A novel function for the conserved glutamate residue in the walker B motif of replication factor C.

摘要: In all domains of life, sliding clamps tether DNA polymerases to increase the processivity synthesis. Clamp loaders load onto in a multi-step process that requires ATP binding and hydrolysis. Like other AAA+ proteins, clamp contain conserved Walker A B sequence motifs, which participate hydrolysis, respectively. Mutation glutamate residue motifs (or DExx-boxes) proteins typically reduces hydrolysis by as much couple orders magnitude, but has no effect on binding. Here, Glu each four active sites eukaryotic loader, RFC, was mutated Gln Ala separately, binding- hydrolysis-dependent activities quadruple mutant were characterized. Fluorescence-based assays used measure individual reaction steps required for loading including binding, opening, Our results show mutations affect ATP-binding-dependent interactions RFC with addition reducing ligand-dependent activity. we is ATP-dependent ligand activity, previously unknown function this RFC.