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

DNA polymerase (pol) delta is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol delta has been implicated in the Okazaki fragment maturation process for the extensio n 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 an nealed RNA/DNA primer, we could measure strand displacement by pol delta co upled to DNA synthesis. Our results suggested that pol delta alone can disp lace 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 delta, thus increasing the pr ocessivity of both synthesis and strand displacement, whereas replication p rotein A (RP-A) limited the size of the displaced fragment down to 20-30 nu cleotides, by generating a "locked" flap DNA structure, which was a substra te for processing of the displaced fragment by Fen 1 into a ligatable produ ct. Our data support a model for Okazaki fragment processing where the stra nd displacement activity of DNA polymerase delta is modulated by the concer ted action of PCNA, RP-A and Fen 1.