Cyclin regulation by the s phase checkpoint

Abstract

In eukaryotic cells a surveillance mechanism, the S phase checkpoint, detects and responds to DNA damage and replication stress, protecting DNA replication and arresting cell cycle progression. We show here that the S phase cyclins Clb5 and Clb6 are regulated in response to genotoxic stress in the budding yeast Saccharomyces cerevisiae. Clb5 and Clb6 are responsible for the activation of the specific Cdc28 cyclin-dependent kinase activity that drives the onset and progression of the S phase. Intriguingly, Clb5 and Clb6 are regulated by different mechanisms. Thus, the presence of Clb6, which is eliminated early in an unperturbed S phase, is stabilized when replication is compromised by replication stress or DNA damage. Such stabilization depends on the checkpoint kinases Mec1 and Rad53. The stabilization of Clb6 levels is a dynamic process that requires continued de novo protein synthesis, because the cyclin remains subject to degradation. It also requires the activity of the G(1) transcription factor Mlu1 cell cycle box-binding factor (MBF) in the S phase, whereas Dun1, the checkpoint kinase characteristically responsible for the transcriptional response to genotoxic stress, is dispensable in this case. On the other hand, two subpopulations of endogenous Clb5 can be distinguished according to turnover in an unperturbed S phase. In the presence of replication stress, the unstable Clb5 pool is stabilized, and such stabilization requires neither MBF transcriptional activity nor de novo protein synthesis.

FIGURE 1.

Clb6 is directly phosphorylated by Rad53 in vitro. GST-Rad53 and GST-Clb6 were separately expressed in Escherichia coli and purified by affinity chromatography using glutathione-Sepharose beads. The GST moieties were removed by incubation with PreScission protease. Hyperphosphorylated, active Rad53 (71, 72) was incubated with Clb6 in the presence of 100 μm ATP and 5 μCi of [γ-³²P]ATP. The samples were resolved by SDS-PAGE and Coomassie Blue-stained (Coomassie), and the phosphorylated proteins were detected by autoradiography (³²P). The positions of the molecular mass markers and the approximate sizes of Rad53 and Clb6 are indicated. The additional bands visible in the Coomassie stain correspond to E. coli proteins that co-elute either with GST-Rad53 or with GST-CLB6. Significantly, none of the contaminant proteins is phosphorylated by Rad53 in the assay.

FIGURE 2.

Clb6 is stabilized in response to replication stress. A log phase culture (strain YGP17) was synchronized with α-factor (G1). The cells were then released from the G₁ arrest by several washes with medium lacking α-factor and allowed to progress into the S phase either in the absence (YPD) or in the presence of HU. The samples were taken at the indicated time points. Upper panels, fluorescence-activated cell sorting analysis of DNA content. Unreplicated (1C) and replicated (2C) haploid DNA contents are indicated. Budding indexes (BI) are shown as measures of synchronicity and cell cycle progression and to confirm that cells exposed to replication stress progress into the cell cycle despite the absence of DNA replication. Lower panels, whole cell extracts were analyzed by immunoblot with anti-Myc (Clb6) or anti-Rad53 (Rad53) antibodies. A Ponceau S-stained region of the same membrane used for immunoblot is shown as a loading control.

FIGURE 3.

The stabilization of Clb6 in response to replication stress depends on Rad53. A, the presence of Clb6 correlates with the active form of Rad53. A log phase culture (strain YGP17) was synchronized with α-factor (G1). The cells were then released from the G₁ arrest by several washes with medium lacking α-factor and allowed to progress into the S phase in the presence of hydroxyurea (1h HU). After 1 h of incubation, HU was washed away, and the cells were allowed to recover from replication stress in rich medium (YPD). The samples were taken at the indicated time points. B, rad53 mutants fail to stabilize Clb6 in the presence of replication stress. The same experiment described in the legend to Fig. 2 was carried out with rad53-21 mutant (73) cells (strain YGP37). Upper panels, fluorescence-activated cell sorting analysis of DNA content. Unreplicated (1C) and replicated (2C) haploid DNA contents are indicated. Budding indexes (BI) are shown as measures of synchronicity and cell cycle progression and to confirm that cells exposed to replication stress progress into the cell cycle despite the absence of DNA replication. Lower panels, whole cell extracts were analyzed by immunoblot with anti-Myc (Clb6) antibodies or anti-Rad53 (Rad53) antibodies where indicated. A Ponceau S-stained region of the same membrane used for immunoblot is shown as a loading control.

FIGURE 4.

Clb6 stabilization requires continued de novo protein synthesis. A log phase culture (strain YGP17) was synchronized with α-factor (G1). The cells were then released from the G₁ arrest by several washes with medium lacking α-factor and allowed to progress into the S phase in the presence of hydroxyurea (1h HU). The culture was then split in two. One half was further incubated under the same conditions (HU) (A), whereas cycloheximide was added to the other half (B), keeping the presence of hydroxyurea (HU + CHX). The samples were taken at the indicated time points, and whole cell extracts were analyzed by immunoblot with anti-Myc antibodies (Clb6). A Ponceau S-stained region of the same membrane used for immunoblot is shown as a loading control. Budding indexes (BI) are shown on top as measures of synchronicity and cell cycle progression and to confirm that cells progress into the cell cycle despite the absence of DNA replication or protein synthesis.

FIGURE 5.

