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. 2020 Jul 23;9:e58571. doi: 10.7554/eLife.58571

Figure 6. Steric inhibition of DDK binding to MCM DHs by Rad53.

Figure 6.

(A) DDK binding to purified MCM DHs was monitored in the presence of ATP and in the absence or presence of Rad53-WT or Rad53-kd, as indicated. In lanes 5+6 DDK and Rad53 were co-incubated in the presence of ATP prior to addition to DNA-bound MCM-7 DHs; in lanes 7+8 DDK was incubated with purified MCM DHs before addition of Rad53. DNA-bound material was analyzed SDS-PAGE and silver stain or western blot as indicated. (B) Standard DNA replication reaction using p1017 (4.8 kb) as template. Rad53 and DDK were either co-incubated prior to simultaneous addition after the MCM loading step (lanes 3+4) or Rad53 was added after DDK prior to the addition of activation factors (lanes 5+6). Replication products were analyzed by denaturing agarose gel-electrophoresis and autoradiography. The results of two experiment repeats are plotted in the graph on the right. (C) DDK binding to DNA-bound MCM DHs was monitored in the presence of AMP-PNP. DDK and Rad53 were either co-incubated in the presence of AMP-PNP prior to addition to purified DNA-bound MCM DHs (lane 4), or added sequentially to MCM DHs (lane 5) as indicated.

Figure 6—source data 1. Figure 6A, silver stain.
Figure 6—source data 2. Figure 6A, immunoblot: Mcm7.
Figure 6—source data 3. Figure 6A, immunoblot: Dbf4.
Figure 6—source data 4. Figure 6A, immunoblot: Cdc7.
Figure 6—source data 5. Figure 6B, autoradiograph.
Figure 6—source data 6. Figure 6C, silver stain.
Figure 6—source data 7. Figure 6C, immunoblot: Mcm7, Dbf4.
Figure 6—source data 8. Figure 6C, immunoblot: Cdc7.