Dbf4p, an essential S phase-promoting factor, is targeted for degradation by the anaphase-promoting complex

Abstract

The Dbf4p/Cdc7p protein kinase is essential for the activation of replication origins during S phase. The catalytic subunit, Cdc7p, is present at constant levels throughout the cell cycle. In contrast, we show here that the levels of the regulatory subunit, Dbf4p, oscillate during the cell cycle. Dbf4p is absent from cells during G(1) and accumulates during the S and G(2) phases. Dbf4p is rapidly degraded at the time of chromosome segregation and remains highly unstable during pre-Start G(1) phase. The rapid degradation of Dbf4p during G(1) requires a functional anaphase-promoting complex (APC). Mutation of a sequence in the N terminus of Dbf4p which resembles the cyclin destruction box eliminates this APC-dependent degradation of Dbf4p. We suggest that the coupling of Dbf4p degradation to chromosome separation may play a redundant role in ensuring that prereplicative complexes, which assemble after chromosome segregation, do not immediately refire.

FIG. 1

Immunodetection of Dbf4p. (a) Detection of Dbf4p in whole-cell extracts. Extracts prepared from asynchronous populations of either W303-1a or the epitope-tagged derivative (yMIG07-DBF4myc) were probed with the 9E10 monoclonal antibody (lanes 1 and 2, respectively). In lanes 3 and 4, the same extracts were probed with polyclonal antibody to Dbf4p. Arrows indicate the positions of Dbf4p and Dbf4p-myc. The positions of nonspecific cross-reacting polypeptides are designated a, b, and c (asterisks). The relative levels of these background bands are variable due to differences in the times of incubation with primary antibody. (b) Dbf4p is absent from α factor-arrested cells. Cultures of cells harboring the epitope-tagged Dbf4p (DBF4myc) and the parental strain (W303-1a) were arrested in G₁ (by α factor), S (by hydroxyurea [α→HU]) and in G₂/M (by nocodazole [NOC]), and Dbf4p-myc was detected by immunoblotting with the 9E10 monoclonal antibody.

FIG. 2

Dbf4p is a phosphoprotein. Absence of Dbf4p from α factor-blocked cells is independent of the epitope tag. Cultures of W303-1a were arrested in G₁ (α factor) or G₂/M (Noc) and extracts were probed with the polyclonal antibody to Dbf4p. (b) Dbf4p is phosphorylated in nocodazole-blocked cells. Extracts from nocodazole-blocked cells were treated with potato acid phosphatase as described previously (51) in either the absence (lane 2) or presence (lane 3) of phosphatase inhibitor (50 mM NaF) prior to electrophoresis and immunoblotting with anti-Dbf4p polyclonal antibody. Background bands are designated as in Fig. 1.

FIG. 3

Kinetics of Dbf4p expression during the cell cycle. (a) Dbf4p levels after α factor block and release. Cells expressing Dbf4p-myc under its endogenous promoter (yMIG07) were grown to mid-log phase and blocked by α factor. The cells were harvested and washed several times to remove α factor and returned to fresh medium to allow cell cycle progression. Samples were taken every 10 min, and individual protein levels were determined by immunoblotting with the 9E10 monoclonal antibody (Dbf4p), 9H8/5 (Cdc6p), and anti-actin antibody. In addition, the Dbf4p blot was stained with amido black after autoradiography (21). The region between 66 and 116 kDa is shown. At each time point, the percentage of budded cells (□) and binucleate cells (⧫) was assessed by microscopy of 4′,6-diamidino-2-phenylindole (DAPI)-stained cells. Samples were taken and processed for fluorescence-activated cell sorting. (b) Dbf4p is absent from cells that have undergone anaphase. Cultures of cdc5^ts, cdc14^ts, and cdc15^ts mutants grown to logarithmic phase were arrested in G₁ (α) and in G₂/M (NOC), both at the permissive temperatures, and a third aliquot was arrested at the restrictive temperature (37°C). Dbf4p was detected in extracts by immunoblotting with polyclonal antibody against Dbf4p. Background bands are designated as in Fig. 1.

FIG. 4

Dbf4p is targeted for destruction by APC. (a) Dbf4p is present in cdc16^ts mutants arrested in G₁ at the permissive temperature. Cultures of W303-1a and cdc16^ts mutants grown at 24°C were arrested in G₁ (α) and in G₂/M (Noc). Dbf4p was detected by immunoblotting with polyclonal antibodies to Dbf4p. Background bands are designated as in Fig. 1. (b) Dbf4p degradation during G₁ requires the APC. W303-1a and cdc16^ts cell cultures harboring tagged Dbf4p under the GAL1-10 promoter were grown in galactose medium and arrested in G₁ with α factor or in G₂/M with nocodazole at the permissive temperature. The cells were then incubated at the restrictive temperature for 30 min, after which the promoter was repressed by harvesting the cells and releasing them into glucose medium containing cycloheximide. Cell aliquots were taken prior to changing the carbon source (0 min) and at different times points thereafter. Dbf4p levels were investigated on immunoblots with the 9E10 monoclonal antibody or the anti-polymerase α monoclonal antibody. The Dbf4p blot was stained with amido black as described in the legend to Fig. 3. The apparent distortion in the amido black pattern (Amido) is due to warping of the membrane during destaining (see Materials and Methods).

FIG. 5

Dbf4p possesses a destruction box sequence responsible for APC-dependent degradation in G₁. An experiment identical to that performed with wild-type Dbf4 in Fig. 4 was performed with wild-type Dbf4p (a) and the Dbf4p R62A L65A mutant (b).

FIG. 6

Dbf4p degradation in the cell cycle. The details of this model are discussed in the text. Dbf4p levels (solid black lines) decrease at the metaphase (Meta)-anaphase (Ana) transition as a result of APC activation. After this, the APC targets Clb2p (dotted black lines) for degradation, which allows pre-RCs (solid grey lines) to assemble. Passage through Start inactivates the APC and allows Dbf4 as well as the S phase-promoting Clb5 and Clb6 to reaccumulate. This triggers the activation of pre-RCs at individual origins during S phase.