CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage
2020; Springer Nature; Volume: 21; Issue: 8 Linguagem: Inglês
10.15252/embr.201948920
ISSN1469-3178
AutoresMichael D. Rainey, Aisling Quinlan, Chiara Cazzaniga, Sofija Mijic, Oliviano Martella, Jana Krietsch, Anja Göder, Massimo Lopes, Corrado Santocanale,
Tópico(s)Molecular Biology Techniques and Applications
ResumoArticle4 June 2020Open Access Source DataTransparent process CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage Michael D Rainey orcid.org/0000-0001-9777-873X Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Aisling Quinlan orcid.org/0000-0003-2888-0057 Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Chiara Cazzaniga orcid.org/0000-0002-7693-9167 Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Sofija Mijic Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland Search for more papers by this author Oliviano Martella Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Jana Krietsch Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland Search for more papers by this author Anja Göder orcid.org/0000-0001-9743-1656 Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Massimo Lopes orcid.org/0000-0003-3847-8133 Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland Search for more papers by this author Corrado Santocanale Corresponding Author [email protected] orcid.org/0000-0003-1337-5656 Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Michael D Rainey orcid.org/0000-0001-9777-873X Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Aisling Quinlan orcid.org/0000-0003-2888-0057 Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Chiara Cazzaniga orcid.org/0000-0002-7693-9167 Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Sofija Mijic Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland Search for more papers by this author Oliviano Martella Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Jana Krietsch Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland Search for more papers by this author Anja Göder orcid.org/0000-0001-9743-1656 Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Massimo Lopes orcid.org/0000-0003-3847-8133 Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland Search for more papers by this author Corrado Santocanale Corresponding Author [email protected] orcid.org/0000-0003-1337-5656 Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Search for more papers by this author Author Information Michael D Rainey1,‡, Aisling Quinlan1,‡, Chiara Cazzaniga1,‡, Sofija Mijic2,†,‡, Oliviano Martella1, Jana Krietsch2, Anja Göder1, Massimo Lopes2 and Corrado Santocanale *,1 1Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland 2Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland †Present address: Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden ‡These authors contributed equally to this work *Corresponding author. Tel: +35391495174; E-mail: [email protected] EMBO Rep (2020)21:e48920https://doi.org/10.15252/embr.201948920 [The copyright line of this article was changed on 6 October 2020 after original online publication.] PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The CDC7 kinase is essential for the activation of DNA replication origins and has been implicated in the replication stress response. Using a highly specific chemical inhibitor and a chemical genetic approach, we now show that CDC7 activity is required to coordinate multiple MRE11-dependent processes occurring at replication forks, independently from its role in origin firing. CDC7 localizes at replication forks and, similarly to MRE11, mediates active slowing of fork progression upon mild topoisomerase inhibition. Both proteins are also retained on stalled forks, where they promote fork processing and restart. Moreover, MRE11 phosphorylation and localization at replication factories are progressively lost upon CDC7 inhibition. Finally, CDC7 activity at reversed forks is required for their pathological MRE11-dependent degradation in BRCA2-deficient cells. Thus, upon replication interference CDC7 is a key regulator of fork progression, processing and integrity. These results highlight a dual role for CDC7 in replication, modulating both initiation and elongation steps of DNA synthesis, and identify a key intervention point for anticancer therapies exploiting replication interference. Synopsis CDC7 kinase acts at paused forks where it coordinates MRE11-dependent fork processing, contributing to fork restart and modulating fork speed. Upon prolonged arrest and compromised fork protection, CDC7 promotes fork degradation contributing to replication-dependent chromosome breakage. CDC7 promotes fork