Biochemical characterisation of the clamp/clamp loader proteins from the euryarchaeon Archaeoglobus fulgidus

Abstract

Replicative polymerases of eukaryotes, prokaryotes and archaea obtain processivity using ring-shaped DNA sliding clamps that are loaded onto DNA by clamp loaders [replication factor C (RFC) in eukaryotes]. In this study, we cloned the two genes for the subunits of the RFC homologue of the euryarchaeon Archaeoglobus fulgidus. The proteins were expressed and purified from Escherichia coli both individually and as a complex. The afRFC subunits form a heteropentameric complex consisting of one copy of the large subunit and four copies of the small subunits. To analyse the functionality of afRFC, we also expressed the A.fulgidus PCNA homologue and a type B polymerase (PolB1) in E.coli. In primer extension assays, afRFC stimulated the processivity of afPolB1 in afPCNA-dependent reactions. Although the afRFC complex showed significant DNA-dependent ATPase activity, which could be further stimulated by afPCNA, neither of the isolated afRFC subunits showed this activity. However, both the large and small afRFC subunits showed interaction with afPCNA. Furthermore, we demonstrate that ATP binding, but not hydrolysis, is needed to stimulate interactions of the afRFC complex with afPCNA and DNA.

Figure 1

Gel filtration of afRFC under denaturing conditions. (A) Elution profile. Shown is the trace at 280 nm, the fraction numbers are indicated at the x-axis. (B) Coomassie brilliant blue-stained 12.5% SDS–PAGE gel. Lane M, molecular mass marker; lane L, 5 µl of the 100 µl loaded sample; lanes 3–8, 20 µl each of column fractions 18–23.

Figure 2

Plots of molecular weight versus concentration for data taken at 7000 r.p.m. The data show a flat profile, distributed around a molecular weight of ∼200 000 Da. This is consistent with a subunit stoichiometry of 1 large:4 small. This result was confirmed by globally fitting the data taken at three concentrations and three speeds (see Results). The loading concentrations were A₂₈₀ = 0.2 (squares), A₂₈₀ = 0.4 (black circles) and A₂₈₀ = 0.6 (grey circles). Molecular weight versus absorbance plots were generated using the software provided with the centrifuge.

Figure 3

Effects of afRFC and afPCNA on DNA synthesis catalysed by afPolB1. Reaction mixtures (20 µl) were as described in Materials and Methods and contained 200 mM NaCl. All reactions contained 25 fmol of singly primed, closed circular M13mp18 DNA and 12 pmol of afPolB1. Where indicated, 4 pmol (lanes 3 and 5) or 12 pmol (lanes 2, 6 and 7) of afPCNA and 4 pmol (lanes 4, 5 and 6) or 12 pmol (lane 7) of afRFC were added. Reactions were incubated for 30 min at 65°C, precipitated and analysed by alkaline gel electrophoresis. An autoradiogram of a dried gel is shown.

Figure 4

ATP binding, but not hydrolysis, is needed to stimulate afRFC–afPCNA interactions. 150 pmol of afRFC were incubated with 400 pmol of afPCNA in the presence of magnesium and various nucleotides. afRFC–afPCNA complexes were captured by incubation with Ni–agarose beads as described in Materials and Methods. Captured proteins were eluted by boiling in SDS sample buffer and aliquots of the reactions were analysed by SDS–PAGE on 15% gels followed by staining with Coomassie brilliant blue dye. To improve quantification, 30% as well as 70% of the eluates were run on the gels. Where indicated above the respective lanes, nucleotides (ATP, AMP-PNP, ATPγS or ADP) at final concentrations of 1 mM were added to both the reaction mixtures and the washing buffers. After correction for non-specifically bound afPCNA [(C), lanes 7 and 8], the amount of afPCNA bound to afRFC in the presence of ATP 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. Lanes 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.

Figure 5

ATP does not stimulate afRFCla–afPCNA interactions. Aliquots of 20 µg of afRFCla were incubated with 400 pmol of afPCNA in the absence (lanes 1–4) or presence of ATP (lanes 5–8). AfRFCla–afPCNA complexes were captured and analysed as described in Materials and Methods and in the legend to Figure 4. 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.

Figure 6

Effects of nucleotides on afRFC–DNA interactions in the absence (A) or presence of 10 pmol afPCNA (B). Increasing amounts of afRFC (0.25–5 pmol) were incubated as described in Materials and Methods with 50 fmol of a 5′-tailed DNA substrate consisting of a 30mer (5′-CTGACCGTCGAGCACCGCTGCGGCTGCACC-3′) annealed to a 60mer (5′-T₃₀GGTGCAGCCGCAGCGGTGCTCGACGGTCAG-3′). In addition, the reactions either contained 2 mM ATP (triangles), 2 mM ATPγS (circles), 2 mM AMP-PNP (diamonds), or were carried out in the absence of nucleotide (squares). Reaction products were separated by native gel electrophoresis in 8% polyacrylamide gels. The amount of shifted DNA (fmol) was quantitated by phosphorimager analysis and plotted against the amount of afRFC (pmol) in the reactions.

Figure 7

Comparison of afRFC–afPCNA binding to single-stranded DNA, single-stranded RNA, a 5′-tailed DNA duplex or an RNA/DNA heteroduplex. Increasing amounts of afRFC (0.25–5 pmol) were incubated as described in Materials and Methods with 50 fmol of the polynucleotides in the presence of 10 pmol afPCNA and 2 mM ATPγS. The single-stranded DNA substrate (circles) was a 30mer (5′-CTGACCGTCGAGCACCGCTGCGGCTGCACC-3′), the RNA substrate (squares) was a 21mer (5′-GAGCACCGCUGCGGCUGCACC-3′), and the RNA/DNA heteroduplex (triangles) consisted of the RNA 21mer annealed to the DNA 60mer already used for the generation of the 5′-tailed DNA substrate of Figure 6. Thus, the RNA/DNA heteroduplex was composed of 21 bp attached to a 5′ (dT)₃₀ tail and a 3′ tail of nine deoxyribonucleotides. The data for the 5′-tailed DNA duplex (diamonds) was obtained from Figure 6B. Reaction products were separated by native gel electrophoresis in 8% polyacrylamide gels. The amount of shifted polynucleotide (fmol) was quantitated by phosphorimager analysis and plotted against the amount of afRFC (pmol) in the reactions.