spectrum match) of the detected proteins was calculated. Among MCM3-binding proteins, all other MCM proteins, PRA1, and FKBP5 interacted with DD-MCM3 in an MCMBP-dependent manner.
Figure 4—figure supplement 2. The nuclear transport of nascent MCM4, but not that of nascent MCM3, depends on MCM-binding protein (MCMBP).

Figure 4—figure supplement 2.

(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 graphs, center lines are medians. The boxes represent 25th and 75th centiles, and the whiskers represent 5 and 95 percentiles. n > 100 cells per condition. AU; arbitrary units. The p values were determined by one-way analysis of variance (ANOVA) with Turkey’s test.
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We further tested whether a functional MCM2–7 hexamer is formed with nascent MCM3. To mimic the expression timing of MCM genes occurring in the late G1 to S phase (Leone et al., 1998; Ohtani et al., 1999), we initially synchronized the cells at the G1 phase using lovastatin and released them in medium with or without 5-Ph-IAA (0 hr) (Figure 4—figure supplement 1c). Subsequently, we added Shield-1 (for expressing DD-MCM3) at 14 hr, when the cells started to enter the S phase. In the presence and absence of MCMBP, the cells progressed through the S, G2 and M phases similarly and came back to the next G1 phase at 25 hr (Figure 4—figure supplement 1d). We confirmed that DD-MCM3 was expressed in the S, G2 and next G1 phases (Figure 4b, c and Figure 4—figure supplement 1e), and noted that the expression level of DD-MCM3 was lower in cells with MCMBP depletion (comparison of absence/presence of MCMBP), suggesting that MCMBP protects nascent MCM3 from degradation (Sedlackova et al., 2020). However, nascent DD-MCM3 was still detected in the nucleus (Figure 4b, c). In the same condition, we extracted soluble proteins before fixation, and DD-MCM3 binding to chromatin was observed (Figure 4d, e and Figure 4—figure supplement 1e). In cells expressing MCMBP, chromatin-bound DD-MCM3 was detected only in the next G1 phase, showing that DD-MCM3 expressed after the S phase was converted to a functional MCM2–7 hexamer, which bound chromatin in the next G1 phase. Conversely, in cells with depletion of MCMBP, chromatin-bound DD-MCM3 was not detected in the next G1 phase, suggesting a defect in the formation of a functional MCM2–7 hexamer with nascent MCM3. Furthermore, we confirmed that DD-MCM3 did not bind to the other MCM subunits in the absence of MCMBP, as assessed by IP-immunoblotting (Figure 4f) and IP-mass spectrometry analyses (Figure 4—figure supplement 1f). The latter also showed that MCM3 did not interact RPA1, a subunit of RPA, without MCMBP. We interpreted these data that DNA replication was defective because the functional MCM2–7 was lost in the absence of MCMBP. Based on these results, we concluded that MCMBP promotes the incorporation of nascent MCM3 into the MCM2–7 hexamer.

Among the six MCM subunits, MCM2 and MCM3 alone contain an NLS (Kimura et al., 1996). Recently, MCMBP, which also contains an NLS, was reported to be required for the translocation of nascent MCM4 into the nucleus (Sedlackova et al., 2020). To confirm this observation, we introduced DD-fused MCM4 (MCM4-DD) and induced its expression by adding Shield-1 (Figure 4—figure supplement 2a). Consistent with the previous report, MCM4-DD did not accumulate in the nucleus in the absence of MCMBP (Figure 4—figure supplement 2b), whereas DD-MCM3 accumulated in the nucleus possibly by using the NLS within MCM3 (Figure 4—figure supplement 2c). In the absence of MCMBP, the association of MCM3 and MCM5 with DD-MCM4 was reduced, supporting our notion that MCMBP promotes the incorporation of MCM3 into the MCM2–7 hexamer (Figure 4—figure supplement 2d, compare lanes 6 and 8).

MCMBP depletion leads to loss of cell viability in P53-negative cells

The results reported above showed that MCMBP is required for the formation of a functional MCM2–7 by promoting the incorporation of nascent MCM3 (Figure 4). Reduced levels of the MCM2–7 hexamer lower DNA replication licensing and promote genome instabilities (Ge et al., 2007; Ibarra et al., 2008; Kawabata et al., 2011; Shima et al., 2007; Woodward et al., 2006). We investigated cell proliferation after MCMBP depletion and found a gradual slowing of cell growth (Figure 5a, MCMBP p53+/+). In these cells, chromatin-bound MCM2 was downregulated with time (Figure 5—figure supplement 1a), and 5-ethynyl-2′-deoxyuridine (EdU) uptake within a fixed period was increased, suggesting increased fork speed (Figure 5—figure supplement 1b; Sedlackova et al., 2020). As expected, the levels of p53 and p21 were increased on Day 5 (Figure 5—figure supplement 1c), suggesting that they slowed cell growth because of the cell-cycle checkpoint. In fact, on Day 8, the MCMBP-depleted cells showed a higher G1 population (Figure 5b, p53+/+, control). Moreover, the addition of RO-3306, which is a CDK1 inhibitor, did not deplete this G1 population (Figure 5b, p53+/+, + RO-3306), supporting the hypothesis that the growth of cells with MCMBP depletion was slowed by the G1 checkpoint. Therefore, we reasoned that the loss of p53 might rescue the growth defect. For this purpose, we deleted the TP53 gene in the MCMBP-mAC background (p53−/−) and investigated cell growth after MCMBP depletion. Unexpectedly, p53−/− cells with MCMBP depletion still exhibited slowed growth, although they showed a slightly better growth than the p53+/+ cells (Figure 5a, MCMBP). Interestingly, p53−/− cells showed increased G2/M populations after MCMBP depletion (Figure 5b and p53−/−, control). This observation implies that p53−/− cells with depletion of MCMBP accumulated more DNA damage than did p53+/+ cells. In fact, p53−/− cells showed a greater number of nuclear 53BP1 foci and higher levels of H2AX phosphorylation than did p53+/+ cells (Figure 5c and Figure 5—figure supplement 1d, e).

Figure 5. 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 = 100). (b) Cell-cycle distribution on Day 8 after the addition of 1 μM 5-Ph-IAA. RO-3306 was added to the culture medium at 16 hr before fixation. (c) Quantification of 53BP1 foci on Day 5 after the addition of 1 μM 5-Ph-IAA. In the graphs, the central lines are medians. The boxes represent 25th and 75th percentiles, and the whiskers represent 5th and 95th percentiles. n > 200 cells per condition. p values were determined using one-way analysis of variance (ANOVA) with Turkey’s test. (d, e) Cell viability after the temporal depletion of MCMBP was determined using colony formation assay. Two hundred cells were plated and cultured for 4 or 8 days in the presence of 1 μM 5-Ph-IAA, and then released into medium without 5-Ph-IAA. The plating efficiency is shown as a relative value (Day 0 = 1). Data represent the mean of three independent experiments. p values were determined using two-way ANOVA with Šídák’s test. Model of MCM2–7 hexamer formation mediated by MCMBP (f) and the phenotypes induced after MCMBP depletion (g).

Figure 5.

Figure 5—figure supplement 1. The p53 checkpoint prevents DNA damage accumulation after the depletion of MCM-binding protein (MCMBP).

Figure 5—figure supplement 1.

(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 chromatin-bound MCM2 was detected using a BD Accuri C6 flow cytometer. 50,000 cells were analyzed for each sample. The p values were determined by one-way analysis of variance (ANOVA) with Turkey’s test. (b) EdU incorporation after MCMBP depletion. The MCMBP-mAC cells were treated with 1 μM 5-Ph-IAA for 3 or 5 days, and incubated with 10 μM EdU for 30 min. After staining the cell using the Click-iT kit, the incorporated EdU was detected by a BD Accuri C6 flow cytometer. 50,000 cells were analyzed for each sample. (c) The levels of p53 and p21 after MCMBP depletion. The indicated antibodies were used for immunoblotting. (d) 53BP1 foci after MCMBP depletion. The MCMBP-mAC cells were treated with 1 μM 5-Ph-IAA for 5 days, and then immunostained with anti-53BP1 antibody. (e) The H2AX phosphorylation after MCMBP depletion was detected by immunoblotting using anti-γH2AX antibody.
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If p53−/− cells with MCMBP depletion enter the S phase and accumulate more DNA damage, and p53+/+ cells undergo arrest in the G1 phase, we would expect that the viability of p53−/− cells should decrease more sharply than that of p53+/+ cells. To test this hypothesis, we initially depleted MCMBP for 4 or 8 days in the presence of 5-Ph-IAA and subsequently reexpressed MCMBP in medium without 5-Ph-IAA (Figure 5d). We found that p53−/− cells showed a pronounced viability loss compared with p53+/+ cells (Figure 5e). These results indicate that the p53-dependent checkpoint prohibited the cells from entering the S phase and protected genome integrity in the absence of MCMBP.

Discussion

Although a recombinant MCM2–7 hexamer has successfully been reconstructed using an overexpression system based on yeast, insect, and human cells (Jenkyn-Bedford et al., 2021; Rzechorzek et al., 2020; Yeeles et al., 2015), little is known about how the MCM2–7 hexamer is assembled in living cells. In this study, we revealed that MCMBP played a crucial role in the assembly of the MCM2–7 hexamer in human cells. MCMBP formed a complex with MCM3 via its N-terminal region and promoted the formation of the MCM2–7 hexamer (Figures 2 and 3). In the absence of MCMBP, nascent MCM3 did not bind to any other of the MCM proteins, whereas the nascent MCM4 formed an MCM2/4/6/7 complex (Figure 4 and Figure 4—figure supplement 2). As a result, the licensing level was gradually reduced as the cells proliferated, and the pre-existing MCM2–7 hexamer was distributed into the two daughter cells. The reduced licensing observed after MCMBP depletion caused a slow-growth phenotype in cells with and without p53 (Figure 5). However, the p53-negative cells became more sensitive to MCMBP depletion than did the p53-positive cells (discussed below). MCMBP is conserved in many species, including Trypanosoma, fission yeast, plants and mammalian cells (Ding and Forsburg, 2011; Kim et al., 2013; Sakwe et al., 2007; Santosa et al., 2013; Takahashi et al., 2010). We expect that the MCMBP homologs in other species might play a similar role in MCM2–7 hexamer assembly. The variable phenotypes of MCMBP mutants observed in these organisms, such as defects in sister-chromatid cohesion, cell cycle, and nuclear deformation, might stem from DNA replication with reduced licensing. Budding yeast does not possess an MCMBP ortholog, and Cdt1 strongly associates with the MCM2–7 hexamer before replication licensing and is required for the accumulation of MCM2–7 in the nucleus (Sun et al., 2013; Tanaka and Diffley, 2002). Cdt1 may also help the assembly of the hexamer in this species.

Here, we propose that MCMBP promotes the assembly of the MCM2–7 hexamer by incorporating MCM3 and MCM5 into the MCM2/4/6/7 subcomplex (Figure 5f). MCMBP may help the assembly of the MCM2–7 hexamer as a chaperone. Previous reports have indicated that overproduction of MCMBP reduces the levels of the MCM2–7 hexamer complex in Xenopus egg extracts, fission yeast, and in vitro using human proteins (Ding and Forsburg, 2011; Kusunoki and Ishimi, 2014; Nishiyama et al., 2011), showing that the amount of MCMBP regulates the equilibrium between the hexamer and the subcomplexes. Therefore, MCMBP may be a molecular chaperone for MCM2–7. Another possibility is that MCMBP co-operates with other chaperones for the assembly of the MCM2–7 hexamer. MCMBP was reported to associate with a prolyl-isomerase, that is FKBP5 (also known as FKBP51) (Taipale et al., 2014), which is involved in protein folding (Pirkl and Buchner, 2001). We found that MCM3 was associated with FKBP5 in an MCMBP-dependent manner (Figure 4—figure supplement 1f). These findings indicate that MCMBP assists the binding of FKBP5 to MCM3, resulting in MCM2–7 hexamer assembly. It has been shown that MCM3 binds to Keap1 (Mulvaney et al., 2016; Tamberg et al., 2018), which is a substrate adaptor for a CUL3 E3 ubiquitin ligase. Therefore, MCMBP may also protect nascent MCM3 from degradation via CUL3-Keap1. In fact, the expression of nascent MCM3 was reduced after MCMBP depletion (Figure 2c).

MCMBP was originally identified as a protein that binds to multiple MCM subunits, with the exception of MCM2 (Sakwe et al., 2007). However, later reports showed that MCMBP interacted with all MCM subunits with different affinities (Kusunoki and Ishimi, 2014; Nguyen et al., 2012). We confirmed this notion in the immunoprecipitation of 2HA-MCMBP (Figure 1—figure supplement 1e). Notably, in the absence of MCMBP, we detected negligible changes in the levels of MCM2, 4, 6, and 7 (Figure 2c, soluble), indicating they formed an MCM2/4/6/7 subcomplex without MCMBP. On the contrary, the levels of MCM3 and 5 were significantly reduced in this condition (Figure 2c, soluble). Taking the fact that nascent MCM3 did not interact with MCM5 in the absence of MCMBP (Figure 4—figure supplement 1f), MCM3 and MCM5 do not form a stable subcomplex and become unstable without MCMBP (Figure 5f).

Among the components of the MCM2–7 hexamer, only MCM2 and MCM3 contain NLS sequences (Kimura et al., 1996). A previous work showed that, in fission yeast, the localization of MCM2–7 in the nucleus requires hexamer formation (Pasion and Forsburg, 1999). In line with this observation, nascent MCM4 did not efficiently translocate into the nucleus when MCM2–7 hexamer formation was disturbed by MCMBP depletion (Figure 4—figure supplement 2b). Recently, Sedlackova et al. reported that the nascent MCM subcomplexes are translocated into the nucleus with the help of the NLS signal in MCMBP, and subsequently form the MCM2–7 hexamer (Sedlackova et al., 2020). We found that the NLS within MCMBP was not required for MCM2–7 hexamer formation nor for maintaining cell growth (Figure 3d, e). This implies that, in human cells, the MCM2–7 hexamer can form in the cytoplasm, and subsequently translocate into the nucleus. However, it is also possible that MCMBP ΔNLS is associated with the localization of MCM3 in the nucleus and promotes hexamer formation in that cell compartment. In support of this hypothesis, MCMBP ΔNLS was localized both in the cytoplasm and nucleus (Figure 3—figure supplement 1c). Our data support the idea that the formation of the MCM2–7 hexamer can take place both in the cytoplasm and the nucleus.

When the loading of the MCM2–7 hexamer onto DNA is reduced by knockdown of ORC, Cdc6, Cdt1, or MCM2–7, the ‘licensing checkpoint’ arrests the cell cycle at the G1 phase (McIntosh and Blow, 2012). The licensing checkpoint causes the p53-dependent activation of the CDK inhibitors p21 and p27, whereas the mechanism via which the checkpoint senses the licensing level remains unknown. When MCMBP was depleted for more than 5 days, we found that the cells were arrested in the G1 phase following the activation of the p53–p21 axis (Figure 5b and Figure 5—figure supplement 1c, e). We observed that the number of 53BP1 foci (indicators of DNA double-strand breaks [DSBs]) was slightly, albeit significantly, increased after MCMBP depletion (Figure 5c and Figure 5—figure supplement 1d, compare MCMBP + and − in p53+/+, p = 0.035). Therefore, we hypothesized that, when cells enter the S phase with reduced levels of the MCM2–7 hexamer, they accumulate small amounts of DSBs. Considering that one DSB alone is sufficient to elicit cell-cycle arrest at the G1 phase (Barlow et al., 2008), the small number of DSBs generated after reduced licensing might be a mechanism of the licensing checkpoint.

In the absence of p53, cells with MCMBP depletion entered the S phase with reduced levels of the MCM2–7 hexamer, and then lost their viability (Figure 5g). A recent article showed that MCMBP accumulates at high levels in cancer cells (Quimbaya et al., 2014), which may be related to the overexpression of MCM2–7 observed in many cancerous tissues (Gonzalez et al., 2005; Tachibana et al., 2005). Therefore, MCMBP is a potential target for cancer chemotherapy, to kill p53-deficient cancers by allowing them to enter the S phase with an insufficient number of licensed origins (McIntosh and Blow, 2012), whereas normal cells are temporarily arrested in the G0 or G1 phase.

Materials and methods

Cell culture

HCT116 cells (ATCC, #CCL-247) were cultured in McCoy’s 5A medium supplemented with 10% FBS (Sigma-Aldrich), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2. Transfection was carried out as described previously (Yesbolatova et al., 2020). For inducing the degradation of MCMBP fused with mAID-Clover, 5-Ph-IAA (BioAcademia, 30-003) was added to the culture medium at 1 μM. For introducing the expression of DD-fused proteins, Shield-1 (CheminPharma, S1) was added to the culture medium at 1 μM.

Cell lines

The original HCT116 cell line was obtained from ATCC. All HCT116 cell lines used in this study are mycoplasma negative and are listed in Table 1. To tag endogenous genes, we transfected a CRISPR plasmid targeting the N- or C-terminus cording region of gene of interest with a donor plasmid(s) to HCT116 cells constitutively expressing OsTIR1(F74G) as described previously (Saito and Kanemaki, 2021). For generating the MCMBP-mAC cells, we used a CRISPR plasmid targeting the C-terminus coding region of the MCMBP gene (target sequence: 5′-GTTTGCCCATTACTCTTCAT-3′) and two donors encoding mAID-Clover with a Neo/Hygro selection marker. After the selection in the presence of G418 (700 µg/ml) and hygromycin (100 µg/ml), the clones were isolated and the biallelic insertion was confirmed by genomic PCR. Subsequently, the expression of the MCMBP-mAID-Clover protein was confirmed by western blotting. For introducing MCMBP transgenes, we transfected a plasmid encoding HA-tagged MCMBP (full length or mutants) with a PiggyBac transposon plasmid to the MCMBP-mAC cells. After culturing the cells for a week in the presence of blasticidin S (10 µg/ml), the surviving cells were used. For introducing a MCM transgene fused with the DD, we transfected a plasmid encoding DD-tagged MCM3 or MCM4 with a PiggyBac transposon plasmid to the MCMBP-mAC cells. After the selection in the presence of blasticidin S (10 µg/ml), the clones were isolated and the expression was confirmed by Western blotting. For knocking out p53, we transfected a CRISPR plasmid targeting exon 3 of p53 gene (target sequence: 5′-CGGACGATATTGAACAATGG-3′) to the MCMBP-mAC cells. Clones were isolated and p53 expression loss was confirmed by western blotting.

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
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Plasmids

All plasmids used in this study are listed in Table 2.

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
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Preparation of cell extracts

HCT116 cells were incubated with ice-cold IP buffer (20 mM HEPES(2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic Acid)–KOH, 150 mM KOAc, 1 mM EDTA, 5% glycerol, 5 mM Mg(OAc)2, 10 mM ATP, and protease inhibitor cocktail [Merck, 11873580001]) containing 0.2% Nonidet P(NP)-40 for 2 min on ice. After low-speed centrifugation at 1200 × g, the supernatant was collected, further centrifuged at 16,000 × g and used as the soluble extract. The pellet obtained after the initial low-speed centrifugation was incubated in ice-cold IP buffer containing 1% NP-40 and 25–50 U of Benzonase (Merck, 70746) for 30 min on ice. After centrifugation at 16,000 × g, the supernatant was collected and used as the chromatin extract.

Gel filtration

Gel filtration was carried out as described previously (Coster et al., 2014). Briefly, gel filtration was performed using a Superose 6 10/300 GL column (GE Healthcare) fitted to an AKTA explorer system (GE Healthcare) at a flow speed of 0.25 ml/min at 4°C. Next, 0.5 ml of soluble and chromatin extracts was applied to the column, which had been pre-equilibrated in gel filtration buffer (40 mM HEPES–KOH, 300 mM KOAc, 5 mM Mg(OAc)2, 0.02% NP-40, 5% glycerol, 5 mM 2-mercaptoethanol and 0.1 mM PMSF(Phenylmethylsulfonyl fluoride)). Three fractions were mixed in a tube (e.g., #1–3, #4–6, etc.) and boiled in a sodium dodecyl-sulfate (SDS) sample buffer. The sizes of proteins in the fractions were estimated using thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), and ovalbumin (44 kDa).

Immunoblotting

Proteins were separated using a TGX Stain-Free gel (BioRad, 4568026) or a SuperSep Ace gel (Fujifilm Wako, 197-15011), and transferred onto an Amersham Protran 0.45 μm NC membrane (Cytiva, 10600003). The membrane was incubated with the primary antibody at 4°C overnight, and subsequently with the secondary antibody at 25°C for 1 hr. Images were acquired using a ChemiDoc Touch MP imaging system (BioRad). The antibodies used in this study are listed in Table 3.

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
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Native PAGE

Proteins extracted as described above were mixed with a loading buffer (20 mM KOAc, 0.006% bromophenol blue, and 10% glycerol) and kept on ice. Proteins were separated on a SuperSep Ace gel using the Novex Tris-Glycine Native Running Buffer (Thermo, LC2672). The gel was incubated in a transfer buffer containing 0.05% SDS and subjected to protein transfer as described in Immunoblotting.

