Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells

Abstract

To prevent rereplication of genomic segments, the eukaryotic cell cycle is divided into two nonoverlapping phases. During late mitosis and G1 replication origins are "licensed" by loading MCM2-7 double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin-a "double fork stall" (DFS)-replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges, and G1-specific p53-binding protein 1 (53BP1) nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agreed with theoretical predictions of DFSs. The 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins were removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle.

Keywords: 53BP1; DNA replication; MCM; UFB; cell cycle.

Fig. 1.

Potential mechanism for resolution of DFSs. (A) Distribution of replicon sizes in HeLa cells, based on data from ref. . The red bar represents the average replicon size of ∼31 kb. (B) Mean number of DFSs predicted using a mathematical model (4), and a computational model that uses origin data from HeLa and IMR-90 (13) when origins are added or depleted randomly. (C) Model for segregation of unreplicated DNA to daughter cells for resolution in the next cell cycle. (D) The 53BP1 nuclear bodies in untreated and aphidicolin-treated cells. (E) Frequency of G1-specific 53BP1 nuclear bodies (n = 100, three replicates). χ² test for a fitted Poisson, P = 0.771. (F) Frequency of G1-specific 53BP1 nuclear bodies at the times indicated after nocodazole treatment and mitotic shake-off (n = 150, three replicates, error bars are SEM). χ² test, P = 0.924. (G) Frequency of 53BP1 nuclear bodies at different stages of the replication timing program, as defined by O’Keefe et al. (32) (n = 150, three replicates, error bars are SEM). χ² test, P = 4.998 × 10⁻⁴.

Fig. S1.

Dynamics of 53BP1 nuclear bodies. (A) Mean number of G1-specific 53BP1 nuclear bodies in untreated and aphidicolin-treated cells. Error bars are SEM of three replicates. (B) Representative image of 53BP1 nuclear body identification at different stages of the cell cycle. Cells were labeled for 53BP1 (green), EdU (orange), and Cyclin A (red). (C) Frequency of 53BP1 nuclear bodies at different stages of the cell cycle. Only G1-specific nuclear bodies fit a Poisson distribution. (D) Frequency of G1-specific 53BP1 nuclear bodies in untreated and aphidicolin-treated cells. n = 100. (E) Frequency of cells at different stages of the cell cycle after release from nocodazole arrest. Cells in G1 were assessed for the frequencies of 53BP1 nuclear bodies from T2–T8 (green area) and cells from T8–T12 were used to identify the S-phase pattern (yellow area).

Fig. 2.

Origin number and the frequency of 53BP1 nuclear bodies. (A) Immunoblot of total and chromatin-bound MCM5 in HeLa cells after MCM5 RNAi. (B) Three-dimensional FACS of HeLa cells labeled with EdU (Left) and MCM2 (Right). Red, G1 phase: EdU negative and G1 DNA content. Blue, early S phase: incorporation of EdU without significant increase in total DNA content. Orange, late S phase: EdU positive cells with >G1 DNA content. Green, G2 phase: EdU negative and G2 DNA content. (C) FACS of chromatin-associated MCM2 signal in early S phase HeLa cells with indicated periods of MCM5 RNAi. (D) Frequency of G1-specific 53BP1 nuclear bodies (y-axis values) after MCM5 knockdown versus relative number of replication origins quantified by 3D FACS of DNA-bound MCM2 (x-axis values). Each point represents the mean of 100 cells. (E and F) CDC6-inducible HBEC cells. Immunoblot of CDC6 and tubulin in whole-cell lysates (E, Top) and MCM5 and Lamin B1 in chromatin samples (E, Bottom). Frequency distribution of 53BP1 nuclear bodies in HBEC cells (F) (n = 100, three replicates). χ² tests for fitted Poissons, P > 0.87. The two conditions are significantly different (Wilcoxon rank sum test, P = 2.843 × 10⁻⁶). (G) Compilation of the predicted number of DFSs using the mathematical model and the computer simulation (from Fig. 1B) and the mean number of 53BP1 nuclear bodies in vivo (from D and F and Fig. S2E). (H) Frequency of G1-specific 53BP1 nuclear bodies in control and MCM5 RNAi-treated IMR-90 cells (n = 150, three replicates, error bars are SEM). t test, P = 1.79 × 10⁻⁴. (I) Immunoblot to show the depletion of MCM5 after RNAi. Quantification of band intensity is indicated below the blot.

Fig. S2.

Quantification of origin reduction. (A) Western blot for replication origin depletion in HeLa cells using RNAi against Cdt1. Cells were transfected for the indicated time before harvesting. (B) Representative FACS plot of chromatin-associated Mcm2 in early S-phase HeLa cells after Cdt1 depletion. (C) PI/EdU FACS profiles for Mcm5 and Cdt1 depletion in HeLa cells. (D) Frequency distribution of 53BP1 nuclear bodies in Mcm5 RNAi-treated cells (Left) and Cdt1 RNAi-treated cells (Right). (E) Mean number of G1-specific 53BP1 nuclear bodies in response to varying degrees of licensing knockdown as measured in B after Cdt1 RNAi. The 53BP1 nuclear bodies were quantified in triplicate in at least 100 cells. (F) Mean number of G1-specific 53BP1 nuclear bodies in U2OS cells after treatment with control or MCM5 RNAi. Error bars are SEM of three replicates, P = 1.4 × 10⁻¹². (G) Total U2OS cell extract after treatment with control or MCM5 RNAi, immunoblotted for MCM5.

