FIG. 2.

Genetic control of Rad55p phosphorylation in response to DNA damage. (A) Overview of the DNA damage and replication block checkpoint in S. cerevisiae and the proposed functions of the genes used in panel B (18, 24, 68). DNA damage in G₁ and G₂ cells is sensed and/or processed by Rad9p, Rad17p, Rad24p, and Mec3p. Replication blocks are sensed by the DNA polymerase ɛ (Pol2p) (57). Both branches feed into the Mec1p kinase, which controls activation of the Rad53p kinase (69, 76). Rad53p controls some but not all checkpoint responses (12) and leads to the activation of the Dun1p kinase (93). Dun1p kinase is involved in the activation of some DNA damage-inducible genes (93) and, in one pathway with Rad53p, in G₂/M cell cycle arrest parallel to a pathway acting through Chk1p kinase and Pds1p (24, 68). As shown here, Dun1p kinase is required for full phosphorylation of Rad55p in response to DNA damage (labeled DNA repair). (B) Rad55p phosphorylation in cycling cells depends on some but not all DNA damage checkpoint functions. Cells (0 or 90 min in 0.1% MMS at 24°C) were analyzed as described in the legend to Fig. 1; wild-type (TWY12; lanes 1 and 2), mec1-1 (TWY308; lanes 3 and 4), rad53 (TWY312; lanes 5 and 6), mec3-1 (TWY316; lanes 7 and 8), rad24-1 (TWY399; lanes 9 and 10), wild-type (Y300; lanes 11, 12, 15, 16, 21, and 22), dun1-Δ100 (Y286; lanes 13 and 14), tel1Δ (WDHY1227; lanes 17 and 18), rad17Δ (WDHY1234; lanes 19 and 20), rad9 (Y438; lanes 23 and 24), pol2-12 (Y439; lanes 25 and 26), and rad9 pol2-12 (Y440; lanes 27 and 28). The wild-type control is shown for each individual experiment. Bars refer to the different forms of Rad55p.