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

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.

Keywords: AAA+ ATPase; ATP hydrolysis; DNA replication; Walker B motif; clamp loader; sliding clamp.

Figure 1

Structural features of replication factor C (RFC) and its interaction with ATP. (a) Ribbon diagram of the S. cerevisiae Rfc4 subunit bound to ATPγS (gray spheres) with conserved ATP site residues including the Walker A Lys (cyan), Walker B Glu (red), Sensor 1 Asn (green) and sensor 2 Arg (orange) shown as sticks. (b) Walker B sequence motifs in each of the RFC subunits. (c) The clamp loading reaction can be divided into two phases based on ATP requirements: (1) formation of a ternary clamp loader clamp DNA complex promoted by ATP binding, and (2) release of the clamp on DNA promoted by ATP hydrolysis. The diagram illustrates a structural model for RFC and PCNA based on crystal structures of clamp loaders and clamps [18,19,30,31]. Individual protein domains represented by spheres or ovals; note that Rfc1 contains an extra C-terminal domain that lies in the gap between Rfc1 and Rfc5.

Figure 2

Clamp binding activities of WT RFC and Walker B mutants. Binding of WT RFC to PCNA-MDCC was measured in the presence of ATP (black squares) or ATPγS (green inverted triangles), and binding of WB-EQ (red circles) and WB-EA mutants (blue triangles) to PCNA-MDCC was measured in the presence of ATP. The relative intensity of MDCC at 470 nm is plotted as a function of RFC concentration for solutions containing 10 nM PCNA-MDCC and 0.5 mM ATP or ATPγS. Data shown are the average of three independent experiments. Error bars represent standard deviation.

Figure 3

Clamp opening activities of WT RFC and Walker B mutants. Binding of WT RFC to PCNA-AF488 was measured in the presence of ATP (black squares) or ATPγS (green inverted triangles), and binding of the WB-EQ mutant (red circles) and WB-EA mutant (blue triangles) to PCNA-AF488 was measured in the presence of ATP. The relative intensity of AF488 at 517 nm is plotted as a function of RFC concentration for solutions containing 10 nM PCNA-AF488 and 0.5 mM ATP or ATPγS. Data shown are the average of three independent experiments. Error bars represent standard deviation.

Figure 4

ATP binding to WT RFC and WB mutants measured by PCNA opening and TNP-ATP fluorescence. (a) Binding of WT RFC (black), the WB-EQ RFC mutant (red) and the WB-EA RFC mutant (blue) to PCNA-AF488 was measured in assays using the ATP concentrations indicated. The relative intensity of AF488 at 517 nm is plotted as a function of ATP concentration for solutions containing 10 nM PCNA-AF488 and 250 nM each RFC. Data shown are the average of three independent experiments (WT and WB-EQ mutant). Error bars represent standard deviation. The average two independent experiments is shown for the WB-EA mutant. (b) Binding of TNP-ATP to WT RFC and RFC WB mutants was measured by measuring the increase in TNP fluorescence that occurs on binding to RFC. The relative fluorescence of TNP was measured in assays containing 50 µM TNP-ATP and increasing concentrations of RFC as indicated. Data shown are the average of three independent experiments with error bars representing the standard deviation.

Figure 5

DNA binding activities of WT RFC and Walker B mutants. The anisotropy of RhX covalently attached to p/t DNA was measured as a function of RFC concentration. Binding of WT RFC (squares), the WB-EQ mutant (circles) and the WB-EA mutant (triangles) to DNA was measured in the presence of 0.5 mM ATPγS and 20 nM p/t DNA-RhX. (a) DNA binding was measured in the absence of PCNA. (b) DNA binding was measured in the presence of 2 µM PCNA. Three independent experiments were done and error bars represent the standard deviation.

Figure 6

Effects of Walker B glutamate mutations on rates of ATP hydrolysis. (a) Rates of ATP hydrolysis were measured for WT RFC (black), the WB-EQ mutant (red) and the WB-EA mutant (blue) in the presence of 0.5 mM ATP and varying concentration of p/t DNA. The concentration of ATP hydrolyzed per second is plotted at several DNA concentrations for solutions containing 450 nM each RFC. (b) Rates of ATP hydrolysis were measured in assays containing 1 µM PCNA in the absence (yellow bars) and presence (magenta bars) of 1 µM DNA. Data from RFC alone (gray bars) and RFC with 1 µM DNA (cyan bars) are taken from panel a. Data shown are the average of three independent experiments. Error bars represent standard deviation.