P50, the Small Subunit of DNA Polymerase Delta, Is Required for Mediation of the Interaction of Polymerase Delta Subassemblies with PCNA

Mammalian DNA polymerase δ (pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and various DNA repair processes. Its function as a chromosomal DNA polymerase is dependent on the association with proliferating cell nuclear antigen (PCNA) which functions as a molecular sliding clamp. All four of the pol δ subunits (p125, p50, p68, and p12) have been reported to bind to PCNA. However, the identity of the subunit of pol δ that directly interacts with PCNA and is therefore primarily responsible for the processivity of the enzyme still remains controversial. Previous model for the network of protein-protein interactions of the pol δ-PCNA complex showed that pol δ might be able to interact with a single molecule of PCNA homotrimer through its three subunits, p125, p68, and p12 in which the p50 was not included in. Here, we have confirmed that the small subunit p50 of human pol δ truthfully interacts with PCNA by the use of far-Western analysis, quantitative ELISA assay, and subcellular co-localization. P50 is required for mediation of the interaction between pol δ subassemblies and PCNA homotrimer. Thus, pol δ interacts with PCNA via its four subunits

Co-nuclear staining of p50 and PCNA.

<p>Hela cells were fixed, permeabilized, and co-stained for p50 and PCNA using indirect immunofluorescence in which an anti-p50 rabbit polyclonal antibody was used for p50 while an anti-PCNA mouse monoclonal antibody PC-10 was used for PCNA. <b>A:</b> Rhodamine-X-conjugated anti-rabbit IgG secondary antibody was used for p50 (red). <b>B:</b> Alexafluor488-conjugated anti-mouse IgG secondary antibody was used for PCNA (green). <b>C:</b> DNA was counterstained with DAPI immunofluorescence (blue). <b>D:</b> Merger for p50 (panel A) and DAPI (panel C). <b>E:</b> Merger for p50 (panel A) and PCNA (panel B). <b>F:</b> Merger for p50 (panel A), PCNA (panel B), and DAPI (panel C). Cells were analyzed and photographed with an Axiovert 200 M fluorescence microscope (Zeiss).</p

Quantitative ELISA assay to verify the interaction of p50 with PCNA.

<p><b>A:</b> Coomassie blue stained SDS-PAGE analysis for the highly purified proteins used in ELISA assays. M, protein marker in kDa; Lane 1, highly purified His-p68. Lane 2, highly purified non-tagged p50. <b>B:</b> Interaction of p50 with PCNA by adding PCNA to coated p50. The assays were performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027092#s2" target="_blank">Materials and Methods</a>”. The 96-well plates were coated with 100 ng of p50, His-p68 and BSA. ELISAs using increasing amounts of PCNA (horizontal axis) were then performed using an antibody PC-10 to detect the bound PCNA to p50. The p68 was taken as positive control while BSA was taken as “noise”. Absorbance readings were taken at 450 nm and the values were plotted after subtraction of the control values with BSA. Each assay was performed three times and the standard deviations are shown by the error bars. <b>C:</b> Interaction of p50 with PCNA by adding p50 to coated PCNA. The 96-well plate was pre-coated with 100 ng of PCNA. Increasing amounts of 50, His-68 (as a control), or BSA were then added (horizontal axis). Bound proteins to PCNA were recognized by an antibody against p50 or p68 to detect the interaction between PCNA and p50 or p68.</p

A model for the network of protein-protein interactions that may be involved in the assembly of the pol δ-PCNA complex.

<p><b>A:</b> Complete structure. The inter-subunit protein-protein contacts for both pol δ and PCNA are shown as solid lines. Interactions between pol δ subunits and PCNA subunits are shown by the tripled lines. PCNA is shown as having the ability to act as a trivalent molecule. <b>B and C:</b> The protein-protein interaction network for the p125/p50/p68 trimer (panel B) and for the p125/p50/p12 trimer (panel C). In such models, p50 provides an alternative interaction with one monomer of PCNA homotrimer retaining three trivalent interactions of PCNA monomers. <b>D:</b> The protein-protein interaction network for 125/p50. Only two PCNA monomers retain trivalent interactions.</p

Far-Western analysis to detect the binding of PCNA to p50.

<p>Highly purified non-tagged p50 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027092#pone-0027092-g006" target="_blank">Figure 6A</a>, Lane 2) and pol δ4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027092#pone.0027092-Zhou1" target="_blank">[29]</a> were run on 12% SDS-PAGE gel. Separated proteins were transferred onto nitrocellulose membrane and stained with Ponceau S. <b>A:</b> Lane 1-3 shows loaded 0.25, 0.5, 1 µg of p50. <b>C:</b> Lane 1-3 shows loaded 0.125, 0.25, 0.5 µg (based on p50) of recombinant pol δ4. The membranes were then subjected to PCNA-DIG overlay assay as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027092#s2" target="_blank">Materials and Methods</a>”. The signals were detected by ECL and the membranes were exposed to the films. <b>B:</b> Exposure of the film for p50 which exactly corresponds to Ponceau S stained membrane of panel A. <b>D:</b> Exposure of the films for individual four subunits of pol δ, respectively, which exactly correspond to Ponceau S stained membrane of panel C. Exposure of the film for p50 in both panel B and panel D was slightly higher than those used for other subunits. Protein marker is indicated in kDa by “M”. Four subunits of pol δ are indicated by arrows.</p

Analysis for the preparation of the immunogen for the production of polyclonal antibody against small subunit p50 of human DNA pol δ.

<p><b>A:</b> Agarose gel electrophoresis. M, DNA marker in bp; Lane 1, a selected colony of pGEX-5X-3-p50 digested with <i>Bam</i>HI and <i>Eco</i>RI. <b>B:</b> Coomassie Blue stained SDS-PAGE analysis for the purification of GST-p50 protein on glutathione-Sepharose 4B column. The lysates (BC), flow-through (FT), and eluted fractions were analyzed on 12% SDS-PAGE followed by Coomassie Blue staining. Protein maker is indicated in lane M as kDa. The position of GST-p50 protein is indicated by an arrow. <b>C:</b> Coomassie Blue stained SDS-PAGE analysis for release of non-tagged p50 by Factor Xa. Lane 1-4: fractions of released p50; Lane 5: the beads containing GST and unreleased p50 after digestion reaction. <b>D:</b> Coomassie Blue stained SDS-PAGE analysis for the peak fractions of released p50 protein further purified on Mono Q column.</p

Analysis for the production of polyclonal antibody.

<p><b>A:</b> Coomassie Blue stained SDS-PAGE analysis for the purification of polyclonal antibody against p50 after affinity chromatography on a 5-ml Protein A/G Plus column. The dialyzed sample in PBS after ammonium sulfate precipitation (Bc), flow-through (Ft), wash (W₁ and W₂), and eluted fractions were analyzed on 12% SDS-PAGE followed by Coomassie Blue staining. Protein marker in kDa is indicated by “M”. The heavy and light chains are marked by arrows. <b>B:</b> Measurement of sensitivity and specificity of purified antibody by Western blotting with Hela cell extracts. The six lanes show decreasing concentrations of antibody contained 0.8, 0.4, 0.2, 0.1, 0.05 (lane 2-6), and 0 µg/ml (lane 1) of antibody/slice membrane, respectively. The detected endogenous p50 is marked by an arrow.</p

MALDI spectra of tryptic digestion of recombinant p50 subunit of human DNA pol δ.

<p>The identified protein, score, amino acid sequence coverage and the number of identified peptides are shown. The sequences of identified peptides shown in bold red covered 34% sequences against the deduced amino acid sequence of p50.</p