Abstract
In all domains of life, sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders load clamps onto DNA in a multi-step process that requires ATP binding and hydrolysis. Like other AAA+ proteins, clamp loaders contain conserved Walker A and Walker B sequence motifs, which participate in ATP binding and hydrolysis, respectively. Mutation of the glutamate residue in Walker B motifs (or DExx-boxes) in AAA+ proteins typically reduces ATP hydrolysis by as much as a couple orders of magnitude, but has no effect on ATP binding. Here, the Walker B Glu in each of the four active ATP sites of the eukaryotic clamp loader, RFC, was mutated to Gln and Ala separately, and ATP binding- and hydrolysis-dependent activities of the quadruple mutant clamp loaders were characterized. Fluorescence-based assays were used to measure individual reaction steps required for clamp loading including clamp binding, clamp opening, DNA binding and ATP hydrolysis. Our results show that the Walker B mutations affect ATP-binding-dependent interactions of RFC with the clamp and DNA in addition to reducing ligand-dependent ATP hydrolysis activity. Here, we show that the Walker B glutamate is required for ATP-dependent ligand binding activity, a previously unknown function for this conserved Glu residue in RFC.
Highlights
DNA replication requires the coordinated activity of many enzymes and proteins
In this work, we show that the Walker B Glu residue in replication factor C (RFC) contributes to ATP-dependent binding to PCNA and DNA
RFC contains five subunits and each subunit contains sequences that are conserved among AAA+ proteins including the Walker B motif or DExx-box
Summary
DNA replication requires the coordinated activity of many enzymes and proteins. Members of the. Sliding clamps are assembled onto DNA by the activity of clamp loaders, which are AAA+ family members This clamp loading reaction requires clamp loaders to bind clamps, open clamps, place open clamps around DNA, close clamps, and release the clamps to allow binding of DNA polymerases. AAA+ proteins contain a second conserved acidic residue that is usually Glu, and the consensus sequence is hhhhDExx in which h is a hydrophobic residue and xx residues are not well conserved but often AD [16]. Mutation of the Walker B Glu residue would be predicted to decrease the ATP hydrolysis activity of clamp loaders and hydrolysis-dependent steps such as release of the clamp on DNA, and this has been shown for an archaeal clamp loader [28]. Individual protein domains represented by spheres or ovals; note that Rfc contains an extra C-terminal domain that lies in the gap between Rfc and Rfc
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