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. 2007 Dec 15;21(24):3331-41.
doi: 10.1101/gad.457807.

Dormant Origins Licensed by Excess Mcm2-7 Are Required for Human Cells to Survive Replicative Stress

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Free PMC article

Dormant Origins Licensed by Excess Mcm2-7 Are Required for Human Cells to Survive Replicative Stress

Xin Quan Ge et al. Genes Dev. .
Free PMC article

Abstract

In late mitosis and early G1, Mcm2-7 complexes are loaded onto DNA to license replication origins for use in the upcoming S phase. However, the amount of Mcm2-7 loaded is in significant excess over the number of origins normally used. We show here that in human cells, excess chromatin-bound Mcm2-7 license dormant replication origins that do not fire during normal DNA replication, in part due to checkpoint activity. Dormant origins were activated within active replicon clusters if replication fork progression was inhibited, despite the activation of S-phase checkpoints. After lowering levels of chromatin-bound Mcm2-7 in human cells by RNA interference (RNAi), the use of dormant origins was suppressed in response to replicative stress. Although cells with lowered chromatin-bound Mcm2-7 replicated at normal rates, when challenged with replication inhibitors they had dramatically reduced rates of DNA synthesis and reduced viability. These results suggest that the use of dormant origins licensed by excess Mcm2-7 is a new and physiologically important mechanism that cells utilize to maintain DNA replication rates under conditions of replicative stress. We propose that checkpoint kinase activity can preferentially suppress initiation within inactive replicon clusters, thereby directing new initiation events toward active clusters that are experiencing replication problems.

Figures

Figure 1.
Figure 1.
HU activates the firing of additional origins. (AC) U2OS cells were incubated ±HU for 4 h and pulsed with BrdU. DNA was isolated and spread on glass slides, and BrdU-labeled tracks were detected with fluorescent antibodies. Clusters containing four or more tracks were selected. (A) The distance between the center points of adjacent tracks in a cluster was measured and averaged to give the mean intracluster fork spacing. (B) The mean intracluster fork spacings were determined for at least 100 clusters ±200 μM HU. (C) The mean intracluster fork spacings were averaged to give mean fork spacing in each sample ±SEM for the indicated concentrations of HU. (Inset) Cells treated with 0–500 μM HU for 4 h were immunoblotted for phospho-Chk1 and actin. (D,E) U2OS cells were incubated ±HU for 4 h and pulsed with BrdU followed by biotin-dUTP. (D) DNA was isolated and spread on glass slides, and labeled tracks were detected with fluorescent antibodies. (E) The distribution of origin-to-origin distances were determined ±200 μM HU; the overall mean and SEM are also indicated. (F) Mean intracluster fork spacing in cells treated with Chk1 siRNA or control siRNA for 96 h, followed by ±200 μM HU for 4 h. Overall means ± SEM (in kilobases) are control, 24.663 ± 0.511, control+ HU, 16.08 ± 0.33; Chk1, 20.42 ± 0.38; Chk1 + HU, 13.71 ± 0.29.
Figure 2.
Figure 2.
Mcm5 knockdown cells proliferate and replicate DNA normally. (A) U2OS cells were transfected with 2 or 4 nM Mcm5-2i siRNA or 4 nM control siRNA and their proliferation rate was analyzed 48–144 h after transfection. (BD) Analysis was carried out 96 h after 2 nM Mcm5-2i siRNA. (B) Cells were pulsed with BrdU for 30 min, and their rate of DNA synthesis as well as DNA content were analyzed by FACS. The percentage S-phase cells and their mean BrdU incorporation are indicated. (C) Immunoblot showing the levels of total Mcm5 and Mcm2. (D) Immunoblot showing the levels of chromatin-bound Mcm2, Mcm3, Mcm5, Mcm6, and Mcm7.
Figure 3.
Figure 3.
Excess Mcm2–7 are required for dormant origin firing. U2OS cells were transfected with 2 nM Mcm5-2i siRNA or control siRNA optionally plus Chk1 siRNA. Ninety-six hours later they were optionally treated for 4 h with 200 μM HU or for 2 h with 5 mM caffeine. (A,B) Cells were pulsed with BrdU and then biotin-dUTP, and interorigin distances were measured. (C–F) Cells were pulsed with BrdU, and intracluster fork spacing was measured. The mean and standard error of the mean for each sample are indicated.
Figure 4.
Figure 4.
Mcm5 knockdown reduces DNA synthesis in response to HU. Cells were transfected with 2 nM Mcm5-2i siRNA. (A,B) Cells were incubated in HU for 4 h (starting 92 h after siRNA) or 40 h (starting 56 h after siRNA), followed by a 30-min BrdU pulse. (A) HU was used at 200 μM. DNA and BrdU content were analyzed by FACS. (B) Total BrdU incorporation after 4-h treatment with HU. (C,D) Fifty hours post-siRNA transfection, cells were incubated ±HU for 48 h then immunoblotted for the indicated checkpoint proteins in isolated chromatin (C) or whole-cell lysates (D). PCNA-L is a light exposure of a PCNA blot to show relative PCNA levels; PCNA-H is a heavy exposure of the same blot, showing an additional band (arrow) migrating at the position of ubiquitinated PCNA.
Figure 5.
Figure 5.
Mcm5 knockdown cells are hypersensitive to HU. Cells were treated with 2 nM Mcm5-2i siRNA or control siRNA. Forty-eight hours or 72 h later they were treated with HU for a further 48 h. Cell number was then assessed; alternatively, cells were subject to a colony-forming assay. (A) Time scale of assays. (B) Cell number was measured 96 h after siRNA and was expressed relative to the cell number plated 40 h after siRNA. (C,D) Surviving colonies were examined 2 wk after siRNA and HU treatment. (C) An example of colonies ±0.5 mM HU treatment. (D) Quantification of colony numbers.
Figure 6.
Figure 6.
Mcm5 knockdown causes hypersensitivity to aphidicolin and camptothecin. (A,B) Fifty hours post-transfection with 2 nM Mcm5-2i siRNA or control siRNA, cells were incubated with 0.1 μg/mL aphidicolin or 10 nM camptothecin for 48 h. DNA content was measured by FACS (A) and whole-cell lysates were immunoblotted for p53, p53 phospho-Ser15, and actin (B). (C,D) The clonogenic assay described in Figure 5A was applied to cells treated with aphidicolin (C) or camptothecin (D).
Figure 7.
Figure 7.
Models of how replicative stress could activate dormant origins. Unfired replication origins licensed by Mcm2–7 (green circles) are shown distributed on DNA (black line) within a replicon cluster. Origins can become inactivated (unlicensed) as a consequence either of initiating (red circles) or of being passively replicated (gray circles). (A) A cluster before it has become activated for initiation. (B) The cluster soon after activation in the absence (left) or presence (right) of HU. (C) By slowing down fork progression, HU (or another replication inhibitor) gives dormant origins more time to fire, despite the increased repression of initiation by checkpoint kinases such as Chk1. However, Chk1 represses initiation more strongly in later-firing (inactive) clusters than in clusters that are already active.

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