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. 2018 Jul 5;71(1):117-128.e3.
doi: 10.1016/j.molcel.2018.05.026. Epub 2018 Jun 28.

DNA Replication Determines Timing of Mitosis by Restricting CDK1 and PLK1 Activation

Bennie Lemmens  1 , Nadia Hegarat  2 , Karen Akopyan  3 , Joan Sala-Gaston  3 , Jiri Bartek  4 , Helfrid Hochegger  5 , Arne Lindqvist  6
Affiliations

DNA Replication Determines Timing of Mitosis by Restricting CDK1 and PLK1 Activation

Bennie Lemmens et al. Mol Cell. .

Abstract

To maintain genome stability, cells need to replicate their DNA before dividing. Upon completion of bulk DNA synthesis, the mitotic kinases CDK1 and PLK1 become active and drive entry into mitosis. Here, we have tested the hypothesis that DNA replication determines the timing of mitotic kinase activation. Using an optimized double-degron system, together with kinase inhibitors to enforce tight inhibition of key proteins, we find that human cells unable to initiate DNA replication prematurely enter mitosis. Preventing DNA replication licensing and/or firing causes prompt activation of CDK1 and PLK1 in S phase. In the presence of DNA replication, inhibition of CHK1 and p38 leads to premature activation of mitotic kinases, which induces severe replication stress. Our results demonstrate that, rather than merely a cell cycle output, DNA replication is an integral signaling component that restricts activation of mitotic kinases. DNA replication thus functions as a brake that determines cell cycle duration VSports手机版. .

Keywords: CDK1; DNA replication; G2 phase; PLK1; S phase; cell cycle; mitosis; replication checkpoint V体育安卓版. .

