Spontaneous Slow Replication Fork Progression Elicits Mitosis Alterations in Homologous Recombination-Deficient Mammalian Cells

Home , Chromosome segregation

Spontaneous slow replication fork progression elicits mitosis alterations in homologous recombination-deficient mammalian cells

Therese Wilhelma,b,c,d,1, Indiana Magdaloua,e,f,1, Aurélia Barascua,e, Hervé Técherb,c,d, Michelle Debatisseb,c,d, and Bernard S. Lopeza,e,f,2

aUniversité Paris Sud, F-91405 Orsay, Cedex, France; bCentre de Recherche, Institut Curie, 75248 Paris Cedex 05, France; cUniversité Pierre et Marie Curie, 75005 Paris, France; dCentre National de la Recherche Scientifique, Unité Mixte de Recherche 3244, F-75248 Paris, France; eCentre National de la Recherche Scientifique, Unité Mixte de Recherche 8200, Institut de Cancérologie Gustave-Roussy, 94805 Villejuif, France; and fCentre National de la Recherche Scientifique, Unité Mixte de Recherche 217, Commissariat à l’Energie Atomique, Direction des Sciences du Vivant, Institut de Radiobiologie Cellulaire et Moléculaire, Fontenay aux Roses, F-92265, France

Edited by James E. Haber, Brandeis University, Waltham, MA, and approved November 25, 2013 (received for review June 19, 2013) Homologous recombination deficient (HR−) mammalian cells spon- account for these two phenotypes. First, replication stress leads taneously display reduced replication fork (RF) movement and mi- to chromosome alteration at incomplete replicated regions and totic extra centrosomes. We show here that these cells present chromosome rearrangements (29). However, centrosomes do not a complex mitotic phenotype, including prolonged metaphase ar- contain DNA, and if extra centrosomes at mitosis [mitotic extra rest, anaphase bridges, and multipolar segregations. We then centrosome (MEC)] are active, unbalanced chromosome segre- asked whether the replication and the mitotic phenotypes are in- gation should lead to global chromosome instability, even for fully terdependent. First, we determined low doses of hydroxyurea replicated chromosomes. Second, HR proteins are associated that did not affect the cell cycle distribution or activate CHK1 with supernumerary centrosomes; therefore, centrosome dupli- phosphorylation but did slow the replication fork movement of HR− cation defects may directly result from HR misregulation (30, 31). wild-type cells to the same level than in cells. Remarkably, In this study, we addressed whether spontaneous MECs result − these low hydroxyurea doses generated the same mitotic defects from slow replication fork movement in HR cells. The data (and to the same extent) in wild-type cells as observed in unchal- − presented here underline the importance of HR at the molecular lenged HR cells. Reciprocally, supplying nucleotide precursors to − interface between replication and chromosome segregation to HR cells suppressed both their replication deceleration and mitotic extra centrosome phenotypes. Therefore, subtle replication protect against spontaneous genomic instability. stress that escapes to surveillance pathways and, thus, fails to Results prevent cells from entering mitosis alters metaphase progres- The Impact of Low Hydroxyurea Doses on the Cell Cycle and RF Speed. sion and centrosome number, resulting in multipolar mitosis. GENETICS We used cell lines derived from hamster V79 cells because Importantly, multipolar mitosis results in global unbalanced − chromosome segregation involving the whole genome, even several extensively characterized HR cell lines exist in this fully replicated chromosomes. These data highlight the cross- background. Notably, we used cells that were affected by a loss- talk between chromosome replication and segregation, and of-function mutation in the endogenous BRCA2 gene (V-C8 the importance of HR at the interface of these two processes cells) (26) and cells that express a dominant negative form of for protection against general genome instability. Significance NA is continuously subjected to injury by exogenous and Dendogenous sources. The faithful transmission of genetic Faithful genome duplication requires the precise coordination material relies on the DNA damage response (DDR), which of DNA replication, repair/recombination and chromosome coordinates a network of pathways, including DNA replication- segregation. Homologous recombination (HR) plays a pivotal − repair-recombination, the cell cycle checkpoint, and chromosome role in the resumption of blocked replication forks, and HR segregation. A defect in any of these pathways causes genetic cells exhibit both spontaneous slower genome-wide replica- instability and cancer predisposition. Strikingly, both spontaneous tion fork speed and mitotic extra centrosomes (MECs). We DDR activation as a consequence of endogenous replication show that MECs result from slow replication kinetics and that stress and centrosome abnormalities, which cause uneven chro- MECs are associated with multipolar mitosis leading to general mosome segregation, have been reported in precancerous and unbalanced chromosome segregation. Thus, low levels of rep- early-stage malignancies (1–10). Therefore, endogenous stresses lication stress, which are not detected by cell surveillance, allow must play a key role in spontaneous chromosome instability and cells to progress through the cell cycle, resulting in aberrant in cancer etiology. mitosis and chromosome instability. These data underline the Homologous recombination (HR) is an evolutionarily con- essential role of HR facing endogenous stress at the interface served process that controls the balance between genetic stability between replication and mitosis for protection against spontaneous general chromosome instability. and diversity. Specifically, HR plays a pivotal role in the reac-