restart, HU-dependent fork collapse and chromosomal breakage. CDC7's role at forks can be uncoupled from its role in origin activation. MRE11 and CDC7 activities are required to limit fork progression upon mild topological stress. CDC7 promotes the degradation of reversed forks in BRCA2-depleted cells. Introduction In eukaryotic cells, DNA replication initiates from multiple origins 1, 2. CDC7 kinase is required for the firing of replication origins, by phosphorylating multiple subunits of the MCM2–7 helicase complex 3-5, thus initiating bidirectional replication 6, 7. In addition, CDC7 kinase has important and yet poorly characterized roles in the replication stress response. For instance, in human cells CDC7-dependent phosphorylation of the mediator protein CLASPIN is important for full activation of CHK1 by ATR and for maintaining cell viability in the presence of drugs that affect replication fork progression 8-11. More recently, CDC7 has been shown to play a role in processing stalled replication forks by promoting limited resection that may be important for efficient replication fork restart and possibly by controlling EXO1 nuclease 12. Furthermore, the dephosphorylation of the MCM complex—which is mediated by protein phosphatase 1 and RIF1, counteracting CDC7—has been associated with loss of replisome stability in Xenopus and human cells 13. RIF1/PP1 also protects stalled forks from DNA2-dependent degradation 13-15. The idea that CDC7 may contribute to the regulation of nucleases is supported by work in budding yeast where CDC7 directly controls the activation of the Mus81 nuclease in mitosis by direct phosphorylation of the Mms4 subunit of the Mus81–Mms4 complex, thus promoting the disentanglement of DNA joint molecules 16. Furthermore, several laboratories have elucidated the role of yeast CDC7 kinase in controlling the formation of DNA double-strand breaks (DSBs) during meiotic DNA replication to promote meiotic recombination 17-22. However, whether CDC7 plays a direct role at forks and how it contributes to the control of replication fork processing are largely elusive. Many CDC7 inhibitors have been developed as potential anticancer agents; among these, PHA-767491 and XL413 have become tool compounds that have also been widely used to test CDC7 functions. Direct comparison of these two compounds in biochemical assays showed that XL413 is more specific than PHA-767491 23. In order to better elucidate CDC7 functions, we have recently generated and characterized an engineered cell line, based on the principle of the analogue-sensitive kinase 24, in which CDC7 activity can be partially but very specifically reduced using a bulky ATP competitor, 3MB-PP1. Importantly, all the phenotypes observed by treating WT cells with 10 μM XL413 are recapitulated by treatment with 10 μM 3MB-PP1 in this AS-CDC7 cell line, including reduction of MCM2 phosphorylation at CDC7-dependent sites and a mild delay in progressing through S-phase 25. Drugs that target DNA replication fork progression such as hydroxyurea (HU), DNA polymerases and DNA topoisomerase inhibitors, and DNA-damaging compounds slow down forks causing their reversal into a four-branched structure that can be detected by electron microscopy, also described as chicken-foot structure 26-29. Formation of these structures is promoted by several enzymatic activities, leading to BRCA1- and BRCA2-dependent deposition of a RAD51 filament on the regressed arm of the fork (reviewed in 28, 29). Fork reversal also occurs at endogenous obstacles in the absence of genotoxic drugs and can be triggered by oncogene activation 30, 31. It was thus proposed that the overall speed of each individual fork is determined by the combination of processive DNA synthesis, pausing into a chicken-foot conformation and subsequent restart 28. Once regressed arms are formed, they are stabilized by BRCA2-dependent assembly of RAD51 filaments on their ssDNA portion, thus preventing nucleolytic attack and extensive fork degradation that is mediated by either CTIP, MRE11 or EXO1 (reviewed in 32, 33). Lack of reversed fork protection in BRCA2-deficient cells leads to a high level of genome instability and in particular to chromosomal breakage, as well as sensitivity to various chemotherapeutic drugs 34, 35. Thus, in genetic backgrounds with compromised fork stability, reversed forks represent crucial entry points for processing events that mediate chemosensitivity. In this work, using the specific inhibitor XL413 and the AS-CDC7 cell line, we describe the role of the CDC7 kinase in the dynamics of established replication forks. We find that CDC7 kinase promotes MRE11-dependent processing of reversed replication