Figure 6.

Coordination of Rad50 ATPase sites is required for ATM activation. (A) In vitro kinase assays with recombinant human ATM (0.2 nM) were performed with WT or S1202W hMR complexes (20 nM), Nbs1 (20 nM) and linear DNA using GST-p53 (50 nM) as a substrate. Reactions were separated by polyacrylamide gel electrophoresis, transferred to a PVDF membrane and probed with an antibody specific for phospho-Ser15 phosphorylation. Membranes were also probed for ATM and Rad50 for comparison as shown. (B) In vitro kinase assays were performed as in (A) but with homodimeric hMR (WT, K42A or D1231A) complexes. (C) In vitro kinase assays were performed as in (A) but with WT and heterodimeric (KA–SW) hMR.