Figure 4. Pol δ’s Enzymatic Activity Regulates the Assembly and Disassembly Dynamics of the Holoenzyme.

(A) Schematic diagram of SNAP-Pol δ Dead p125 fusion protein (PIP, PCNA interacting protein). In this mutant, two essential active site residues that catalyze DNA synthesis (Asp602 and Asp757) and a residue essential for the 3′ –5′ exonuclease proofreading activity of Pol δ (Asp402) were mutated to alanine, resulting in a catalytically dead protein.

(B) Immunoblot of cell lysates of LOX cell lines stably expressing doxycycline-inducible SNAP-Pol δ Dead p125. The percentage of the total Pol δ p125 protein represented by SNAP-tagged (top band) and untagged endogenous (bottom band) proteins is shown below each lane.

(C) Growth curve of LOX stable cell lines co-expressing SNAP-Pol δ Dead p125 and Halo-PCNA proteins. Cultures of three clonal lines, C2, C5, and C7 (82%, 52%m and 20% of total Pol δ p125 is SNAP-tagged, respectively), were grown in the absence or presence (+ dox) of 1 μg/ml doxycycline and cells counted at time points indicated (cell number is relative to initial seeding). Values were obtained from two independent cultures. Error bars indicate SD.

(D) Histogram of the arrival and departure order of SNAP-Pol δ and Halo-PCNA molecules during 188 observed nuclear colocalization events (from SNAP-Pol δ Dead line C2). The percentage of SNAP-Pol δ and Halo-PCNA molecules arriving and departing first or at the same time are indicated. Error bars indicate SD as determined by Bootstrap analysis (1,000 iterations) of observed colocalizations. A 2-fold greater percentage of SNAP-Pol δ Dead molecules arrive before Halo-PCNA and depart after PCNA than SNAP-Pol δ WT.

(E) Scatterplots of SNAP-Pol δ arrival (binding) and departure (dissociation) relative to Halo-PCNA binding and dissociation during nuclear colocalization events. Timelines (blue and orange-brown bars) shown in each quadrant indicate the binding order of SNAP-Pol δ and Halo-PCNA and time interval between arrival and departure of Pol δ/PCNA, as described in Figure 3. The 95% confidence interval of median times is reported in Figure S2.

(F) Boxplots comparing the duration of Halo-PCNA colocalizations with SNAP-Pol δ WT (C11) versus SNAP-Pol δ Dead (C2). A total of 59 nuclei from SNAP-Pol δ WT cells from 4 independent experiments and 13 nuclei from SNAP-Pol δ Dead cells from 2 independent experiments were analyzed for colocalization. A Kruskal-Wallis test was used to determine pairwise significance. ****p = 2.21 × 10⁻⁶.