tivation of replication forks that have been blocked, contributing Author contributions: M.D. and B.S.L. designed research; T.W., I.M., A.B., and H.T. per- to DNA replication accuracy (11–16). Replication forks are formed research; T.W., I.M., A.B., H.T., M.D., and B.S.L. analyzed data; and M.D. and B.S.L. routinely inactivated by endogenous stress (17, 18); therefore, wrote the paper. HR should play an essential role to protect cells against these The authors declare no conflict of interest. types of stresses, and HR deficiency should reveal endogenous This article is a PNAS Direct Submission. − replication stress. Remarkably, unchallenged HR-deficient (HR ) 1T.W. and I.M. contributed equally to this work. cells display both a genome-wide decrease in replication fork 2To whom correspondence should be addressed. E-mail: [email protected]. speed (19) and a spontaneous increase in the frequency of cells This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. containing extra centrosomes (20–28). Two hypotheses may 1073/pnas.1311520111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1311520111 PNAS | January 14, 2014 | vol. 111 | no. 2 | 763–768 Downloaded by guest on September 30, 2021 RAD51 (V79SM24), which specifically inhibits gene conversion for the forks slowing, consistently with the moderate effect on without affecting cell viability (20, 32–34). cell cycle distribution. These data demonstrate that at least 10- In an attempt to reproduce the level of replication fork (RF) fold lower HU doses than the levels classically used (1–5 mM) − slowing observed in untreated V-C8 and V79SM24 (HR ) cells strongly impair fork progression without causing S phase arrest. − (19), we analyzed genome-wide RF speed following treatment Surprisingly, 10 μM HU reduced the RF speed in HR pro- with increasing doses of hydroxyurea (HU), an inhibitor of the ficient cells (Fig. 1), although this concentration did not signifi- ribonucleotide reductase that depletes deoxyribonucleotide (dNTP) cantly induce CHK1 phosphorylation (Fig. S2A) or significantly pools (35). Doses between 1 and 5 mM are commonly used to affect the cell cycle distribution (Fig. S1). Even a lower HU dose arrest cells in S phase, but we expected that lower doses would (5 μM) affected genome-wide RF elongation kinetics (Fig. 1). decelerate rather than block RF progression. Interestingly, the RF speed observed in WT cells treated with Flow cytometry analysis demonstrated that increasing the HU 5or10μM HU was similar to that of untreated V-C8 and − dose progressively leads to the accumulation of cells in S phase, V79SM24 (HR ) cells (Fig. 1B). Interorigin distances were also which was accompanied by fewer cells in the G phase (Fig. S1). significantly reduced in 5 or 10 μM HU-treated WT cells to 1 − Overall, 1 mM HU provoked a strong accumulation of cells in S values similar to that of V-C8 and V79SM24 (HR ) cells (Fig. phase, which was less pronounced with 250 or 500 μM HU. S3B). These data show that the treatment of WT cells with 5–10 Remarkably, 100 μM HU moderately affected the cell cycle μM HU mimics the replication dynamics of unchallenged V-C8 − distribution, and 10 μM HU did not significantly affect the cell and V79SM24 (HR ) cells. cycle distribution (Fig. S1), indicating that the cells were actively replicating their DNA and efficiently progressing through S Slowing the Replication Forks Generates MECs. Next, we analyzed phase. Consistently, the level of CHK1 phosphorylation (pCHK1) the impact of low HU doses on mitosis in WT cells. We first progressively increased in a HU dose-dependent manner (Fig. monitored the number of centrosomes per mitotic cells that were S2A). Ten micromolar HU did not impact the pCHK1 status in identified by chromosome condensation (Fig. 2A and Fig. S4). − WT and V-C8 and V79SM24 (HR ) cells. CHK1 phosphorylation The centrosomes were monitored by immunofluorescence by was detectable in all cell lines except V-C8#13 upon treatment using antibodies raised against two different centrosomal mark- with 50 μM HU, and high HU doses strongly activated CHK1 ers, namely, γ-tubulin (Fig. 2A) and centrobin (Fig. S4), the latter (Fig. S2A). Taken together, these data suggest that HU treatment of which is a centriole-associated protein that is required for progressively affects replication in a dose-dependent manner. centriole elongation and stability and regulates functional mi- HR− totic spindles (36–38). We first confirmed with both antibodies Interestingly, untreated V-C8 and V79SM24 ( ) cells exhibi- − ted higher levels of phosphorylated CHK1 than their respective that V-C8 and V79SM24 (HR ) mutants displayed a spontane- A B ous increase in the frequency of mitotic cells bearing MECs controls (Fig. S2 and ), revealing endogenous replication − stress in this genetic context. compared with untreated HR proficient cells. Strikingly, the RF speed was monitored by using single molecule analysis treatment of WT cells with 5 or 10 μM HU increased the frequency of cells with MECs by 2.7- to 5.5-fold, which is compa- (molecular combing). We found that the replication tracts are − much shorter in cells grown in the presence of 100 μM HU than rable to the frequency in V-C8 and V79SM24 (HR ) cells (Fig. in untreated cells, demonstrating that genome-wide RF pro- 2B and Fig. S4B). Remarkably, the centrobin analysis suggested gression was strongly affected by this treatment (Fig. 1). How- that these MECs may be functional. Importantly, interphase ever, interorigin distance was strongly reduced (Fig. S3), showing cells did not display abnormal centrosome number (Fig. S5), that more replication origins were active, partially compensating suggesting that extra centrosomes accumulate specifically during mitosis. The analysis of the MEC distribution demonstrated that three centrosomes were more frequently observed both in un- − treated V-C8 and V79SM24 (HR ) cells and WT cells treated with low doses of HU (Fig. 2C and Fig. S4C). In addition, although treatment with 10 μMHUgenerated15–30% of cells with MECs (Fig. 2B), these values increased to 50–70% upon exposure to 100 μMHU(Fig. S6).Thesedataarguethatthefrequencyof MECs is correlated with the intensity of replication stress. Finally, exposure to low doses of aphidicolin (an inhibitor of DNA polymerases α, e, and δ) also increased the frequency of MEC-positive cells, specifically mitotic cells with three centrosomes (Fig. S7). This result demonstrates that MECs result from different types of replication stresses, which excludes the possibility that HU may exert an unexpected side effect on MECs.

