G1 cyclin driven DNA replication

<p>The mitotic cell cycle is driven by Cyclin-Dependent Kinases (CDK). CDK activation requires the binding of activatory subunits termed cyclins. Different waves of cyclins are expressed during the cell cycle, enabling CDKs to trigger phase specific events. For instance, S phase cyclins promote the initiation of DNA replication but not chromosome segregation. There are at least 2 explanations for how such regulation is achieved. According to one of the visions, cyclins confer intrinsic substrate specificity to the CDK catalytic subunit. Alternatively a quantitative model has been proposed, according to which ever-increasing CDK activity is required to trigger cell cycle events from G1 to M. If a quantitative control prevails, then an early cyclin should trigger later cycle events if accumulated at high enough levels at the right time and place. We show here that a G1 phase cyclin bears the potential to trigger DNA replication and promote S and G2 phase specific transcription.</p

Neither unrestrained M-CDK activity alone nor lack of Pds1/securin alone are enough to abrogate the block of chromosome segregation in the presence of DNA damage.

<p>(A) Mutant <i>rad53 swe1</i> cells remain competent to block the segregation of damaged chromosomes. Percentage of cells showing segregated masses of DNA. Wild type (WT, strain YGP20), <i>rad53 swe1</i> (strain YGP121), <i>rad53</i> (strain YGP38), and <i>swe1</i> (strain YGP98) cells were grown to mid-exponential phase (Log), synchronized in G1 phase with the pheromone alpha-factor (G1), then released into S phase in the presence of 0.033% MMS. Cells were collected at the indicated times (min). Fixed cells were stained with DAPI to visualize DNA by fluorescence microscopy. 120 cells were counted in each of 3 independent experiments. Data are represented as mean ± SD (error bars). (B) Representative cells 240 minutes after the release from G1. (C-D) The absence of Pds1/securin is not sufficient to allow chromosome segregation in the presence of DNA damage in S phase. Wild type cells (WT, YGP20) and null <i>pds1</i> cells (strain YRP33) were treated and analyzed as in (A-B).</p

Three different pathways prevent chromosome segregation in the presence of genotoxic stress.

<p>Molecular diagram showing the three pathways that block Mitotic Cyclin Dependent Kinase activity and anaphase in response to genotoxic stress. Swe1 and the S phase checkpoint effector kinase Rad53 are individually sufficient to block M-CDK activity. Our results place Swe1 as a downstream effector of the S phase checkpoint. M-CDK inhibition and Pds1/securin stabilization are individually sufficient to prevent anaphase. Only when the three pathways are disrupted cells fail to block the segregation of damaged or incompletely replicated chromosomes. In grey, regulatory pathways taken from previous works [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005468#pgen.1005468.ref023" target="_blank">23</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005468#pgen.1005468.ref028" target="_blank">28</a>]. The Spindle Assembly Checkpoint (SAC) is included in the model as it may also play a role in Pds1 stabilization [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005468#pgen.1005468.ref076" target="_blank">76</a>].</p

Three Different Pathways Prevent Chromosome Segregation in the Presence of DNA Damage or Replication Stress in Budding Yeast

<div><p>A surveillance mechanism, the S phase checkpoint, blocks progression into mitosis in response to DNA damage and replication stress. Segregation of damaged or incompletely replicated chromosomes results in genomic instability. In humans, the S phase checkpoint has been shown to constitute an anti-cancer barrier. Inhibition of mitotic cyclin dependent kinase (M-CDK) activity by Wee1 kinases is critical to block mitosis in some organisms. However, such mechanism is dispensable in the response to genotoxic stress in the model eukaryotic organism <i>Saccharomyces cerevisiae</i>. We show here that the Wee1 ortholog Swe1 does indeed inhibit M-CDK activity and chromosome segregation in response to genotoxic insults. Swe1 dispensability in budding yeast is the result of a redundant control of M-CDK activity by the checkpoint kinase Rad53. In addition, our results indicate that Swe1 is an effector of the checkpoint central kinase Mec1. When checkpoint control on M-CDK and on Pds1/securin stabilization are abrogated, cells undergo aberrant chromosome segregation.</p></div

Swe1 and Cdk1 phosphorylation at Tyr19 are required for the regulation of M-CDK activity in response to replication stress.

