Figure 3.

Requirement of checkpoint proteins for DNA damage-induced Rev1 phosphorylation. The Rev1 protein tagged at the C terminus with 4HA was expressed from plasmid pVP64 in the wild-type or the checkpoint mutant strain. Exponentially growing cells were treated (+) or not (−) with 4NQO (2 μg/mL) for 15 min. Cells were then washed with water and total protein extract was obtained in the presence of protease inhibitors and phosphatase inhibitors. Protein extracts were analyzed on 6% acrylamide gels (77:1 acrylamide:bis-acrylamide), and Rev1 was detected by Western blotting with anti-HA antibody using standard techniques. (A) Rev1 phosphorylation is inhibited in the absence of Mec1–Ddc2 kinase but not in the absence of Rad53 kinase. (B) Rev1 phosphorylation does not occur in mec3Δ, rad17Δ, ddc1Δ, and rad24Δ strains.