Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair
2016; Springer Nature; Volume: 36; Issue: 1 Linguagem: Inglês
10.15252/embj.201593540
ISSN1460-2075
AutoresMaría Teresa Villoria, Facundo Ramos, Encarnación Dueñas, Peter Faull, Pedro R. Cutillas, Andrés Clemente‐Blanco,
Tópico(s)Plant Genetic and Mutation Studies
ResumoArticle16 November 2016Open Access Source DataTransparent process Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair María Teresa Villoria María Teresa Villoria Cell Cycle and Genome Stability Group, Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca (USAL), Salamanca, Spain Search for more papers by this author Facundo Ramos Facundo Ramos Cell Cycle and Genome Stability Group, Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca (USAL), Salamanca, Spain Search for more papers by this author Encarnación Dueñas Encarnación Dueñas Cell Cycle and Genome Stability Group, Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca (USAL), Salamanca, Spain Search for more papers by this author Peter Faull Peter Faull Biological Mass Spectrometry and Proteomics Laboratory, Medical Research Council, Clinical Science Centre, Imperial College, London, UK Search for more papers by this author Pedro Rodríguez Cutillas Pedro Rodríguez Cutillas Biological Mass Spectrometry and Proteomics Laboratory, Medical Research Council, Clinical Science Centre, Imperial College, London, UK Search for more papers by this author Andrés Clemente-Blanco Corresponding Author Andrés Clemente-Blanco [email protected] orcid.org/0000-0002-3674-0671 Cell Cycle and Genome Stability Group, Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca (USAL), Salamanca, Spain Search for more papers by this author María Teresa Villoria María Teresa Villoria Cell Cycle and Genome Stability Group, Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca (USAL), Salamanca, Spain Search for more papers by this author Facundo Ramos Facundo Ramos Cell Cycle and Genome Stability Group, Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca (USAL), Salamanca, Spain Search for more papers by this author Encarnación Dueñas Encarnación Dueñas Cell Cycle and Genome Stability Group, Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca (USAL), Salamanca, Spain Search for more papers by this author Peter Faull Peter Faull Biological Mass Spectrometry and Proteomics Laboratory, Medical Research Council, Clinical Science Centre, Imperial College, London, UK Search for more papers by this author Pedro Rodríguez Cutillas Pedro Rodríguez Cutillas Biological Mass Spectrometry and Proteomics Laboratory, Medical Research Council, Clinical Science Centre, Imperial College, London, UK Search for more papers by this author Andrés Clemente-Blanco Corresponding Author Andrés Clemente-Blanco [email protected] orcid.org/0000-0002-3674-0671 Cell Cycle and Genome Stability Group, Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca (USAL), Salamanca, Spain Search for more papers by this author Author Information María Teresa Villoria1,‡, Facundo Ramos1,‡, Encarnación Dueñas1, Peter Faull2, Pedro Rodríguez Cutillas2,3 and Andrés Clemente-Blanco *,1 1Cell Cycle and Genome Stability Group, Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca (USAL), Salamanca, Spain 2Biological Mass Spectrometry and Proteomics Laboratory, Medical Research Council, Clinical Science Centre, Imperial College, London, UK 3Present address: Integrative Cell Signalling and Proteomics Group, Centre for Haemato-Oncology, Barts Cancer Institute, John Vane Science Centre, Queen Mary University of London, London, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +34 923 294 887; E-mail: [email protected] The EMBO Journal (2017)36:79-101https://doi.org/10.15252/embj.201593540 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 Cells are constantly threatened by multiple sources of genotoxic stress that cause DNA damage. To maintain genome integrity, cells have developed a coordinated signalling network called DNA damage response (DDR). While multiple kinases have been thoroughly studied during DDR activation, the role of protein dephosphorylation in the damage response remains elusive. Here, we show that the phosphatase Cdc14 is essential to fulfil recombinational DNA repair in budding yeast. After DNA double-strand break (DSB) generation, Cdc14 is transiently released from the nucleolus and activated. In this state, Cdc14 targets the spindle pole body (SPB) component Spc110 to counterbalance its phosphorylation by cyclin-dependent kinase (Cdk). Alterations in the Cdk/Cdc14-dependent phosphorylation status of Spc110, or its inactivation during the induction of a DNA lesion, generate abnormal oscillatory SPB movements that disrupt