Low Replication Stress Elicits Prolonged Metaphase Arrest. Extra centrosomes are frequently but not systematically associated with aberrant mitosis; therefore, we determined whether moderate reductions of RF speed that still allows progression through S and G2 phases actually cause altered mitosis and aberrant − chromosome segregation in V-C8 and V79SM24 (HR ) cells and in WT cells exposed to low HU doses. Monitoring mitotic cells using phosphorylated histone H3 Fig. 1. The impact of HR deficiency or hydroxyurea on replication fork antibody or condensed chromosomes by DAPI staining demon- speed. (A) Examples of combed DNA fibers with replication tracts: IdU strated that the mitotic index increased in HR-proficient cells (green), CldU (red), and ssDNA (blue) in nontreated (NT) conditions or after exposed to 5 μM HU as well as in untreated V-C8 and V79SM24 HU exposure. (B) RF speed distribution in V79 cells and derivatives (Left) and HR− V-C8 cells and derivatives (Right). HR-deficient cells are monitored in red. ( ) cells (Fig. S8). This finding is consistent with the delay in Median and P values are indicated (*P < 0.05; **P < 0.01; ***P < 0.001). chromosome condensation resulting from delayed replication tim- Median values are represented as horizontal black lines. Approximately, ing (39). To determine which stage of mitosis was most impacted, 100–120 fibers were scored per condition. ns, not significant. we performed time-lapse video microscopy analyses of mitotic

764 | www.pnas.org/cgi/doi/10.1073/pnas.1311520111 Wilhelm et al. Downloaded by guest on September 30, 2021 demonstrate that the presence of MECs is associated with ab- − normal mitosis both in V-C8 and V79SM24 (HR ) cells and in WT cells treated with low HU doses. Consistent with the centrobin analysis, the increased multipolar segregations in the situations analyzed here demonstrate that the MECs are functional. Thus, MECs represent a tractable predictive marker of aberrant mitosis upon moderate levels of replication stress.

The Addition of Deoxynucleotide Precursor to V-C8 and V79SM24 − (HR ) Cells Restores Both RF Speed and MEC Frequency. Next, we attempted to rescue the fork speed in V-C8 and V79SM24 − (HR ) cells. In various mutant cell types, spontaneous RF slowing has been corrected by adding deoxynucleotide precursors (dNs) to the culture media (42–45). Particularly in co- lorectal cancer cells, supplying dNs corrects slow replication and complex chromosome segregation errors, which were generated by the silencing of CIN (cancer chromosomal instability) sup- pressor genes, although centrosome number was not affected (46). Here, in our cell lines, the addition of dNs did not alter RF progression in WT cells but rescued RF speed in V-C8 and − V79SM24 (HR ) cells (Fig. 5A). Remarkably, exogenous dNs did not affect the frequency of cells with MECs in unchallenged WT cells, but they decreased their frequency in V-C8 and V79SM24 − (HR ) cells (Fig. 5B). Moreover, dNs specifically decreased the frequency of mitotic cells with three centrosomes (Fig. 5C). Treatments with low HU doses or aphidicolin primarily induced cells with three centrosomes; therefore, these data support the conclusion that MECs result from RF slowing. Discussion The data presented here demonstrate that moderate decreases in RF movement do not detectably delay the progression in S μ Fig. 2. The impact of HR deficiency or very low HU doses (5 or 10 M) on and G2 phases and mitotic entry; however, they cause severe metaphases with aberrant centrosome number monitored with γ-tubulin mitosis defects via MEC formation, resulting in multipolar mi- antibody. (A) Examples of labeled centrosomes in mitotic cells (chromosomal tosis and uneven chromosome segregation. The model in Fig. 6 GENETICS = – DAPI staining). (Left) Normal centrosome number ( 2); columns 2 5, aber- summarizes the mechanisms that may link HR, replication, rant centrosomes number (unequal 2), causing metaphase alterations (see and mitosis to protect against global chromosome instability: DNA labeling). (Scale bars: 10 μm.) (B) Frequency of mitotic cells with aberrant centrosome number. Left histograms, V79 cells and derivatives; right histograms, V-C8 cells and derivatives. The mean value ± SD was calculated from at least three independent experiments: *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. In total, 150 mitoses were scored for each experi- ment and condition. (C) Centrosome number distribution in mitotic cells. Upper histograms, V79 cells and derivatives; lower histograms, V-C8 cells and derivatives. In total, 100–150 mitotic cells per cell line were analyzed per condition.

progression by using cells expressing histone H2B-GFP (Movies S1–S3). This analysis revealed that the mitotic delay specifically results from prolonged metaphase arrest both in V-C8 and − V79SM24 (HR ) cells (Fig. 3 A and B and Movie S1) and in WT cells treated with HU doses that mimic the fork speed of V-C8 − and V79SM24 (HR ) cells (Fig. 3B).