forks and, by doing so, contributes to the modulation of replication fork speed and to chromosomal instability upon fork stalling, which is a hallmark of BRCA2-deficient cells. Results CDC7 promotes replication fork processing and collapse in HU CDC7 kinase is known to be involved in DNA replication initiation and checkpoint signalling 8, 9, 36-39. To further characterize CDC7-dependent events in the replication stress response, we treated U2OS osteosarcoma-derived cancer cells and MCF10A breast-derived immortalized cells with HU in the presence or absence of the CDC7 inhibitor XL413. With both cell lines, we observed that HU-induced phosphorylation of RPA2—the middle subunit of the ssDNA-binding protein RPA—was greatly reduced, as assessed by either altered electrophoretic mobility or specific anti-phosphopeptide antibodies. In a time-course experiment, the suppression of RPA phosphorylation in CDC7i-treated cells was observed as early as 2 h post-treatment and throughout the course of the experiment (Fig 1A). Intriguingly, the phosphorylation of H2AX at serine 139 (γH2AX) that occurs upon fork stalling and is further amplified upon fork collapse and formation of DNA double-strand breaks (DSBs) 40 was also partially suppressed in XL413-treated U2OS (Fig 1A). Similar results were obtained with breast-derived immortalized MCF10A cells (Fig EV1A). In order to confirm that the suppression of RPA2 and H2AX phosphorylation in response to HU was specifically due to CDC7 kinase inhibition, we used an analogue-sensitive CDC7 cell line in which the activity of the kinase can be partially downregulated with the bulky ATP competitive inhibitor 3MB-PP1 25. Treatment of AS-CDC7 cells with 3MB-PP1 reduced both RPA2 and H2AX phosphorylation occurring in HU-treated cells to a similar extent as XL413 in the isogenic MCF10A cells (Fig EV1A and B), suggesting that when CDC7 is inhibited, fewer DNA DSBs are generated and/or stalled forks are differently processed. Figure 1. CDC7 inhibition suppresses histone H2AX and RPA2 phosphorylation and DNA double-strand break formation upon fork stalling U2OS cells were either mock-treated or treated with 10 μM XL413 for 30 min, at which point 4 mM HU was added and cells further incubated for the indicated times. Whole-cell extracts were then analysed by Western blotting with the indicated antibodies. Reduction of phosphorylation of Ser40/41 on MCM2 is indicative of CDC7 inhibition by XL413. Data are representative of two independent experiments. U2OS cells were either mock-treated or treated with 10 μM XL413, 4 mM HU or both for 24 h before performing neutral comet assays. Representative images of comets are shown. Scale bar = 100 μm. In the dot plots, ˜800 comets per each condition were analysed, means are indicated with a red line, and their values are shown above the plot. Data are from two independent experiments. U2OS cells were either untreated or treated with 10 μM XL413 or with 5 μM AZD6738 alone or in combination and, where indicated, after 30 min, 4 mM HU was added for a further 2 h. Whole-cell extracts were analysed by Western blotting with the indicated antibodies. Total protein stain (TPS) is as a loading control. Data are representative of two independent experiments. U2OS cells were either untreated or treated with 4 mM HU for 5 h in the presence or absence of 10 μM XL413, which had been added to cells either 30 min prior to (pre-treatment) or during (cotreatment) the addition of HU. Whole-cell extracts were analysed by Western blotting with the indicated antibodies, and total protein stain (TPS) is displayed as a loading control. Data are representative of two independent experiments. Source data are available online for this figure. Source Data for Figure 1 [embr201948920-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. (related to Fig 1): Inhibition of CDC7 with XL413 in MCF10A cells and with 3MB-PP1 in AS-CDC7 cells suppresses histone H2AX and RPA2 phosphorylation and DNA double-strand break formation in HU A, B. MCF10A or AS-CDC7 cells were either mock-treated or treated with 10 μM XL413 for 30 min, at which point 4 mM HU was added and cells further incubated for the indicated times. Whole-cell extracts were then analysed by Western blotting with the indicated antibodies. Data are representative of at least two independent experiments. C. AS-CDC7 cells were either mock-treated or treated with 10 μM 3MB-PP1, 4 mM HU or both for 24 h before performing neutral comet assays. Representative images of cells are shown. Scale bar = 100 μm. In the dot plots, ˜400 comets per each condition were analysed, means are indicated with red lines, and their values are shown above the