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Results: 7

1.
Figure 1

Figure 1. The N-terminus of Dbf4p interacts with the Polo PBD.. From: Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase.

Cells co-transformed with various bait GAL4 DNA binding domain fusions (GBD-DBF4) and prey GAL4 activation domain fusions (GAD-CDC5) constructs were spotted at 10-fold serial dilutions on SCM–Trp –Leu and SCM–Trp–Leu–His plates containing 2 mM 3AT (A, C) or 0.5 mM 3AT (B) and incubated at 30°C for 2 to 3 days. The GBD-Dbf4 protein levels are shown in Figure S1.

Charles T. Miller, et al. PLoS Genet. 2009 May;5(5):e1000498.
2.
Figure 4

Figure 4. Dbf4p is a Polo inhibitor.. From: Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase.

(A) cdc5-1 and mcm2-1 strains expressing the indicated Dbf4 N-terminal regions from the GAL1 promoter were plated onto glucose (repressing conditions) or galactose to induce Dbf4p expression. (B) Dbf4p Western blot of total cell extracts induced for GAL1-DBF4 expression at 0, 3, and 6 hours. Asterisks indicate N-terminal Dbf4 peptides. (C) High copy vectors expressing wild type DBF4 and mutant genes were transformed into cdc5-1 (M1614) and spotted at 10-fold serial dilutions at the indicated temperatures.

Charles T. Miller, et al. PLoS Genet. 2009 May;5(5):e1000498.
3.
Figure 2

Figure 2. Dbf4p residues 67–109 interact directly with the Polo PBD.. From: Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase.

(A) HA-Cdc7p-Dbf4p kinase or HA-Cdc7p plus Dbf4p truncation mutants were expressed in Sf9 cells together with Polo and immunoprecipitated using 12CA5 antibody. Blots were probed with anti-Myc (Cdc5p), anti-Cdc7p and anti-Dbf4p polyclonal antibodies. (B) Endogenous HA3-Cdc7p-Dbf4p complexes were immunoprecipitated using 12CA5 antibody from HA3-CDC7 DBF4 CDC5-Myc15 (M2741) and HA3-CDC7 dbf4-NΔ109 CDC5-Myc15 (M2743) yeast strains following nocodazole arrest and probed for Cdc7, Dbf4, and Cdc5-Myc. (C) Purified Sumo and Sumo-Dbf467–109 proteins were co-incubated with purified GST-PBD or GST alone; proteins were pulled down using glutathione-Sepharose beads and blotted with antibodies against GST or Sumo. (D) In vitro phosphorylation of GST-PBD using purified Cdc7p-Dbf4p kinase.

Charles T. Miller, et al. PLoS Genet. 2009 May;5(5):e1000498.
4.
Figure 6

Figure 6. DBF4 regulates mitotic exit independently of BFA1-BUB2.. From: Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase.

(A) Deletion of the DBF4 polo-box interacting region does not cause rebudding. W303-1A, M1652, M1656 and M1860 were synchronized with alpha-factor and released into YPD containing 15 ug/ml of nocodazole at 30°C. Samples were quantitated for the percentage of rebudded cells (large budded cells with a new bud). (B) Deletion of BUB2 and the DBF4 PIR cooperate to suppress the growth defect of cdc5-2. Strains of the indicated genotypes were spotted at 5-fold serial dilutions on YPD and grown at increasing temperature. Two isolates are shown for the cdc5-2 recombinants.

Charles T. Miller, et al. PLoS Genet. 2009 May;5(5):e1000498.
5.
Figure 3

Figure 3. The Dbf4p N-terminus inhibits Polo activity.. From: Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase.

(A) dbf4-NΔ109 rescues the cdc5-1 temperature sensitivity. Indicated strains were spotted at ten-fold serial dilution on YPD and grown at increasing temperatures. (B) Cell cycle progression of wild type and dbf4-NΔ109 at 30°C by flow cytometry of alpha-factor arrested (t = 0) and released cells (t = 10 to 180 mins). (C) The indicated diploid strains were spotted at increasing temperatures (D) DBF4 cdc5-1 was transformed with the indicated ARS CEN plasmids and spotted at 25°C and 32°C (E) Representative tetrads from a dbf4-NΔ65 (M2007) cross to cdc5-1 (M1614).

Charles T. Miller, et al. PLoS Genet. 2009 May;5(5):e1000498.
6.
Figure 5

Figure 5. DBF4 inhibits Cdc14p nucleolar release and the MEN pathway.. From: Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase.

(A) Deletion of the Dbf4p PIR rescues Cdc14p nucleolar release at high temperature. M1992, M2005, M2139 and M2287 were arrested with alpha-factor at 25°C and released into YPD at 34°C followed by alpha-factor re-addition at 60 minutes. Entry and exit from the cell cycle was analyzed as a percentage of budded cells. Cdc14-EGFP release was analyzed in at least 100 cells at each time point and is reported as a percentage of cells with diffuse Cdc14p-EGFP nuclear staining (absence of nucleolar Cdc14p). (B) dbf4-NΔ109 rescues the growth defect of the cdc5-1 and dbf2-1 strains at 35°C. Single and double mutant strains were spotted at 10-fold dilutions on YPD and incubated at increasing temperatures for 2 to 3 days.

Charles T. Miller, et al. PLoS Genet. 2009 May;5(5):e1000498.
7.
Figure 7

Figure 7. Deletion of the DBF4 PIR allows exit from mitosis in cells with misoriented nuclei.. From: Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase.

(A, B) Representative cells showing nuclear positioning and mitotic arrest defects. Nuclear position was analyzed after DAPI staining. Cells with anaphase spindles located exclusively in the mother cell body are noted (white arrow). Anucleate (*), binucleate (**) and multinucleate (***) cells were frequently observed in kar9Δ dbf4-NΔ109 (A) and dyn1Δ dbf4-NΔ109 (B) cells. (C, E) Quantitation of nuclear segregation defects. >300 cells were counted for each strain and nuclear position was represented as a percentage of the total cells in culture. Black bars represent cells with nuclei segregated between mother and daughter cells; gray bars, divided nuclei in the mother cell; white bars, multinucleate and anucleate cells. (D, F) Spindle morphology of strains with misoriented nuclei and spindles. >50 cells were counted for each genotype. Black bars represent cells with mitotic spindles. Gray bars represent cells that have exited mitosis.

Charles T. Miller, et al. PLoS Genet. 2009 May;5(5):e1000498.

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