Immunoprecipitation

Immunoprecipitation was carried out as described previously (Hustedt et al., 2019). To precipitate FLAG-tagged proteins, 15 μl of an anti-FLAG M2 affinity gel (Sigma-Aldrich, A2220) was added to extracts and incubated at 4°C for 2 hr with rotation. To precipitate HA-tagged proteins, 4 μg of an HA-tag antibody (MBL, M132-3) was mixed with 20 μl of Dynabeads Protein G (Invitrogen, #10,004D) and subsequently added to extracts. Beads were washed three times with an IP buffer containing 1% NP-40 and boiled in SDS sample buffer. The supernatant was used for immunoblotting.

Measurement of cell proliferation

Cells were plated onto a 6-well plate and cultured in the presence or absence of 1 μM 5-Ph-IAA. Growth curves were obtained by passaging cells and measuring cell density every 2 days. Relative cell density was calculated by taking the density recorded on Day 0 as 100.

Cell-cycle synchronization

Cell-cycle synchronization was carried out as described previously (Natsume et al., 2016). Briefly, cells were plated onto a 6-well plate and cultured for 2 days. Lovastatin (LKT Laboratories, M1687) was added to a final concentration of 20 μM. After 24 hr, the cells were washed once with a lovastatin-free medium and then grown in a medium containing 2 mM (±) mevalonolactone (Sigma-Aldrich, M4667) with or without 1 μM 5-Ph-IAA. The cells were collected at 14 hr (G1/S), 17 hr (S), 21 hr (G2), or 25 hr (next G1).

Survival assay

Two hundred cells were plated onto a 6-well plate and cultured in the presence or absence of 1 μM 5-Ph-IAA for 4 or 8 days. The cells were washed three times with a 5-Ph-IAA-free medium and then grown in fresh medium for 10 days. Colonies were counted after staining with crystal violet.

Microscopy

Cells grown in a poly-d-lysine-coated glass-bottom dish (MATTEK, P35GC-1.5-14C) were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde phosphate buffer (Fujifilm Wako, 161-20141) at 4°C for 1 hr. The cells were imaged on a Delta Vision deconvolution microscope (GE Healthcare) after DNA staining with Hoechst 33,342 (Invitrogen, H1399). Images were analyzed using the Volocity 6.3.1 software. For detecting all or chromatin-bound DD-MCM3, cells were trypsinized and incubated in PBS with or without 0.2% NP-40 for 2 min on ice. After washing with ice-cold PBS, the cells were fixed in 4% paraformaldehyde phosphate buffer at 4°C for 0.5 hr, mounted on a glass slide using CytoSpin 4 (Thermo) and stained with Hoechst 33,342. For the formation of 53BP1 foci, the cells were fixed in 4% paraformaldehyde phosphate buffer and incubated with the primary antibody at 25°C for 2 hr. After washing, the cells were incubated with the secondary antibody at 25°C for 1 hr and stained with Hoechst 33,342.

Flow cytometry

Cells were plated onto a 6-well plate and cultured in the presence or absence of 1 μM 5-Ph-IAA. For RO-3306 treatment, RO-3306 (Sigma-Aldrich, SML0569) was added to 9 μM for 16 hr before fixation. The cells were fixed in 70% ethanol, washed with PBS and resuspended in PBS containing 1% BSA(Bovine Serum Albumin), 50 μg/ml of RNase A and 40 μg/ml of propidium iodide. For detecting MCM2, cells were incubated in PBS with 0.2% NP-40 for 2 min on ice. After washing with ice-cold PBS, the cells were fixed in 4% paraformaldehyde phosphate buffer and incubated with the primary antibody at 25°C for 2 hr. After washing, the cells were incubated with the secondary antibody at 25°C for 1 hr and resuspended in PBS containing 1% BSA, 50 μg/ml of RNase A, and 40 μg/ml of propidium iodide. For detecting DNA synthesis, cells were incubated with 10 μM EdU for 0.5 hr. EdU was visualized using the Click-iT Plus EdU Imaging Kit (Thermo, C10640), according to the manufacturer’s instructions. After DNA staining at 37°C for 0.5 hr, the cells were analyzed using a BD Accuri C6 flow cytometer (BD Biosciences) and the FCS4 Express Cytometry software (DeNovo Software); 10,000 (for cell cycle) or 50,000 (for detecting MCM2 and DNA synthesis) cells were analyzed for each sample.

Mass spectrometry

To purify FLAG-tagged DD-MCM3, 3 × 107 cells were incubated with ice-cold IP buffer containing 1% NP-40 and 150 U of Benzonase for 30 min on ice. After centrifugation at 16,000 × g, the supernatant was collected and subjected to the IP experiment. Next, 5 μg of a FLAG M2 antibody (Sigma-Aldrich) was mixed with 50 μl of Dynabeads Protein G, and subsequently processed through an extraction step and incubated at 4°C for 2 hr with rotation. Beads were washed three times with an IP buffer containing 1% NP-40. Bound proteins were eluted using IP buffer containing 0.25 mg/ml of 3× FLAG peptides (Sigma-Aldrich) at 25°C for 5 min. The elution step was repeated a total of three times and the eluent was collected into a tube. Proteins were precipitated with acetone by centrifugation at 16,000 × g, washed with methanol and dissolved in SDS sample buffer. Proteins were run for a few centimetres in SDS–polyacrylamide gel electrophoresis (PAGE). Gel bands containing proteins were excised, cut into approximately 1-mm-sized pieces and used for in-gel digestion by trypsinization before mass spectrometry. Proteins in the gel pieces were reduced with DTT (20291, Thermo Fisher Scientific), alkylated with iodoacetamide (90034, Thermo Fisher Scientific) and digested with trypsin and lysyl endopeptidase (Promega, USA) in a buffer containing 40 mM ammonium bicarbonate, pH 8.0, overnight at 37°C. The resultant peptides were analyzed on an Advance UHPLC system (AMR/Michrom Bioscience) coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific), and the raw mass spectrum was processed using Xcalibur (Thermo Fisher Scientific). The raw LC–MS/MS data were analyzed against NCBI nonredundant protein and UniProt using Proteome Discoverer version 1.4 (Thermo Fisher Scientific) using the Mascot search engine, version 2.5 (Matrix Science). A decoy database comprising either randomized or reversed sequences in the target database was used for false discovery rate (FDR) estimation, and the Percolator algorithm was used to evaluate false positives. Search results were filtered against 1% global FDR for a high confidence level.

Acknowledgements

We thank Ms Mayumi Takahashi, Tomoko Suzuki, and Shizuoko Endo for technical support. We also thank Drs Yumiko Kurokawa, Yasuto Murayama, and Yasushi Saeki for discussion, and Dr Naoki Tani for the MS analyses. This research was supported by JSPS KAKENHI grants (JP21K15021 to YS; JP20H05396 and JP21H04719 to MTK), a JST CREST grant (JPMJCR21E6 to MTK), a research grant from the Tokyo Biomedical Research Foundation to MTK and the Joint Usage/Research Centre for Developmental Medicine, IMEG, Kumamoto University.

Funding Statement

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Contributor Information

Masato T Kanemaki, Email: mkanemak@nig.ac.jp.

Michael R Botchan, University of California, Berkeley, United States.

Kevin Struhl, Harvard Medical School, United States.

Funding Information

This paper was supported by the following grants:

  • Japan Society for the Promotion of Science JP21K15021 to Yuichiro Saito.

  • Japan Society for the Promotion of Science JP20H05396 to Masato T Kanemaki.

  • Japan Society for the Promotion of Science JP21H04719 to Masato T Kanemaki.

  • Japan Science and Technology Agency JPMJCR21E6 to Masato T Kanemaki.

  • Tokyo Biochemical Research Foundation research grant to Masato T Kanemaki.

  • Kumamoto University research grant to Masato T Kanemaki.

Additional information

Competing interests

No competing interests declared.

No competing interests declared.

Author contributions

Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing - review and editing.

Investigation, Writing - review and editing.

Data curation, Investigation, Writing - review and editing.

Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – original draft.

Additional files

Transparent reporting form
Source data 1. Underlying data for all graphs.
elife-77393-data1.xlsx (355.1KB, xlsx)
Source data 2. All original immunoblot images.
elife-77393-data2.zip (11.8MB, zip)

