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Equilibrium between nascent and parental MCM proteins protects replicating genomes

Abstract

Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2–7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45–MCM–GINS (CMG) helicases that are required for genome duplication1,2,3,4. It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes5,6. Here we show that, for daughter cells to sustain error-free DNA replication, their mother cells build up a nuclear pool of MCMs both by recycling chromatin-bound (parental) MCMs and by synthesizing new (nascent) MCMs. Although all MCMs can form pre-RCs, it is the parental pool that is inherently stable and preferentially matures into CMGs. By contrast, nascent MCM3–7 (but not MCM2) undergo rapid proteolysis in the cytoplasm, and their stabilization and nuclear translocation require interaction with minichromosome-maintenance complex-binding protein (MCMBP), a distant MCM paralogue7,8. By chaperoning nascent MCMs, MCMBP safeguards replicating genomes by increasing chromatin coverage with pre-RCs that do not participate on replication origins but adjust the pace of replisome movement to minimize errors during DNA replication. Consequently, although the paucity of pre-RCs in MCMBP-deficient cells does not alter DNA synthesis overall, it increases the speed and asymmetry of individual replisomes, which leads to DNA damage. The surplus of MCMs therefore increases the robustness of genome duplication by restraining the speed at which eukaryotic cells replicate their DNA. Alterations in physiological fork speed might thus explain why even a minor reduction in MCM levels destabilizes the genome and predisposes to increased incidence of tumour formation.

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Fig. 1: Nascent MCMs continuously replenish parental MCMs in mother cells and form functionally distinct pre-RCs in daughter cells.
Fig. 2: MCMBP safeguards inheritance of nascent MCMs in daughter cells.
Fig. 3: MCMBP stabilizes and translocates nascent MCM3–7 to the nucleus.
Fig. 4: Reduced contribution of nascent MCMs to pre-RCs accelerates replication forks and causes DNA damage.

Data availability

Primary imaging data in Figs. 1d, 2d, 3b–d and Extended Data Figs. 1a, e, 4f, 5e, g, 6b, 7c, 9c have been deposited at the European Bioinformatics Institute (EBI) BioStudies database (https://www.ebi.ac.uk/biostudies/) with accession number S-BSST445 and made accessible as an open resource. All data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

Research funding was provided by the Novo Nordisk Foundation (grant NNF14CC0001) and the Danish Cancer Society (grant R204 A12615) to J.L. H.S. was supported by the Novo Nordisk Foundation (grant NNF16CC0020906). K.S. was supported by the Danish Council for Independent Research (grant EDFF-FSS 82262) and the Lundbeck Foundation (grant R264-2017-2819). R.G. was supported by a European Molecular Biology Organization long-term postdoctoral fellowship (ALTF271-2014). C.C. was supported by the Hallas Møller Investigator Fellowship from the Novo Nordisk Foundation (NNF14OC0008541), the Danish Cancer Society (grant R204-A12286) and by the European Union’s Horizon 2020 research and innovation programme (grant 648039). We thank J. Bulkescher, J. Dreier and G. Karemore from the Protein Imaging Platform for their assistance with microscopy and image analysis. FACS analyses were carried out at the Center for Protein Research and the Danstem Flow Cytometry Platform. The pX335 and pX458 plasmids were a gift from F. Zhang, and MLN4924 was a gift from J. Duxin. We thank L. Toledo and D. Gilbert for providing critical reagents. We thank C. Lukas for conceptual and technical inputs to this study and members of the Lukas laboratory for discussions and critical comments on the manuscript.

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Contributions

H.S., K.S. and J.L. conceived the project and planned the study. H.S. performed experiments and prepared figures. H.S generated all the cell lines with the help of M.-B.R. K.S. performed LFD analysis. R.G. carried out proteomic data acquisition under the supervision of C.C. H.S., K.S. and J.L. analysed the data and wrote the manuscript. All authors read and commented on the manuscript.

Corresponding authors

Correspondence to Kumar Somyajit or Jiri Lukas.

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The authors declare no competing interests.