Fig. S3.

Cdc6 overexpression. (A) Mean number of 53BP1 nuclear bodies in uninduced and overexpression of Cdc6 in HBEC cells. Error bars are SEM of three replicates, P = 0.00662. (B) FACS profiles of the distribution of chromatin-associated Mcm2 and the EdU incorporation of uninduced and Cdc6 overexpressing HBEC cells.

Fig. 3.

The 53BP1 is enriched at genomic loci that correspond to large replicons. (A) Representative image of the colocalization between G1-specific 53BP1 nuclear bodies and RPA foci. (B) Percentage of total cellular 53BP1 nuclear bodies that colocalize with RPA after treatment with control or MCM5 RNAi (n = 100, three replicates, error bars are SEM). t test, P = 3.01 × 10⁻³. (C) Mean frequency of G1-specific γ-H2AX foci in HeLa cells after MCM5 RNAi (n = 150, three replicates, error bars are SEM). t test, P = 0.585. (D) Plot of the average 53BP1/IgG signal ratio per kilobase against replicon size. A strong and significant correlation is observed (Spearman ρ = 0.91, P < 10⁻¹⁵). (E) Distribution of the size of 53BP1+ and 53BP1− replicons. t test, P < 10⁻¹⁵. (F) Frequency distribution of 53BP1+ and 53BP1− replicons across different replicon sizes. χ² test, P < 10⁻¹⁵.

Fig. S4.

G1-specific γ-H2AX foci. (A) Representative staining of γ-H2AX foci in asynchronous HeLa cells. Intense γ-H2AX foci were quantified in cells that stained negatively for EdU and Cyclin A. (B) Transfected cells used for microscopy were tested for successful depletion by immunoblotting for Mcm5. Quantification of Mcm5 reduction is indicated below the corresponding treatments.

Fig. S5.

Details of 53BP1 ChIP sequencing. (A) Experiment names, key, and number of reads. Details of the alignment can be found in SI Materials and Methods. (B and C) ChIP sequencing quality analysis for 53BP1 (B) and IgG (C). Quality score with respect to the length of the reads (Left) and quality distribution (Right). (D) Plot of the average 53BP1/IgG signal ratio per kilobase against replicon size including SEM (error bars not reported when fewer than three values are present).

Fig. S6.

53BP1 ChIP sequencing distributions. (A) Total intensity of 53BP1 in the nucleus and in nuclear bodies obtained by quantifying microscopy images of cells expressing GFP-53BP1. (B) Immunoblot of 53BP1 in CSK-extracted pellets and CSK-solubilized fractions of HeLa cells. (C) Distribution of ChIP sequencing reads for 53BP1 and control IgG across the entire human genome, with DNA grouped into 1-kb bins. (D) ChIP sequencing 53BP1/IgG ratio for common fragile sites. (E) Mean replication timing computed for each 1-kb genomic region enriched in 53BP1 using timing data from Weddington et al. (35). The null distribution, using all of the values reported for HeLa, is plotted for comparison (Left). The distributions are significantly different (Wilcoxon signed rank test P < 10⁻¹⁰). (F) Frequency of 53BP1+ and 53BP1− replicons of different sizes (χ² test, P = 5 × 10⁻⁴). (G) Plating efficiency of cells for the clonogenic assay. Error bars are SEM of three independent experiments for untreated cells and for the four different genotypes used in the clonogenic assays.

Fig. 4.

MCM5 RNAi effects on mitosis. (A) Representative image of early-mitotic HeLa cell with foci of EdU incorporation. (B) Quantification of foci of EdU incorporation during prophase and prometaphase HeLa cells after MCM5 RNAi (n = 100, three replicates, error bars are SEM). t test, P = 3.43 × 10⁻⁸. (C) Immunoblot to show depletion of MCM5 after MCM5 RNAi. Quantification of band intensity is indicated below the blot. (D) Representative image of UFBs stained with BLM in an anaphase HeLa cell. (E) Frequency of UFBs after 48-h treatment with MCM5 RNAi (n = 75, three replicates, error bars are SEM). t test, P = 0.0473. (F) Frequency distribution of UFBs (n = 100 cells, four replicates). χ² tests for Poissons, P > 0.85. A significant difference was observed. Wilcoxon rank sum test, P = 5.095 × 10⁻³. (G) Immunoblot showing the knockdown of MCM5 and 53BP1 by RNAi in HeLa cells. (H) Clonogenic assay after treatment, as seen in G, with increasing HU (three replicates, error bars are SEM).