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Figures

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Graphical abstract
Figure 1
Figure 1
Plk1 Activation Correlates to Completion of DNA Replication (A) Schematic of hypothesis. (B) Example of RPE cell expressing PLK1-FRET and PCNA-cb in S phase, G2 phase, and mitosis. Time between images is 20 min. Please note negative correlation between nuclear PLK1 activity and presence of PCNA-cb foci. (C) S phase cells expressing PCNA-cb foci were imaged every 20 min and either mock treated or exposed to 2.5 mM thymidine. (Top) Single-cell examples of PLK1 activity and PCNA foci quantifications are shown. (Bottom) Color-coded heatmap of PLK1 activity and PCNA-cb quantifications of multiple single cells are shown. Dotted line highlights temporal correlation between DNA replication completion and PLK1 activation. Further characterization of thymidine-induced S phase arrest is described in Figure S1. (D) U2OS, RPE, or BJ cells were fixed after a 1-hr EdU pulse and monitored by high-content microscopy. Cells were sorted based on cyclin A2 levels and nuclear size and plotted versus estimated time (Akopyan et al., 2016). Graphs show moving median and SD of EdU signal and pTCTP signal from >1,600 single cells. EdU incorporation is used to measure DNA replication in single cells. pTCTP signal is corrected by treating a control population with the Plk1 inhibitor BI2536. A stepwise scheme of simultaneous cell cycle and TCTP phosphorylation analysis is described in Figure S2.
Figure 2
Figure 2
Suppression of DNA Replication Initiation by Targeting CDC6 and CDC7 (A) Schematic of approach to target CDC6d degradation by a double-degron system. (B) RPE CDC6d cells were treated as outlined, and cell lysates were probed against indicated proteins. (C) RPE CDC6d cells were treated as in (B) yet fixed 48 hr post-AUX addition. EdU was added 1 hr prior to fixation. Upper panel shows representative images of EdU and cyclin A2 expression, and examples of G2 or S phase cells (dotted circles) are indicated. EdU incorporation is used to measure DNA replication in single cells. Lower panel depicts quantification of EdU intensities of S phase cells, as determined by nuclear cyclin A2 levels. Boxplots indicate 10, 25, 50, 75, and 90th percentiles of at least 200 cells per condition. ∗ indicates p < 0.01; Student’s t test. Light or dark gray bars represent data from 2 independent CDC6d clones. (D) RPE CDC6d cells were treated as outlined in top panel, and anticipated cell cycle events are shown in middle panel. EdU incorporation is used to measure DNA replication in single cells. Lower graph depicts quantification of EdU staining versus cyclin A2 levels to detect early and late S phase cells. Please note rapid induction of EdU incorporation in early S phase cells. (E) RPE CDC6d cells were treated as outlined in top panel, and anticipated cell-cycle-dependent effects of combined CDC6d degradation and CDC7 inhibition are shown in middle panels. Lower graph depicts quantification of EdU staining versus cyclin A2 levels to detect early and late S phase cells. Please note that targeting CDC6 and CDC7 specifically inhibits initiation of DNA replication in early S phase cells.
Figure 3
Figure 3
DNA Replication Controls Cell Cycle Duration (A) Asynchronous RPE CDC6d cells were treated as outlined and either fixed after a 2-hr pulse of EdU or followed by time-lapse microscopy. The level of DNA replication initiation, measured by EdU incorporation in single early S phase cells, is correlated directly to cell cycle speed. (B) Schematic of anticipated effect of CDC6d degradation and/or CDC7 inhibition. (C) Cells were treated as outlined in (A) and (B), and nuclear EdU intensity was plotted versus nuclear cyclin A2 levels to monitor DNA replication during cell cycle progression. Please note that combined CDC6 degradation and CDC7 inhibition prevents EdU incorporation in early S phase cells. Lower panels illustrate a single cell entering mitosis after CDC6 degradation and/or CDC7 inhibition (i.e., upon addition of AUX/CDC7i). Illustration depicts relative timings of empirical tests (fix or live) as well as anticipated cell fate. (D) Tracks of single cells passing through mitosis within 6 hr after addition of auxin or CDC7i were followed from mitosis to the following mitosis. Grey bars indicate mitosis, and red bars indicate interphase. See Figure S4 for example images of mitotic cells.
Figure 4
Figure 4
DNA Replication Restricts Activation of PLK1 (A) Examples of U2OS cells expressing PLK1-FRET and PCNA-cb followed in the absence or presence of combined CDC6/CDT1 RNAi. Time between images is 45 min. Please note absence of PCNA foci despite proficient PCNA-cb expression and nuclear PLK1 activation within 5 hr. (B) U2OS cells expressing PLK1-FRET were treated as outlined in left panel and fixed after a 2-hr pulse of EdU or followed by time-lapse FRET microscopy. (Top) EdU quantification of >600 single cells; nuclear EdU intensity was plotted versus nuclear cyclin A2 levels to monitor DNA replication during cell cycle progression. Outlined boxes indicate EdU-negative cells; please note that the three most right conditions generate cells that have intermediate cyclin A2 levels but fail to incorporate EdU, which corresponds to the conditions that have premature PLK1 activation. (Bottom) Live-cell traces of single cells that entered mitosis within 2 hr upon addition of CDC7i were followed for 16 hr. Graphs show quantified PLK1 activity of 25 single cells. (C) Representative single-cell traces of the different classes observed upon Cdc6/Cdt1 co-depletion. U2OS cells expressing PLK1-FRET and PCNA-cb were transfected with control or Cdc6/Cdt1 small interfering RNA (siRNA) for 24 hr and mitotic cells were followed by live-cell imaging. The distinct classes include (class I) cells similar to controls, having discrete PCNA foci and PLK1-FRET activity only upon PCNA foci resolution, (class II) cells maintaining dim PCNA foci and no PLK1-FRET activity, and (classes III and IV) cells with no or very few PCNA foci and premature PLK1-FRET activity (4–16 hr post), which either undergo S/G2 arrest (class III) or premature mitosis (class IV). (D) Pie charts depict distribution of classes I–IV among cells depicted in (B). Whereas targeting licensing or firing can cause premature mitosis, we find the latter approach to be most effective.