DSB-SPB interactions. Remarkably, these defects impair DNA repair by homologous recombination indicating that SPB integrity is essential during the repair process. Together, these results show that Cdc14 promotes spindle stability and DSB-SPB tethering during DNA repair, and imply that metaphase spindle maintenance is a critical feature of the repair process. Synopsis In yeast, DNA damage causes re-localization of DNA breaks to the nuclear periphery for efficient repair. New work implicates the Cdk-counteracting phosphatase Cdc14 in promoting spindle-dependent recruitment of DNA lesions to spindle pole bodies. Cdc14 is partially released from the nucleolus into the nucleoplasm upon DNA damage. Cdc14-dependent Spc110 dephosphorylation contributes to stabilization of the metaphase spindle. Spindle integrity is important to stimulate the tethering of DNA lesions to the spindle pole bodies. Recruitment of DNA breaks to spindle pole bodies enhances their repair by homologous recombination. Introduction Maintenance of genomic integrity is an essential part of cellular physiology. Genotoxic insults that induce DNA lesions must be repaired in order to avoid propagation of mutations that can lead to the appearance of a malignant transformation. DNA damage can arise following a wide range of stimuli, including ionizing radiation, ultraviolet radiation, replication stress, chemicals and reactive oxygen species generated during the cell metabolism. Of the various forms of damage that are caused by these mutagens, the most hazardous is arguably the DNA double-strand break (DSB), generated when the two complementary DNA strands of the double helix are severed simultaneously. The capacity to deal with these DNA lesions is critical for cellular survival and for the maintenance of genome stability, since errors during the repair process might result in mutations, cancer or even cell death. To combat this threat, cells have evolved a series of mechanisms collectively known as DDR for DNA damage response to detect the lesion, signal its presence and promote its repair. DSBs may be repaired by either non-homologous end joining (NHEJ) (Burma et al, 2006) or homologous recombination (HR) (Paques & Haber, 1999). Repair by NHEJ is generally considered to be an error-prone pathway because it requires direct rejoining of the broken DSB ends. By contrast, HR involves a genomic search for sequences to be used as template for repair and is considered an error-free repair pathway (Branzei & Foiani, 2008; Mathiasen & Lisby, 2014; Ceccaldi et al, 2016). At the DNA level, HR requires 5′-to-3′ resection of the DSB ends to generate a single-stranded DNA tail (ssDNA). This ssDNA is used to stimulate homologous pairing and strand invasion with a DNA template that serves as donor, forming a structure called the displacement loop (D-loop). The D-loop can be extended by DNA polymerases, copying the information that might be missing at the break site. In order to resolve this DNA structure, cells can use different strategies: (i) synthesis-dependent strand annealing (SDSA), by which the invading strand of the DNA can be displaced and re-annealed to the other broken chromosome end; or (ii) formation of a double Holliday junction (dHJ), whereby the displaced strand of the D-loop anneals with ssDNA on the other end of the break (second end capture) and the 3′ end primes DNA synthesis. Many steps of the HR pathway require cyclin-dependent kinase (Cdk) activity. Early steps are particularly sensitive to Cdk1 inhibition because of its role in DSB-end resection (Chen et al, 2011). In later steps, Cdk1 is important as it recruits Rad52 to sites of DNA damage (Barlow & Rothstein, 2009) and promotes the correct function of the Srs2 helicase during SDSA (Saponaro et al, 2010). It has also been recently shown that the Cdk controls the decision to use NHEJ or HR depending on the cell cycle stage at which the lesion occurs (Zhang et al, 2009). Consequently, repair by HR is favoured during late S and G2 phases, while NHEJ is preferentially used in G1 (Aylon et al, 2004). Cdk1 function therefore influences the cellular response to DNA damage at multiple steps of the repair process. However, it is currently unknown whether specific phosphatases of Cdk phosphorylation are also involved in the regulation of the DNA damage response. The dual-specificity Cdc14 phosphatase was originally identified in Saccharomyces cerevisiae for its role in reversing Cdk1 phosphorylation during mitotic exit (Visintin et al, 1998). Further studies on Cdc14 orthologues from yeast to humans have characterized additional roles for this family of phosphatases in cytokinesis (Meitinger et al, 2010, 2012), chromosome segregation (Clemente-Blanco et al, 2009; Mocciaro & Schiebel, 2010), transcription (Clemente-Blanco et al, 2009, 2011; Guillamot et al, 2011), centrosome duplication (Mocciaro & Schiebel, 2010), ciliogenesis (Clement et al, 2011) and in resolving linked DNA intermediates (Blanco et al, 2014; Eissler et al, 2014; Garcia-Luis et al, 2014). In budding yeast, Cdc14 activity is regulated by dynamic changes in the subcellular localization throughout the cell cycle. Cdc14 is sequestered in the nucleolus until early anaphase, when it migrates to the nucleus and cytoplasm, a process regulated by the FEAR and MEN networks, respectively (Stegmeier et al, 2002). Surprisingly, Clp1/Flp1, the Schizosaccharomyces pombe Cdc14 orthologue, and Cdc14B, its mammalian counterpart, exit the nucleolus during interphase upon DNA replication stress or damage, implicating Cdc14 phosphatases in response to genotoxic insults (Diaz-Cuervo & Bueno, 2008; Mocciaro & Schiebel, 2010). Despite the evidences of an evolutionary conserved function of Cdc14 in response to DNA damage, there is not a consensus agreement about the molecular function of the phosphatase during DDR activation. Flp1 exclusion from the nucleolus after a DNA replication arrest induced by the addition of hydroxyurea (HU) is crucial to promote a fully checkpoint activation (Diaz-Cuervo & Bueno, 2008). Similarly, Cdc14B translocation from the nucleolus to the nucleoplasm in response to genotoxic stress is responsible for Plk1 degradation by the ubiquitin ligase APC/CCdh1. This results in the stabilization of the DNA damage checkpoint activator Claspin and the cell cycle inhibitor Wee1, with the subsequent initiation of the G2 checkpoint (Bassermann et al, 2008). Intriguingly, by using both chicken and human cells, Mocciaro et al have shown that Cdc14A/B-KO mutants arrest efficiently in G2 with normal levels of Chk1 and Chk2 activation in response to irradiation. However, γ-H2A.X foci and DSBs persist longer in Cdc14A-KO or Cdc14B-KO cells than controls, suggesting that both Cdc14 phosphatases are required for efficient DNA repair (Mocciaro et al, 2010). Supporting this work, another study has shown that Cdc14b-deficient mouse embryonic fibroblasts cells accumulate more endogenous DNA damage, and more cells enter senescence in response to exogenous DNA damage, suggesting that the function of this phosphatase is restricted to enhance efficient DNA damage repair (Wei et al, 2011). Here, we show that in budding yeast, Cdc14 is transiently released from the nucleolus in response to genotoxic stress to enhance recombinational repair. This function is attained by stimulating the recruitment of the broken DNA to the vicinity of SPBs (spindle pole bodies) in a process that requires the SPB component Mps3 and a competent DNA damage checkpoint activation. Inactivation of Cdc14 during the induction of the DNA lesion causes continuous misalignment of the metaphase spindle, increases oscillatory SPB movements and impairs DSB-SPB tethering, suggesting a role of the phosphatase in DNA repair by promoting spindle stability. In a screen looking for Cdc14 substrates at the SPBs during the induction of a DNA lesion, we identified Spc110, the intra-nuclear receptor for the γ-tubulin complex. Remarkably, the steady-state phosphorylation of Spc110 during the induction of a DNA lesion is essential to promote DSB-SPB interaction and DNA repair by HR. Taken together, our results point to the function of Cdc14 in DNA repair by promoting SPB stabilization and DSB-SPB interaction and suggest that the relocation of damage sites to the SPBs proximities stimulates homologous recombination in a naturally occurring repair process that minimizes genome instability. Results Cells lacking Cdc14 activity exposed to genotoxic stress are compromised in cell viability but retain DNA damage checkpoint proficiency As a first approach to analyse the role of Cdc14 in the DNA damage response in S. cerevisiae, we tested the effect of the DNA-damaging agent methyl methanesulphonate (MMS) on the growth of cells carrying the temperature-sensitive allele cdc14-1 (Fig 1A). Because Cdc14 is an essential gene in S. cerevisiae, we carried out tests on a range of temperatures. At 30°C, both the cdc14-1 mutant and its isogenic wild-type strain grew in the absence of DNA damage. On the contrary, a severe defect in cell growth was observed when the mutant was plated on MMS, indicating that Cdc14 function is important when cells are exposed to DNA damage (Fig 1A). To further characterize the essential role of Cdc14 when grown on different genotoxic compounds, we plated both wild-type and cdc14-1 backgrounds in the presence of the UV-mimic 4-nitroquinoline-1-oxide (4NQO), the ribonucleotide reductase