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

References

  1. Barlow JH, Lisby M, Rothstein R. Differential regulation of the cellular response to DNA double-strand breaks in G1. Molecular Cell. 2008;30:73–85. doi: 10.1016/j.molcel.2008.01.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bleichert F, Botchan MR, Berger JM. Mechanisms for initiating cellular DNA replication. Science. 2017;355:eaah6317. doi: 10.1126/science.aah6317. [DOI] [PubMed] [Google Scholar]
  3. Bochman ML, Schwacha A. The Mcm complex: unwinding the mechanism of a replicative helicase. Microbiology and Molecular Biology Reviews. 2009;73:652–683. doi: 10.1128/MMBR.00019-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chong JP, Mahbubani HM, Khoo CY, Blow JJ. Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature. 1995;375:418–421. doi: 10.1038/375418a0. [DOI] [PubMed] [Google Scholar]
  5. Coster G, Frigola J, Beuron F, Morris EP, Diffley JFX. Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase. Molecular Cell. 2014;55:666–677. doi: 10.1016/j.molcel.2014.06.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ding L, Forsburg SL. Schizosaccharomyces pombe minichromosome maintenance-binding protein (MCM-BP) antagonizes MCM helicase. The Journal of Biological Chemistry. 2011;286:32918–32930. doi: 10.1074/jbc.M111.282541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Edwards MC, Tutter AV, Cvetic C, Gilbert CH, Prokhorova TA, Walter JC. MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts. The Journal of Biological Chemistry. 2002;277:33049–33057. doi: 10.1074/jbc.M204438200. [DOI] [PubMed] [Google Scholar]
  8. Evrin C, Clarke P, Zech J, Lurz R, Sun J, Uhle S, Li H, Stillman B, Speck C. A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. PNAS. 2009;106:20240–20245. doi: 10.1073/pnas.0911500106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ge XQ, Jackson DA, Blow JJ. Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress. Genes & Development. 2007;21:3331–3341. doi: 10.1101/gad.457807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gonzalez MA, Tachibana KK, Laskey RA, Coleman N. Control of DNA replication and its potential clinical exploitation. Nature Reviews. Cancer. 2005;5:135–141. doi: 10.1038/nrc1548. [DOI] [PubMed] [Google Scholar]
  11. Hills SA, Diffley JFX. DNA replication and oncogene-induced replicative stress. Current Biology. 2014;24:R435–R444. doi: 10.1016/j.cub.2014.04.012. [DOI] [PubMed] [Google Scholar]
  12. Hustedt N, Saito Y, Zimmermann M, Álvarez-Quilón A, Setiaputra D, Adam S, McEwan A, Yuan JY, Olivieri M, Zhao Y, Kanemaki MT, Jurisicova A, Durocher D. Control of homologous recombination by the HROB-MCM8-MCM9 pathway. Genes & Development. 2019;33:1397–1415. doi: 10.1101/gad.329508.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ibarra A, Schwob E, Méndez J. Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication. PNAS. 2008;105:8956–8961. doi: 10.1073/pnas.0803978105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jagannathan M, Sakwe AM, Nguyen T, Frappier L. The MCM-associated protein MCM-BP is important for human nuclear morphology. Journal of Cell Science. 2012;125:133–143. doi: 10.1242/jcs.089938. [DOI] [PubMed] [Google Scholar]
  15. Jenkyn-Bedford M, Jones ML, Baris Y, Labib KPM, Cannone G, Yeeles JTP, Deegan TD. A conserved mechanism for regulating replisome disassembly in eukaryotes. Nature. 2021;600:743–747. doi: 10.1038/s41586-021-04145-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kawabata T, Luebben SW, Yamaguchi S, Ilves I, Matise I, Buske T, Botchan MR, Shima N. Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression. Molecular Cell. 2011;41:543–553. doi: 10.1016/j.molcel.2011.02.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kim H-S, Park SH, Günzl A, Cross GAM. MCM-BP is required for repression of life-cycle specific genes transcribed by RNA polymerase I in the mammalian infectious form of Trypanosoma brucei. PLOS ONE. 2013;8:e57001. doi: 10.1371/journal.pone.0057001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim HS. Genome-wide function of MCM-BP in Trypanosoma brucei DNA replication and transcription. Nucleic Acids Research. 2019;47:634–647. doi: 10.1093/nar/gky1088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kimura H, Ohtomo T, Yamaguchi M, Ishii A, Sugimoto K. Mouse MCM proteins: complex formation and transportation to the nucleus. Genes to Cells. 1996;1:977–993. doi: 10.1046/j.1365-2443.1996.840284.x. [DOI] [PubMed] [Google Scholar]
  20. Kubota Y, Mimura S, Nishimoto S, Takisawa H, Nojima H. Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Cell. 1995;81:601–609. doi: 10.1016/0092-8674(95)90081-0. [DOI] [PubMed] [Google Scholar]
  21. Kunnev D, Rusiniak ME, Kudla A, Freeland A, Cady GK, Pruitt SC. DNA damage response and tumorigenesis in Mcm2-deficient mice. Oncogene. 2010;29:3630–3638. doi: 10.1038/onc.2010.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kusunoki S, Ishimi Y. Interaction of human minichromosome maintenance protein-binding protein with minichromosome maintenance 2-7. The FEBS Journal. 2014;281:1057–1067. doi: 10.1111/febs.12668. [DOI] [PubMed] [Google Scholar]
  23. Leone G, DeGregori J, Yan Z, Jakoi L, Ishida S, Williams RS, Nevins JR. E2F3 activity is regulated during the cell cycle and is required for the induction of S phase. Genes & Development. 1998;12:2120–2130. doi: 10.1101/gad.12.14.2120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li J, Schnick J, Hayles J, MacNeill SA. Purification and functional inactivation of the fission yeast MCM(MCM-BP) complex. FEBS Letters. 2011;585:3850–3855. doi: 10.1016/j.febslet.2011.10.033. [DOI] [PubMed] [Google Scholar]
  25. Maric M, Maculins T, De Piccoli G, Labib K. Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication. Science (New York, N.Y.) 2014;346:1253596. doi: 10.1126/science.1253596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Masai H, Matsumoto S, You Z, Yoshizawa-Sugata N, Oda M. Eukaryotic chromosome DNA replication: where, when, and how? Annual Review of Biochemistry. 2010;79:89–130. doi: 10.1146/annurev.biochem.052308.103205. [DOI] [PubMed] [Google Scholar]
  27. McIntosh D, Blow JJ. Dormant origins, the licensing checkpoint, and the response to replicative stresses. Cold Spring Harbor Perspectives in Biology. 2012;4:a012955. doi: 10.1101/cshperspect.a012955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moreno SP, Bailey R, Campion N, Herron S, Gambus A. Polyubiquitylation drives replisome disassembly at the termination of DNA replication. Science (New York, N.Y.) 2014;346:477–481. doi: 10.1126/science.1253585. [DOI] [PubMed] [Google Scholar]
  29. Mulvaney KM, Matson JP, Siesser PF, Tamir TY, Goldfarb D, Jacobs TM, Cloer EW, Harrison JS, Vaziri C, Cook JG, Major MB. Identification and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate. The Journal of Biological Chemistry. 2016;291:23719–23733. doi: 10.1074/jbc.M116.729418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors. Cell Reports. 2016;15:210–218. doi: 10.1016/j.celrep.2016.03.001. [DOI] [PubMed] [Google Scholar]
  31. Nguyen T, Jagannathan M, Shire K, Frappier L. Interactions of the human MCM-BP protein with MCM complex components and Dbf4. PLOS ONE. 2012;7:e35931. doi: 10.1371/journal.pone.0035931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nishiyama A, Frappier L, Méchali M. MCM-BP regulates unloading of the MCM2-7 helicase in late S phase. Genes & Development. 2011;25:165–175. doi: 10.1101/gad.614411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ohtani K, Iwanaga R, Nakamura M, Ikeda M, Yabuta N, Tsuruga H, Nojima H. Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F. Oncogene. 1999;18:2299–2309. doi: 10.1038/sj.onc.1202544. [DOI] [PubMed] [Google Scholar]
  34. Pasion SG, Forsburg SL. Nuclear localization of Schizosaccharomyces pombe Mcm2/Cdc19p requires MCM complex assembly. Molecular Biology of the Cell. 1999;10:4043–4057. doi: 10.1091/mbc.10.12.4043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pirkl F, Buchner J. Functional analysis of the Hsp90-associated human peptidyl prolyl cis/trans isomerases FKBP51, FKBP52 and Cyp40. Journal of Molecular Biology. 2001;308:795–806. doi: 10.1006/jmbi.2001.4595. [DOI] [PubMed] [Google Scholar]
  36. Prokhorova TA, Blow JJ. Sequential MCM/P1 subcomplex assembly is required to form a heterohexamer with replication licensing activity. The Journal of Biological Chemistry. 2000;275:2491–2498. doi: 10.1074/jbc.275.4.2491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pruitt SC, Bailey KJ, Freeland A. Reduced Mcm2 expression results in severe stem/progenitor cell deficiency and cancer. Stem Cells (Dayton, Ohio) 2007;25:3121–3132. doi: 10.1634/stemcells.2007-0483. [DOI] [PubMed] [Google Scholar]
  38. Quimbaya M, Raspé E, Denecker G, De Craene B, Roelandt R, Declercq W, Sagaert X, De Veylder L, Berx G. Deregulation of the replisome factor MCMBP prompts oncogenesis in colorectal carcinomas through chromosomal instability. Neoplasia (New York, N.Y.) 2014;16:694–709. doi: 10.1016/j.neo.2014.07.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Remus D, Beuron F, Tolun G, Griffith JD, Morris EP, Diffley JFX. Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Cell. 2009;139:719–730. doi: 10.1016/j.cell.2009.10.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rzechorzek NJ, Hardwick SW, Jatikusumo VA, Chirgadze DY, Pellegrini L. CryoEM structures of human CMG-ATPγS-DNA AND CMG-AND-1 complexes. Nucleic Acids Research. 2020;48:6980–6995. doi: 10.1093/nar/gkaa429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Saito Y, Kanemaki MT. Targeted Protein Depletion Using the Auxin-Inducible Degron 2 (AID2) System. Current Protocols. 2021;1:e219. doi: 10.1002/cpz1.219. [DOI] [PubMed] [Google Scholar]
  42. Sakwe AM, Nguyen T, Athanasopoulos V, Shire K, Frappier L. Identification and characterization of a novel component of the human minichromosome maintenance complex. Molecular and Cellular Biology. 2007;27:3044–3055. doi: 10.1128/MCB.02384-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Santosa V, Martha S, Hirose N, Tanaka K. The fission yeast minichromosome maintenance (MCM)-binding protein (MCM-BP), Mcb1, regulates MCM function during prereplicative complex formation in DNA replication. The Journal of Biological Chemistry. 2013;288:6864–6880. doi: 10.1074/jbc.M112.432393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sedlackova H, Rask MB, Gupta R, Choudhary C, Somyajit K, Lukas J. Equilibrium between nascent and parental MCM proteins protects replicating genomes. Nature. 2020;587:297–302. doi: 10.1038/s41586-020-2842-3. [DOI] [PubMed] [Google Scholar]
  45. Shima N, Alcaraz A, Liachko I, Buske TR, Andrews CA, Munroe RJ, Hartford SA, Tye BK, Schimenti JC. A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice. Nature Genetics. 2007;39:93–98. doi: 10.1038/ng1936. [DOI] [PubMed] [Google Scholar]
  46. Sun J, Evrin C, Samel SA, Fernández-Cid A, Riera A, Kawakami H, Stillman B, Speck C, Li H. Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA. Nature Structural & Molecular Biology. 2013;20:944–951. doi: 10.1038/nsmb.2629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tachibana K-EK, Gonzalez MA, Coleman N. Cell-cycle-dependent regulation of DNA replication and its relevance to cancer pathology. The Journal of Pathology. 2005;205:123–129. doi: 10.1002/path.1708. [DOI] [PubMed] [Google Scholar]
  48. Taipale M, Tucker G, Peng J, Krykbaeva I, Lin Z-Y, Larsen B, Choi H, Berger B, Gingras A-C, Lindquist S. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways. Cell. 2014;158:434–448. doi: 10.1016/j.cell.2014.05.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Takahashi N, Quimbaya M, Schubert V, Lammens T, Vandepoele K, Schubert I, Matsui M, Inzé D, Berx G, De Veylder L. The MCM-binding protein ETG1 aids sister chromatid cohesion required for postreplicative homologous recombination repair. PLOS Genetics. 2010;6:e1000817. doi: 10.1371/journal.pgen.1000817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tamberg N, Tahk S, Koit S, Kristjuhan K, Kasvandik S, Kristjuhan A, Ilves I. Keap1-MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa. Scientific Reports. 2018;8:12136. doi: 10.1038/s41598-018-30562-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tanaka S, Diffley JFX. Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2-7 during G1 phase. Nature Cell Biology. 2002;4:198–207. doi: 10.1038/ncb757. [DOI] [PubMed] [Google Scholar]
  52. Todorov IT, Attaran A, Kearsey SE. BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication. The Journal of Cell Biology. 1995;129:1433–1445. doi: 10.1083/jcb.129.6.1433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Woodward AM, Göhler T, Luciani MG, Oehlmann M, Ge X, Gartner A, Jackson DA, Blow JJ. Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress. The Journal of Cell Biology. 2006;173:673–683. doi: 10.1083/jcb.200602108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yeeles JTP, Deegan TD, Janska A, Early A, Diffley JFX. Regulated eukaryotic DNA replication origin firing with purified proteins. Nature. 2015;519:431–435. doi: 10.1038/nature14285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano R, Nakaoka H, Fukui K, Gamo K, Tominari Y, Takeuchi H, Saga Y, Hayashi K-I, Kanemaki MT. The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice. Nature Communications. 2020;11:5701. doi: 10.1038/s41467-020-19532-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zeman MK, Cimprich KA. Causes and consequences of replication stress. Nature Cell Biology. 2014;16:2–9. doi: 10.1038/ncb2897. [DOI] [PMC free article] [PubMed] [Google Scholar]

Editor's evaluation

Michael R Botchan 1

This study is an important advance in the DNA replication field as the work clearly shows for the first time that the protein MCMBP is essential for the assembly of the MCM 2-7 hexamer. The levels of the hetero-hexamer are critical for genome maintenance and while more hexamers may be loaded to the DNA fiber during one cell cycle than used, such an excess is required as back-ups for replication completion requires these dormant and licensed origins.

