Comparison of the activities and PCNA stimulation of Pol δ and its subassemblies on poly(dA)/oligo(dT) primer-templates.

<p>Panel A. “In-gel” determination of p125 content of each Pol δ assemblies. The recombinant p125 alone (lane a), core enzyme of p125/p50 (lane b), core+p68 trimer (lane c), core+p12 trimer (lane d), and Pol δ4 (lane e) were expressed and purified to near-homogeneity by immunoaffinity chromatography and FPLC on Mono-Q ion exchange as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039156#s2" target="_blank">Materials and Methods</a>”. Protein concentrations were determined based on the in-gel analysis of p125 content for each of the complexes using catalase as a protein standard. The peak fraction of each enzyme from the Mono Q step was separated on 12.5% SDS-PAGE and followed by silver staining. The four left lanes show the catalase standards (0.5, 1, 2, and 4 μg). Lane “M” contains the protein markers in kDa. The four subunits are marked by arrows. Panel B. Pol δ activity assay. Increasing amounts of each enzyme in fmoles of p125 per reaction were analyzed for their activities in the standard poly(dA)/oligo(dT) assay in the presence of PCNA. The vertical axis indicates the incorporated dTMP in CPMs and the horizontal axis shows the enzyme concentrations in fmoles of p125. Panel C. PCNA stimulation. The graph shows the activities (in triplicate) on poly(dA)/oligo(dT) with increasing PCNA. The vertical axis indicates the incorporated dTMP in fmoles per minute and the horizontal axis shows the PCNA concentrations in nM trimer. The recombinant enzyme complexes are indicated by the lines.