plots. Data are from two independent experiments. Source data are available online for this figure. Download figure Download PowerPoint To directly assess if CDC7 promotes formation of DNA DSBs from stalled forks, we then performed neutral comet assays on U2OS cells. Following exposure of cells to HU for 24 h with or without XL413, we observed that CDC7 inhibition significantly reduced the tail moment, which is proportional to the number of DNA DSBs in the cell (Fig 1B). The formation of DNA DSBs in HU was also suppressed when CDC7 was inhibited with 3MB-PP1 in the AS-CDC7 cell line (Fig EV1C). As DNA DSBs in HU can arise from replication fork collapse, which is actively prevented by ATR signalling 41, 42, we tested the effects of CDC7i in the presence of the ATR inhibitor AZD6738 finding that the addition of XL413 strongly decreased RPA2 and H2AX hyper-phosphorylation (Fig 1C). In all previous experiments, the CDC7 inhibitor was added 30 min before HU, as we had previously shown that CDC7 inhibition can only delay the onset of checkpoint signalling (i.e. CHK1 phosphorylation) before it is established 8. Consistent with these early findings, we found that unlike a short pre-treatment with the CDC7i, simultaneous cotreatment of cells with CDC7i and HU was unable to prevent the phosphorylation of neither RPA2 nor H2AX (Fig 1D). These observations suggest that CDC7 activity is required for replication fork processing or collapse upon sustained fork arrest, particularly in the absence of proper checkpoint signalling. However, as CDC7 is also required for origin firing, they may also reflect fewer active forks present at the moment of HU addition in cells cotreated with CDC7i, thus limiting fork-associated phosphorylation events. CDC7 drives H2AX phosphorylation in HU independently from origin firing and in a MRE11-dependent manner In order to possibly uncouple CDC7 function in initiation and at elongating forks, we first performed a titration from 0.123 to 10 μM of the CDC7 inhibitor XL413 in U2OS cells and accurately measured by flow cytometry the amount of DNA synthesis occurring at different doses as well as the amount of H2AX phosphorylation when CDC7i-treated cells were also challenged with HU. This analysis indicated that very low levels of CDC7i had almost no effects on the rate of EdU incorporation which decreased then in a dose-dependent manner at higher concentrations. Conversely, already at the lowest concentration of XL413, we detected a profound reduction on HU-induced H2AX phosphorylation. These data suggest a different level of requirement for CDC7 activity in promoting efficient DNA synthesis and in driving HU-dependent H2AX phosphorylation and suggest that key targets of CDC7 activity in initiation and at forks may be different or differently regulated (Fig EV2). Click here to expand this figure. Figure EV2. Partial inhibition of CDC7 has a minor effect on DNA synthesis but markedly affects pS139 H2AX phosphorylationU2OS cells were treated with the indicated concentrations of XL413 in the presence or absence of 4 mM HU for 5 h and then labelled with 10 μM EdU 30 min prior to harvest. Samples were processed for flow cytometry analysis. Mono-parametric analysis of cell count against EdU intensity. Histograms are overlaid to appreciate changes in EdU intensity upon treatment with XL413 (blue lines) relative to the untreated control (grey lines). Mono-parametric analysis of cell count against pS139 histone H2AX intensity in HU-treated cells. Histograms are overlaid to appreciate changes in pS139 histone H2AX intensity upon treatment (red lines) relative to untreated controls (grey lines). Whole-cell extracts were analysed by Western blotting with the indicated antibodies, and total protein stain (TPS) is used as a loading control. Reduction of phosphorylation of Ser40/41 on MCM2 is indicative of CDC7 inhibition. Data are representative of two repeat experiments. Source data are available online for this figure. Download figure Download PowerPoint If CDC7 is indeed required to promote fork collapse independently from its role in initiation, we reasoned that allowing cells to enter S-phase and be held in HU in the presence of CDC7i would allow the following events to occur: (i) the arrest of existing forks; (ii) checkpoint activation, which would prevent further origin firing and further loading of the initiator factor CDC45, as well as checkpoint-dependent contribution in protecting existing forks from collapse; and (iii) further fork stabilization caused by CDC7 inhibition. Under these conditions, the subsequent removal of the CDC7i would possibly cause fork destabilization, while keeping strong checkpoint