Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication

Abstract

The six main minichromosome maintenance proteins (Mcm2-7), which presumably constitute the core of the replicative DNA helicase, are present in chromatin in large excess relative to the number of active replication forks. To evaluate the relevance of this apparent surplus of Mcm2-7 complexes in human cells, their levels were down-regulated by using RNA interference. Interestingly, cells continued to proliferate for several days after the acute (>90%) reduction of Mcm2-7 concentration. However, they became hypersensitive to DNA replication stress, accumulated DNA lesions, and eventually activated a checkpoint response that prevented mitotic division. When this checkpoint was abrogated by the addition of caffeine, cells quickly lost viability, and their karyotypes revealed striking chromosomal aberrations. Single-molecule analyses revealed that cells with a reduced concentration of Mcm2-7 complexes display normal fork progression but have lost the potential to activate "dormant" origins that serve a backup function during DNA replication. Our data show that the chromatin-bound "excess" Mcm2-7 complexes play an important role in maintaining genomic integrity under conditions of replicative stress.

Fig. 1.

Cell proliferation after MCM downregulation. (A) Proliferation curves after transfection of siRNA oligonucleotides targeting each one of the Mcm2–7 subunits (Materials and Methods). (B) Levels of individual MCM subunits on chromatin were determined by immunoblotting at 48, 72, and 96h after transfection of the indicated siRNAs. Orc2 is shown as loading control. (C) Quantification of the efficiency of Mcm3 knockdown. The amount of Mcm3 remaining in a total cell extract prepared 72 h after Mcm3 siRNA was determined by immunoblotting using different amounts of extract from untreated cells as reference. Orc2 levels are shown as loading control.

Fig. 2.

S-phase dynamics after MCM downregulation. (A) DNA content, analyzed by flow cytometry 48 h after transfection with the indicated siRNA. The percentage of cells in each phase of the cell cycle was calculated by using ModFit software. (B) Cellular BrdU incorporation assay, carried out 24 or 72 h after transfection with the indicated siRNA, after a 30-min pulse with BrdU (Materials and Methods). (C) Fork progression rates. Control cells or cells treated with the indicated siRNA were pulsed with BrdU for 30 min and processed for DNA combing analysis 48 h after RNAi (Materials and Methods). The length of >100 BrdU tracks were measured in each case. In the box plot, the horizontal line represents the full range of experimental data, the box spans the interquartile range, leaving out the lower and upper quartiles, and the vertical line within the box indicates the median value.

Fig. 3.

Hypersensitivity to replicative stress and activation of the DDR. (A) Proliferation curves of control or Mcm3 siRNA-treated cells growing in regular medium (black lines) or medium supplemented with 0.1 μM aphidicolin (red lines). (B) DNA content of control cells (i) or cells treated with Mcm3 siRNA (ii), analyzed 120 h after RNAi. iii and iv show the same analyses in cells treated with 5 mM caffeine for 5 h before cell collection. v–viii are similar to i–iv, but cells were grown in the presence of 0.1 μM aphidicolin. (C) Detection of the activated forms of Chk1 and Chk2 kinases and the phosphorylated form of H2AX at days 1, 3, and 5 after Mcm3 siRNA treatment, in the absence or presence of 0.1 μM aphidicolin. The uppermost row of blots shows the reduction of Mcm3 levels. Mek2, a cytosolic kinase, is shown as loading control. (D) Immunostaining of Mcm3 (green) and RPA (red) at 120 h after RNAi. DNA was stained with DAPI (blue). The bar graph indicates the percentage of cells that score positive for two or more RPA foci (n > 200 in each case) either in the absence or presence of 0.1 μM aphidicolin.

Fig. 4.

Increased chromosomal instability under conditions of limited licensing. Metaphase spreads of control or Mcm3 siRNA-treated cells (96 h after siRNA), grown in the absence or in the presence of 0.1 μM aphidicolin, were scored for chromosome gaps (dark gray boxes), breaks (light gray boxes), and aberrant rearrangements (white boxes). Ten metaphases of each cell condition were analyzed.

Fig. 5.

Loss of backup replication origins. (A) Relative frequency of origin activation in control cells or cells treated for 48 h with the indicated siRNA, as determined by single-molecule analyses. (B) Fold-increase in origin activation when the same cells were treated with UCN-01 for 5 h. (C) Schematic of DNA replication under full licensing conditions: MCM complexes are located in multiple sites within a replicon. The activation of one “main origin” (black arrow) sends a signal, mediated by Chk1, to inhibit the activation of nearby origins (open arrows). In the eventuality of a fork collapse, the activation of a previously “silent” origin rescues the stretch of nonreplicated DNA. (D) DNA replication under limited licensing conditions. Fewer potential origins are licensed than in the control situation, and the rescue of stalled forks is compromised by the lack of backup origins.