Low Replication Stress Generates Anaphase Chromatin Bridges and Aberrant Chromosome Segregation. Chromatin bridges at ana- − phase have been described in other HR cell systems (27, 40, 41). Here, we confirmed these observations and, in addition, showed that the percentage of cells displaying anaphase chromatin − bridges increased similarly in V-C8 and V79SM24 (HR ) cells and in WT cells exposed to 10 μM HU (Fig. 4A). Moreover, we Fig. 3. The impact of HR deficiency or 5 μM HU on mitosis duration. (A) also observed aberrant mitosis and uneven chromosome segre- Chromosome segregation kinetics. Example of time-lapse video microscopy gation (Fig. 4B), which primarily corresponded to multipolar of y-H2B-GFP–tagged wild-type (Top) and HR defective (Middle and Bottom) mitosis (Fig. 3A, Bottom and Movie S3), at similar frequencies cells during a complete mitotic cycle. (Scale bars: 10 μm.) (B) Median kinetics − in both V-C8 and V79SM24 (HR ) cells, and WT cells exposed of the different mitosis phase as measured by time-lapse video microscopy. B For analysis, mitosis was clustered into prophase to metaphase and meta- to low levels of HU (Fig. 4 ). Remarkably, multipolar mitosis phase to anaphase. Left histogram, V79 cells and derivatives; right histo- was generally associated with chromatin bridges (see examples gram, V-C8 cells and derivatives. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not in Fig. 3A, Bottom and Movie S3). Taken together, the data significant. At least 100 cells were analyzed per condition.

Wilhelm et al. PNAS | January 14, 2014 | vol. 111 | no. 2 | 765 Downloaded by guest on September 30, 2021 Centrosome reduplication arises when centrosome duplication is uncoupled from cell cycle progression when replication is com- pletely arrested by high doses of HU for a cell division cycle (51). Centrosome reduplication should primarily cause a paired number of extra centrosomes; however, we essentially observed cells with three centrosomes. In addition, interphase cells did not display an increased number of centrosomes, which would be expected for centrosome reduplication. Finally, WT cells treated with low doses − of HU and V-C8 and V79SM24 (HR ) mutant cells cycled nor- mally; therefore, centrosome reduplication does not account for the phenomenon we observed here. Alternatively, centrosome splitting has been associated with DNA damage accumulation (52). HR proteins are associated with centrosomes, and centrosome duplication defects are thought to directly result from HR misregulation (30, 31). In addition, CHK1 activation controls centrosome alterations and amplification (25, 53–55). However,

Fig. 4. The impact of HR deficiency or very low HU doses (5 or 10 μM) on chromosome segregation. (A) Chromatin bridges. (A Upper) Example of anaphase chromatin bridge (see also Fig. 3A, Middle). (Scale bars: 10 μm.) (A Lower) Frequency of mitotic cells with chromatin bridges. Shown are V79 cells and derivatives (ALeft) and V-C8 cells and derivatives (ARight). The mean value ± SD from three independent experiments was calculated. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. In total, 150 mitoses were scored per condition. (B) The frequency of aberrant mitosis in V79 cells and derivatives (Left) and V-C8 cells and derivatives (Right). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. The mean value ± SD of three independent experiments was calculated. In total, 150 mitoses were scored per condition.

Different causes decelerate RF (HR defect, dNTP shortage, polymerase inhibition); the firing of origins compensates for RF slowing upon moderate levels of stress, allowing maintenance of the global replication rate and normal cell cycle progression (47); however, the origins cannot be activated in several genomic regions, resulting in mitotic entry with incomplete replicated chromosomes (48). This process should be facilitated by the fact that low levels of replication stress escape to the surveillance pathway. Incomplete replicated regions impair chromatid segregation (29), generating chromatin bridges that activate mitotic arrest. Indeed, in this study, we demonstrate the increased frequency of mitotic cells and metaphase delay. The capacity to bypass mitotic arrest without resolving the causes of the arrest has been described in yeast in a process called adaptation (49, 50). We propose that anaphase chromatin bridges are revealed in cells bypassing the mitotic arrest in a process that is reminis- cent of the adaptation process described in yeast. In addition, it is tempting to speculate that during the mitotic arrest bypass, opposite forces between chromosome migration and metaphase arrest lead to the splitting of centrosomes, resulting in active Fig. 5. The effect of dNs addition on replication fork speed and the fre- MECs and multipolar segregations. This model is consistent with quency of mitosis with aberrant centrosome number. (A) Replication fork speed distribution in V79 cells and derivatives (Left) and V-C8 cells and the fact that extra centrosomes are detectable during mitosis but derivatives (Right) is presented. The numbers correspond to the median not in interphase; that these centrosomes are functional, causing replication speed. Median and P values are indicated (*P < 0.05; **P < 0.01; multipolar segregations; and that almost all multipolar segrega- ***P < 0.001). Median values are represented as horizontal black lines. Se- tions are associated with chromatin bridges. Incomplete repli- venty to 145 fibers were scored per condition. (B) The quantification of mi- cation results in local chromosome abnormalities during mitosis totic cells with aberrant centrosome number (unequal 2) using γ-tubulin (29). Here, very moderate replication stress lead to MEC for- labeling. Left histogram, V79 cells and derivatives; right histogram, V-C8 cells ± mation, which do not contain DNA but amplify the signal to the and derivatives. The mean value SD of three independent experiments was calculated. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. In total, 150 whole genome by generating multipolar mitosis and aberrant mitoses were scored per condition. (C) Centrosome number distribution in chromosome segregation. Therefore, very low replication stress mitotic cells monitored by immunofluorescence using γ-tubulin labeling. V79 not detected by the cell cycle checkpoint leads to global chromo- cells and derivatives (Left); V-C8 cells and derivatives (Right). At least 100 some missegregation involving even fully replicated chromosomes. mitotic cells were analyzed per condition.