<p>Cells were grown to mid-exponential phase (Log), synchronized in G1 phase with the pheromone alpha-factor (G1), then released into S phase in the presence of 200 mM hydroxyurea (HU). Cells were collected at the indicated times (min). Whole cell extracts were immunoblotted against Pol12, Swe1 or with antibodies that specifically recognize Cdk1 phosphorylated at tyrosine 19 (pY19-Cdk1), as indicated. Ponceau S-stained regions of the same membranes used for Western blotting are shown as a loading control. Budding indexes (BI %) of the cell cultures are shown as a measure of synchronicity and cell cycle progression. (A) A Swe1-AQ allele mimics the <i>swe1</i> deletion when combined with the <i>rad53</i> mutation (strain YRP100). A <i>rad53</i> mutant strain (YGP117) is used as a control of wild type Swe1. Cell density and flow cytometry analysis of DNA content show that cells released in the presence of 200 mM hydroxyurea fail to replicate and divide despite they bud normally. (B) A non-phosphorylatable Cdk1-19F allele mimics the <i>swe1</i> deletion when combined with the <i>rad53</i> mutation (strain YRP48) but fails to abrogate M-CDK inhibition on its own (strain YRP70).</p

Mutant <i>rad53 swe1 pds1</i> cells elongate spindles in the presence of DNA damage.

<p>Cells were grown to mid-exponential phase, synchronized in G1 phase with the pheromone alpha-factor, then released into S phase in the presence of 0.033% MMS. Results correspond to cells 240 minutes after the release from G1. (A) Spindle lengths were measured in wild type (WT, strain YGP20), <i>swe1</i> (YGP98), <i>pds1</i> (strain YRP33), <i>rad53 swe1</i> (YGP121), <i>rad53 pds1</i> (strain YGP208), and <i>rad53 swe1 pds1</i> (strain YGP201) cells. Cells were fixed, probed with anti-tubulin antibody, to visualize spindles, and stained with Hoechst 33258, to visualize DNA by fluorescence microscopy. Spindle length in 200 cells for each strain were measured and represented as box-and-whisker plots. (B) Representative cells obtained by double fluorescence with wild type (WT, strain YRP117), <i>swe1</i> (YRP118), <i>pds1</i> (strain YRP159), <i>rad53 swe1</i> (YRP165), <i>rad53 pds1</i> (strain YRP164), and <i>rad53 swe1 pds1</i> (strain YRP144) cells. Spindles (Tub1-GFP) and chromatin (Htb2-mCherry) and were visualized by fluorescence microscopy.</p

M-CDK activity is inhibited in response to replication stress in a Mec1 dependent manner.

<p>(A) Pol12 phosphorylation is inhibited in response to replication stress. Wild type cells (strain YGP20) were grown to mid-exponential phase (Log), synchronized in G1 phase with the pheromone alpha-factor (G1), then released into S phase in the absence (YPD) or in the presence of 200 mM hydroxyurea (HU). Cells were collected at the indicated times (min). Whole cell extracts were immunoblotted against Pol12 and Clb2. A Ponceau S-stained region of the same membrane used for Western blotting is shown as a loading control. Budding indexes (BI %) and cell density of the culture are shown as a measure of synchronicity and cell cycle progression. Cells in rich medium (YPD) are able to divide upon completion mitosis, as assessed by the increase in cell density. Cells in the presence of replication stress bud normally but fail to replicate, as assessed by flow cytometry analysis of DNA content. (B) Rad53 is dispensable to inhibit Pol12 phosphorylation in response to replication stress. Null <i>rad53</i> cells (strain YGP24) were treated and analyzed as in (A). (C) Mec1 dependent inhibition of Pol12 phosphorylation in response to replication stress. Null <i>mec1</i> cells (strain YGP123) were treated and analyzed as in (A).</p