Jonathan Baxter, John F.X. Diffley  Molecular Cell 

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Topoisomerase II Inactivation Prevents the Completion of DNA Replication in Budding Yeast  Jonathan Baxter, John F.X. Diffley  Molecular Cell  Volume 30, Issue 6, Pages 790-802 (June 2008) DOI: 10.1016/j.molcel.2008.04.019 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Degradation of Top2 Prior to Entry into the Cell Cycle Results in Catenation and Chromosome Missegregation without Affecting Chromosome Resolution or Cell Cycle Progression (A) Degron-mediated proteolysis of Top2 causes rapid degradation of Top2 and loss of viability. (Top panel) Western blot of myc-tagged top2p-td protein in the top2-td strain in YP raffinose 25°C, following induction of GAL-UBR1 and 1, 2, and 3 hr after shifting to the restrictive conditions (YP gal 50 μg/ml doxycycline, 37°C). (Bottom panel) top2-td is inviable at 37°C, dependent on expression of UBR1. (B) Episomal plasmid becomes catenated during S phase in top2-td. Parental and top2-td strains containing the plasmid pRS316 were blocked in G1 and Top2p-td degraded. Genomic DNA was prepared from cells isolated prior to blocking (exp 25), at release (0), and 50 min following release as the cells began to bud and analyzed by Southern blot (left panel). The sample was also treated with recombinant mammalian topoII α (right panel) to confirm that catenated plasmid could be resolved to relaxed monomer. Resolution of these samples in ethidium bromide gels induced positive supercoiling of both catenanes and decatenated monomers, confirming these isoforms were covalently closed. Supercoiled monomeric pRS316 isolated from E. coli was analyzed under the same conditions. (C) Missegregation of chromosomes following passage of one cell cycle without Top2. Parental and top2-td strains were blocked in α factor and top2p-td degraded. Samples were taken at midlog phase (25°C exp), prior to alpha factor release (0), and at time points shown for analysis by flow cytometry to assess DNA content. (D) Chromosome resolution by PFGE is not significantly affected by loss of Top2. Prepared chromosomal samples from the same cells as analyzed in (A) were resolved by pulse field gel electrophoresis and revealed with ethidium bromide staining. (E) Loss of Top2 does not delay anaphase onset or prevent nuclear division. Nuclear cytologies of cells following block, degradation, and release were assessed, and the number that were single or double nucleated at each time point was counted. Histograms are overlaid with the numbers of singularly nucleated cells that were undergoing anaphase (elongated nucleus). (F) Loss of Top2 generated the “cut” phenotype at mitotic exit. Representative images of DAPI-stained top2-td cells are shown at each indicated time point. Arrowheads indicate anaphase chromosomes; arrows, “cut” chromosomes. Molecular Cell 2008 30, 790-802DOI: (10.1016/j.molcel.2008.04.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Loss of Top2 Causes DNA Damage and Cell Inviability Only as Cells Exit Mitosis (A) Loss of Top2 activates the DNA damage checkpoint at a late stage of the cell cycle. Exponentially growing parental (WT) and top2-td cells were arrested with alpha factor and shifted to the restrictive conditions to degrade Top2p-td. Cells were then released into the cell cycle, and samples were collected for FACS analysis of DNA content and immunoblotting for Rad53 protein. WT cells treated ± HU were blotted as a control for Rad53 activation status. Whole protein visualized by Ponceau S staining is shown in the bottom panel. (B) Blocking entry into mitosis prevents DNA damage in the absence of Top2. As in (A) except cells were released into media containing nocodazole. (C) Blocking exit from mitosis prevents DNA damage in the absence of Top2. As in (A) except both parental and top2-td strains were crossed onto the cdc15-2 background. (D) Active cytokinesis machinery is required for DNA damage in the absence of Top2. As in (A) except both parental and top2-td strains were crossed onto the cdc12-6 background. (E) Blocking entry or exit of mitosis rescues the lethality associated with loss of Top2. Following arrest, degradation, and release for 160 min, the viability of the cells was assessed by plating a known number of cells onto YPD plates. The percentage of viable cells for each condition/genotype from three independent experiments is shown with standard deviation. Molecular Cell 2008 30, 790-802DOI: (10.1016/j.molcel.2008.04.