766 | www.pnas.org/cgi/doi/10.1073/pnas.1311520111 Wilhelm et al. Downloaded by guest on September 30, 2021 Fig. 6. RF deceleration alters global chromosome segregation. Different causes such as dNTP shortage, polymerase inhibition, and HR deficiency cause RF

deceleration, which elicits the firing of RFs. At such low or endogenous stresses, cells do not arrest and, thus, progress through the G2 phase with incompletely replicated DNA. RF deceleration exacerbates these processes leading to chromatin bridges in nonreplicated regions. However, these cells are blocked at metaphase. Bypassing this arrest (in an “adaptation-like” process) causes abnormal mitosis including MEC, anaphase chromatin bridges and multipolar cells, which results in uneven chromosome segregation and aneuploidy in the whole genome, even for replicated chromosomes.

we observed MECs in the HR proficient cells treated with HU or Molecular Combing. Molecular combing was performed as described (64). aphidicolin doses that did not trigger CHK1 phosphorylation, Iododeoxyuridine (IdU) labeling and chlorodeoxyuridine (CldU) labeling which argues against these hypotheses. Rather, the data pre- (20-min pulse labeling for each) were performed as described (42). Briefly, sented here demonstrate that HR protects against endogenous chromatin fibers were incubated with rat anti-BrdU (1:25, OBT0030, clone stress arising upon RF slowing. Thus, deciphering the molecular BU1/75 ICR1; AbD Serotec) and FITC-conjugated mouse anti-BrdU (1:5, mechanism at the origin of the endogenous replication stress in 347583, clone B44; BD Biosciences) for 1 h at 37 °C. After a short wash step − HR cells represents an exciting challenge. (0.5 M NaCl, 20 mM Tris at pH 7.8, and 0.5% Tween 20), the fibers were incubated with goat anti-mouse Alexa 488 (1:50, A-11029; Invitrogen) and Theodor Boveri hypothesized one century ago that tumors goat anti-rat Alexa 594 (1:50, A-11007; Invitrogen) for 1 h. DNA counter- originate from improper chromosome segregation, thus creating staining was performed by using anti-DNA antibody (1:20, MAB3034, clone aneuploid cells that undergo clonal expansion. He proposed that 16–19; Millipore) and the following two secondary antibodies: goat-anti in some cases, abnormal chromosome segregation and aneu- mouse Cy5.5 (1:100, ab6947; Abcam) and donkey anti-goat Cy5.5 (1:100, ploidy are caused by extracentrosome generation, leading to ab6951; Abcam). For each condition, ∼100 fibers with symmetrical green-red multipolar cells (56, 57). Our data suggest that modest RF labeling were analyzed. Images were captured by using a 63× oil immersion slowing imbalances general chromosome segregation. Among objective of a motorized Axio Imager.Z2 epifluorescence microscope (Carl the stresses responsible for RF slowing, dNTP pools appear to be Zeiss) that had been equipped with a high-sensitivity cooled interline CCD very sensitive limiting factors. Two hypothesis can account for camera (Cool SNAP HQ2; Roper Scientific) and a PIEZO stage (Physik Instru- − the data in HR deficient cells: (i) the nucleotide pools are mente). Image acquisition was performed with MetaMorph software (Mo- spontaneously affected by processes that remain to be identified; lecular Devices). and (ii) part of the dNTP production can be channeled to the − endogenous damages, which should be more persistent in HR Centrosome Analysis. Cells were fixed in methanol for 15 min at –20°C and deficient cells, resulting in a dNTP shortage for replication. permeabilized with acetone. After three washes in PBS, the samples were GENETICS Because the nucleotide pool is a tight limiting factor for repli- incubated in 2% (wt/vol) BSA for 30 min and washed three times in PBS. γ cation but is not expendable in mammalian cells after genotoxic Centrosomes were stained by using -tubulin antibody (Sigma T3559, 1/300) stress, this outcome