A Switch in p53 Dynamics Marks Cells That Escape from DSB-Induced Cell Cycle Arrest

© 2020 The Author(s) Cellular responses to stimuli can evolve over time, resulting in distinct early and late phases in response to a single signal. DNA damage induces a complex response that is largely orchestrated by the transcription factor p53, whose dynamics influence whether a damaged cell will arrest and repair the damage or will initiate cell death. How p53 responses and cellular outcomes evolve in the presence of continuous DNA damage remains unknown. Here, we have found that a subset of cells switches from oscillating to sustained p53 dynamics several days after undergoing damage. The switch results from cell cycle progression in the presence of damaged DNA, which activates the caspase-2-PIDDosome, a complex that stabilizes p53 by inactivating its negative regulator MDM2. This work defines a molecular pathway that is activated if the canonical checkpoints fail to halt mitosis in the presence of damaged DNA

A Cohesin-Based Partitioning Mechanism Revealed upon Transcriptional Inactivation of Centromere

<div><p>Transcriptional inactivation of the budding yeast centromere has been a widely used tool in studies of chromosome segregation and aneuploidy. In haploid cells when an essential chromosome contains a single conditionally inactivated centromere (<i>GAL-CEN</i>), cell growth rate is slowed and segregation fidelity is reduced; but colony formation is nearly 100%. Pedigree analysis revealed that only 30% of the time both mother and daughter cell inherit the <i>GAL-CEN</i> chromosome. The reduced segregation capacity of the <i>GAL-CEN</i> chromosome is further compromised upon reduction of pericentric cohesin (<i>mcm21∆</i>), as reflected in a further diminishment of the Mif2 kinetochore protein at <i>GAL-CEN</i>. By redistributing cohesin from the nucleolus to the pericentromere (by deleting <i>SIR2</i>), there is increased presence of the kinetochore protein Mif2 at <i>GAL-CEN</i> and restoration of cell viability. These studies identify the ability of cohesin to promote chromosome segregation via kinetochore assembly, in a situation where the centromere has been severely compromised.</p></div

Cohesin concentration in the pericentric region in <i>GAL-CEN3</i>, <i>mcm21Δ</i> and <i>mcm21Δ</i>, <i>sir2Δ</i> mutants.

<p>A. Cells containing <i>GAL-CEN3</i> as the only centromere in chromosome 3 were transferred from lactose to galactose and grown on galactose for over 6 hours to inactivate the centromere. Chromatin immunoprecipitation was performed as described in Snider et al., (2014) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006021#pgen.1006021.ref027" target="_blank">27</a>], using a ChIP grade antibody against GFP to immunoprecipitate the only copy of Smc3 fused to GFP at the C-terminus of Smc3 in the genome. Oligonucleotide primers against <i>CEN3</i> (114,800), 8 kb (Stp22, position 105696..106853), 10 kb (Ilv6, position 104619..105548) and 87 kb (kar4 27929..28936) were utilized. Primers were designed to amplify a 600 bp fragment for each of the 4 reactions. Titrations of template were performed to ensure the analysis was in the linear range of amplification. For <i>GAL-CEN3</i> glucose, the integrated intensity ranged from 5.5 x 10^6 to 6.1 x 10^6 (indicated by glucose text inset in graph)(n = 4). The average was 5.78 x 10^6 ± 2.5 x 10^5 (STD). For GAL-CEN3 galactose, integrated intensity ranged from 1.2 x 10^6 to 3.3 x 10^6 (indicated by galactose text inset in graph)(n = 4). The average was 2.33 x 10^6 ± 8.3 x 10^5 (STD). For <i>STP22</i> (8 kb), <i>ILV6</i> (10 kb) and <i>KAR4</i> (87 kb) the galactose and glucose products ranged from 2.4 x 10^5 to 5.6 x 10^5 (n = 3). There was no significant difference between glucose and galactose grown samples for the 8, 10 or 87 kb products. B. The concentration of Smc3-GFP was determined in <i>GAL-CEN3</i> WT, <i>mcm21</i>∆ and <i>mcm21</i>∆ <i>sir2</i>∆ mutants. The concentration of pericentric cohesin is reduced in <i>mcm21</i>∆ cells (from 30,540 to 18,509 arbitrary fluorescence units). In the double mutant, <i>mcm21</i>∆ <i>sir2</i>∆, the concentration of cohesin in the pericentromere is increased to almost wild-type levels (29,848 vs 30,540). C. Representative images of Smc3-GFP in <i>GAL-CEN3</i> WT (left), <i>mcm21</i>∆ (middle) and <i>mcm21</i>∆ <i>sir2</i>∆ (right). Spindle poles are visualized using Spc29-RFP, cohesin with Smc3-GFP. The rightmost image in each strain is an overlay of the spindle poles with Smc3-GFP. White arrows indicate the cohesin barrel concentrated in the pericentric chromatin between the spindle poles (red). Note the absence of the cohesin barrel in <i>mcm21Δ</i>.</p

