Figure 1. RFC-mediated loading of PCNA onto DNA.

(A) Schematic representation of the Cy3-P/T DNA substrate used in this study. The primer had a Cy3 dye at the 5′ end and biotin was attached to the 3′ end of the template. Sequences for the primer, template, and flap constructs of all substrates used in this study are shown in Table 1. The recombinant human proteins used in this study are shown in Figure 1—figure supplement 1 .(B) Model of human PCNA generated using Pymol from PDB code 1AXC (Gulbis et al., 1996). PCNA subunits are shown in ribbon form in green, orange, and blue. The asparagine 107 residue, shown in red for one PCNA monomer in space-filling form, was mutated to cysteine for dye labeling. On average, each PCNA trimer has at least one labeled monomer. The mutations nor the labeling of PCNA had any effect on its ability to interact of RFC (Figure 1—figure supplement 2). Frontal and side views are shown. (C) Schematic representation of RFC-catalyzed loading of PCNA onto DNA. The N107C residue of PCNA is located on the face opposite that which interacts with RFC and faces the Cy3 FRET donor on the P/T DNA. (D) Fluorescence emission spectra obtained by exciting the Cy3-P/T DNA with a 514-nm light source. Cy5-PCNA can be excited through FRET from Cy3 only when the two dyes are in close proximity (<∼10 nm). Cy5 fluorescence intensity peaks at 665 nm.

DOI: http://dx.doi.org/10.7554/eLife.00278.003

Figure 1—figure supplement 1. SDS–PAGE analysis of recombinant human proteins used in this study.

(Left) RFCp140ΔN555 (Lane 1, 12.5 pmol), wtPCNA (Lane 2, 25 pmol), mutPCNA (Lane 3, 25 pmol), Cy5-PCNA (Lane 4, 25 pmol), and polδ (Lane 5, 12.5 pmol) were loaded on a SDS 10% polyacrylamide gel and stained with Coomassie Blue. (Right) wtPCNA (Lane 1, 25 pmol), mutPCNA (Lane 2, 25 pmol), and Cy5-PCNA (Lanes 3–4, 25 pmol), were loaded on a SDS 10% polyacrylamide gel and imaged with UV trans illumination to visualize Cy5-label (Lane 4) prior to staining with Coomassie Blue.

DOI: http://dx.doi.org/10.7554/eLife.00278.004

Figure 1—figure supplement 2. Activities of labeled PCNA mutants in stimulating human RFC ATPase activity.

The ATPase activity was determined at 25°C in an assay solution containing 125 nM RFC, 125 nM wtPCNA, mutPCNA, or Cy5PCNA, 125 nM P/T DNA (500 nM Neutravidin), 1 mM ATP, 3 mM phosphoenolpyruvate, 200 mM NADH, and 6–8 units of phosphoenolpyruvate kinase-lactate dehydrogenase mix. For each condition, the initial rates of ATP hydrolysis are reported as an average of three independent experiments ±SEM.

DOI: http://dx.doi.org/10.7554/eLife.00278.005