Fig 2. The rad50-V1269M mutation impairs Rad50 ATPase and binding to DNA in vitro.

(A) Purified Rad50, Rad50-V1269M, MRX, and MR^V1269MX, 1 μg each, were analyzed by SDS-PAGE and stained with Coomassie Blue. (B) The ATPase activity of wild-type MRX and mutant MR^V1269MX complexes was determined as described in [27]. Plotted values are the mean value with error bars denoting standard deviation (s.d.) (n = 3). (C) Rad50 and Rad50-V1269M proteins (50, 100, and 200 nM) were incubated with ³²P-labeled DNA (10 nM) in the presence of ATP. In lane 5 and 9, the reaction mixture was deproteinized with SDS and proteinase K (PK) prior to analysis. Plotted values are the mean value with error bars denoting s.d. (n = 3). (D) MRX and MR^V1269MX complexes (10, 20, and 40 nM) were treated as in (C).