Multi-step loading of human minichromosome maintenance proteins in live human cells

Abstract

Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2-7 complex onto chromatin during G1 phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G1 phase. The immobile fraction of MCM2 and MCM4 increases during G1 phase, suggestive of reiterative licensing. In late G1 phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G1 phase progresses and do not colocalize with sites of DNA synthesis in S phase.

Keywords: Cell Biology; Cell Cycle; Chromatin; DNA Replication; Fluorescence Recovery after Photobleaching; Genome Stability; Imaging; Licensing; Live-cell Imaging; Minichromosome Maintenance Complex.

FIGURE 1.

Characterization of MCF7 cells stably expressing GFP-MCM2, GFP-MCM4 or GFP-NLS. A, GFP-MCM2 and GFP-MCM4 expression levels are similar to the endogenous ones. Total cell extracts from parental MCF7 cells and cells stably expressing GFP-NLS, GFP-MCM2 or GFP-MCM4 were analyzed by Western blotting using antibodies against MCM2, MCM4, GFP, and tubulin as a loading control. B and C, GFP-MCM2 and GFP-MCM4 proteins are confined to the nucleus and are absent from the nucleoli. Parental MCF7 cells and cells stably expressing GFP-MCM2, GFP-NLS or GFP-MCM4, as indicated, were analyzed by immunofluorescence using antibodies against MCM2 (B) or MCM4 (C), respectively. DNA was stained with TOTO-3. D and E, stable cells exhibit a physiological cell cycle profile. Cells were fixed; stained with propidium iodide; subjected to FACS analysis (2N refers to diploid DNA content and 4N refers to tetraploid DNA content) (D) or immunostained with antibodies against Cdt1, Geminin, or cyclin A; and the number of cells positive for each staining was recorded (E). F, GFP-MCM2 and GFP-MCM4 form complexes with endogenous MCM proteins. Extracts from parental MCF7, stable GFP-NLS, GFP-MCM2 and GFP-MCM4 cells were prepared, immunoprecipitated (IP) using an antibody against GFP, and immunoblotted with antibodies against GFP, MCM2, MCM4 and MCM7.

FIGURE 2.

GFP-MCM2 and GFP-MCM4 bind to chromatin during G₁ phase, similar to endogenous MCM2 and MCM4 proteins. A and B, stable GFP-MCM2 (A) and GFP-MCM4 (B) cells were synchronized in M phase by nocodazole block and mitotic shake-off, and cells were harvested at different time points (40, 50, 150, 300, and 600 min) as they progressed into G₁ phase. Total cell extracts were prepared (Total) and further fractionated into a soluble (S100) and a chromatin-enriched fraction (P100) (see “Experimental Procedures”). Fractions were subjected to Western blot analysis using antibodies against MCM2, MCM4 and Cdt1. Tubulin and Coomassie Brilliant Blue (CBB) staining were used as loading controls.

FIGURE 3.

Fluorescence recovery after photobleaching shows GFP-MCM2 and GFP-MCM4 dynamics in live cells. Asynchronous MCF7 cells stably expressing GFP-MCM2, GFP-MCM4, Cdt1-GFP, or GFP-NLS were analyzed by FRAP. Recovery of fluorescence in the photobleached region as a function of time is depicted. Immobile fraction (Imm. Frac.) and t½ of the recovery of the mobile fraction were calculated for each curve after fitting the data as described under “Experimental Procedures” using easyFRAP (52). N represents the number of cells analyzed in each condition. Mean values are given for the calculated immobile fraction and half-time of recovery, with corresponding standard deviations.

FIGURE 4.

GFP-MCM2 and GFP-MCM4 bind to chromatin in a Cdt1-dependent manner and exhibit different binding properties through the cell cycle. A, stable GFP-MCM2, GFP-MCM4 and GFP-NLS cells were treated with non-target siRNA (siLuc) or siRNA for Cdt1 (siCdt1) followed by FRAP analysis. Imm. Frac., immobile fraction. N represents the number of cells analyzed for each condition. B, PCNA localization enables discrimination of different cell cycle phases. Stable GFP-MCM4 cells transiently transfected with PCNA-RFP were fixed, and representative images of characteristic non-S phase and S phase patterns (early S phase, middle S phase, and late S phase) were taken. C and D, stable GFP-MCM2 (C), GFP-MCM4 (D) and GFP-NLS cells were transiently transfected with PCNA-RFP. Cells in early, middle, and late S phase as well as non-S phase cells were identified on the basis of the localization of PCNA and analyzed by FRAP. Recovery of fluorescence in the photobleached region as a function of time is depicted. Mean values are given for the calculated immobile fraction and half-time of recovery for all conditions tested, with corresponding standard deviations. N represents the number of cells analyzed for each condition.

