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

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) eta and kappa replicate CFS-derived sequences more efficiently than the replicative Pol delta. 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 delta holoenzyme, Pol eta, and Pol kappa in polymerase mixing assays. We discovered that Pol eta and kappa are both able to exchange with Pol delta 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 delta holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol eta and kappa were still proficient in rescuing this stalled Pol delta synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol eta and Pol kappa-deficient cells. In total, our data support a model wherein Pol delta stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA.