FIG. 7.

Dbf4p is modified in cdc13-1 cells in a MEC1-dependent manner. (a) DBF4-proA cdc13-1 (YCH216) cells were incubated at 37°C, the nonpermissive temperature, for 4 h. Samples for extract preparation were taken at 1-h intervals. (b) DBF4-proA cdc13-1 (YCH216) cells were grown at 37°C for 4 h. Dbf4-ProA was immunoprecipitated from extract by using IgG-Sepharose, washed into CIP buffer, and divided into two samples. One hundred units of CIP was added to one of the samples (+) but not to the other (−). Dbf4-ProA was also immunoprecipitated from extracts derived from DBF4-proA cdc13-1 (YCH216) cells grown at the permissive temperature. This sample was not exposed to CIP (−∗). All three tubes were placed at 37°C for 30 min. The samples were loaded onto an SDS–7% polyacrylamide gel, and immunoblot analysis was performed with anti-IgG to detect Dbf4-ProA. (c) DBF4-proA cdc13-1 mec1-1 (YCH317) cells were incubated at 37°C for 6 h. Samples for extract preparation were taken at 1-h-intervals. (d) DBF4-proA CDC13 MEC1 (YCH201) cells were incubated at 37°C. DBF4-proA cdc13-1 MEC1 (YCH216) and DBF4-proA cdc13-1 mec1-1 (YCH324) cells were incubated at 37°C for 4 and 6 h, respectively. Samples for extract preparation were taken at these times. Dbf4-ProA and actin were detected by immunoblotting with anti-IgG and antiactin antibodies, respectively.