Dissecting the role of PCNA and Pif1 in replication of individual DNA molecules by DNA polymerase δ

PCNA loading and stabilization at primer-template junctions is crucial for processive DNA synthesis by replicative polymerases. It is also essential for strand displacement synthesis during Break Induced Replication (BIR). In this study, we employed a single molecule approach to directly visualize the role of PCNA, DNA polymerase δ and the Pif1 DNA helicase in these pathways. We first dissected the preferential loading of PCNA by RFC to 3'-junctions of gaps, nicks and flapped substrates. Moreover, we found that Pol δ and Pif1 both stabilize loaded PCNA and reduce its diffusion coefficient along DNA. We further show that the interaction of PCNA with Pol δ is essential for gap-filling synthesis. On a 5'-flapped substrate, we show that both Pol δ and Pif1 are required for strand displacement synthesis, while PCNA is not; although, it stimulates the process.