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Peer review information Nature thanks Olivier Hyrien and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Development of tools and characterization of endogenous MCM proteins.

a, Left, representative images of U2OS cells with endogenously tagged MCM2–GFP and ectopically expressing RFP–chromoPCNA were classified by cell cycle stages (S1–S3 denote early, mid and late S, respectively); scale bar, 12 μm. Right, cells depicted in the image on the left were subjected to single bleach pulse and followed by the real-time recording of MCM2–GFP fluorescence recovery kinetics. Normalized MCM2–GFP FRAP curves reflect a gradual increase of the MCM2 mobile fraction from G1 to G2 phase; each data point indicates mean ± s.e.m., n = 14 cells per cell cycle stage. b, Left and middle, immunoblots of U2OS cells or their derivative with endogenously tagged MCM4–Halo immunoblotted with MCM4 or Halo antibody. Right, junction PCR showing homozygous MCM4–Halo tagging; n = 2 biological replicates. c, QIBC of MCM4–Halo cells pulsed with JF549 HaloTag ligand (200 nM) for the indicated time points. Nuclear DNA was counterstained with DAPI. Lines denote medians; n ≈ 3,500 cells per condition. d, QIBC of MCM4–Halo cells pulsed with JF549 HaloTag ligand (200 nM) for 20 min, immunostained for MCM4 and DAPI without pre-extraction (left, n ≈ 16,000 cells per condition) or with pre-extraction (right, n ≈ 12,000 cells per condition). e, Maximum intensity projection images of U2OS cells endogenously expressing MCM4–Halo and ectopically expressing GFP–PCNA with dual labelling to discriminate parental and nascent MCMs. Dotted circles show representative trajectories in an individual cell monitored by confocal time-lapse microscopy at the indicated time points for one complete cell cycle marked by PCNA. See also quantification in Fig. 1d and Methods for cell cycle classifications. For optimal image presentation, brightness and contrast were adjusted for each colour channel with identical settings across all time points. Scale bar, 15 μm. Gel source data for b are provided in Supplementary Fig. 1.

Source data

Extended Data Fig. 2 Newly synthesized MCMs replenish the declining pool of parental MCMs.

a, Top left, dual-HaloTag labelling protocol in MCM4–Halo cells. Bottom left, MCM4–Halo cell counting for indicated time points. Right, SDS–PAGE of whole-cell lysates of MCM4–Halo cells labelled for nascent and parental MCM4 at indicated time points (with indicated cell count). Total protein staining at each time point is shown below; the dashed box marks the cell doubling time. b, Top, dual-HaloTag labelling protocol in MCM4–Halo cells. Middle and bottom, QIBC of MCM4–Halo cells immunostained for parental and nascent MCM4 without pre-extraction. The dashed box indicates the cell doubling time. Lines denote medians; n ≈10,000 cells per condition. c, Top, dual-HaloTag labelling protocol in MCM4–Halo cells treated as indicated with cycloheximide (CHX; 12.5 μg ml−1). Middle and bottom, QIBC of MCM4–Halo cells immunostained for parental and nascent MCM4 without pre-extraction at the indicated time points after the indicated treatments. Lines denote medians; n ≈ 9,000 cells per condition. d, Top, dual-HaloTag labelling protocol in MCM4–Halo cells treated as indicated with MG132 (2 μM). Middle and bottom, QIBC of MCM4–Halo cells immunostained for parental and nascent MCM4 without pre-extraction before fixation after indicated treatments. Lines denote medians; n ≈ 3,500 cells per condition. Gel source data for a are provided in Supplementary Fig. 1.

