Prolonged cell cycle arrest in response to DNA damage in yeast requires the maintenance of DNA damage signaling and the spindle assembly checkpoint

  1. Felix Y Zhou
  2. David P Waterman
  3. Marissa Ashton
  4. Suhaily Caban-Penix
  5. Gonen Memisoglu
  6. Vinay V Eapen
  7. James E Haber  Is a corresponding author
  1. Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, United States
  2. Department of Molecular Genetics & Cell Biology, University of Chicago, United States
10 figures, 2 tables and 2 additional files

Figures

Measuring checkpoint arrest in 1-DSB and 2-DSB strains.

(A) Morphological categories of budding yeast cells using brightfield microscopy and DAPI staining were used to determine G2/M arrest. Cells that arrest at G2/M shift toward a large bud state. G2/M-a…

Figure 2 with 3 supplements
Checkpoint maintenance requires Ddc2, Rad9, Rad24, and Rad53 activity.

(A) Above: percentage of G2/M-arrested cells in a 2-DSB DDC2-AID strain after DNA damage induction in a liquid culture. Cultures were split 4 hr after galactose treatment to induce DNA damage by …

Figure 2—source data 1

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The 7 hr ± IAA samples were added to the last two wells.

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Figure 2—figure supplement 1
Adaptation assay of AID-tagged checkpoint activation proteins.

(A) Adaptation assay of 50 G1 cells on a YEP-Gal plate after 24 hr for 2-DSB (WT), 2-DSB DDC2-AID, 2-DSB RAD9-AID, 2-DSB RAD24-AID, and 2-DSB RAD53-AID with TIR1. (B) Adaptation assay of 50 G1 cells …

Figure 2—figure supplement 2
AID-tagged checkpoint proteins readily degrade with auxin.

(A) Morphological profile of a 2-DSB background where 1 mM auxin (IAA) was added 2 hr before DSB induction with galactose. Western blot of a 2-DSB strain probed with α-Rad53. α-Rad53 shows both an …

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Figure 2—figure supplement 3
Cell cycle profile as determined by budding and DAPI staining in Ddc2-AID and Rad53-AID mutants ±IAA 4 hr after galactose.

(A) Profile of DAPI-stained cells in a 2-DSB DDC2-AID strain after HO induction. Cultures were split 4 hr after Gal-HO induction. Cells were divided based on cell morphology and number of DAPI …

Figure 3 with 1 supplement
Chk1 is dispensable for activation of the cell cycle arrest, but essential for its maintenance.

(A) Percentage of G2/M cells in a 2-DSB chk1∆ strain following DNA damage. Data are shown from three independent experiments, with error bars representing the standard error of the mean (SEM). …

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Figure 3—figure supplement 1
Tel1 is not required for DDC activation or Rad53 phosphorylation.

(A) Adaptation assay for a tel1∆ strain. Cultures were grown in YEP-Lac and put on a YEP-Gal plate. 50 G1 cells were selected to monitor their morphology after 4 and 24 hr on the YEP-Gal plate. (B) …

Dun1 is not required for checkpoint maintenance.

(A) Adaptation assay of 50 G1 cells on a YEP-Gal plate with 2-DSB dun1∆. G2/M arrest was determined based on cell morphology as shown in Figure 1A. Data is shown from three trials with standard …

Figure 5 with 1 supplement
Ddc2 and Rad53 are dispensable for >24 hr checkpoint arrest.

(A) Percentage of G2/M-arrested cells for 2-DSB DDC2-AID after HO induction. Data is shown from three trials with standard error of the mean (SEM). Western blot probed with α-Rad53 and α-Myc. …

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Figure 5—figure supplement 1
Relative levels of Ddc2 decrease after DSB induction.

(A, B) Western blots probed with α-Myc for Ddc2-9xMyc and Ddc2-9xMyc-AID in a 1-DSB and 2-DSB, respectively. α-Pgk1 is used as a loading control. (C) Relative levels of Ddc2 in a 1-DSB and 2-DSB …

Figure 6 with 3 supplements
Degradation of Mad2 or Mad1 at 15 hr releases cells from checkpoint arrest.

(A) Percentage of G2/M-arrested cells for 2-DSB MAD2-AID after HO induction. Data is shown from three trials with standard error of the mean (SEM). Western blot probed with α-Rad53 and α-Myc. …

Figure 6—figure supplement 1
Mad2 is required for permanent arrest in a 2-DSB strain.