inhibitor hydroxyurea (HU), the radiomimetic drug phleomycin and the microtubule-destabilizing drug benomyl at the semipermissive temperature of 30°C (Fig 1B). Remarkably, cdc14-1 cells presented a substantial sensitivity in all media tested, extending the essential role of this phosphatase to a great variety of DNA damage stresses. Figure 1. Cdc14 is required for intra-chromosomal DNA repair by HR Tenfold serial dilutions from overnight cultures of wild-type and cdc14-1 cells dropped and grown on solid rich media or media containing MMS at 25, 28, 30 or 33°C. Note that cdc14-1 cells exhibit growth sensitivity to MMS at 28 and 30°C compared to wild-type cells. Tenfold serial dilution from mid-log phase cultures of wild-type and cdc14-1 cells grown on solid rich media or media containing mock DMSO (as non-treated control), 4NQO, HU, phleomycin and benomyl at 30°C. Note that cdc14-1 cells present a marked sensitivity to all DNA damage agents tested. Left panel: Schematic representation showing relevant genomic structure of the strain used to assess intra-chromosomal repair. The location of a MAT-specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of intra-chromosomal repair in wild-type and cdc14-1 cultures at the semipermissive temperature. After DSB formation by the expression of the HO, glucose was added to repress it, thus allowing repair with HM donor sequences. Genomic DNA was digested with StyI, separated on agarose gel and blotted. Blots were hybridized with a probe corresponding to the MATa-distal sequence. A second probe for the actin gene was used to control the amount of genomic DNA loaded at each time point. Immunoblot analysis of Rad53 during mating-type switching experiments is shown. Coomassie blue staining is depicted as a loading control. Graphs show quantification of gene conversion (GC) leading to the re-establishment of MATa or switching to MATα and DSB formation. The data were normalized with the actin as a loading control. Graphs show the mean ± SD from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a two-tailed unpaired Student's t-test. Data information: MMS, methyl methanesulphonate; DMSO, dimethyl sulphoxide; 4NQO, 4-nitroquinoline-1-oxide; HU, hydroxyurea; Raf, raffinose; Gal, galactose; Glu, glucose; DSB, double-strand break; GC, gene conversion. Source data are available online for this figure. Source Data for Figure 1 [embj201593540-sup-0006-SDataFig1.pdf] Download figure Download PowerPoint Cells containing a defect either in DNA damage checkpoint or in DSB repair fail to proliferate after induction of DNA damage. To test for a putative function of Cdc14 in DNA damage checkpoint activation, we took advantage of strains containing the homothallic endonuclease HO gene under the control of the inducible galactose promoter GAL1. Addition of galactose to the media induces expression of the nuclease and the subsequent production of a single DSB in the genome at the MAT locus on chromosome III. We generated a DNA break by continuous expression of the HO endonuclease in both wild-type and cdc14-1 strains and tested their efficiency to block in G2/M due to the DNA damage checkpoint activation. Southern blots of both wild-type and cdc14-1 showed the same kinetics of DSB formation and stabilization during the entire experiment (Fig EV1A). After 8 h in the presence of galactose, both cultures presented more than 90% of mono-nucleated G2/M arrested cells, indicating that Cdc14 is not required for activation of the DNA damage checkpoint pathway induced by a single DSB. This finding suggests that the sensitivity to genotoxic compounds observed in cdc14-1 mutants could be a consequence of defective repair rather than a checkpoint deficiency. Click here to expand this figure. Figure EV1. Cdc14 is required for recombinational DNA repair Constitutive expression of HO produces a constant DSB at the MAT locus in wild-type and cdc14-1 cultures at semipermissive temperature. Southern blot of wild-type and cdc14-1 cells showing the efficiency and stability of the DSB. A diagram with the genomic information, the restriction sites used and the location of the MAT-distal probe is shown. Micrographs depict cultures blocked in G2/M 8 h after DSB induction. Scale bar: 5 μm. Graphs show quantification of gene conversion (GC) leading to the re-establishment of MATa and the percentage of cells blocked in G2/M after 8 h from the galactose addition. Analysis of mating-type switching through formation of G1 shmoo cells in wild-type and cdc14-1 cultures at semipermissive temperature. Glucose was added to the cultures after DSB formation to repress HO, thus allowing repair with HM donor sequences (top diagram). FACS analysis was performed on cells