Source data

Extended Data Fig. 3 Nascent and parental MCMs are equally proficient in pre-RC formation.

a, Top, dual-HaloTag labelling protocol in MCM4–Halo cells. Middle and bottom, QIBC of MCM4–Halo cells stained for parental and nascent MCM4 and DAPI after pre-extraction. The dashed box marks the cell doubling time; n ≈ 2,000 cells per condition. b, Top, MCM4–Halo dual labelling protocol. Middle and bottom, QIBC of cells transfected with control siRNA or siRNA against CDC6 and stained for chromatin-bound parental and nascent MCM4 and DAPI. n ≈ 9,500 cells per condition. c, Top, QIBC-based sub-stratification of G1 phase by CDT1 immunostaining in pre-extracted MCM4–Halo cells; DAPI indicates DNA content. Bottom, quantification of chromatin bound parental and nascent MCM4 at the indicated G1 stages defined at the top and according to the labelling protocol depicted in b. Each data point indicates mean ± s.d. of mean intensity of parental and nascent MCM pools normalized individually as 100% with respect to the G1-phase gate 7; n = 3 biological replicates with n ≈ 4,500 cells per replicate. d, Left, dual-HaloTag labelling protocol in MCM2–Halo cells. Middle and right, QIBC of MCM2–Halo cells stained for parental and nascent MCM2 without pre-extraction. The dashed box marks the cell doubling time. Lines denote medians; n ≈ 4,400 cells per condition. e, Top and bottom, QIBC of MCM2–Halo cells stained for parental and nascent MCM2 and DAPI after pre-extraction according to the labelling protocol depicted in d. The dashed box marks the cell doubling time; n ≈ 3,800 cells per condition.

Source data

Extended Data Fig. 4 The inherited pools of nascent and parental MCMs display distinct unloading patterns during S phase.

a, Top, dual-HaloTag labelling protocol. Bottom, QIBC of total nuclear CDT1 in MCM4–Halo cells after treatment with DMSO or MLN4924 (5 μM, 6 h); DAPI indicates DNA content; n ≈ 5300 cells per condition. b, QIBC of chromatin-bound parental and nascent MCM4 under conditions depicted in a; the colour gradient marks chromatin-bound TIMELESS; n ≈ 7,500 cells per condition. c, Quantification of chromatin-bound parental and nascent MCM4 in TIMELESS-positive or -negative cells from b. In box plots, centre lines are medians, the boxes indicate the 25th and 75th centiles, the whiskers indicate 5 and 95 per cent values. P values were determined by one-way ANOVA with Tukey’s test; n = 2,000 cells per condition. d, Left, MCM4–Halo dual-labelling protocol. Middle and right, QIBC of chromatin-bound parental and nascent MCM4 and DAPI at indicated time points; n ≈ 4,700 cells per condition. e, Left, QIBC-based sub-stratification of S phase by chromatin-bound CDC45–GFP in MCM4–Halo–CDC45–GFP cells; DAPI indicates DNA content. Middle and right, quantification of chromatin-bound parental and nascent MCM4 at indicated stages of S phase defined in e (left) and under conditions depicted in d. Each data point indicates mean ± s.d. of mean intensity of parental and nascent MCM pools normalized individually as 100 per cent with respect to the S-phase gate 1; n = 3 biological replicates with around 4,700 cells per replicate. f, Images of chromatin-bound parental and nascent MCM4 and CDC45–GFP throughout daughter-cell S phase sub-stratified as in e (left) at the 24-h time point. Scale bar, 20 μm. g, QIBC of chromatin-bound CDC45–GFP in CDC45–GFP cells treated with DMSO or ATR inhibitor (5 μM; 1 h); n ≈ 3,300 cells per condition.

Source data

Extended Data Fig. 5 MCMBP associates with the CMG-independent pool of MCMs.