(A) Adaptation assay of 1-DSB and 2-DSB where morphology is measured for up to 24 hr and 48 hr, respectively. A second copy of Ddc2 with a GAL1,10 promotor, Ddc2 overexpression (Ddc2oe), was …

Figure 6—figure supplement 2
Mad1 and Mad2 are required for permanent arrest in a 2-DSB strain.

(A) Morphological profile of MAD2-AID after HO induction on a YEP-Gal or YEP-Gal-IAA plate. Galactose was added to an overnight culture of Mad2-AID in YEP-Lac. 4 hr after adding galactose, cells …

Figure 6—figure supplement 3
Degradation of Ddc2 and Mad2 at 15 hr releases cells from checkpoint arrest.

(A) Morphological profile of a 2-DSB MAD2-AID and DDC2-AID MAD2-AID strains after HO induction. Cultures were added onto YEP-Gal ± IAA plates 15 hr after adding HO induction. Ctrl samples were …

Figure 7 with 1 supplement
Degradation of Bub2 but not Bfa1 at 15 hr releases cells from checkpoint arrest.

(A) Percentage of G2/M-arrested cells for 2-DSB BUB2-AID after HO induction. Data is shown from three trials with standard error of the mean (SEM). Western blot probed with α-Rad53 and α-Myc. …

Figure 7—figure supplement 1
Bub2 but not Bfa1 is required for prolonged arrest.

(A) Morphological profile of a 2-DSB BUB2-AID strain with the auxin-Gal plating assay. Cultures were added onto YEP-Gal ± IAA plates 4 hr after HO induction. Western blot probed with α-Rad53 and …

Adaptation assay of different 2-DSB strains.

(A) Adaptation assay of 1-DSB and 2-DSB strains tracking the morphology of 50 G1 cells on a YEP-Gal plate. The percentage of G2/M-arrested cells was shown 4 hr and 24 hr after placement of YEP-Gal …

Phosphomimetic and non-phosphorylatable mutants of histone H2A and H2B do not affect the growth rate of cells.

Growth rate of strains were measured in YPD (2% dextrose) in H2A and H2B mutants for up to 10 hr. Cultures were grown in YPD until they reached an OD600 of 0.1. The OD of each strain was then …

Activation and maintenance of checkpoint arrest in response to a DSB.

The Mre11-Rad50-Xrs2 (MRX) complex is one of the first complexes recruited to DSBs and initiates the resection of dsDNA to ssDNA. ssDNA is then coated with RPA which recruits Ddc2. Mec1 is the …

Tables

Table 1
Comparison of the percentage of large-budded cells back to baseline levels.
FigureStrainTimepoint comparison*p-ValueSignificancePost hoc test
Figure 2ADDC2-AID0 vs 5 + IAA<0.0001****Sidak
0 vs 6 + IAA<0.0001****Sidak
0 vs 7 + IAA<0.0001****Sidak
0 vs 8 + IAA0.0009***Sidak
0 vs 9 + IAA0.054nsSidak
Figure 3DDDC2-AID CHK1∆0 vs 5 + IAA<0.0001****Sidak
0 vs 6 + IAA<0.0001****Sidak
0 vs 7 + IAA0.10nsSidak
0 vs 8 + IAA0.25nsSidak
0 vs 9 + IAA0.072nsSidak
Figure 2BRAD9-AID0 vs 5 + IAA<0.0001****Sidak
0 vs 6 + IAA<0.0001****Sidak
0 vs 7 + IAA<0.0001****Sidak
0 vs 8 + IAA0.0055**Sidak
0 vs 9 + IAA1.00nsSidak
Figure 3ERAD9-AID CHK1∆0 vs 5 + IAA0.00050***Sidak
0 vs 6 + IAA0.0052**Sidak
0 vs 7 + IAA0.21nsSidak
Figure 2CRAD24-AID0 vs 5 + IAA<0.0001****Sidak
0 vs 6 + IAA<0.0001****Sidak
0 vs 7 + IAA0.00***Sidak
0 vs 8 + IAA0.36nsSidak
0 vs 9 + IAA0.90nsSidak
Figure 3FRAD24-AID CHK1∆0 vs 5 + IAA0.00010***Sidak
0 vs 6 + IAA0.00020***Sidak
0 vs 7 + IAA0.10nsSidak
Figure 5DRAD53-AID TIR1(F74G)18 vs 18 + IAA0.35nsSidak
21 vs 21 + IAA0.96nsSidak
24 vs 24 + IAA0.42nsSidak
Figure 5ERAD9-AID pRAD9-AID18 vs 18 + IAA0.80nsSidak
21 vs 21 + IAA0.99nsSidak
24 vs 24 + IAA0.84nsSidak
Figure 6—figure supplement 3ADDC2-AID MAD2-AID AND MAD2-AID18 + IAA vs 18 + IAA0.95nsSidak
21 + IAA vs 21 + IAA0.64nsSidak
24 + IAA vs 24 + IAA0.97nsSidak
  1. *