Decision letter

Editor: Michael R Botchan1
Reviewed by: Michael R Botchan2, Karim Labib3

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "MCMBP maintains genome integrity by protecting the MCM subunits from degradation" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

However, the reviewers recognized the potential importance of the observation that MCMBP might control the stability of the MCM2-7 helicase subunits, and indeed there was some data to support the conclusion in the paper, but it was not definitive. The reviewers noted that the conclusion that MCMBP stabilizes the MCM2-7 subunits was not demonstrated to the level that would allow acceptance of the current manuscript. If the authors can provide additional data that clearly demonstrate that MCMBP specifically stabilizes MCM2-7 subunits, then a revised manuscript will be considered, but the reviewers comments copied below will need to be addressed.

Reviewer #1:

The authors report that MCMBP, a protein that is known to bind to some subunits of the MCM helicase complex, is involved in maintaining the stability of some of the MCM subunits. The authors created a cell line that has auxin-inducible degradation of a tagged MCMBP protein and they find that in the presence of auxin, MCMBP disappears by 6 hr. The cells keep proliferating for 4 days with little effect, but then start to slow down compared to the absence of auxin. They also report that MCMBP is associated with subcomplexes of MCM subunits, particularly MCM5, 6 and 7, which is a potentially interesting observation. They find that upon depletion of MCMBP, some MCM subunits are unstable after a long time (from 24-96 hr). They show that the MCMBP depleted cells have a difficulty progressing through S phase.

The paper suffers from overinterpretation of the data because of the lack of any specificity of connecting MCMBP to the stability of MCM subunits. As noted below, it could be simply a loss of cell proliferation that causes selective loss of MCM subunits. The authors did not attempt to assess the levels of other pre-RC protein levels upon addition of auxin (e.g., ORC subunits, CDC6, CDT1). They did not perform critical control experiments by treating the parent HCT116-OsTIR cells with auxin and measuring any effect of auxin on these cells over the 24 to 96 hr time courses. They also do not describe the Methods well.

1. There is no information about the tagging of the MCMBP gene in the paper, except a theoretical diagram in Figure S2. There are presumably two alleles of the MCMBP gene in HCT116 cells and it was not clear if both alleles were tagged. Further, the methods describe the use of neomycin or hygromycin as selection agents, but the actual cell line used for the experiments is not described in any detail. Was it neomycin resistant, or hygromycin resistant?

2. The Methods section does not describe the HCT116 cells with OsTIR expressed and whether this expression is constitutive or induced. Only HCT116 cells are mentioned in the Methods.

3. The authors should use the appropriate figure labeling for eLife papers, such as Figure 1, supplement 1, etc.

4. Figure 2, panel A. What are #1 and #2? Are they independent cell lines? If so, describe them and whether they are really independent, or clines form the same isolation.

5. The results in Figure 2 show the effect of adding auxin on MCMBP protein levels, but only a 24 hr time point is shown. Since the protein has a short half life of ~ 3 hr when cycloheximide was added, the protein should disappear faster than 24 hr. A time course of protein levels after auxin treatment must be shown. It is noted that the time course is provided in Figure 3, but it should be presented first.

6. DNA staining: Figure 2 say the DNA was stained with SiR-DNA but the Methods state that Hoechst33342 stain was used for microscopy. Which is correct?

7. Figure 2B. Which antibody was used to detect MCMBP-mAC, or was the fluorescent protein detected? The Figure legend does not describe what is shown.

8. Figure 2C. It is necessary to perform the time course over a longer period than 5 days since it is not clear if MCMBP is essential, or its absence just slows proliferation. Only a longer proliferation time course will clarify this point, assuming that auxin removes all of the MCMBP-mAC, which is also not clear. 10% protein remaining would not be detected in an experiment shown in Figure 2A.

9. Figure 3. Why is MCM2, MCM4 and MCM detected in the HCT116-mAC cells (but nit MCM5) and then in RPE and HeLa cells MCM5 is added to the list. Why not detect all MCM subunits.

10. A control for the experiment in Figure 3A s needed. Treating HCT116-OsTIR cells with auxin over a time course for 48 hrs is needed since this cell line should not be sensitive to auxin. It is formally possible that auxin slows cell proliferation independent of loss of MCMBP. For example, auxin lowers MCM4 mRNA levels in HCT116-OsTIR cells (Figure S3). This is a necessary control.

11. Page 8, lines 13-15. This conclusion that MCMBP protects MCM5, 6 and 7 from degradation is far too premature based on the data in Figure 3. It could simply be that MCMBP depletion slows cell proliferation, and this indirectly affects MCM subunit levels. If this is specific, then other pre-RC protein such as ORC, CDC6 and CDT1 should not change. These must be measured.

12. A major problem is in Figure 4C. The cells were treated for 4 days with auxin (one day in the presence of lovastatin) and yet the reduction in MCM7 and MCM7 is minimal, just the same as MCM2. But in Figure 3A, a dramatic reduction is shown at 48 hours. The auxin treatment for 96 hrs in Figure 4C should show at least a reduction as great as shown at 48 he in Figure 3A, but it does not. The is clearly a difference on chromatin, but this could be a secondary effect of cell proliferation.

13. Figure 4C and 4D. Again, a control experiment using auxin treatment of HCT116-OsTIR cells for 96 hr is needed.

14. Page 10, lines 3-4 and Figure 4F. This conclusion is far too strong based on the data. The effect could be indirect since the time between MCMBP depletion upon addition of auxin (stated to be 3 hr) and the time of depletion of MCM subunits at least 24 hr for partial depletion and in Figure 4, weak depletion after 96 hr, makes it possible that the effect is due to loss of cell proliferation and not a direct effect.

Reviewer #2:

Significance. In addition to the individual subunits that comprise the Mcm2-7 helicase, many eukaryotes contain additional genes that encode protein that are evolutionarily related to Mcm2-7. One such proteins has been named MCMBP (Mcm binding protein) which is found I the genomes of most eukaryotes except for budding yeast. Despite its prevalence, the function of this protein has remained obscure and controversial. In this report, the authors generate and use an auxin-inducible degron to generate a conditional allele of the MCMBP gene in human cell culture to provide evidence that the role of MCMBP in this system is to prevent the inappropriate degradation of Mcm subunits in actively growing cells.

Experimental design. The unspoken assumption in this paper as well as this area of study is that since MCMBP was first identified as an MCM2-7 binding protein, MCMBP likely exerts its sole function through physical interaction with all or some of the MCM2-7 subunits. Although a reasonable conjecture, I'd be surprised that the reality is this tidy. Although to date the main effects of various types of MCMBP mutations can be rationalize as some type of replication-related defect, could this be through modulation of Mcm8 or 9? Perhaps due to its similarity to an Mcm2-7 subunit, could it be that MCMBP exerts its effect by interacting with the replication factors that Mcm2-7 normally interacts with?

The above issues extend beyond the scope of the current study. However, minimally one should ascertain if the observed defects of the MCMBP mutant are direct (i.e., require physical interaction between MCMBP and Mcm2-7) or are indirect (that binding between MCMBP and Mcm2-7 is not required). Given our current knowledge of MCMBP, one should be able generate and test MCMBP mutants that specifically do not bind Mcm subunits yet lack other collateral problems. Such data would considerably strengthen the paper and help elevate its significance from descriptive to mechanistic.

Nature of the conditional auxin-inducible MCMBP degron. In this study, the MCMBP gene was tagged with both an auxin-inducible degron construct as well as with GFP CLOVER. The expression level of the tagged MCMBP, in the presence and absence of auxin, are shown in a western blot in Figure 2A. The good news is that auxin addition results in rapid depletion of MCMBP. However, relative to the non-degron parent cell line, the expression level of the MCMBP degron construct is vastly reduced relative to the wild type protein (levels not quantified, but by eye may be <25% of the parent cell line). This problem is massaged as the authors typically show subsequent results {plus minus} auxin, not with respect to the parental cell line lacking the auxin construct.

The nature of this construct raises the potential problem that it may be producing off-target effects. In the absence of auxin, how functional is the degron mutant compared to the wild type parent cell line (e.g., growth, viability, cell cycle progression, DNA damage)? It is very difficult to evaluate the loss of MCMBP upon auxin addition unless one has carefully evaluated the original construct in the absence of auxin.

Auxin control. In addition to the above considerations, it is important to show that in the original non-degron parental cell line, that auxin addition has no effect on the stability of either MCMBP or MCM2-7. This essential control is not provided.

HeLa cell experiment. In Figures 3C and D, the key experiment using the auxin cell line (resulting in a reduction in the level of Mcm subunits after McmBP is degraded), is replicated by siRNA experiments in 2 independent cell lines (hTERT RPE and HeLa). However, only the experiment in the hTERT RPE cell line appears to replicate the basic results in the auxin cell line (i.e., that addition of the MCBP siRNA reduces the levels of Mcm subunits). To my eye, I see no reduction in the levels of MCM subunits in the parallel experiment using the HeLa cell line. This unfortunate little problem is conveniently not mentioned in the text.

Reviewer #3:

MCMBP is a poorly characterised protein found in most eukaryotes, which was previously shown to associate with the MCM2-7 proteins that form the core of the replicative helicase at DNA replication forks. Various functions of MCMBP have been suggested previously, such as a role in unloading MCM2-7 from chromatin in late S-phase. This manuscript provides interesting evidence to argue that MCMBP is actually a form of chaperone, which is important for proliferating cells to maintain the very high level of expression of MCM2-7. The data are generally of high quality and the study should be of considerable interest to those in the chromosome duplication and genome integrity fields.

