Okazaki fragment processing: Modulation of the strand displacement activity of DNA polymerase δ by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1

DNA polymerase (pol) δ is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol δ has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA primer, we could measure strand displacement by pol δ coupled to DNA synthesis. Our results suggested that pol δ alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol δ, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20–30 nucleotides, by generating a “locked” flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase δ is modulated by the concerted action of PCNA, RP-A and Fen 1.