Cells retain the ability to restore the presence of Clb6 for as long as replication stress persists. A log phase culture (strain YGP17) was synchronized with α-factor (G1). The cells were then released from the G₁ arrest by several washes with medium lacking α-factor and allowed to progress into the S phase in the presence of hydroxyurea (1h HU). After 1 h of incubation cycloheximide was added, keeping the presence of hydroxyurea (CHX + HU). After 30 min the cycloheximide was washed away while keeping the HU to allow protein synthesis to resume in the presence of replication stress. The samples were taken at the indicated time points, and the whole cell extracts were analyzed by immunoblot with anti-Myc antibodies (Clb6). A Ponceau S-stained region of the same membrane used for immunoblot is shown as a loading control. Budding indexes (BI) are shown on top as measures of synchronicity and cell cycle progression and to confirm that cells progress into the cell cycle despite the absence of DNA replication or protein synthesis.

FIGURE 6.

CLB6 mRNA is stabilized in response to replication stress. A log phase culture (strain YGP20) was synchronized with α-factor (G1). The cells were then released from the G₁ arrest by several washes with medium lacking α-factor and allowed to progress into the S phase either in the absence (YPD) or in the presence of HU. The samples were taken at the indicated time points. CLB6 mRNA levels were quantified by quantitative reverse transcription-PCR, using ACT1 mRNA as reference. The results are expressed as mean fold changes relative to α-factor arrested pre-Start cells (G1). The error bars are the standard deviations of three independent preparations. The results were checked to be reproducible in two independent time course sets of samples.

FIGURE 7.

MBF function is required for the stabilization of CLB6 levels in response to replication stress. Log phase cultures of GAL-NRM1ΔN (strain YGP70) or control cells (strain YGP27) grown in YP raffinose were synchronized with α-factor (G1). The cells were then released from the G₁ arrest by several washes with medium lacking α-factor and allowed to progress into the S phase in the presence of hydroxyurea (1h HU). After 1 h of incubation, galactose was added to a final concentration of 0.05% while keeping the presence of hydroxyurea (HU + GAL). Upper panel, fluorescence-activated cell sorting analysis of DNA content. Unreplicated (1C) and replicated (2C) haploid DNA contents are indicated. Budding indexes (BI) are shown as measures of synchronicity and cell cycle progression and to confirm that cells exposed to replication stress progress into the cell cycle despite the absence of DNA replication. Lower panel, whole cell extracts were analyzed by immunoblot with anti-Myc (Clb6 and Nrm1ΔN) or anti-Clb5 (Clb5) antibodies. A Ponceau S-stained region of the same membrane used for immunoblot is shown as a loading control.

FIGURE 8.

Persistent nuclear localization of Swi6 in a compromised S phase. A log phase culture of Swi6–13Myc cells (strain YGP39) was synchronized in G₁ phase with α-factor. The cells were then released from the G₁ arrest by several washes with medium lacking α-factor and allowed to progress into the S phase either in the presence of hydroxyurea (S-phase + HU) or in the absence of hydroxyurea (Unperturbed S-phase). The samples were taken after a 60-min incubation in HU or 30 min after the release into an unperturbed S phase. Epifluorescence microscopy was used to detect the immunofluorescence Swi6 signal and the 4′,6-diamidino-2-phenylindole-stained DNA signal. Cell morphology was visualized by Nomarski contrast microscopy.

FIGURE 9.

Dun1 is not required for the transcriptional stabilization of Clb6 levels. The same experiment described in the legend to Fig. 2 was carried out with a dun1Δ mutant (strain YGP54). Upper panel, fluorescence-activated cell sorting analysis of DNA content. Unreplicated (1C) and replicated (2C) haploid DNA contents are indicated. Budding indexes (BI) are shown as measures of synchronicity and cell cycle progression and to confirm that cells exposed to replication stress progress into the cell cycle despite the absence of DNA replication. Lower panel, whole cell extracts were analyzed by immunoblot with anti-Myc (Clb6) antibodies or anti-Rad53 (Rad53) antibodies. A Ponceau S-stained region of the same membrane used for immunoblot is shown as a loading control.

FIGURE 10.

An unstable pool of Clb5 is stabilized in response to replication stress. A log phase culture (strain YGP23) was synchronized with α-factor and released under the conditions described below. The samples were taken at the indicated time points. Whole cell extracts were analyzed by immunoblot with anti-Clb5 antibodies (Clb5) or anti-Rad53 (Rad53) antibodies where indicated. A Ponceau S-stained region of the same membranes used for immunoblot is shown as a loading control. Budding indexes (BI) are shown on top as measures of synchronicity and cell cycle progression and to confirm that cells progress into the cell cycle despite the absence of DNA replication or protein synthesis. A, stable Clb5 levels in an unperturbed and in a compromised S phase. The cells were released from the G₁ arrest by several washes with medium lacking α-factor and allowed to progress into the S phase either in the absence (YPD) or in the presence of HU. B, two pools of Clb5 can be distinguished according to turnover in an unperturbed S phase. The cells were released from the G₁ arrest by several washes with medium lacking α-factor and allowed to progress into an unperturbed S phase (S-phase). 30 min later cycloheximide was added to block protein synthesis (CHX). C, the whole Clb5 population is stabilized in response to replication stress. The cells were released from the G₁ arrest by several washes with medium lacking α-factor and allowed to progress into the S phase in the presence of hydroxyurea (S-phase + HU). 60 min later cycloheximide was added to block protein synthesis while keeping the presence of hydroxyurea (HU + CHX).

FIGURE 11.

CLB5 mRNA persists in an unperturbed S phase and is not increased in the presence of replication stress. The samples in Fig. 6 were quantified for CLB5 mRNA levels (see details in Fig. 6).