Fig 2: Lamin B2 increases MCM7 activity(A) Chromatin association experiments demonstrate that LMNB2 knockdown decreases MCM7 chromatin association, whereas LMNB2 overexpression increases MCM7 chromatin association than in control cells (P<0.01). Note: The chromatin (Chr) and non-chromatin (Non-Chr) fractions were purified and immunoblotted with antibodies specific for MCM7. Antibodies against histone H3 and glyceraldehyde-3-phosphate dehydrogenase were used as chromatin purity controls (P<0.01) (B) Helicase activity of MCM7 is decreased in LMNB2-RNAi transfected cells and increased in GFP-LMNB2 transfected cells than in control cells (P<0.01). MCM7 was immunoprecipitated with anti-MCM7 antibodies from A549 cells transfected with shLMNB2 or GFP-LMNB2 vectors and their corresponding controls. Helicase assays were performed with a Cy5-labeled DNA duplex template and biotin-labeled oligocapture probe. The probes were then captured onto streptavidin-agarose beads (Thermo Fisher Scientific, USA) and washed by 1X TBST buffer thrice and analyzed the fluorescence intensity by Thermo Varioskan Flash. (P<0.01).

Fig 3: 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).

Fig 4: 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.

Fig 5: 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).