Ability of afRFC wild-type or mutant complexes to stimulate DNA synthesis catalysed by afPolB1. Reaction mixtures (20 μl) were supplemented with 200 mM NaCl and singly primed, closed circular M13mp18 DNA (25 fmol). After incubation for 30 min at 65°C, the DNA was precipitated and analysed by alkaline gel electrophoresis. Autoradiograms of dried gels are shown.

Model for clamp loading by afRFC. A proposed mechanism for ATP utilisation by the archaeal clamp loader includes the following (clockwise, starting from the top): (A) initial binding of two molecules of ATP by the afRFC small subunits, (B) upon interaction with PCNA, binding of two additional molecules of ATP (one of which binds to the large subunit), (C) loading of PCNA onto DNA, (D) hydrolysis of ATP at the afRFC small subunits, which results in both release of PCNA onto DNA and release of the clamp loader from the PCNA–DNA complex, thereby enabling the polymerase to associate with PCNA, and (E) hydrolysis of ATP at the afRFC large subunit, which results in recycling of afRFC and in initiation of a new cycle of clamp loading.

Nucleotide binding by wild-type and mutant afRFC complexes in the absence or presence of DNA and/or PCNA. The amount of bound [³⁵S]ATPγS was measured using equilibrium dialysis (A) or spin column assays (B–E) as described in the Experimental procedures. (A, B) Wild-type afRFC was incubated with [³⁵S]ATPγS in the presence and absence of DNA and PCNA. The data were fitted by nonlinear regression assuming equivalent binding sites to the equation shown in Experimental procedures. Similar studies were carried out with the mutant complexes, (C) in the presence of PCNA, (D) in the presence of DNA, and (E) in the absence of cofactors. Theoretical curves for 1, 2, 3, and 4 bound nucleotides are overlaid for reference in panels C, D, and E.

Release of PCNA from afRFC is necessary for interaction with afPolB1. (A) ATP bound to the afRFC small subunits inhibits release of afRFC and blocks access of afPolB1 to PCNA. Proteins in the eluates of the spin columns were visualised after electrophoresis in a 12% SDS–PAGE gel by silver staining. Reactions contained 5 pmol nicked pUC19 DNA, 8 pmol afPolB1 (lanes 4–8), 16 pmol afRFC (lanes 5, 6, and 9–11), 16 pmol LargeB_mut/Small_wt (lanes 7 and 8), 16 pmol pk-PCNA (lanes 5–8, 10, and 11), 1 mM ATP (lanes 4, 5, 7, and 10), or 1 mM ATPγS (lanes 6, 8, 9, and 11). In addition, 5% of the input afPolB1 and PCNA (lane 1), afRFC (lane 2), or LargeB_mut/Small_wt (lane 3) proteins were loaded onto the gel. (B) In all, 1 nmol PCNA and, where indicated, 3 nmol afPolB1 and 3 nmol afRFC were separated at room temperature by gel filtration in the presence or absence of 5 mM ATP. A measure of 15 μl of the 750 μl fractions 17–26 were separated in a 12% SDS–PAGE gel stained with Coomassie brilliant blue.

ATP binding at both the afRFC large and small subunits is necessary and sufficient to promote stable PCNA–DNA complexes, whereas ATP hydrolysis at only the afRFC small subunits is necessary for PCNA release. Loading assays were performed using a spin column assay. Reactions contained 8 pmol ³²P-PCNA, 4 pmol nicked pUC19 DNA (reactions 1–9), 4 pmol supercoiled pUC19 DNA (reaction 10), 4 pmol linearised pUC19 DNA (reaction 11), or 4 pmol nicked, linearised pUC19 DNA (reactions 12–17). Reactions 1–8, 10–12, 14, and 16 contained 5 mM ATP. Reactions 9, 13, 15, and 17 contained 5 mM ATPγS. The mixtures were substituted with 4 pmol wild-type (reactions 2, 9–11, 14, and 15) or mutant (reactions 3–8 and 16–17) afRFC complexes. The spin columns were pre-equilibrated in 200 mM NaCl. (A) Control of DNA retention on the spin columns. In all, 10% of the input DNA (load) and 10% each of the eluates of reactions 1–8 were loaded onto a 1% agarose–TBE gel stained with ethidium bromide. (B) Analysis of ³²P-afPCNA in the eluates by SDS–PAGE. In all, 5% of the input ³²P-PCNA (load) or 90% of the eluates of reactions 1–8 were separated by SDS–PAGE in a 15% gel and visualised on a phosphorimager. (C) Quantitative depiction of results obtained by phosphorimager analysis.

ATP binding to both the large and small subunits is required to stabilise afRFC–PCNA complexes. In all, 150 pmol of the afRFC proteins was incubated with 400 pmol of PCNA in the absence (A, B) or presence of 1 mM ATP (C, D). AfRFC–PCNA complexes were captured by incubation with Ni-agarose beads (via the His-tag located at the N-terminus of the large subunit), eluted by boiling in SDS sample buffer and analysed by SDS–PAGE in 15% gels stained with Coomassie brilliant blue dye. To improve quantification, 30% as well as 70% of the eluates were run on the gels. In the presence of 1 mM ATP, after subtraction of nonspecific PCNA binding (E), the total amount of PCNA in the eluates bound to the 60 pmol of afRFC recovered from the beads was estimated to be 80 pmol. This ratio of bound PCNA per afRFC was taken to be 100% and all other values were normalised to this value. The results of these calculations are shown below the respective lanes. Lane M, molecular mass markers; lanes 1 and 5, 10% of reaction mixtures; lanes 2 and 6, 10% of supernatants after incubation with Ni-agarose beads; lanes 3 and 7, 30% of eluates; lanes 4 and 8, 70% of eluates.