Pericentric sister chromatid separation in the presence or absence of tethered Sir2.

<p>The percent of separated sister chromatids in cells containing LexA/LacO binding sites proximal to the endogenous centromere on Chr 3 either with (right) or without (left) plasmid expressing Sir2-LexA fusion protein. Top panel: Chromosomes with intact <i>CEN3</i>. Bottom panel: Chromosomes with excised <i>CEN3</i>. Representative images (green lacI-GFP, red spindle poles Spc29-RFP) of cells in mitosis. Scale bar 1 micron.</p

Centromere-linked LacO position in wildtype and anaphase cells.

<p>GAL-CEN3 proximal lacO arrays in metaphase (A) (n = 84 to 92 cells) and anaphase (B) (n = 34 to 51 cells) cells. Cells were grown on galactose for 3h prior to image analysis. The fraction of replicated spots that appeared as one or two foci between the spindle poles and along the spindle axis (on axis), versus the fraction of replicated spots that appeared as one or two foci displaced from the spindle axis was determined. Representative images are shown to the right. In wildtype cells with endogenous CEN3 as the sole centromere in Chr 3, the lacO array appeared as a single focus (30%) or separated foci (70%) on the spindle axis of metaphase cells. In mcm21Δ cells with GAL-CEN3 as the sole centromere in Chr 3, the distribution of GAL-CEN3 lacO arrays was comparable to cells with GAL-CEN3 on glucose (metaphase 2 spots on axis 62%, one spot on axis 30%, two spots off axis 2%, one spot off axis 7%; anaphase on axis 2 spots 100%, (metaphase n = 60, anaphase n = 22). In mcm21Δ sir2Δ cells with GAL-CEN3 as the sole centromere in Chr 3, the distribution of GAL-CEN3 lacO arrays was comparable to cells with GAL-CEN3 on glucose (metaphase 2 spots on axis 57%, one spot on axis 34%, two spots off axis 1%, one spot off axis 7%; anaphase on axis 2 spots 100%, (metaphase n = 82, anaphase n = 25). The fraction of cells with two spots in late anaphase is shown (bottom panel). In wild type cells with endogenous CEN3 as the sole centromere in Chr 3, the lacO array appeared as two foci, one at each pole of the anaphase spindle in 100% of cells. In mcm21Δ cells with GAL-CEN3, 100% of spots in late anaphase appeared in mother and daughter cells on glucose (n = 22). In mcm21Δ sir2Δ cells with GAL-CEN3, 100% of spots in late anaphase appeared in mother and daughter cells on glucose (n = 25). Panel C indicates the percent of cells from each sample in which lacO arrays segregate to mother and daughter in late anaphase. 17 and 12% of GAL-CEN3 WT (n = 18 cells) and mcm2∆ sir2∆ mutants (n = 26 cells) contain two foci segregated to mother and daughter, while only 5% of mcm21∆ cells (n = 18 cells) exhibit this phenotype.</p

Pedigree analysis of <i>mcm21Δ</i>.

<p>G1 cells (<i>mcm21</i>Δ <i>GAL-CEN3</i>) were micromanipulated on YEP-galactose plates and after the first division mothers and daughters were separated and allowed to grow approximately 24 h. Images of microcolonies were photographed. A. Both mother and daughter cells divided multiple times. B. Only mother cells divided multiple times. C. Only mother cell divided and grew into an observable colony. D. Neither mother nor daughter progressed beyond 2–3 divisions.</p

Strain list.

<p>Strain list.</p