might lead to replication fork deceleration or centrobin antibody (Abcam 70448, 1/40) diluted in 0.5% BSA and 0.05% without affecting the global intracellular nucleotide pool (58– Tween 20 for 1 h at 37°C. The cells were washed three times in 0.05% Tween 20 in PBS followed by incubation with Alexa Fluor 594-conjugated goat anti- 60). Remarkably, deoxynucleotide deficiencies promote genomic rabbit antibodies (Molecular Probes), which were diluted 1:400 in 0.5% BSA instability at early oncogenic stages (43). Consistent with our and 0.05% Tween 20 for 1 h at 37 °C. After three washes in 0.05% Tween 20 data, both replication stress and centrosome abnormalities have in PBS, the cells were incubated with DAPI (1 μg/mL). In each case, 100–150 also been reported at early stages of malignancy. Interestingly, metaphase cells were analyzed per condition. Images were captured by HR protects against spontaneous endogenous replication stress, using a 63× oil immersion objective of a motorized Axio Imager.Z2 epi- is affected in most familial breast cancers (61), and likely occurs fluorescence microscope (Carl Zeiss) that had been equipped with a high- in a high frequency of sporadic cases (62). Therefore, the data sensitivity cooled interline CCD camera (Cool SNAP HQ2; Roper Scientific) presented here shed light on the importance of HR at the mo- and a PIEZO stage (Physik Instrumente). Image acquisition was performed lecular interface between replication and mitosis when cells with MetaMorph software (Molecular Devices). face spontaneous endogenous or low genotoxic stresses that do not trigger cell cycle checkpoints or prevent the cells from en- Time-Lapse Video Microscopy. Cells lines expressing GFP-tagged H2B were tering mitosis. cultured for 48 h before video microscopy on glass coverslips. The cells were enclosed in a Ludin chamber, and a photo was taken every 2 min with a 40× Materials and Methods objective for at least 16 h. Image acquisition was performed with a motorized inverted microscope (Olympus IX81) that had been wired to a CoolSNAP Cells and Treatments. Cell lines were cultured at 37 °C in 5% CO2 in Eagle’s minimal essential medium supplemented with 10% (vol/vol) FBS, 2 mM HQ camera (Princeton Instruments). During image capture, the carbon di- glutamine, 200 IU/mL penicillin, and 200 μg/mL streptomycin. V79SM24 is oxide levels were 5% and the temperature was 37 °C, which are typical cell a derivative of the V79 cell line that stably expresses the RAD51 dominant culture conditions. MetaMorph software (Molecular Devices) was used for negative SMRAD51; V79puro are V79 cells that were transfected with the image capture and analysis. empty expression vector (21, 32). We also used the V-C8 BRCA2-defective cell line and its V-C8#13 counterpart in which BRCA2 is complemented by human Statistical Analysis. RF speed, interorigin distance, and mitosis duration were chromosome 13 (26, 63). V-C8#13 cells were used as a second WT cell line. compared by using the Mann–Whitney test. Mitotic cells and cells with MEC Cells were exposed to the indicated HU or aphidicolin doses for 48 h. percentages were analyzed by Fisher’s exact test. All of the tests were two- Nucleotide precursor supply analysis was performed by adding a mix of sided, and P < 0.05 was considered statistically significant. the four dNs (20 μM each) for 48 h. Deoxycytidine (Sigma D0776) was solubilized in 1 M NaOH (100 mM). Deoxyadenosine (Sigma D8668) was ACKNOWLEDGMENTS. We thank Dr. Laurent Gauthier for assistance with solubilized in 0.1 M NaOH (20 mM). Thymidine (Sigma T1895) was solu- video microscopy. This work was supported by l’Association pour la Recherche bilizedinH2O (50 mM). Deoxyguanosine (Sigma D7145) was solubilized Contre le Cancer and l’Institut National du cancer. I.M. received a fellowship in 1 M NH4OH (100 mM). from La Ligue Nationale Contre le Cancer.

