Fig. 6
Interactions between PCNA and various PolD mutants. a SPR analyses were performed to detect the physical interactions of PCNA with various PolD mutants (wt, ΔPIP, ΔKR, and ΔPIPΔKR). Purified PCNA was immobilized on a sensor chip, and six different concentrations (50, 100, 200, 300, 500, 1000 nM) of purified PolDs were analyzed. The sensorgrams at 500 nM of PolDs are overlaid. b The kinetic parameters were calculated from the sensorgrams as shown in Figure S10. ka, the association rate constant; kd, the dissociation rate constant; KD, the apparent equilibrium constant. c Complex formation analyzed by gel filtration chromatography. A primed DNA composed of d29 mer (5′-GGTACCGGGCCCCCCCTCGAGGTCGACGGam-3′)/d45 mer (5′-ATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACC-3′) was mixed with various PolDs and PCNA and subjected to the column. The elution profiles, monitored by the absorbance at 260 and 280 nm, are shown in the dashed red and solid blue lines, respectively. The peak positions of the marker proteins are indicated on the top. Aliquot (5 μl of injected mixture and 8 μl of each elution fraction were subjected to 10% SDS-PAGE followed by Coomassie Brilliant Blue staining. The mutant proteins are indicated by the following notations. ΔPIP, DP2(Δ1314-1324); ΔKR, DP1(K326A K328A) and DP2(K432A R434A R781A). d The primer extension activity of PolD variants were measured in the presence or absence of PCNA. For each PolD variant, the amount of incorporated dNTPs in the absence of PCNA was normalized to 1 and the relative activities were shown to compare the stimulation by PCNA for each PolD variant. Three independent experiments were carried out in succession for each PolD variant, and the S.E.M. values are shown as vertical lines on the plots in each graph