FIGURE 5.

GFP-MCM2 and GFP-MCM4 display maximal binding to chromatin during late G₁ phase. A and B, stable GFP-MCM2 (A) and GFP-MCM4 (B) cells were synchronized in M phase by nocodazole (noc) block and mitotic shake-off. GFP-MCM2/4 cells in early G₁ phase (3 h post-mitotic release) and in middle to late G₁ phase (9 h post-mitotic release) as well as unsynchronized (unsynch) stable Cdt1-GFP and GFP-NLS cells were analyzed by FRAP. C and D, stable GFP-MCM2 (C) and GFP-MCM4 (D) cells were synchronized in late G₁ phase after mimosine treatment (mimosine), and in early S phase by a double thymidine block (thymidine) or hydroxurea (HU) treatment and analyzed by FRAP in parallel to unsynchronized GFP-MCM2/4 and GFP-NLS cells. E and F, stable GFP-MCM2 (E) and GFP-MCM4 cells were synchronized in mitosis by monastrol (mon), and time points were taken in early, middle, and late G₁ phase (3, 7, and 13 h following release). Recovery of fluorescence in the photobleached region as a function of time is depicted for all conditions. G and H, following curve fitting of individual FRAP curves, immobile fraction (Imm. Frac.) and t½ of the recovery of the mobile fraction were computed for all cells analyzed in A–F using easyFRAP (52). p.m., postmonastrol; p.n., postnocodazole. N represents the number of cells analyzed for each condition. Data are mean ± S.D.

FIGURE 6.

GFP-MCM2/4 display transient binding during telophase. A, GFP-MCM4 is absent from chromatin during prophase, metaphase, and anaphase but colocalizes with chromatin during telophase. Stable GFP-MCM4 cells were synchronized in M phase after nocodazole block, fixed, and then DNA was stained with DAPI. B–D, stable GFP-MCM2 (B), GFP-MCM4 (C) and control GFP-NLS cells (D) were transiently transfected with a plasmid expressing H2B-RFP that marks chromosomes. Cells in the different stages of M phase were identified on the basis of morphology and analyzed by FRAP. Fluorescence recovery is shown as a function of time. Immobile fraction (Imm. Frac.) and t½ of the recovery of the mobile fraction were computed. N represents the number of cells analyzed for each condition.

FIGURE 7.

Subnuclear distribution of chromatin-bound GFP-MCM2. A, stable GFP-MCM2 cells were either synchronized in M phase after nocodazole (noc) block and mitotic shake-off and released into G₁ phase or synchronized in late G₁ phase by mimosine treatment. After 3 h (Post noc 3h) and 7 h (Post noc 7h) following nocodazole release and in late G₁ phase (Mimosine block), cells were subjected to FLIP analysis, and representative images are shown before and after the bleaching step. B, stable GFP-MCM2 cells were synchronized in mitosis by monastrol and released into a synchronous G₁ phase. 3, 7 and 13 h post-release (early, middle, and late G₁ phase, respectively), cells were analyzed by FLIP. Pre- and post-bleach images are shown for representative cells. C, GFP-MCM2 does not colocalize with PCNA foci in the various stages of S phase. Stable GFP-MCM2 cells were transiently transfected with PCNA-RFP. Cells were discriminated according to PCNA patterning, FLIP was carried out, and representative images were taken. Cells before bleaching are displayed on the left (Pre bleach), and cells after bleaching are displayed on the right (Post bleach). In all panels, the bleached regions are marked by a red line in the post-bleached images. In A and B, red arrowheads indicate immobile structures of GFP-MCM2. In C, part of the post-bleach images in early and middle S phase cells has been enlarged (red squares) to highlight the lack of colocalization between PCNA and GFP-MCM2 foci. The difference in fluorescence intensity between A and B is due to differences in recording and bleaching conditions.

FIGURE 8.

GFP-MCM2 resides on chromatin for ∼60–80 min. A, stable GFP-MCM2 cells were transiently transfected with a plasmid expressing PCNA-RFP. Cells in G₁, early S and middle S phase were identified according to PCNA patterning and subjected to FLIP. Z-stack sections of the volume of each cell were captured every 5 min for a total duration of 2 h, and mesh plots were created as described under “Experimental Procedures.” A subnuclear area of each cell containing GFP-MCM2 foci was defined (red boxes), and the fluorescence intensity (B) as well as the corresponding standard deviation (C) were plotted throughout the course of the experiment.