Wilhelm et al. PNAS | January 14, 2014 | vol. 111 | no. 2 | 767 Downloaded by guest on September 30, 2021 1. Bartkova J, et al. (2005) DNA damage response as a candidate anti-cancer barrier in 33. Lambert S, Lopez BS (2001) Role of RAD51 in sister-chromatid exchanges in mam- early human tumorigenesis. Nature 434(7035):864–870. malian cells. Oncogene 20(45):6627–6631. 2. Deng G, Lu Y, Zlotnikov G, Thor AD, Smith HS (1996) Loss of heterozygosity in normal 34. Lambert S, Lopez BS (2002) Inactivation of the RAD51 recombination pathway stim- tissue adjacent to breast carcinomas. Science 274(5295):2057–2059. ulates UV-induced mutagenesis in mammalian cells. Oncogene 21(25):4065–4069. 3. Gorgoulis VG, et al. (2005) Activation of the DNA damage checkpoint and genomic 35. Skoog L, Nordenskjöld B (1971) Effects of hydroxyurea and 1-beta-D-arabinofur- instability in human precancerous lesions. Nature 434(7035):907–913. anosyl-cytosine on deoxyribonucleotide pools in mouse embryo cells. Eur J Biochem 4. Krämer A (2005) Centrosome aberrations—hen or egg in cancer initiation and pro- 19(1):81–89. gression? Leukemia 19(7):1142–1144. 36. Gudi R, Zou C, Li J, Gao Q (2011) Centrobin-tubulin interaction is required for cen- 5. Lakhani SR, et al. (1999) Genetic alterations in ‘normal’ luminal and myoepithelial triole elongation and stability. J Cell Biol 193(4):711–725. cells of the breast. J Pathol 189(4):496–503. 37. Jeffery JM, Urquhart AJ, Subramaniam VN, Parton RG, Khanna KK (2010) Centrobin 6. Larson PS, de las Morenas A, Bennett SR, Cupples LA, Rosenberg CL (2002) Loss of regulates the assembly of functional mitotic spindles. Oncogene 29(18):2649–2658. heterozygosity or allele imbalance in histologically normal breast epithelium is dis- 38. Zou C, et al. (2005) Centrobin: A novel daughter centriole-associated protein that is tinct from loss of heterozygosity or allele imbalance in co-existing carcinomas. Am J required for centriole duplication. J Cell Biol 171(3):437–445. Pathol 161(1):283–290. 39. Smith L, Plug A, Thayer M (2001) Delayed replication timing leads to delayed mitotic 7. Larson PS, de las Morenas A, Cupples LA, Huang K, Rosenberg CL (1998) Genetically chromosome condensation and chromosomal instability of chromosome trans- abnormal clones in histologically normal breast tissue. Am J Pathol 152(6):1591–1598. locations. Proc Natl Acad Sci USA 98(23):13300–13305. 8. Li Z, et al. (2002) Increased risk of local recurrence is associated with allelic loss in 40. Lahkim Bennani-Belhaj K, et al. (2010) The Bloom syndrome protein limits the le- normal lobules of breast cancer patients. Cancer Res 62(4):1000–1003. thality associated with RAD51 deficiency. Mol Cancer Res 8(3):385–394. 9. Nigg EA (2002) Centrosome aberrations: Cause or consequence of cancer progression? 41. Laulier C, Cheng A, Stark JM (2011) The relative efficiency of homology-directed re- Nat Rev Cancer 2(11):815–825. pair has distinct effects on proper anaphase chromosome separation. Nucleic Acids 10. Sluder G, Nordberg JJ (2004) The good, the bad and the ugly: The practical con- Res 39(14):5935–5944. sequences of centrosome amplification. Curr Opin Cell Biol 16(1):49–54. 42. Anglana M, Apiou F, Bensimon A, Debatisse M (2003) Dynamics of DNA replication in 11. Cox MM, et al. (2000) The importance of repairing stalled replication forks. Nature mammalian somatic cells: Nucleotide pool modulates origin choice and interorigin 404(6773):37–41. spacing. Cell 114(3):385–394. 12. Michel B, et al. (2001) Rescue of arrested replication forks by homologous re- 43. Bester AC, et al. (2011) Nucleotide deficiency promotes genomic instability in early combination. Proc Natl Acad Sci USA 98(15):8181–8188. stages of cancer development. Cell 145(3):435–446. 13. Lomonosov M, Anand S, Sangrithi M, Davies R, Venkitaraman AR (2003) Stabilization 44. Chabosseau P, et al. (2011) Pyrimidine pool imbalance induced by BLM helicase de- of stalled DNA replication forks by the BRCA2 breast cancer susceptibility protein. ficiency contributes to genetic instability in Bloom syndrome. Nat Commun 2:368. – Genes Dev 17(24):3017 3022. 45. Gay S, et al. (2010) Nucleotide supply, not local histone acetylation, sets replication 14. Petermann E, Orta ML, Issaeva N, Schultz N, Helleday T (2010) Hydroxyurea-stalled origin usage in transcribed regions. EMBO Rep 11(9):698–704. replication forks become progressively inactivated and require two different RAD51- 46. Burrell RA, et al. (2013) Replication stress links structural and numerical cancer – mediated pathways for restart and repair. Mol Cell 37(4):492 502. chromosomal instability. Nature 494(7438):492–496. 15. Saintigny Y, et al. (2001) Characterization of homologous recombination induced by 47. Kawabata T, et al. (2011) Stalled fork rescue via dormant replication origins in un- – replication inhibition in mammalian cells. EMBO J 20(14):3861 3870. challenged S phase promotes proper chromosome segregation and tumor suppres- 16. Schlacher K, et al. (2011) Double-strand break repair-independent role for BRCA2 in sion. Mol Cell 41(5):543–553. – blocking stalled replication fork degradation by MRE11. Cell 145(4):529 542. 