Figure 4.

Cdk and Cdc14 Phosphatase Regulate the Nuclease Activity and Subcellular Localization of Yen1

(A) Schematic representation of the experimental conditions employed in the Cdk inhibition experiments (B and C).

(B) Cdk inhibition in S phase cells increases Yen1 activity. cdc28-as1 cells carrying galactose-inducible YEN1-myc9 were treated with HU. To one-half of the culture, galactose was added before DMSO (lane 1) or the Cdc28-as1 kinase inhibitor 1NM-PP1 (lane 2). The other half was split, and galactose was added after DMSO (lane 3) or 1NM-PP1 (lane 4). Yen1-myc9 was immunoprecipitated and assayed for HJ resolution.

(C) Yen1^ON activity is refractory to Cdk inhibition. As in (B), except that YEN1^ON-myc9 was expressed after addition of DMSO (3) or 1NM-PP1 (4). The amounts of immunoprecipitated protein were reduced compared with (B) in order to limit DNA cleavage.

(D) cdc14-1 mutants fail to activate Yen1 at restrictive temperature. Yen1-myc9 IPs from the indicated strains (grown at 25°C and then shifted to 37°C for 2 hr) were tested for nuclease activity.

(E) Yen1^ON activity is refractory to Cdc14 inactivation. As in (D), but employing cdc14-1 strains carrying either YEN1-myc9 or YEN1^ON-myc9.

(F) Cdc14 is required for nuclear enrichment of Yen1 at anaphase. The immunofluorescence analysis of Yen1-myc18 or Yen1^ON-myc18 during anaphase in CDC14 or cdc14-1 strains at the restrictive temperature was carried out as in Figure 3. The percentage of the cells displaying the most representative type of Yen1 distribution (nuclear or cellular) is shown. The contours of the cells, determined from DIC images, are depicted by a dotted line.