1. The authors present evidence to indicate that MCMBP protects MCM2-7 proteins from degradation, but also "do not rule out the possibility that MCMBP helps transport nascent MCM subunits from the cytoplasm to the nucleus" (page 10, lines 19-21). Does it really take 5 days for MCM2-7 proteins to be lost from the nucleus upon rapid degradation of MCMBP-AID (as in Figure 3B), or is the effect also seen at earlier times (which might then be distinguishable from the effect of MCMBP on MCM2-7 stability)?

2. The authors suggest that MCMBP also works as a chaperone for MCM8-9 (page 11, lines 19-23), which are distantly related to MCM2-7 and function during homologous recombination. The authors mention unpublished data to show that MCMBP interacts with MCM8-9, and presumably they have already tested whether levels of MCM8-9 drop after depletion of MCMBP. If so, this would be a nice addition to the manuscript, so the authors might consider including these findings?

3. Comparing Figure 3D and 3C, the depletion by siRNA of MCMBP in HeLa cells appears to be just as efficient as in RPE1 cells, yet the effect on MCM2-7 levels is apparently much weaker in HeLa than in RPE1 cells. Do the authors have any explanation for this?

4. The authors suggest that MCMBP functions together with the chaperone FKBP51 to "promote the formation of MCM2-7 hexamers in the nucleus" (page 11, lines 4-6). Have they tested the effect of siRNA depletion of FKBP51 on the level of MCM2-7 proteins and MCMBP?

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "MCMBP promotes the assembly of the MCM2-7 hetero-hexamer to ensure robust DNA replication in human cells" for further consideration by eLife. Your revised article has been evaluated by Kevin Struhl (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Two of the 4 reviewers thought the manuscript could be published without any revisions whilst Reviewers 1 and 4 had found similar questions that could readily be addressed with minor new additions or clarifications of the text.

1) When found in extracts the MCMBP fractionates into two forms one where Mcm3 is found and the other a high molecular weight form. It may be that the low molecular weight form has other mcm subunits aside from 3. Please address this point as described below. Further, a perhaps minor one is the nature of high molecular weight form is it MCMBP and the mcm2-7?

2) Figure 4 the supplement needs some clarification and what do you make of the fact that RPA levels decrease when MCMBP is lost?

Reviewer #1:

The MCM Binding Protein (MCMBP) has been implicated in many processes in cell cycle progression including DNA replication and DNA repair. The work presented in this paper shows for the first time that loss of function of MCMBP by classical cell and molecular biological methods lead to lower levels of pools of the MCM 2-7 hexamer. Moreover, MCMBP binds directly to Mcm3 and through deletions and biochemical methods in vitro, the authors show that loss of this interaction leads to lower levels of the hexamer when such defective alleles are expressed in cells. Given that the hexamer is the core of the helicase during elongation and numbers of origins are established through double hexamer formation these results will be widely impactful. Genome integrity depends upon an abundance of potential origins, particularly under stressed conditions where back -up or late origins need to finish the replication domain. The methods applied are good to establish these points but provide little direct insights into how the MCMBP actually works mechanistically in establishing these normal levels of hexamers. Is it through prevention of degradation of Mcm 3 or in an actual pathway to assembly? Nevertheless, such questions raised by the present study actually raise questions that will provide impetus for future work.

1. My only recommendation of importance for the authors is to clarify with further studies the suggestion that other Mcm's are present within the low molecular weight sub-complexes found in extracts. Figure 1 surely shows Mcm3 and also 5 and 7. There are many ways to clarify this point including a further column step or immunoprecipitation/western.

Reviewer #2:

MCMBP is a distant relative of the MCM2-7 proteins, which form the catalytic core of the essential DNA helicase at eukaryotic DNA replication forks. MCMBP was previously reported to associate with MCM2-7 proteins and a recent paper (Sedlackova et al, 2020, Nature, 587, 297-302) indicated that human MCMBP stabilises nascent MCM3-7 (but not MCM2) and helps translocate these helicase subunits to the nucleus. Thereby, MCMBP was proposed to be a chaperone that safeguards genome integrity by ensuring that cells contain enough MCM2-7 complexes to be loaded onto chromatin across the genome.

The manuscript by Saito et al revisits the role of MCMBP in human cells and argues that MCMBP promotes the assembly of functional MCM2-7 complexes, by association of MCMBP with MCM3, which thereby promotes the association of MCM3 with MCM2/4/6/7 (and MCM5). The authors use the recently described 'AID2' version of the auxin degron system to induce rapid degradation of MCMBP in human cells. Thereby, they show that the association of MCM3 with other MCM2-7 proteins is lost upon degradation of MCMBP, correlating with destabilisation of MCM3 and its major partner MCM5. In contrast, other MCM2-7 proteins are not destabilised upon acute depletion of MCMBP. Importantly, the NLS of MCMBP is dispensable for its role in promoting formation of the MCM2-7 hexamer. Moreover, MCMBP is shown to be required for newly synthesised MCM3 to associate with other MCM2-7 proteins, but MCMBP is not essential for the nuclear accumulation of MCM3 (that has its own NLS sequence, unlike several of the other MCM2-7 proteins that require complex formation for nuclear accumulation). Finally, the manuscript shows that depletion of MCMBP, and the associated reduction in functional MCM2-7 complexes, causes cell cycle arrest in human cells that contain p53, but DNA damage and loss of viability in cells that lack p53. These findings make an interesting contribution to our understanding of an important protein in human cells and should be of considerable interest to those in the chromosome replication and genome integrity fields.

The authors have dealt comprehensively with the points that I raised previously in relation to their original submission in 2019. The new version of the manuscript contains a large amount of new data, which are of high quality and provide strong support for the authors' conclusions. The data indicate that MCMBP associates with MCM3 and is important for MCM3 to associate with other MCM2-7 proteins, which in turn is an essential prerequisite for loading of the MCM2-7 proteins onto chromatin. The data in this revised manuscript build on the recent findings by Sedlackova et al (2020) but go considerably further and lead to important new insights. Whereas Sedlackova et al interpreted their data in terms of MCMBP promoting nuclear accumulation of nascent MCM2-7, the data in the present manuscript indicate that the key contribution of MCMBP is to promote the association of MCM3 with other MCM2-7 proteins. In the absence of MCMBP, MCM3 is still able to enter the nucleus (via its own NLS) but does not form productive MCM2-7 complexes (so nuclear accumulation of other MCM2-7 subunits is impaired).

In summary, I think that the present manuscript merits publication in eLife.

Reviewer #3:

Excellent manuscript revision and reviewer concerns on the original manuscript were well addressed. I support the publication of this revised manuscript in eLife.

Reviewer #4:

In the work described here, biochemical and cell biological approaches are used to show that MCMBP binds to MCM3 and that this complex is required for the assembly of MCM2-7 hexamer. Truncation constructs were used to define some of the important binding regions between MCMBP and MCM3. This study clarifies some of the previous data involving the role of MCMBP in supporting DNA replication and shows the importance of the protein to cell cycle control and genetic integrity. The experimental design is straightforward, and the reported observations largely appear to support the primary conclusions of the work. Whether MCMBP passively stabilizes MCM3 to protect it from degradation or actively helps catalyzes MCM2-7 hexamer formation is not resolved by the study.

Line 133 and figure 1a. The data suggest that Mcm5 and maybe Mcm7 can also be part of the Mcm3 complex with MCMBP? Figure 2d in particular indicates that Mcm5 is destabilized without MCMBP. These data should be elaborated upon in the paper and potentially incorporated into the model shown in Figure 5f.

In Figures 2d and 4a, when MCMBP was present in cells, MCM3 was found to be present in two populations, one around 600kDa and a second around 160kDa. It seems logical to presume that the two species correspond to the MCM2-7 hexamer and an MCM3-MCMBP complex (or perhaps an MCM3-MCM5-MCMBP ternary complex), respectively. However, in the absence of MCMBP (in Figures 2d and 4a), although MCM3 was present only in lower size species (fraction 37-45), the size of the lower species remained constant, similar to +MCMBP sample. One would expect that in the absence of MCMBP, MCM3 would be largely monomeric (or perhaps dimeric with MCM5) and appear in lower-size fractions. Please comment.

In Figure 1b, what are the identities of the subcomplexes? These should be resolved by mass spectrometry analysis.

Figure 4, supplement 1, panel f and accompanying text. This experiment and the data need a clearer explanation. Also, what is to be made of the loss of RPA when MCMBP is absent? And what is the identity of the gray circle next to NME1?

eLife. 2022 Apr 19;11:e77393. doi: 10.7554/eLife.77393.sa2

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

The authors report that MCMBP, a protein that is known to bind to some subunits of the MCM helicase complex, is involved in maintaining the stability of some of the MCM subunits. The authors created a cell line that has auxin-inducible degradation of a tagged MCMBP protein and they find that in the presence of auxin, MCMBP disappears by 6 hr. The cells keep proliferating for 4 days with little effect, but then start to slow down compared to the absence of auxin. They also report that MCMBP is associated with subcomplexes of MCM subunits, particularly MCM5, 6 and 7, which is a potentially interesting observation. They find that upon depletion of MCMBP, some MCM subunits are unstable after a long time (from 24-96 hr). They show that the MCMBP depleted cells have a difficulty progressing through S phase.

The paper suffers from overinterpretation of the data because of the lack of any specificity of connecting MCMBP to the stability of MCM subunits. As noted below, it could be simply a loss of cell proliferation that causes selective loss of MCM subunits. The authors did not attempt to assess the levels of other pre-RC protein levels upon addition of auxin (e.g., ORC subunits, CDC6, CDT1). They did not perform critical control experiments by treating the parent HCT116-OsTIR cells with auxin and measuring any effect of auxin on these cells over the 24 to 96 hr time courses. They also do not describe the Methods well.

The new manuscript showed that MCMBP directly bound MCM3, and this interaction was required for maintaining the MCM2–7 hexamer (Figure 3). Furthermore, we revealed that MCMBP promoted the hexamer assembly of MCM2–7 using nascent MCM3 (Figure 4). Because the reduced expression of the MCM2–7 hexamer occurred before defective cell growth (compare Figure 2c and Figure 3e/5a), it was likely that the defective MCM2–7 formation caused the proliferation defect. To prove this point, we carried out rescue experiments using wild-type and mutant MCMBP (Figure 3). The wild-type MCMBP restored the hexamer formation and cell growth, whereas an MCMBP mutant lacking the interaction with MCM3 failed (Figure 3c, d, e). These data demonstrated that the proliferation defect after MCMBP depletion was caused by losing the functional MCM2–7 hexamer.