signalling and HU-mediated prevention of further initiation events. Conversely, checkpoint inhibition would simultaneously cause both fork collapse and origin activation. To test these hypotheses, U2OS cells were treated with XL413 and HU for 24 h; then, still in the presence of HU, XL413 was either kept or washed off and cells incubated for a further 2 h (Fig 2A). As a control for the checkpoint-dependent suppression of both origin firing and replication fork stabilization, the ATRi AZD6738 was included. Proteins were then analysed by Western blotting (Fig 2B) and the levels of H2AX phosphorylation quantitatively assessed by flow cytometry (Fig 2C). In this assay, treatment with HU and XL413 for 24–26 h arrested cells in S-phase with active checkpoint and low levels of γH2AX (Fig 2B, lanes 4 and 6; and Fig 2D). However, removal of CDC7i after 24 h clearly caused 2 h later a marked increase in γH2AX and CHK1 phosphorylation (Fig 2B, lanes 4 and 5; and Fig 2D). Importantly, this was not accompanied by an increase in chromatin-bound CDC45, which is a surrogate marker for origin firing. Conversely, CDC45 was clearly and expectedly increased upon treatment with the ATRi AZD6738 and occurred in the absence of detectable EdU incorporation (Appendix Fig S1). Figure 2. CDC7 inhibition suppresses histone H2AX phosphorylation independently from origin firing A. Outline of experimental procedure. U2OS cells were either untreated or treated with 10 μM XL413 or with 4 mM HU in the presence or absence of XL413 for 24 h. Cells were EdU-labelled for 30 min before harvesting. Cells that had been treated with both XL413 and HU for 24 h were washed with equilibrated media and retreated for a further 2 h. All further treatments included 4 mM HU, to prevent further DNA synthesis in either the absence or presence of CDC7 (XL413), MRE11 (Mirin) or ATR (AZD6738) inhibitors. B. CSK soluble and chromatin-enriched protein fractions were analysed by Western blotting with the indicated antibodies. Data are representative of two independent experiments. C, D. Flow cytometry analysis to assess the levels of pS139 histone H2AX (c). Histograms show the mono-parametric analysis of cell count against pS139 histone H2AX intensity. Histograms are overlaid to appreciate changes in pS139 histone H2AX intensity upon treatment (red lines) relative to appropriate experimental baseline controls (grey lines). Data are representative of two independent experiments. E. Graphical concept: cells treated with HU and XL413 for 24 h would have stalled forks, which exist in a stable state due to inhibition of CDC7 and with an active ATR-dependent origin firing checkpoint. Upon washing and retreating of cells in the presence of HU, the late origin checkpoint is maintained, in an ATR-dependent manner, and accumulation of DNA damage (pS139 H2AX) can be monitored independently from origin firing in the absence or presence of CDC7 (XL413) or MRE11 (Mirin) inhibitor. AZD6738 was used as a control to investigate molecular events occurring upon loss of checkpoint caused by both fork collapse and loss of the inhibition of origin firing. The increase in chromatin binding of CDC45 was used as a surrogate marker for origin (ori) activation, pS139 histone H2AX was used to monitor fork stability, pS345 CHK1 was used to monitor the ATR-checkpoint signalling, and pS40/41 MCM2 was used to monitor CDC7 kinase activity. Source data are available online for this figure. Source Data for Figure 2 [embr201948920-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Fork collapse and DSB formation are thought to be due to the loss of protection from the attack of several active nucleases, including MRE11 and EXO1. While EXO1 levels were previously reported to be downregulated by a promiscuous CDC7 inhibitor, PHA-767491 12, these were not affected by XL413 (Appendix Fig S2). We therefore tested MRE11 involvement in H2AX phosphorylation after the removal of CDC7 inhibition in HU. Indeed, MRE11 inhibition by mirin 43 strongly limited histone H2AX and CHK1 phosphorylation to almost similar levels as maintaining the CDC7 inhibition (Fig 2B, lanes 5–7; and Fig 2D). Altogether, these experiments strongly support the notion that CDC7 activity promotes MRE11-dependent processing of stalled replication forks, independently from its role in regulating origin firing (Fig 2E), which can lead to checkpoint signalling amplification. CDC7 and MRE11 associate with nascent DNA, and CDC7 is required for maintenance of MRE11 localization at replication factories To further corroborate the hypothesis that CDC7 and MRE11 may act directly at replication forks, we performed a series of experiments to assess whether