a, MCM interactions with MCMBP obtained by SILAC-based mass spectrometry of Flag immunoprecipitation (Flag IP) from control U2OS cells (grown in light media) or its derivative ectopically expressing Flag-tagged MCMBP (grown in heavy media). The enrichments were obtained by averaging heavy/light SILAC ratio. Data represent mean ± s.d.; n = 3 technical replicates. b, Left, immunoblotting of Flag IP from U2OS cells or their derivative ectopically expressing Flag-tagged MCMBP. Middle and right, immunoblotting of GFP-immunoprecipitation (GFP IP) from U2OS cells or its derivative with endogenous GFP-tagged CDC45 or GFP-tagged MCM4. c, Sub-cellular fraction (500 mM NaCl) of U2OS cells or their derivative ectopically expressing Flag-tagged MCMBP followed by immunoblotting of H3 or alpha-tubulin denoting chromatin-bound (CB) and soluble (S) fraction, respectively. d, Immunoblotting of GFP IP from sub-cellular fractions of U2OS cells or its derivative with endogenous GFP-tagged MCM4. e, Representative maximum intensity projections of U2OS cells with endogenously tagged MCM4–Halo and MCMBP–AID–GFP treated with DMSO or IAA (0.5 mM) following a dual labelling of parental and nascent MCM4–Halo at the indicated time points. For optimal image presentation, brightness and contrast were adjusted individually for each treatment and colour channel with identical settings across time points. Scale bar, 15 μm. See also quantification in Fig. 2d. f, Left, dual-HaloTag labelling protocol. Middle and right, QIBC of MCM4–Halo cells stained for parental and nascent MCM4 without pre-extraction after the indicated treatments. Lines denote medians; n ≈ 4,000 cells per condition. g, Left, dual-HaloTag labelling protocol. Middle and right, total intensities of parental and nascent MCM4. Fluorescence intensities at the start of time-lapse microscopy were pooled separately for U2OS MCM4–Halo and MCMBP-knockout MCM4–Halo cells as 100%. Data represent mean ± s.d.; n = 15 cells per condition tracked over indicated time-points. Gel source data in bd are provided in Supplementary Fig. 1.

Source data

Extended Data Fig. 6 MCMBP fosters nuclear accumulation of nascent but not parental MCMs.

a, QIBC of U2OS cells expressing endogenously tagged MCM4–Halo and MCMBP–AID–GFP stained for parental and nascent MCM4 and counterstained by DAPI after pre-extraction and following the indicated IAA treatment (0.5 mM). Staining of parental and nascent MCM4 was performed according to the labelling protocol in Extended Data Fig. 5f. The dashed box marks the cell doubling time; n ≈ 2,900 cells per condition. b, Top, representative images of immunostained MCMs in U2OS and MCMBP-knockout cells without pre-extraction. For optimal image presentation, pseudocolour was used and brightness and contrast were adjusted for each MCM and kept identical in U2OS and MCMBP-knockout cells. The colour gradient indicates the mean MCM intensity. Scale bar, 20 μm. Bottom, quantification of mean fluorescence intensity (MFI) of cytoplasmic MCMs. c, Left, QIBC of RPE cells treated with indicated siRNAs and stained for MCMBP and indicated MCMs without pre-extraction. Lines denote medians; n ≈ 2,000 cells per condition. d, Right, MFI of cytoplasmic MCM4 and MCM5 derived from experiments in c. In the box plots (b, d), centre lines are medians, the boxes indicate the 25th and 75th centiles, the whiskers indicate 5 and 95 per cent values. P values were determined by two-tailed unpaired t-test (b, d); n = 500 cells per condition (b, d).

Source data

Extended Data Fig. 7 MCMBP possesses an autonomous NLS motif that fosters nuclear import of MCM3–7.

