Investigation of the mechanistic basis for the role of Rad50 in double-strand break repair

Members of the Rad52 epistasis group, which includes a heterotrimeric complex, formed by Mre11, Rad50 and Nbs1 (Xrs2 in yeast) helps in the protection of cell’s genetic content from DNA doublestrand breaks. The Rad50 component of the human Mre11/Rad50/Nbs1 (Xrs2 in yeast) complex (MRN(X)) belongs to the ABC superfamily of ATPases and is conserved among all organisms and contains Walker A (N-Terminus) and Walker B (C-Terminus) ATPase domains connected by a long coiled-coil region. We show for the first time that Rad50 shows adenylate kinase activity (ATP + AMP ↔ 2ADP) and that this activity is important for tethering of DNA ends. We further show that Rad50 can catalyze “reverse” adenylate kinase activity (2ADP → ATP + AMP) and this activity is stimulated in the presence of linear DNA ends. We also show that the signature motif of Rad50 is essential for all ATP-dependent activities in vivo and in vitro.