In the current manuscript, we revealed that MCMBP depletion initially affected the level

MCM3 and MCM5 in soluble extracts prepared from the cytoplasm and nucleoplasm

(Figure 2c, d). Therefore, we do not think that the replication proteins involved in preRC formation (such as ORC and CDT1) affected the loss of MCM3 and MCM5 in the cytoplasm and nucleoplasm. Please note that we are now more focusing on how the MCM2–7 assembly occurs in the current manuscript.

To avoid a side-effect caused by auxin (500 µM IAA), we employed an improved degron system, AID2, recently reported by us (Yesbolatova et al. Nat. Commun. 2020). The ligand concentration was drastically reduced using AID2. In this publication, we extensively tested the cytotoxicity of the new ligand, 5-Ph-IAA (no changes in the transcriptome and cell growth with 1 µM 5-Ph-IAA; the same experimental condition). In the current manuscript, we added this ligand in the rescue experiments (Figure 3c, d, e) and observed no problem with the MCM2–7 expression and cell growth in the MCMBP-degron cells rescued by the wild-type MCMBP transgene. Furthermore, we treated the parental HCT116 cells (without modified MCMBP) with 5-Ph-IAA for 8 days and did not find any growth defects (Figure 5a).

1. There is no information about the tagging of the MCMBP gene in the paper, except a theoretical diagram in Figure S2. There are presumably two alleles of the MCMBP gene in HCT116 cells and it was not clear if both alleles were tagged. Further, the methods describe the use of neomycin or hygromycin as selection agents, but the actual cell line used for the experiments is not described in any detail. Was it neomycin resistant, or hygromycin resistant?

2. The Methods section does not describe the HCT116 cells with OsTIR expressed and whether this expression is constitutive or induced. Only HCT116 cells are mentioned in the Methods.

As we mentioned above, we changed the degron system to AID2. The new MCMBPdegron cell line continuously expressed OsTIR1(F74G) from the AAVS1 locus and MCMBP-mAID-Clover from both endogenous MCMBP alleles. The details were described in Methods and our protocol publication (Saito and Kanemaki, Current Protocol, 2021). All cell lines used in this study are summarized in Table S1.

3. The authors should use the appropriate figure labeling for eLife papers, such as Figure 1, supplement 1, etc.

We understand that the format can be different from the eLife style at the point of initial submission. Therefore, we will change the detailed format when necessary.

4. Figure 2, panel A. What are #1 and #2? Are they independent cell lines? If so, describe them and whether they are really independent, or clines form the same isolation.

We did not include any data from the previous submission.

5. The results in Figure 2 show the effect of adding auxin on MCMBP protein levels, but only a 24 hr time point is shown. Since the protein has a short half life of ~ 3 hr when cycloheximide was added, the protein should disappear faster than 24 hr. A time course of protein levels after auxin treatment must be shown. It is noted that the time course is provided in Figure 3, but it should be presented first.

We took time course samples after inducing degradation of MCMBP to monitor the expression levels of MCM2–7 (Figure 2c, Figure S2c, d). Please note that we did not add cycloheximide in these experiments. MCMBP was rapidly degraded within 4 h (Figure S2a), and the expression level of MCM2–7 gradually decreased because MCMBP was required to assemble the new complex as shown in Figure 4.

6. DNA staining: Figure 2 say the DNA was stained with SiR-DNA but the Methods state that Hoechst33342 stain was used for microscopy. Which is correct?

We used Hoechst33342 and changed the text accordingly.

7. Figure 2B. Which antibody was used to detect MCMBP-mAC, or was the fluorescent protein detected? The Figure legend does not describe what is shown.

We now include all antibody information used for detection in Table S3.

8. Figure 2C. It is necessary to perform the time course over a longer period than 5 days since it is not clear if MCMBP is essential, or its absence just slows proliferation. Only a longer proliferation time course will clarify this point, assuming that auxin removes all of the MCMBP-mAC, which is also not clear. 10% protein remaining would not be detected in an experiment shown in Figure 2A.

We extended the time course to 8 days (Figure 5a) and found that the MCMBPdepleted cells stopped growing. Depletion efficiency of MCMBP-mAC was the undetectable level (Figures 2b, S2a). We concluded MCMBP was essential for normal cell proliferation in HCT116.

9. Figure 3. Why is MCM2, MCM4 and MCM detected in the HCT116-mAC cells (but nit MCM5) and then in RPE and HeLa cells MCM5 is added to the list. Why not detect all MCM subunits.

We did not include these data using siRNA knockdown in this new submission.

10. A control for the experiment in Figure 3A s needed. Treating HCT116-OsTIR cells with auxin over a time course for 48 hrs is needed since this cell line should not be sensitive to auxin. It is formally possible that auxin slows cell proliferation independent of loss of MCMBP. For example, auxin lowers MCM4 mRNA levels in HCT116-OsTIR cells (Figure S3). This is a necessary control.

12. A major problem is in Figure 4C. The cells were treated for 4 days with auxin (one day in the presence of lovastatin) and yet the reduction in MCM7 and MCM7 is minimal, just the same as MCM2. But in Figure 3A, a dramatic reduction is shown at 48 hours. The auxin treatment for 96 hrs in Figure 4C should show at least a reduction as great as shown at 48 he in Figure 3A, but it does not. The is clearly a difference on chromatin, but this could be a secondary effect of cell proliferation.

13. Figure 4C and 4D. Again, a control experiment using auxin treatment of HCT116-OsTIR cells for 96 hr is needed.

14. Page 10, lines 3-4 and Figure 4F. This conclusion is far too strong based on the data. The effect could be indirect since the time between MCMBP depletion upon addition of auxin (stated to be 3 hr) and the time of depletion of MCM subunits at least 24 hr for partial depletion and in Figure 4, weak depletion after 96 hr, makes it possible that the effect is due to loss of cell proliferation and not a direct effect.

Please look at our response above. The rescue experiments shown in Figure 3c, d, e should clear your concern about the side effects caused by the new ligand, 5-Ph-IAA, and the impact on the cell proliferation after MCMBP depletion.

11. Page 8, lines 13-15. This conclusion that MCMBP protects MCM5, 6 and 7 from degradation is far too premature based on the data in Figure 3. It could simply be that MCMBP depletion slows cell proliferation, and this indirectly affects MCM subunit levels. If this is specific, then other pre-RC protein such as ORC, CDC6 and CDT1 should not change. These must be measured.

Please look at our response above. Moreover, our data indicated that MCMBP protected MCM3 from degradation and promoted the MCM2–7 assembly in the cytoplasm and nucleoplasm (Figures 2c, d and S2c, d).

Reviewer #2:

Significance. In addition to the individual subunits that comprise the Mcm2-7 helicase, many eukaryotes contain additional genes that encode protein that are evolutionarily related to Mcm2-7. One such proteins has been named MCMBP (Mcm binding protein) which is found I the genomes of most eukaryotes except for budding yeast. Despite its prevalence, the function of this protein has remained obscure and controversial. In this report, the authors generate and use an auxin-inducible degron to generate a conditional allele of the MCMBP gene in human cell culture to provide evidence that the role of MCMBP in this system is to prevent the inappropriate degradation of Mcm subunits in actively growing cells.

Experimental design. The unspoken assumption in this paper as well as this area of study is that since MCMBP was first identified as an MCM2-7 binding protein, MCMBP likely exerts its sole function through physical interaction with all or some of the MCM2-7 subunits. Although a reasonable conjecture, I'd be surprised that the reality is this tidy. Although to date the main effects of various types of MCMBP mutations can be rationalize as some type of replication-related defect, could this be through modulation of Mcm8 or 9? Perhaps due to its similarity to an Mcm2-7 subunit, could it be that MCMBP exerts its effect by interacting with the replication factors that Mcm2-7 normally interacts with?

The above issues extend beyond the scope of the current study. However, minimally one should ascertain if the observed defects of the MCMBP mutant are direct (i.e., require physical interaction between MCMBP and Mcm2-7) or are indirect (that binding between MCMBP and Mcm2-7 is not required). Given our current knowledge of MCMBP, one should be able generate and test MCMBP mutants that specifically do not bind Mcm subunits yet lack other collateral problems. Such data would considerably strengthen the paper and help elevate its significance from descriptive to mechanistic.

We removed the discussion related to MCM8/9 because we agreed that it would be beyond the scope of this manuscript. We found that the N-terminus of MCMBP interacted with MCM3 and generated an MCMBP mutant lacking this interaction (Figure 3a, b). This MCMBP∆N mutant could not rescue the hexamer formation (Figure 3c, d) and growth defect after MCMBP-mAC depletion (Figure 3e), whereas wild-type MCMBP rescued both phenotypes. These results clearly showed the MCMBP directly functioned in the MCM2–7 hexamer formation to maintain the hexamer level.

Nature of the conditional auxin-inducible MCMBP degron. In this study, the MCMBP gene was tagged with both an auxin-inducible degron construct as well as with GFP CLOVER. The expression level of the tagged MCMBP, in the presence and absence of auxin, are shown in a western blot in Figure 2A. The good news is that auxin addition results in rapid depletion of MCMBP. However, relative to the non-degron parent cell line, the expression level of the MCMBP degron construct is vastly reduced relative to the wild type protein (levels not quantified, but by eye may be <25% of the parent cell line). This problem is massaged as the authors typically show subsequent results {plus minus} auxin, not with respect to the parental cell line lacking the auxin construct.

Please note that we employed an improved degron system, AID2, in this manuscript. We did not see any leaky degradation with this new system without the inducing ligand, 5-Ph-IAA (Figure 2b). Therefore, we did not have the raised issue anymore.

The nature of this construct raises the potential problem that it may be producing off-target effects. In the absence of auxin, how functional is the degron mutant compared to the wild type parent cell line (e.g., growth, viability, cell cycle progression, DNA damage)? It is very difficult to evaluate the loss of MCMBP upon auxin addition unless one has carefully evaluated the original construct in the absence of auxin.

We compared cell proliferation, cell cycle distribution and DNA damage between the parental and MCMBP-mAC lines (p53+/+) in Figure 5a, b, c and did not observe a significant difference between the two lines. A slight increase of the G2 phase in MCMBP-mAC (p53+/+) might be because of a minor functional defect of the MCMBPmAC fusion protein.

Auxin control. In addition to the above considerations, it is important to show that in the original non-degron parental cell line, that auxin addition has no effect on the stability of either MCMBP or MCM2-7. This essential control is not provided.

As shown in Figure 5a, the parental cells were treated with 5-Ph-IAA for 8 days, and we did not observe any growth defects. As shown in Figure 3c, d, the MCMBP-mAC cells rescued by adding the wild-type MCMBP gene restored the MCM2–7 hexamer level. These data provided good controls, showing that the ligand did not affect the MCM2–7 level and cell growth.