a, Quantification of MFI of cytoplasmic MCM5 and MCM7. b, QIBC of MCMBP-knockout cells ectopically expressing MCMBP–tGFP or MCMBP(∆NLS)–tGFP stained with indicated MCMs without pre-extraction. Lines denote medians; n ≈ 3,000 cells per condition. c, Left, representative images of MCMBP-knockout cells transiently expressing MCMBP–tGFP variants. Scale bar, 20 μm. Right, quantification of nuclear/cytoplasmic ratio of MCMBP–tGFP. d, tGFP IP followed by immunoblotting of U2OS cells or their derivative transiently expressing MCMBP variants. e, Quantification of nuclear/cytoplasmic ratio of MCM4 and MCM5. f, Whole-cell extracts from U2OS and MCMBP-knockout cells (clones 1 and 2) immunoblotted with indicated antibodies. g, qPCR analysis of mRNA levels for MCM2, MCM5 and MCM7 in indicated cells, normalized to control cells (100%). Data represents mean ± s.d.; n = 4 technical replicates. h, Immunoblotting of U2OS and MCMBP-knockout cells treated with control siRNA or siRNA against PSMD14. i, Top, MFI of cytoplasmic MCM4 and MCM2 for indicated cells treated with DMSO or MG132 (2 μM; 6 h) or siRNA against PSMD14 as indicated. Bottom, QIBC of indicated cells treated with DMSO or MG132 (2 μM; 6 h) or siRNA against PSMD14 and stained for MCM2 or MCM4 without pre-extraction. Lines denote medians. Left, n ≈ 7,000 cells per condition; middle, n ≈ 10,000 cells per condition; right, n ≈ 4,700 cells per condition. In box plots (a, c, e, i), centre lines are medians, the boxes indicate the 25th and 75th centiles, the whiskers indicate 5 and 95 per cent values. P values were determined by one-way ANOVA with Tukey’s test, not significant (n.s.) denotes P > 0.05; n = 500 cells per condition (a, i) and n = 100 cells per condition (c, e). Gel source data for d, f, h are provided in Supplementary Fig. 1.

Source data

Extended Data Fig. 8 Excess nascent MCM2 in daughter MCMBP-knockout cells does not engage in pre-RC formation in the absence of complementary MCM subunits.

a, MCM4–Halo dual labelling protocol. b, Left, QIBC of U2OS cells stained for parental and nascent MCM2–Halo and DAPI (top), and parental and nascent MCM4–Halo and DAPI (bottom) without pre-extraction; n ≈ 3,000 cells per condition. Right, graphical summary of the inherited parental and nascent MCMs in daughter cells. c, Left, QIBC of MCMBP-knockout cells analysed as in b. Right, graphical summary of the inherited parental and nascent MCMs in MCMBP-knockout daughter cells. The boxes in b, c mark the excess nascent MCM2 or MCM4 over the parental MCM2 or MCM4 in G1 and at the G1/S boundary. d, QIBC of U2OS cells stained for chromatin-bound parental and nascent MCM2–Halo and DAPI (top, n ≈ 3,000 cells per condition) and chromatin-bound parental and nascent MCM4–Halo and DAPI (bottom, n ≈ 2,000 cells per condition). e, QIBC of MCMBP-knockout cells processed and analysed as in d. The boxes in d, e mark the excess nascent MCM2 or MCM4 over the levels of parental MCM2 or MCM4 in G1 and at the G1/S boundary. f, QIBC of chromatin-bound MCM7 and PCNA in indicated cells. Box plots, centre lines are medians, the boxes indicate the 25th and 75th centiles, the whiskers indicate 5 and 95 per cent values. n = 1,000 cells per condition. g, Frequency of micronucleation in the indicated cells and represented as percentage of all counted nuclei (500 nuclei per condition). Values denote mean ± s.d.; n = 3 biological replicates. h, Relative plating efficiency in the indicated cells. Values denote mean ± s.d.; n = 6 technical replicates. P values were determined by one-way ANOVA with Tukey’s test (f, g), not significant (n.s.) denotes P > 0.05.

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Extended Data Fig. 9 Reduced pre-RCs accelerate forks and trigger DNA damage without major alterations of CMG activity.