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Expression of Enzymatically Inactive Top2 Arrests the Cells in G2 with an Activated DNA Damage Checkpoint (A) Expression of inactive Top2Y-F in top2-td triggers checkpoint activation in G2. Strains top2-td, top2-tdGALTOP2 and top2-tdGALTOP2Y-F were arrested, Top2p-td degraded, and released with samples taken for FACS analysis of DNA content. (B) Samples in (A) were immunoblotted for exogeneously expressed Top2 (HA tag) or Rad53. Whole protein in samples was visualized by Ponceau S staining of blotted membranes. (C) Inhibition of nuclear division following expression of Top2Y-F. Nuclear cytologies of the indicated strains following block, degradation, and release were assessed, and the number with one or two nuclei at each time point was counted. Histograms are overlaid with the numbers of singularly nucleated cells, which were undergoing anaphase. Molecular Cell 2008 30, 790-802DOI: (10.1016/j.molcel.2008.04.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Rad53 Activation Is Dependent on the DNA Damage Checkpoint and DNA Replication (A) Inactive Top2-dependent G2 Rad53 activation requires MEC1. The indicated strains were arrested in G1, Top2p-td was degraded, Top2Y-F was induced, and cells were released into media containing nocodazole. Samples were taken for FACS, Rad53 in situ kinase assay, and immunoblot analysis. (B) Checkpoint activation occurs before entry into mitosis. top2-td GALTOP2Y-F was released into restrictive media ± nocodazole and samples taken for immunoblotting analysis. (C) Inactive top2-dependent Rad53 activation requires CDC45. The double degron strain cdc45-td top2-td was arrested in G1, Top2p-td and Cdc45p-td were degraded, and the cells were released into media containing nocodazole. Samples were taken for FACS and immunoblot analysis of Rad53 (top) and Top2p-td (myc tag) and Top2Y-Fp (HA tag) (bottom). Molecular Cell 2008 30, 790-802DOI: (10.1016/j.molcel.2008.04.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Enzymatically Inactive Top2 Disrupts Termination of DNA Replication (A) Expression of Top2Y-F generates an excess of gaps and nicks in genomic DNA. Prepared postreplicative (80 min postrelease) genomic DNA was subjected to nuclease treatment with the indicated units of either S1 single-strand nuclease (top panel) or 3′–5′ exonuclease ExoIII (bottom panel). DNA was visualized by probing of the URA3 locus. (B) Top2Y-F prevents normal replication termination. Samples of blocked and released top2-td and top2-tdGALTOP2Y-F cultures were taken every 7.5 min throughout S phase and analyzed by FACS (bottom panel) for total DNA content and Southern blot analysis for pRS316 sequences (top panel). Above top panel, long exposure of top of blot containing last replication intermediate (L.R.I). (C) Top2Y-F-dependent isoforms of pRS316 are not dependent on mitotic spindles. Strain top2-tdGALTOP2Y-F was arrested, Top2p-td degraded, and Top2Y-F expressed and then released into media ± nocodazole. Samples were taken prior to blocking (exp 25°C), prior to release (0), and at intervals indicated thereafter for Southern blot analysis. Molecular Cell 2008 30, 790-802DOI: (10.1016/j.molcel.2008.04.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Expression of Enzymatically Inactive Top2 Generates Gaps in Replicated DNA Molecules (A) Form ϕ is composed of open catenated molecules. Postreplicative DNA (100 min postrelease) was either mock treated or treated with recombinant topoII α before resolving by 1D native electrophoresis and Southern blot analysis. To ascertain whether the resulting products were open or covalently closed, an identical sample was additionally run in a second dimension of EtBr-saturated agarose to supercoil any closed plasmid isoforms. ∗See text for details. (B) Form ϕ has exposed 3′ strand ends. DNA isolated during bulk genomic replication (50 min postrelease) that contained both pre- and postreplicated forms of the plasmid was subjected to increasing amounts of ExoIII. (C) Form ϕ is composed of gapped DNA molecules. Genomic DNA samples from an S phase fraction were resolved under native conditions. The lane was excised and run in the second dimension under denaturing alkaline conditions (1.6% agarose). (Right panel) Purified linearized pRS316 and undigested pR316 were run on the second dimension as size controls. (Left panel) Long exposure of LRI signal. Molecular Cell 2008 30, 790-802DOI: (10.1016/j.molcel.2008.04.019) Copyright © 2008 Elsevier Inc. Terms and Conditions