48. Letessier A, et al. (2011) Cell-type-specific replication initiation programs set fragility 17. Hyrien O (2000) Mechanisms and consequences of replication fork arrest. Biochimie of the FRA3B fragile site. Nature 470(7332):120–123. – 82(1):5 17. 49. Lee SE, et al. (1998) Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate 18. Branzei D, Foiani M (2010) Maintaining genome stability at the replication fork. Nat adaptation to G2/M arrest after DNA damage. Cell 94(3):399–409. Rev Mol Cell Biol 11(3):208–219. 50. Toczyski DP, Galgoczy DJ, Hartwell LH (1997) CDC5 and CKII control adaptation to the 19. Daboussi F, et al. (2008) A homologous recombination defect affects replication-fork yeast DNA damage checkpoint. Cell 90(6):1097–1106. progression in mammalian cells. J Cell Sci 121(Pt 2):162–166. 51. Balczon R, et al. (1995) Dissociation of centrosome replication events from cycles of 20. Bertrand P, Lambert S, Joubert C, Lopez BS (2003) Overexpression of mammalian DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary Rad51 does not stimulate tumorigenesis while a dominant-negative Rad51 affects cells. J Cell Biol 130(1):105–115. centrosome fragmentation, ploidy and stimulates tumorigenesis, in p53-defective 52. Hut HM, et al. (2003) Centrosomes split in the presence of impaired DNA integrity CHO cells. Oncogene 22(48):7587–7592. during mitosis. Mol Biol Cell 14(5):1993–2004. 21. Daboussi F, Thacker J, Lopez BS (2005) Genetic interactions between RAD51 and its 53. Bourke E, et al. (2007) DNA damage induces Chk1-dependent centrosome amplifi- paralogues for centrosome fragmentation and ploidy control, independently of the cation. EMBO Rep 8(6):603–609. sensitivity to genotoxic stresses. Oncogene 24(22):3691–3696. 54. Löffler H, et al. (2007) DNA damage-induced accumulation of centrosomal Chk1 22. Deng CX (2002) Roles of BRCA1 in centrosome duplication. Oncogene 21(40): contributes to its checkpoint function. Cell Cycle 6(20):2541–2548. 6222–6227. 55. Robinson HM, Black EJ, Brown R, Gillespie DA (2007) DNA mismatch repair and Chk1- 23. Griffin CS, Simpson PJ, Wilson CR, Thacker J (2000) Mammalian recombination-repair dependent centrosome amplification in response to DNA alkylation damage. Cell genes XRCC2 and XRCC3 promote correct chromosome segregation. Nat Cell Biol Cycle 6(8):982–992. 2(10):757–761. 24. Dodson H, et al. (2004) Centrosome amplification induced by DNA damage occurs 56. Boveri T (1914) [Zur Frage der Entstehung maligner Tumoren] (Gustav Fisher, Jena, during a prolonged G2 phase and involves ATM. EMBO J 23(19):3864–3873. Germany). German. 25. Katsura M, et al. (2009) The ATR-Chk1 pathway plays a role in the generation of 57. Boveri T (2008) Concerning the origin of malignant tumours by Theodor Boveri. – centrosome aberrations induced by Rad51C dysfunction. Nucleic Acids Res 37(12): Translated and annotated by Henry Harris. J Cell Sci 121(Suppl 1):1 84. 3959–3968. 58. Håkansson P, Hofer A, Thelander L (2006) Regulation of mammalian ribonucleotide 26. Kraakman-van der Zwet M, et al. (2002) Brca2 (XRCC11) deficiency results in radio- reduction and dNTP pools after DNA damage and in resting cells. J Biol Chem 281(12): – resistant DNA synthesis and a higher frequency of spontaneous deletions. Mol Cell 7834 7841. Biol 22(2):669–679. 59. Niida H, et al. (2010) Essential role of Tip60-dependent recruitment of ribonucleotide 27. Rodrigue A, et al. (2013) The RAD51 paralogs ensure cellular protection against mi- reductase at DNA damage sites in DNA repair during G1 phase. Genes Dev 24(4): totic defects and aneuploidy. J Cell Sci 126(Pt 1):348–359. 333–338. 28. Plo I, Lopez B (2009) AKT1 represses gene conversion induced by different genotoxic 60. Niida H, Shimada M, Murakami H, Nakanishi M (2010) Mechanisms of dNTP supply stresses and induces supernumerary centrosomes and aneuploidy in hamster ovary that play an essential role in maintaining genome integrity in eukaryotic cells. Cancer cells. Oncogene 28(22):2231–2237. Sci 101(12):2505–2509. 29. Mankouri HW, Huttner D, Hickson ID (2013) How unfinished business from S-phase 61. Walsh T, King MC (2007) Ten genes for inherited breast cancer. Cancer Cell 11(2): affects mitosis and beyond. EMBO J 32(20):2661–2671. 103–105. 30. Cappelli E, Townsend S, Griffin C, Thacker J (2011) Homologous recombination pro- 62. Plo I, et al. (2008) AKT1 inhibits homologous recombination by inducing cytoplasmic teins are associated with centrosomes and are required for mitotic stability. Exp Cell retention of BRCA1 and RAD51. Cancer Res 68(22):9404–9412. Res 317(8):1203–1213. 63. Wiegant WW, Overmeer RM, Godthelp BC, van Buul PP, Zdzienicka MZ (2006) Chinese 31. Lesca C, et al. (2005) DNA damage induce gamma-tubulin-RAD51 nuclear complexes hamster cell mutant, V-C8, a model for analysis of Brca2 function. Mutat Res 600(1-2): in mammalian cells. Oncogene 24(33):5165–5172. 79–88. 32. Lambert S, Lopez BS (2000) Characterization of mammalian RAD51 double strand 64. Michalet X, et al. (1997) Dynamic molecular combing: Stretching the whole human break repair using non-lethal dominant-negative forms. EMBO J 19(12):3090–3099. genome for high-resolution studies. Science 277(5331):1518–1523.

768 | www.pnas.org/cgi/doi/10.1073/pnas.1311520111 Wilhelm et al. Downloaded by guest on September 30, 2021