HeLa cell experiment. In Figures 3C and D, the key experiment using the auxin cell line (resulting in a reduction in the level of Mcm subunits after McmBP is degraded), is replicated by siRNA experiments in 2 independent cell lines (hTERT RPE and HeLa). However, only the experiment in the hTERT RPE cell line appears to replicate the basic results in the auxin cell line (i.e., that addition of the MCBP siRNA reduces the levels of Mcm subunits). To my eye, I see no reduction in the levels of MCM subunits in the parallel experiment using the HeLa cell line. This unfortunate little problem is conveniently not mentioned in the text.

We removed these data because we could not study the assembly of MCM2–7

hexamer with slow depletion by using siRNA.

Reviewer #3:

MCMBP is a poorly characterised protein found in most eukaryotes, which was previously shown to associate with the MCM2-7 proteins that form the core of the replicative helicase at DNA replication forks. Various functions of MCMBP have been suggested previously, such as a role in unloading MCM2-7 from chromatin in late S-phase. This manuscript provides interesting evidence to argue that MCMBP is actually a form of chaperone, which is important for proliferating cells to maintain the very high level of expression of MCM2-7. The data are generally of high quality and the study should be of considerable interest to those in the chromosome duplication and genome integrity fields.

We appreciate that this reviewer gave a very positive comment. We spent almost two years revising all data and now got new results showing the mechanical and biological significance of MCMBP.

1. The authors present evidence to indicate that MCMBP protects MCM2-7 proteins from degradation, but also "do not rule out the possibility that MCMBP helps transport nascent MCM subunits from the cytoplasm to the nucleus" (page 10, lines 19-21). Does it really take 5 days for MCM2-7 proteins to be lost from the nucleus upon rapid degradation of MCMBP-AID (as in Figure 3B), or is the effect also seen at earlier times (which might then be distinguishable from the effect of MCMBP on MCM2-7 stability)?

We wrote that "MCMBP might help the nuclear transport of MCM2–7" in the previous manuscript because we knew that Lukas' paper, which was under review in Nature, focused on this point (Sedlackova et al. Natrure, 2020). However, the current manuscript shows that an MCMBP mutant lacking the NLS sequence rescued the level of MCM2–7 and growth defect (Figure 3c, d, e). These data indicated that the critical function of MCMBP was to promote the assembly of MCM2–7, thus maintaining the level of MCM2–7, rather than to promote the MCM subunits to the nucleus. Furthermore, we looked at the localization of nascent MCM3 after MCMBP depletion (Figure 4b). It localized in the nucleus, possibly using NLS within MCM3, but its chromatin binding was defective (Figure 4d), supporting the idea that the hexamer formation was defective without MCMBP. In the current manuscript, we discussed what the critical function of MCMBP is and where the MCM2–7 hexamer formation occurs.

2. The authors suggest that MCMBP also works as a chaperone for MCM8-9 (page 11, lines 19-23), which are distantly related to MCM2-7 and function during homologous recombination. The authors mention unpublished data to show that MCMBP interacts with MCM8-9, and presumably they have already tested whether levels of MCM8-9 drop after depletion of MCMBP. If so, this would be a nice addition to the manuscript, so the authors might consider including these findings?

We changed all cell lines used in this study. Therefore, we have not explored the relationship between MCMBP and MCM8/9 yet. We wished to leave this issue for future research and decided not to mention MCM8/9 in the current manuscript.

3. Comparing Figure 3D and 3C, the depletion by siRNA of MCMBP in HeLa cells appears to be just as efficient as in RPE1 cells, yet the effect on MCM2-7 levels is apparently much weaker in HeLa than in RPE1 cells. Do the authors have any explanation for this?

As wrote to Reviewer 2, we removed these data because we could not study the assembly of MCM2–7 hexamer with slow depletion by using siRNA.

4. The authors suggest that MCMBP functions together with the chaperone FKBP51 to "promote the formation of MCM2-7 hexamers in the nucleus" (page 11, lines 4-6). Have they tested the effect of siRNA depletion of FKBP51 on the level of MCM2-7 proteins and MCMBP?

The current manuscript showed that the interaction between MCM3 and FKBP5 (also called FKBP51) was lost when MCMBP was depleted (Figure S4f). We wished to clarify whether FKBP5 also promoted the MCM2–7 assembly with MCMBP. For this purpose, we treated the cells with an FKBP5 inhibitor, SAFit2. However, we did not see a defect in the MCM2–7 assembly. So far, we cannot distinguish the two possibilities; FKBP5 is dispensable for the assembly, or the inhibitor is not good enough.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #1 (Recommendations for the authors):

The MCM Binding Protein (MCMBP) has been implicated in many processes in cell cycle progression including DNA replication and DNA repair. The work presented in this paper shows for the first time that loss of function of MCMBP by classical cell and molecular biological methods lead to lower levels of pools of the MCM 2-7 hexamer. Moreover, MCMBP binds directly to Mcm3 and through deletions and biochemical methods in vitro, the authors show that loss of this interaction leads to lower levels of the hexamer when such defective alleles are expressed in cells. Given that the hexamer is the core of the helicase during elongation and numbers of origins are established through double hexamer formation these results will be widely impactful. Genome integrity depends upon an abundance of potential origins, particularly under stressed conditions where back -up or late origins need to finish the replication domain. The methods applied are good to establish these points but provide little direct insights into how the MCMBP actually works mechanistically in establishing these normal levels of hexamers. Is it through prevention of degradation of Mcm 3 or in an actual pathway to assembly? Nevertheless, such questions raised by the present study actually raise questions that will provide impetus for future work.

1. My only recommendation of importance for the authors is to clarify with further studies the suggestion that other Mcm's are present within the low molecular weight sub-complexes found in extracts. Figure 1 surely shows Mcm3 and also 5 and 7. There are many ways to clarify this point including a further column step or immunoprecipitation/western.

Please look at Figure 1—figure supplement 1e, which was added to the revised manuscript. We carried out MCMBP IP from the smaller and larger fractions (about 600 kDa and 160 kDa, respectively), and IP samples were blotted with MCM antibodies. In the smaller fractions, we detected all MCM proteins albeit MCM2 was detected less amount compared to the others. This is consistent with the previous reports, showing that MCMBP interacted with multiple MCM subunits (PMID 22540012 and 24299456). We wrote our interpretations in lines 136 to 146. Please refer to the text in the manuscript.

Reviewer #4 (Recommendations for the authors):

In the work described here, biochemical and cell biological approaches are used to show that MCMBP binds to MCM3 and that this complex is required for the assembly of MCM2-7 hexamer. Truncation constructs were used to define some of the important binding regions between MCMBP and MCM3. This study clarifies some of the previous data involving the role of MCMBP in supporting DNA replication and shows the importance of the protein to cell cycle control and genetic integrity. The experimental design is straightforward, and the reported observations largely appear to support the primary conclusions of the work. Whether MCMBP passively stabilizes MCM3 to protect it from degradation or actively helps catalyzes MCM2-7 hexamer formation is not resolved by the study.

Line 133 and figure 1a. The data suggest that Mcm5 and maybe Mcm7 can also be part of the Mcm3 complex with MCMBP? Figure 2d in particular indicates that Mcm5 is destabilized without MCMBP. These data should be elaborated upon in the paper and potentially incorporated into the model shown in Figure 5f.

We added a new data in Figure 1—figure supplement 1e to investigate MCMBP interactors in the smaller fractions. For this purpose, we carried out MCMBP IP from the smaller fractions and found that all MCM proteins interacted with MCMBP with different affinities, consistent with previous reports (PMID 22540012 and 24299456). Because MCM3 forms a subcomplex with either MCM5 and MCM7 (PMID 9077461 and 10644704), the smaller fractions possibly contained multiple subcomplexes, such as MCMBP/MCMs, MCM3/5 and MCM3/7. However, it appeared that MCMBP did not form a complex of MCM3/5/7/BP because MCM3 was not precipitated with MCMBP from the larger fractions (Figure 1—figure supplement 1e). We updated the model in Figure 5f for clarification.

In Figures 2d and 4a, when MCMBP was present in cells, MCM3 was found to be present in two populations, one around 600kDa and a second around 160kDa. It seems logical to presume that the two species correspond to the MCM2-7 hexamer and an MCM3-MCMBP complex (or perhaps an MCM3-MCM5-MCMBP ternary complex), respectively. However, in the absence of MCMBP (in Figures 2d and 4a), although MCM3 was present only in lower size species (fraction 37-45), the size of the lower species remained constant, similar to +MCMBP sample. One would expect that in the absence of MCMBP, MCM3 would be largely monomeric (or perhaps dimeric with MCM5) and appear in lower-size fractions. Please comment.

We understand your point. However, MCM3 monomer (91 kDa) and MCMBP/MCM3 subcomplex (73 kDa + 91 kDa) cannot be distinguished using Superose 6 column because we cannot achieve fine resolutions at the lower molecular weight. This is possibly why MCM3 in the smaller fractions were detected similarly with and without MCMBP in Figure 4a.

In Figure 1b, what are the identities of the subcomplexes? These should be resolved by mass spectrometry analysis.

Please look at Figure 1—figure supplement 1b. Among the three bands, the smallest band might be MCM3 monomer. The other two appears to colocalize with MCMBP. However, it is difficult to say what these subcomplex are. We are sorry that we cannot inform you conclusively.

Figure 4, supplement 1, panel f and accompanying text. This experiment and the data need a clearer explanation. Also, what is to be made of the loss of RPA when MCMBP is absent? And what is the identity of the gray circle next to NME1?

Following your comments, we changed the figure legend and updated the figure. Essentially, we immunoprecipitated MCM3 with or without MCMBP, and then looked at the differences of proteins coprecipitated with MCM3. As shown in the updated Figure 4—figure supplement f, RPA1, FKBP5 and NME1 were top interactors with MCM3 in a

MCMBP dependent manner. We interpreted that RPA1 was lost in the absence of MCMBP because DNA replication became defective (lines 247 to 249). The interaction between MCMBP and FKBP5 has been reported (PMID 25036637) so that we hypothesize FKBP interacted with MCM3 via MCMBP (lines 338 to 342). NME1 (also known as NM23-H1) is a nucleotide diphosphate kinase producing GTP, CTP and TTP, and also works as a ssDNA nicking enzyme (PMID 16818237). Again, this might be related to defective DNA replication without MCMBP.

Associated Data

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    Supplementary Materials

    Transparent reporting form
    elife-77393-transrepform1.docx (111.6KB, docx)
    Source data 1. Underlying data for all graphs.
    elife-77393-data1.xlsx (355.1KB, xlsx)
    Source data 2. All original immunoblot images.
    elife-77393-data2.zip (11.8MB, zip)

    Data Availability Statement

    All data generated or analysed during this study are included in the manuscript and supporting files.

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