DNA polymerases eta and kappa exchange with the polymerase delta holoenzyme to complete common fragile site synthesis

Abstract

Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA.

Keywords: AT microsatellite; DNA polymerase δ; DNA polymerase η; DNA polymerase κ; PCNA; TLS.

Fig. 1. Plasmids Containing Repetitive Sequences From FRA16D and FRA3B Adopt Non-B DNA Structures

(A) Representative agarose gel of CFS plasmids treated with S1 nuclease for 1–5 minutes. C = plasmid incubated in reaction buffer at 37 °C for 5 minutes without S1 nuclease. See Table 1 for description of CFS insert sequences. Plasmid pJY9 encodes a [TTTC/AAAG]₉ microsatellite with H-DNA potential (29) serves as a positive control. (B) The nicked and linear forms for each plasmid at each timepoint were quantified relative to the supercoiled band.

Fig. 2. Pols η and κ Rescue of Stalled Pol δHE Synthesis within CFS sequences

(A) Schematic of PCNA pre-loading and dual polymerase experiments (see text Materials and Methods for details). (B) Mono-polymerase reactions were conducted on FRA16D Control template to establish equal activity conditions. (C) Representative gel of dual polymerase reaction products on FRA16D AT1 with (+Loaded PCNA) or without (-Loaded PCNA) RFC in the preloading reaction. “+” lane is a hybridization control reaction using Klenow Fragment; “−” lane is a negative control lane with no polymerase added. (D) Quantification of Normalized Replication Efficiency (NRE) for N≥3 independent preloading and polymerase reactions. The % transit is quantified as ([products in 3′ region] ÷ [total products CFS + 3′ regions]) x100%. NRE=%Transit (T₂) minus % Transit (T₁), and directly compares the dual polymerase reaction efficiencies by accounting for variability during the pre-loading and T₁ reactions. Individual data points correspond to independent experiments. (E) %Transit quantification for the Pol δ+δ reactions with of without Loaded PCNA. Data are the mean ± SEM, and were analyzed by ANOVA (D) or one-tailed T-Test (E). Significance relative to δ+δ control indicated by asterisks: * = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001.

Fig. 3. Pol η Errors are Enriched within CFS-derived Repeat Sequences

(A) FRA16D IR1 sequence (black) with errors made by Pol η alone (above, Red) or Pol δ+η (below, Blue). Repetitive elements highlighted yellow. (B): Analysis of errors created during the indicated polymerase reactions and distribution on the template sequence.

Fig. 4. Rescue of Stalled Pol δHE is Independent of PCNA

(A) Representative gels of dual polymerase reaction products on FRA16D IR1 with Loaded unmodified PCNA, Ub-PCNA, or without Loaded PCNA. (B) Quantification of NRE for the Loaded PCNA and Ub-PCNA reactions. Asterisk (*) above columns indicate statistical significance relative to Pol δ+δ control reactions. (C) Comparison of %Transit values for each dual polymerase reaction with or without Loaded PCNA on the IR1 template. (D) Representative gel of dual polymerase reaction products on AT3 with Loaded PCNA or Ub-PCNA. (E) Quantification of NRE for the Loaded PCNA and Ub-PCNA reactions. Data represent the mean of N≥3 reactions with individual points plotted. All data were analyzed by one- or two-way ANOVA with Tukey’s post-hoc.

Fig. 5. Pol η CFS Synthesis Can be Stimulated by PCNA

(A) Representative gels of Pol η reactions performed in high salt buffer to detect stimulation using the AT1 template. All reactions contained Pol η and additional protein(s) as indicated. (B, C) Quantitation of %Transit and Primer Extension at the 15 minute timepoint. Data are the mean of N≥3 ± SEM, with individual reaction points shown, and were analyzed by one-way ANOVA.

Fig. 6. Pols η and κ are Indispensable for CFS Replication in the Presence of Aph

(A) Top: Representative gel of synthesis products for each polymerase with increasing amounts of Aph (control template). PCNA was preloaded as in Fig 5A. Bottom: Quantitation of %Transit relative to EtOH solvent (N=3). (B, C) Top: Representative gels of dual polymerase reactions with Aph added with the second polymerase (after T1). Bottom: NRE (mean ± SEM) for N>3 independent experiments. # = statistical significance relative to EtOH; *, statistical significance relative to δ+δ.