collected at each time point indicated. Graph shows percentage of cells blocked at G1 (shmoos) or mono-nucleated arrested G2/M (by DAPI staining) 4 h after glucose addition. Micrographs show cultures 4 h after incubation with glucose. Scale bar: 5 μm. Wild-type and cdc14-1 mutant strains carrying the ectopic gene conversion assay depicted were grown overnight and exposed to expression of HO through addition of galactose, thus producing a DSB on chromosome V (top diagram). Samples were taken at 0, 2.5, 5, 7.5, 10 and 24 h. FACS analysis of each time point is shown. Graphs show percentage of mono-nucleated G2/M cells of each sample. Micrographs show cultures 24 h after DSB induction. Scale bar: 5 μm. Wild-type and cdc14-1 mutant strains harbouring the repair pathway choice assay described (top diagram) were grown overnight and exposed to expression of HO through addition of galactose for 1.5 h. Samples were taken before and at 1, 2, 3, 4 and 5 h after supplementing with glucose. FACS analysis of each time point is shown. Quantification of mono-nucleated G2/M arrested cells of wild-type and cdc14-1 cells during the time course is shown. Micrographs show cultures 5 h after glucose addition. Scale bar: 5 μm. Data information: DAPI, 4′,6-diamidino-2′-phenylindole dihydrochloride; DSB, double-strand break; Glu, glucose; Raf, raffinose; Gal, galactose; GC, gene conversion. Download figure Download PowerPoint Cdc14 is required for intra- and inter-chromosomal DNA repair by homologous recombination but is not involved in DSB repair pathway choice To determine whether Cdc14 was directly implicated in DNA repair and to investigate which specific process in the DSB response pathway was controlling, we examined DNA repair by using strains harbouring the HO-induced DSB system in three different conditions: intra-chromosomal DNA repair, inter-chromosomal gene conversion and repair pathway choice. It has been proposed that intra-chromosomal DNA repair at the MAT locus occurs mainly by SDSA (reviewed in Haber, 2012). To check whether Cdc14 plays a role in this repair pathway, we induced the DSB formation by supplementing the media with galactose for 2 h. HO was subsequently repressed by glucose addition, and the kinetics of the repair process by HR with the donor sequence HMLα or HMRa was followed (Fig 1C, diagram). Physical analysis by Southern blot showed that cdc14-1 cells repaired the DSB slower and less efficiently than wild-type cells when occurred by restoration of the original MATa or switched to MATα allele (Fig 1C). We also observed that the checkpoint effector kinase Rad53 was significantly phosphorylated during repair in cdc14-1 mutants (Fig 1C). This was not the case for wild-type cells where the DNA damage checkpoint kinases are not active during intra-chromosomal repair (Pellicioli et al, 2001; Kim et al, 2011). Supporting this data, wild-type controls presented 75% of the cells blocked in G1 with shmoo morphology by 4 h of glucose addition (Fig EV1B). Shmoo formation is a good indicator of successful repair as it requires the appearance of cells with the opposite mating type in the culture. By contrast, cdc14-1 mutant cells presented only 25% of shmoo cells, while an increased number of mono-nucleated G2/M arrested cells were observed (Fig EV1B). These results demonstrate that SDSA-mediated DSB repair is defective when Cdc14 function is impaired. In order to assess whether Cdc14 was also required for gene conversion events when the donor sequence is located on a different chromosome, that is inter-chromosomal repair, we used a genetic background containing a MATa sequence in chromosome V that can be cleaved by HO and repaired with an uncleavable MATa-inc sequence located on chromosome III (Ira et al, 2003). This strain lacks the donor HM loci and the MATa-inc mutation renders the repair product insensitive to subsequent HO cleavage (Fig 2A, diagram). By using this system, a single reparable DSB by gene conversion is generated which can occur either with or without an accompanying crossover (Mazon & Symington, 2013). As the restriction fragments have an altered size when a crossover is formed, one can detect these events during the repair process. Gene conversion with no associated crossovers was the predominant form of repair in both wild-type and cdc14-1 cells. However, wild-type cells repaired the break with faster kinetics and more efficiently than cdc14-1 mutants (Fig 2A). We detected no significant differences in the proportion of gene conversion associated with crossing-over between both strains among cells that repaired the DNA lesion (Fig 2A). Accordingly, while wild-type cells repaired the DNA break and re-entered the cell cycle 10 h after the induction