Figure 6.

Esc4 is phosphorylated in response to MMS. (A) Modification of Esc4 with increasing exposure to 0.1% MMS. An asynchronous culture of strain FR467 was grown to early log phase and treated with MMS; cells were removed at the times indicated and processed as described (see Methods). (B) Cells were grown as in (A), but lysed in buffer without phosphatase inhibitors. Lysate from untreated cells is shown in lane 1. Lysate from cells exposed to 0.1% MMS for 90 minutes is shown in lanes 2 and 3. In lane 3, 22 μg of lysate was treated with 600 units of λ protein phosphatase (New England Biolabs) for 20 min at 30 °C prior to analysis. (C) Phosphorylation of Esc4 is Mec1-dependent. Strains FR672 and FR673 cells were grown as in (A) with or without 0.1% MMS for 90 minutes. (D) Phosphorylation of Esc4 is mildly affected by deletion of RAD53, but not other, downstream, damage checkpoint functions. Strains FR713, FR716, FR718, and FR715 were treated as in (C). (E) Phosphorylation of Esc4 is not dependent on the presence of recombination mediator proteins. Strains FR674, K37-3, K37-7, and K37-15 were treated as in (C). (F) Phosphorylation of Esc4 is not dependent on either Mrc1 or Tof1. Strains FR674, FR1007, and FR1009 were treated as in (C). Arrows on the right side of each panel indicate the different forms of Esc4. Lanes contain 3 μg of total cell lysate, separated on 5% SDS-PAGE.