FIG. 1.

Phosphorylation of Rad55p in response to DNA damage and replication blocks. (A) Modification of Rad55p in response to DNA damage was analyzed by immunoprecipitation and immunoblotting. Lanes 1 and 2 contain precipitates from rad55Δ cells (WDHY1089) without MMS (lane 1) and after 60 min of exposure to 0.075% MMS (lane 2). Lanes 3 to 7 contain precipitates from wild-type cells (WDHY1075) without MMS (lane 3) and after increasing exposure time (15 to 120 min) to 0.075% MMS (lanes 4–7). In lane 8, extract of wild-type cells overexpressing Rad55p was directly blotted to show the position of the Rad55 protein. In lanes 5 and 7, accidentally less protein was loaded. (B) Modification of Rad55p in response to replication blocks. Cells were treated with HU and the Rad55p status was analyzed as in panel A. Lanes 1 and 2 contain precipitates from rad55Δ cells without HU (lane 1) and after 60 min of exposure to 200 mM HU (lane 2). Lanes 3 to 7 contain precipitates from wild-type cells without HU (lane 3) and after increasing exposure time (15 to 120 min) to 200 mM HU (lanes 4 to 7). (C) Modification of Rad55p in response to UV and gamma radiation. Exponentially growing wild-type cells (FF18984) were irradiated with gamma rays (¹³⁷Cs source at 8 Gy/min) or UV rays (254 nm) at the indicated doses or mock irradiated. Cell extracts were prepared 1 h postradiation and analyzed for their Rad55p status. (D) Phosphorylation of Rad55p in response to DNA damage. The Rad55p status was analyzed in wild-type cells as in panel A from cells grown in the absence (lane 1) and in the presence (lanes 2 and 3) of MMS (0.075% for 120 min). An immunoprecipitate of a sample from MMS-treated cells was incubated with phosphatase before immunoblot analysis (lane 3). (E) Rad55p is phosphorylated in vivo in response to MMS. Wild-type cells (WDHY1075) were metabolically labeled with ³²P in the presence (0.1%) and absence of MMS. Rad55p was immunoprecipitated and analyzed by autoradiography. Immunoblotting confirmed the position of unphosphorylated and phosphorylated Rad55p. Bars refer to the different forms of Rad55p.