    Timepoints are relative to when galactose was added.

  2. A one-way ANOVA was used to test for significant differences.

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-Rad53;
(mouse monoclonal)
AbcamCat# ab166859;
RRID:AB_2801547
WB (1:1000)
AntibodyAnti-Rad53;
(rabbit polyclonal)
Abcamab104232WB (1:1000)
AntibodyAnti-Myc;
(mouse monoclonal)
AbcamCat# ab16918;
RRID:AB_30256
WB (1:1000)
AntibodyAnti-Pgk1;
(mouse monoclonal)
AbcamCat# ab32;
RRID:AB_30359
WB (1:5000)
AntibodyAnti-Rad9;
(rabbit polyclonal)
Usui et al., 2009N/AWB (1:4000)
AntibodyECL TH Anti-mouse IgG horseradish peroxidase from sheepGE HealthcareNXA931V
Lot 16937010
WB (1:10,000)
AntibodyECL TH Anti-rabbit IgG horseradish peroxidase from donkey
GE HealthcareNA934V lot 6969611WB (1:10,000)
Chemical compound, drugIndole-3-acetic acidSigma-AldrichI3750-25G-A1 mM
Chemical compound, drug5-Ph-IAASigma-AldrichSML3574-25MG1 µM
Chemical compound, drugFormaldehydeSigma-Aldrich476084%
Chemical compound, drugVECTASHIELD Antifade Mounting Medium with DAPIVector LaboratoriesCat# H-12001 μg/ml
Chemical compound, drugPrometheus Protein Biology
Products 20–313 OneBlock Western-CL Blocking
Buffer, For Chemiluminescent Blots
Genesee
Scientific
Cat# 20-313Blocking buffer for western blots
Commercial assay or kitECL Prime Western Blotting SystemMilliporeSigmaGERPN2232
Strain, strain background (Saccharomyces cerevisiae)cerevisiae: strain background S228cSee Supplementary file 1a for full strain listN/AStrains used in this study
Strain, strain background (S. cerevisiae)JKM179Lee et al., 1998Yeast strainMATα ade1 leu2-3 lys5 trp1
::hisG ura3-52 hoΔ
hmlΔ::ADE1 hmr
Δ::ADE1 ade3::
GAL::HO
Strain, strain background (S. cerevisiae)DW184This studyYeast strainTIR1-myc6::URA3
Strain, strain background (S. cerevisiae)DW417This studyYeast strainHOcse6::HPH TIR1
-myc6::URA3
Strain, strain background (S. cerevisiae)DW418This studyYeast strainDdc2-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)DW419This studyYeast strainRad9-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)DW420This studyYeast strainRad24-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)DW421This studyYeast strainRad53-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)DW426This studyYeast strainchk1∆::NAT
Strain, strain background (S. cerevisiae)DW647This studyYeast strainDdc2-AID*–9xMyc::KAN chk1∆::NAT
Strain, strain background (S. cerevisiae)DW427This studyYeast strainRad9-AID*–9xMyc::KAN chk1∆::NAT
Strain, strain background (S. cerevisiae)DW428This studyYeast strainRad24-AID*–9xMyc::KAN chk1∆::NAT
Strain, strain background (S. cerevisiae)DW429This studyYeast strainRad53-AID*–9xMyc::KAN chk1∆::NAT
Strain, strain background (S. cerevisiae)DW625This studyYeast straindun1∆::KAN
Strain, strain background (S. cerevisiae)DW626This studyYeast strainDun1-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)DW641This studyYeast strainDun1-AID*–9xMyc::KAN chk1∆::NAT
Strain, strain background (S. cerevisiae)FZ009This studyYeast strainMad2*–9xMyc-AID::NAT
Strain, strain background (S. cerevisiae)FZ010This studyYeast strainDdc2-AID*–9xMyc
