Cyclin and cyclin-dependent kinase substrate requirements for preventing rereplication reveal the need for concomitant activation and inhibition

Abstract

DNA replication initiation in S. cerevisiae is promoted by B-type cyclin-dependent kinase (Cdk) activity. In addition, once-per-cell-cycle replication is enforced by cyclin-Cdk-dependent phosphorylation of the prereplicative complex (pre-RC) components Mcm2-7, Cdc6, and Orc1-6. Several of these controls must be simultaneously blocked by mutation to obtain rereplication. We looked for but did not obtain strong evidence for cyclin specificity in the use of different mechanisms to control rereplication: both the S-phase cyclin Clb5 and the mitotic cyclins Clb1-4 were inferred to be capable of imposing ORC-based and MCM-based controls. We found evidence that the S-phase cyclin Clb6 could promote initiation of replication without blocking reinitiation, and this activity was highly toxic when the ability of other cyclins to block reinitiation was prevented by mutation. The failure of Clb6 to regulate reinitiation was due to rapid Clb6 proteolysis, since this toxic activity of Clb6 was lost when Clb6 was stabilized by mutation. Clb6-dependent toxicity is also relieved when early accumulation of mitotic cyclins is allowed to impose rereplication controls. Cell-cycle timing of rereplication control is crucial: sufficient rereplication block activity must be available as soon as firing begins. DNA rereplication induces DNA damage, and when rereplication controls are compromised, the DNA damage checkpoint factors Mre11 and Rad17 provide additional mechanisms that maintain viability and also prevent further rereplication, and this probably contributes to genome stability.

Figure 1.—

The survival of cells with disrupted rereplication controls depends on cell-cycle regulators. (A) ORC6-rxl GAL-CDC6ΔNT-HAs cells (top left) rely on SIC1 for their limited survival when GAL-CDC6ΔNT-HAs is induced by galactose. Ten-fold serial dilutions on galactose-containing or glucose-containing plates (YEP-G or YEP-D) were performed. Deletion of SIC1 in ORC6-ps,rxl GAL-CDC6ΔNT-HAm caused synthetic lethality. The lethality was rescued by deleting both CLB5 and CLB6 (top right). The viability of clb5 GAL-CDC6ΔNT-HAs cells (bottom) is rescued by deletion of CLB6 or CDH1 on YEP-G. (B) Viability of various strains with disruptions of mechanisms preventing rereplication in the presence or absence of S-phase cyclins. All strains contain GAL-CDC6ΔNT-HAm and DDC2-GFP (analyzed in Figure 2). Cell viability was tested as in A.

Figure 2.—

Induction of DNA rereplication and recruitment of Ddc2-GFP foci. (A) Cells were grown in YEP-D and transferred to YEP-R for 8 hr. Galactose at the final concentration of 3% was added for 4 hr. Cells were fixed and DNA content was measured by DNA flow cytometry analysis. The approximate position of 2C DNA content was determined on the basis of the major 2C peak in the control (1; GAL-CDC6ΔNT-HAm strain). This position is indicated by a bar. All samples were processed in parallel, and data were collected using the same set up (voltage 780 and total cell number 20,000 cells) and gating. As a rough empirical guide, we consider the DNA flow cytometry profile to represent over-replication if the main peak is shifted rightward compared to the bar. Each histogram is numbered on the basis of the strain numbering in Figure 1B. (B) The same strains, numbered as in A, were induced with galactose for 4 hr, and cells were observed under a DeltaVision microscope. Image stacks were deconvolved and processed at maximum projection. (C) Percentages of the cells showing DDC2-GFP foci in the presence of glucose or galactose. At least 100 cells were counted for each experiment, and three experiments were repeated. Average number and standard deviation were plotted. Open bars show percentages of DDC2-GFP foci when incubated in glucose, and shaded bars show the same when incubated in galactose.

Figure 3.—

Cdh1 can promote inviability and rereplication in the absence of specific replication control mechanisms. (A) Serial dilutions as in Figure 1 for clb5 GAL-CDC6ΔNT-HAm ORC6-ps,rxl and clb5 GAL-CDC6ΔNT-HAm MCM7-NLS, with and without CDH1. (B) DNA flow cytometry analysis performed for selected strains after GAL-CDC6ΔNT-HAm induction, as in Figure 2.

Figure 4.—

Stabilized Clb6 reverses loss of viability and replication control due to absence of Clb5. (A) Serial dilutions for MCM7-NLS clb5 GAL-CDC6ΔNT-HAm with CLB6Δ3P [gene replacement, encoding Clb6 stabilized due to mutation of phosphorylation sites (Jackson et al. 2006)] or CLB6-wt. (B) DNA content was measured by DNA flow cytometry analysis, as in Figure 2A. There is a minor peak at 4C DNA content in the CLB6Δ3P MCM7-NLS clb5 GAL-CDC6ΔNT-HAm strain. Microscopically, we did not detect significant clumping or division problems in this strain (data not shown). At present we do not know the explanation of this peak.

Figure 5.—

Requirement for DNA damage response genes MRE11 and RAD17 in rereplicating cells. (A) Cell viability was tested in the presence or absence of MRE11 or RAD17 in cells of the indicated genotypes, as in Figure 1. (B–D) DNA content was analyzed by DNA flow cytometry after galactose induction as in Figure 2. (E) Cells were grown in YEP-D and transferred to YEP-R for 8 hr. Galactose at the final concentration of 3% was added. The samples were collected every hour after the galactose induction and were subjected to serial dilution experiments. The serial dilution was performed on YEP-D plates after the incubation in YEP-G media. As a control, cells were plated on YEP-G after the incubation in YEP-D media. Genotypes are as indicated.