of the break, cdc14-1 mutants presented a significant delay in cell cycle progression with up to 75% of mono-nucleated dumbbell cells (Fig EV1C). Confirming our previous observations, we found high levels of Rad53 phosphorylation in both wild-type and cdc14-1 strains, ruling out a function of the phosphatase in the DNA damage checkpoint activation (Fig 2A). These data confirm that Cdc14 activity is also required to promote ectopic recombination with homologous sequences. Figure 2. Cdc14 is required for inter-chromosomal DNA repair by HR but is not involved in DSB repair pathway choice Left panel: Schematic diagram displaying relevant genomic structure of the strains used to measure inter-chromosomal DNA repair efficiency. The location of a MAT-specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that crossover and non-crossover products have different restriction fragment sizes that can be differentiated in a Southern blot assay. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 mutant strains carrying the inter-chromosomal gene conversion assay. Cells were grown overnight before adding galactose at semipermissive temperature to induce HO expression, thus producing a DSB on chromosome V. Samples were taken at different time points, and genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were hybridized with a MATa-only DNA sequence, and an actin probe was used as a loading control. Asterisk denotes an overexposed film to visualize crossover formation. Immunoblot of Rad53 was performed as previously described. Coomassie staining is shown as a control for loading. Graphs show the mean ± SD of gene conversion, DSB induction and crossover versus non-crossover ratio from three independent experiments. All data were normalized using actin as a loading control. Replicates were averaged and statistical significance of differences assessed by a two-tailed unpaired Student's t-test. Left panel: Schematic representation depicting the genomic structure of the strains used to evaluate repair pathway choice. The location of a MAT-specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that the use of NHEJ or HR generates different product that can be recognized in Southern blots by the different disposition of the restriction sites for the endonucleases used. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 cells harbouring the repair pathway choice assay. Cells were grown overnight and HO induction was attained through addition of galactose for 1.5 h at semipermissive temperature. After induction of the HO, glucose was added to repress the HO expression and samples were taken to analyse repair efficiency. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Blots were hybridized with a probe corresponding to the MATa-only DNA sequence. A second probe to the HIS3 gene was used to control the amount of genomic DNA loaded at each time point. Graphs show quantification of mating-type switching by gene conversion (HR), restoration of MATa (NHEJ) and the kinetics of the DSB induction. All data were normalized with the HIS3 gene. Graphs show the mean ± SD from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a two-tailed unpaired Student's t-test. Data information: Raf, raffinose; Gal, galactose; Glu, glucose; DSB, double-strand break; GC, gene conversion; CO, crossover; NHEJ, non-homologous end joining. Source data are available online for this figure. Source Data for Figure 2 [embj201593540-sup-0007-SDataFig2.zip] Download figure Download PowerPoint If Cdc14 function is required at the initial steps during repair, this might translate into alteration of DSB repair pathway choice in cdc14-1 mutants. To address this subject, we used a strain where repair of the HO-induced break by HR is obligatorily associated with mating-type switching from MATa to MATα, while repair by NHEJ retains the mating type as MATa (Fig 2B, diagram). We expressed the HO endonuclease for 90 min in logarithmically growing cells and observed the dynamics of the repair process 5 h after repressing the HO expression by glucose addition (Fig 2B). As before, cdc14-1 cells presented a delay in the appearance of the switched product when compared to wild-type cells, indicating a failure in DNA repair by gene conversion. Supporting this observation, an extended G2/M arrest with an increased number in mono-nucleated cells was detected in cdc14-1 cultures (Fig EV1D). We could not detect a significant accumulation of NHEJ repair product when comparing both strains suggesting that Cdc14 does not contribute to DSB repair pathway choice but rather affects downstream events during recombinational repair. Cdc14 is released from the nucleolus
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