::KAN Mad2*–9xMyc
-AID::NAT
Strain, strain background (S. cerevisiae)DW455This studyYeast strainmad2Δ::KAN
Strain, strain background (S. cerevisiae)GM180Memisoglu et al., 2019Yeast strainpGal::Ddc2::LEU2
Strain, strain background (S. cerevisiae)DW648This studyYeast strainHOcse6::HPH pGal::
Ddc2::LEU2
Strain, strain background (S. cerevisiae)DW649This studyYeast strainHOcse6::HPH pGal::
Ddc2::LEU2 mad2∆::NAT
Strain, strain background (S. cerevisiae)DW642This studyYeast strainHOcse6::HPH
Ddc2-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)DW643This studyYeast strainHOcse6::HPH Rad9-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)DW644This studyYeast strainHOcse6::HPH Rad24-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)DW645This studyYeast strainHOcse6::HPH Rad53-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)DW650This studyYeast strainpGal::Ddc2::LEU2
mad2∆::NAT
Strain, strain background (S. cerevisiae)GM539Memisoglu et al., 2019Yeast strainDdc2-9xMyc::KAN
Strain, strain background (S. cerevisiae)FZ001This studyYeast strainMATα HOcse6::HPH
Strain, strain background (S. cerevisiae)JY542This studyYeast strainHOcse6::HPH tel1∆::KAN
Strain, strain background (S. cerevisiae)FZ024This studyYeast strainHOcse6::HPH Rad9-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)FZ025This studyYeast strainHOcse6::HPH Rad24-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)FZ026This studyYeast strainHOcse6::HPH Rad53-AID*–9xMyc::KAN
Strain, strain background (S. cerevisiae)FZ173This studyYeast strainHOcse6::HPH Rad24-AID*–9xMyc::KAN
TIR1(F74G)::URA3
Strain, strain background (S. cerevisiae)FZ174This studyYeast strainHOcse6::HPH Rad9-AID*–9xMyc::KAN TIR1(F74G)::URA3
Strain, strain background (S. cerevisiae)FZ175This studyYeast strainHOcse6::HPH
Rad53-AID*–9xMyc::KAN TIR1(F74G)::
URA3
Strain, strain background (S. cerevisiae)YSL53Lee et al., 1998Yeast strainHOcse5::URA3
Strain, strain background (S. cerevisiae)GEM188This studyYeast strainHOcse2::LYS2
Strain, strain background (S. cerevisiae)FZ201This studyYeast strainRad9-AID*–9xMyc::KAN pRAD9-AID*–9xMyc
Strain, strain background (S. cerevisiae)FZ155This studyYeast strainbfa1∆::KAN
Strain, strain background (S. cerevisiae)yMA11This studyYeast strainH2A-S129A H2B-T129A
Strain, strain background (S. cerevisiae)yMA12This studyYeast strainH2AS129E H2B-T129E
Strain, strain background (S. cerevisiae)yMA13This studyYeast strainH2B-T129A
Strain, strain background (S. cerevisiae)yMA14This studyYeast strainH2B-T129E
Strain, strain background (S. cerevisiae)yBL257This studyYeast strainH2A-S129E
Strain, strain background (S. cerevisiae)yBL259This studyYeast strainH2A-S129A
Strain, strain background (S. cerevisiae)FZ062This studyYeast strainMad1*–9xMyc-AID::NAT
Strain, strain background (S. cerevisiae)FZ165This studyYeast strainBfa1*–9xMyc-AID::NAT
Strain, strain background (S. cerevisiae)FZ167This studyYeast strainBub2*–9xMyc-AID::NAT
Sequence-based reagentGAT1p1BThis paperPCR primersGCTCAGTGTGCGTTATGCTT
Sequence-based reagentGAT1p2BThis paperPCR primersTTCAGGTCTCGGTTGCTCTT