a, Left, DNA-fibre labelling protocol. Middle and right, distance between active forks and local fork density. Hydroxyurea (HU; 0.5 mM) was added 30 min before CldU; n = 50 initiation events per condition except n = 35 for siTRESLIN in MCMBP-knockout cells. b, Immunoblots of U2OS and MCMBP-knockout cells treated as indicated. c, Micronucleation (500 nuclei per condition) in MCMBP–AID–GFP cells after IAA (0.5 mM) treatment; values represent mean ± s.d.; n = 3 biological replicates. Scale bar, 20 μm. d, QIBC of MCM7 and PCNA in MCMBP–AID–GFP cells after treatment with IAA (0.5 mM, 24 h) and siRNA against CDC6 (left and middle) with corresponding immunoblots (right). e, QIBC of total, parental and nascent MCM4 from Fig. 4d. Lines denote medians; n ≈ 6,000 cell per condition. f, QIBC of TIMELESS in cells from Fig. 4d; n ≈1,500 cells per condition g, QIBC of γH2AX in S-phase cells from d. h, Left, DNA fibre protocol and immunoblots; right, replication fork speed in U2OS and MCMBP-knockout cells. i, Left, DNA fibre protocol. Right, fork speed after treatment with CHK1 inhibitor (1 μM, 1 h). j, QIBC of TIMELESS in cells treated as in i. Lines in h, i denote medians; n = 200 fibres per condition (h, i). In box plots (a, d, f, g, j), centre lines denote medians, the boxes indicate the 25th and 75th centiles, the whiskers indicate 10 and 90 (a) or 5 and 95 per cent values (d, f, g, j). P values were determined by one-way ANOVA with Sidak’s (a) or Tukey’s test (d, gj) or two-tailed unpaired t-test (f). Not significant (n.s.) denotes P > 0.05; n = 1,000 cells per condition (d, g, j). Gel source data for b, d, h are provided in Supplementary Fig. 1.

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Extended Data Fig. 10 Enforced deceleration of fast forks in MCMBP-deficient cells mitigates replication stress.

a, Immunoblots of MCMBP-degron cells treated with IAA (0.5 mM, 24 h) control siRNA and siRNA against TIMELESS. b, Left, DNA fibre protocol. Middle, replication fork speed in control and MCMBP-knockout cells treated with control siRNA, siRNA against TIMELESS or aphidicolin (APH; 25 nM, 40 min). Lines denote medians; n = 200 fibres per condition. Right, sister fork ratio; n = 50 bidirectional forks per condition. c, Left, replication fork speed in indicated cells treated with control siRNA and siRNA against TIMELESS. Lines denote medians; n = 200 fibres per condition. Middle, sister fork ratio; n = 50 bidirectional forks per condition. Right, QIBC of γH2AX in S phase; n ≈ 1,000 cells per condition. In box plots (b, c), centre lines are medians, the boxes indicate the 25th and 75th centiles, the whiskers indicate Tukey values (a, c middle) or 5 and 95 per cent values (c right). P values were determined by one-way ANOVA with Tukey’s test (b, c). Not significant (n.s.) denotes P > 0.05. Gel source data for a are provided in Supplementary Fig. 1. d, Model depicting production, nuclear transport and inheritance of MCM2–7, and the role of MCMBP (see text for details). Because both nascent and parental MCMs are activated as CMG helicases in a 1:1 ratio (despite the twofold excess of nascent MCMs), we propose that homotypic double hexamers composed only of parental MCMs are rare; instead, we suggest that the bulk of parental and nascent MCM2–7 rings engage in heterotypic pre-RCs, which—owing to the parental component—are more likely to mature to active CMGs. The remaining excess of nascent MCMs might then form homotypic pre-RCs fully composed of nascent MCMs involved primarily in fork speed regulation. Because MCMBP-deficiency reduces the latter component, such cells still support origin firing but are less efficient in confining fork speed within its physiological range.

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Supplementary information

Source data (uncropped gels and LFD)

Supplementary Figure 1 . This figure contains all uncropped gel and LFD scans shown in the main and extended data figures. Dashed black boxes indicate cropped regions.

Reporting Summary

Chemical reagents, siRNAs and antibodies

Supplementary Table 1 . The table details information on the chemicals, siRNAs, and antibodies used in this study. The information for the siRNA concentration and incubation time is provided corresponding to the data in main and extended data figures.

SILAC-MS-based analysis of MCMBP interactome

Supplementary Table 2 . This table details the SILAC-mass spectrometry-based experimental design and protein list identified as MCMBP interactome after FLAG-IP of FLAG-tagged MCMBP. The data in Extended Data Figure 5a showing the levels of MCM interactions with MCMBP is derived from this table.

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Sedlackova, H., Rask, MB., Gupta, R. et al. Equilibrium between nascent and parental MCM proteins protects replicating genomes. Nature 587, 297–302 (2020). https://doi.org/10.1038/s41586-020-2842-3

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