Basic-patch mutations in bacteriophage T4 Rad50 uncouple ATPase activation from processive nuclease activity.

The Mre11/Rad50 (MR) complex uses adenosine triphosphate (ATP) binding and hydrolysis to coordinate the recognition and processing of DNA double-strand breaks. Although Mre11 and DNA stimulate the relatively slow ATPase activity of Rad50, the mechanism by which this occurs remains incompletely understood. In the present study, we investigated a basic patch on bacteriophage T4 Rad50, consisting of Arg154, Arg155, and Lys156, that was predicted to contribute to DNA binding. Mutation of these residues caused only modest changes in DNA affinity, indicating that this region is unlikely to function primarily as a direct DNA-contact surface. In contrast, the effects on ATP hydrolysis were pronounced. R154A and the TripleA mutant displayed strong ATPase activation in the presence of Mre11 alone, approaching the activity of the wild-type MR complex bound to DNA. DNA titrations further showed that these mutants were relatively insensitive to increasing double-stranded DNA concentrations, consistent with a shift in the conformational equilibrium toward an ATPase-active-like state. However, ATP-dependent stimulation of repetitive nucleotide excision was reduced for all mutants, with the strongest defect observed for TripleA, indicating that enhanced ATP hydrolysis alone is not sufficient to support processive nuclease activity. A mutation at Asp479 had a related but distinct effect, supporting long-range coupling within the T4 MR complex. Overall, the results support a model in which a basic patch near the base of the Rad50 coiled-coils contributes to an allosteric pathway linking Mre11 and DNA engagement with productive ATP hydrolysis and its coupling to nuclease output.