Sequence-based reagentVE162 Ddc2-AID ForThis paperPCR primersATCTAACCACACTAGAGGAGGCCGATTCATTATATATCTCAATGGGACTGCCTAAAGATCCAGCCAAACCTCC
Sequence-based reagentVE163 Ddc2-AID RevThis paperPCR primersATTACAAGGTTTCTATAAAGCGTTGACATTTTCCCCTTTTGATTGTTGCCCAGTATAGCGACCAGCATTCACATAC
Sequence-based reagentDW217 Rad9-AID 1 FThis paperPCR primersGGTTTTCACGATGATATTACGGACAATGATATATACAACACTATTTCTGAGGTTAGACCTAAAGATCCAGCCAAACCTCC
Sequence-based reagentDW218 Rad9-AID 1 RThis paperPCR primersCTAAATTTTTTTTTATTTAATCGTCCCTTTCTATCAATTATGAGTTTATATATTTTTATAATTCAGTATAGCGACCAGCATTCACATAC
Sequence-based reagentDW208 Rad24-AID 1 FThis paperPCR primersCAGATTCAGATCTGGAAATACTCCCTAAAGATCCAGCCAAACCTCC
Sequence-based reagentDW209 Rad24-AID 1 RThis paperPCR primersGTGGAATATTTCCTGGGGTTTTCTCGTCAAATTTAAAGAGTAAAAAGCCTAAAGATCCAGCCAAACCTCC
Sequence-based reagentDW199 Rad53AID 1 FThis paperPCR primersGGTTAAAAGGGCAAAATTGGACCAAACCTCAAAAGGCCCCGAGAATTTGCAATTTTCGCCTAAAGATCCAGCCAAACCTCC
Sequence-based reagentDW200 Rad53AID 1 RThis paperPCR primersCCATCTTCTCTCTTAAAAAGGGGCAGCATTTTCTATGGGTATTTGTCCTTGGCAGTATAGCGACCAGCATTCACATAC
Recombinant DNA reagentpKan-9xMyc-AIDMorawska and Ulrich, 2013pJH2892Backbone: pSM409
Recombinant DNA reagentpNAT-9xMyc-AIDMorawska and Ulrich, 2013pJH2899Backbone: pSM409
Recombinant DNA reagentsTIR1::URA3Nishimura et al., 2009pNHK53
Recombinant DNA reagentGAL-DDC2Paciotti et al., 2000pML100Backbone: pML95
Recombinant DNA reagentADH1-OsTIR1(F74G)Yesbolatova et al., 2020pMK420
Recombinant DNA reagentbRA90Anand et al., 2017bRA90
Recombinant DNA reagentbG059This studybG059Backbone: bRA90
Recombinant DNA reagentbG060This studybG060Backbone: bRA90
Recombinant DNA reagentpRad9-3HALazzaro et al., 2008pFL36.1Backbone: pRS306
Recombinant DNA reagentpRad9-9xMyc-AIDThis studypFZ052Backbone: pRS306
Recombinant DNA reagentpBL15 – HTA1 gRNA1This studypBL15Backbone: BRA89
Recombinant DNA reagentpBL16 – HTA2 gRNA2This studypBL16Backbone: BRA89
Recombinant DNA reagentpKL004 – HTB1 gRNA1This studypKL004Backbone: BRA89
Recombinant DNA reagentpKL005 – HTB2 gRNA1This studypKL005Backbone: BRA89
Software, algorithmPrism 7.00GraphPad Software, Inc.N/A
Software, algorithmImage LabBio-RadN/A
Software, algorithmFiJiImageJN/A
Gene (S. cerevisiae)Ddc2Saccharomyces Genome DatabaseSystematic name
YDR499W
Gene (S. cerevisiae)Rad9Saccharomyces Genome DatabaseSystematic name
YDR217C
Gene (S. cerevisiae)Rad24Saccharomyces Genome DatabaseSystematic name
YER173W
Gene (S. cerevisiae)Rad53Saccharomyces Genome DatabaseSystematic name
YPL153C
Gene (S. cerevisiae)Chk1Saccharomyces Genome DatabaseSystematic name
YBR274W
Gene (S. cerevisiae)Dun1Saccharomyces Genome DatabaseSystematic name
YDL101C
Gene (S. cerevisiae)Tel1Saccharomyces Genome DatabaseSystematic name
YBL088C
Gene (S. cerevisiae)Mad2Saccharomyces Genome DatabaseSystematic name
YJL030W
Gene (S. cerevisiae)Mad1Saccharomyces Genome DatabaseSystematic name
YGL086W
Gene (S. cerevisiae)Bub2Saccharomyces Genome DatabaseSystematic name
YMR055C
Gene (S. cerevisiae)Bfa1Saccharomyces Genome DatabaseSystematic name
YJR053W

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