these proteins could be specifically captured on nascent DNA by DNA-mediated chromatin pull-down (Dm-ChP), a technique which is very similar to iPOND 44, 45. Firstly, in a pulse chase experiment we observed that the two proteins could be efficiently cross-linked to newly synthesized DNA, while their levels were substantially reduced on mature chromatin (Fig 3A), which is also consistent with previous studies coupled to mass spectrometry 45, 46. Both proteins could be still detected at replication forks when their progression was arrested by HU up to 24 h, but CDC7 inhibition notably reduced their retention at forks (Fig 3B and C). Figure 3. CDC7 is a replisome-associated protein, and its inhibition affects the localization of MRE11 at replication factories U2OS cells were labelled with EdU for 30 min; then, EdU was washed, and cells were further incubated for either 1 or 2 h in the presence of thymidine. At the indicated time points, cells were fixed and proteins binding to EdU-labelled DNA captured by the DNA-mediated chromatin pull-down technique (DmChP). Graphical experimental outline is shown above the analysis by Western blot of relevant proteins in both input and captured materials. U2OS cells were labelled with EdU for 30 min and then treated with 4 mM HU for 2 h in the presence or absence of 10 μM XL413. Proteins binding to EdU-labelled DNA captured by DmChP were then analysed by Western blot as above. As in panel B, but incubation with HU was extended to 24 h. Black arrow indicates MRE11 electrophoretic mobility shift. U2OS cells were either mock-treated or treated with 10 μM XL413 for 30 min, at which point 4 mM HU was added and cells incubated for a further 24 h. Extracts prepared from HU-treated cells were then incubated in the presence or absence of λ-phosphatase. Proteins were analysed by Western blotting with anti-MRE11 antibodies. Total protein stain (TPS) is used as a loading control. Black arrow indicates MRE11 electrophoretic mobility shift. U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 h. PCNA (green) and MRE11 (red) were detected by immunofluorescence. Insets I–II represent enlargements of selected region of the merged images. Quantification of PCNA and MRE11 colocalization was assessed with ImageJ in ˜70 randomly selected cells for each condition from four biological replicates and expressed as Pearson's correlation coefficient. Error bars represent SEM. Statistical significance was assessed by Student's t-test (*P ˂ 0.05). Source data are available online for this figure. Source Data for Figure 3 [embr201948920-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint During the course of these experiments, we noticed that after a prolonged arrest in HU, a small fraction of MRE11 protein, which was also captured on stalled forks, was retarded in its electrophoretic mobility, and that its abundance was markedly decreased upon CDC7 inhibition (Fig 3C). Subsequently, we found that MRE11 altered electrophoretic mobility was sensitive to the method of protein extraction and that a mild treatment of cells with PFA before lysis greatly helped in its detection. We reasoned that this could be due to a post-translational modification, most likely due to phosphorylation, which could be easily lost in native extracts. To test this hypothesis, new extracts were prepared from U2OS cells that had been either mock-treated or treated with HU. Again, a slower migrating form of MRE11 was detected in HU-treated cells, which was decreased by cotreatment with XL413. Importantly, MRE11 electrophoretic mobility shift was completely lost upon incubation of these extracts with purified lambda phosphatase (Fig 3D). Altogether, these experiments suggest that CDC7 may control MRE11 by regulating its phosphorylation status. In order to refine our analysis, we assessed MRE11 localization by immunofluorescence microscopy. In these experiments, after drug treatments and fixation, cells were stained with anti-MRE11 and anti-PCNA antibodies. As previously described, MRE11 can be detected in a large number of nuclear foci and in HU-arrested cells with a high degree of colocalization with PCNA, indicative of its association with replication factories 35, 47. Treatment with the CDC7 inhibitor markedly reduced the degree of colocalization of the two proteins (Fig 3E and F). Partial colocalization of MRE11 with RPA2 was also reduced by CDC7 inhibition, albeit to a lesser extent (Fig EV3). Click here to expand this figure. Figure EV3. CDC7 inhibition decreases colocalization of MRE11 with RPA2 U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 h. RPA2 (green) and MRE11 (
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