Figure 5
Figure 5
DNA Replication Restricts PLK1 and CDK1/2 Activity to Prevent Replication Stress (A) Asynchronous U2OS cells expressing PLK1-FRET and APC/CCdh1 substrate reporter were mock treated or treated with 50 nM CHIR-124 at mitosis (±1 hr) and followed by time-lapse microscopy for 24 hr. Each line represents a single cell showing expression of APC/CCdh1 substrate probe (light blue) and/or PLK1 activity (red). Visual appearance of nuclear GEM-RED signal (i.e., stabilization of the APC/CCdh1 substrate probe) was used to assess APC/C inactivation at the G1/S transition (APCOFF). Detection of nuclear PLK1 FRET signal was used to assess PLK1 activation at the S/G2 transition (PLK1ON). (B) Boxplot depicts G1 phase duration of 50 single cells, as determined by the time observed between mitosis and APC/CCdh1 substrate appearance at the G1/S transition. Boxplots indicate 10, 25, 50, 75, and 90th percentiles. n.s. indicates p > 0.5; Student’s t test. (C) As in (B), yet boxplot depicts S phase duration as determined by the time observed between APC/CCdh1 substrate appearance and the detection of nuclear PLK1 activity. Boxplots indicate 10, 25, 50, 75, and 90th percentiles. ∗ indicates p < 0.01; Student’s t test. (D) Images illustrate representative cells in S phase (13 hr post-mitosis) or G2 phase (17 hr post-mitosis). (E) Asynchronous RPE cells were treated with EdU, CHIR-124 (Chk1i), and SB202190 (p38i) for 2 hr and during the last hour with RO-3306 (CDK1i) and NU6140 (CDK2i) as indicated. EdU is used to mark S phase cells and directly correlate the presence of DNA replication with CHK1i-induced CDK activation and DNA damage. Graphs show DAPI, lamin A/C pS22, and H2AX pS139 for >800 cells measured by high-content microscopy. Single-cell illustrations below the graph indicate cell cycle phases and presence of DNA replication stress. (F) RPE cells were treated with CHIR-124 (CHK1i) and SB202190 (p38i) as indicated. To define S phase, cells were incubated with EdU 1 hr prior to fixation. Panels show quantifications of high-content microscopy for H2AX pS139 in single cells. Cell populations were separated according to DAPI and EdU intensity. (G) RPE cells were treated with CHIR-124 (CHK1i), SB202190 (p38i), NU6140 (CDK2i), RO3306 (CDK1i), CDK1/2 inhibitor III (CDK1/2i), and Roscovitine as indicated for 4 hr. Before fixation, cells were incubated for 1 hr with EdU. Boxplots show 90, 75, 50, 25, and 10th percentiles of EdU signal or H2AX pS139 signal of EdU positive cells, as assessed by high-content microscopy. ∗∗∗ indicates p < 0.001, and ns indicates p > 0.1; Student’s t test.
Figure 6
Figure 6
DNA Replication Limits CDK1/2 Activation upon S Phase Entry (A) Single U2OS cells were filmed upon mitotic exit, and cells that were ±1 hr from mitosis upon addition of CHIR-124 (CHK1i) were selected for analysis. Montages depict an example cell expressing CDK1/2 activity sensor and PCNA-cb followed by time-lapse microscopy. Time between images is 45 min. Note the rapid cytoplasmic translocation of the CDK1/2 sensor at the G1/S transition in the presence of CHK1i. Please see Figure S6C for readout details. (B) Quantification of single cells imaged as in (A). Red line indicates relative CDK1/2 activity, and blue line indicates PCNA-cb intensity variation as a surrogate measurement for DNA replication. Grey lines show average CDK1/2 activity of control cells. To illustrate concurrence at G1/S, three single cells with different G1 lengths are depicted. (C) U2OS cells expressing CDK1/2 activity sensor and APC/CCdh1 substrate (GEM-RED) after CHIR-124 treatment and followed as in (A). To illustrate concurrence at G1/S, two single cells with different G1 lengths are depicted. (D) Quantification of single cells imaged as in (A) yet 24 hr after transfection of Cdc6 and Cdt1 siRNA. Red line indicates relative CDK1/2 activity, and blue line indicates PCNA-cb intensity variation as a surrogate measurement for DNA replication. Grey lines show average CDK1/2 activity of control cells. (E) U2OS cells expressing CDK1/2 activity sensor and APC/CCdh1 substrate (GEM-RED) treated and followed as in (D). To illustrate concurrence at G1/S, two single cells with different G1 lengths are depicted per condition. (F) Quantification of CDK1/2 reporter of cells monitored as in (C) and (E). G1 is defined as 1 hr before and S phase as 3 hr after appearance of APC/CCdh1 substrate reporter. Graph shows mean and SD of 14 cells per condition. ∗p < 0.04; Student’s t test.
Figure 7
Figure 7
Model for Integral Role of DNA Replication in Human Cell Cycle Wiring Proposed model in which DNA replication constitutes a functional output as well as an integral component of an incoherent feedforward loop in human cell cycle signaling. DNA replication generates a CHK1- and p38-dependent signal that prevents premature mitosis and CDK1-driven DNA damage in S phase.
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