FIGURE 4.

A role for Dna2 prolines 122 and 132 in checkpoint function. A, titration of the Dna2(1–499) domain or the PP122,132AA mutant domain in the standard Mec1 assay with Rad53-kd as substrate, but with 100 mm NaCl (see “Experimental Procedures”). B, far UV CD spectra of the two domains at 2 μm, in 50 mm Hepes 7.5, 150 mm NaCl buffer at 20 °C. C, 10-fold serial dilutions of strains containing both plasmid pRS316-DNA2 (URA3 DNA2) and the indicated DNA2 mutant plasmid or empty vector (Δ) were plated on 5-FOA medium to evict the complementing URA3 plasmid, and growth scored after 2 days at 30 °C. The relevant genotypes are shown. See “Experimental Procedures” for details. D, 10-fold serial dilutions of strains containing either wild-type DNA2 or the indicated mutants were plated on YPD medium and irradiated with 30 J/m² of UV₂₅₄ where indicated, or plated on YPD containing either 75 mm or 10 mm hydroxyurea (HU). Growth was scored after 2 days at 30 °C. E, strains PY270 (tel1Δ ddc1Δ) containing the indicated DNA2 mutant were synchronized in G1 and released into S phase with or without hydroxyurea. Phosphorylation of Rad53 was measured by Western analysis. See “Experimental Procedures” for details.