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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 98; no. 25; pp. 14298 - 14303
• Main Authors: Maga, Giovanni, Villani, Giuseppe, Tillement, Vanessa, Stucki, Manuel, Locatelli, Giada A., Frouin, Isabelle, Spadari, Silvio, Hübscher, Ulrich
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 04-12-2001; National Acad Sciences; The National Academy of Sciences
• Subjects: Animals; Base Sequence; Biochemistry; Biological Sciences; Cattle; Cell growth; Cell nucleus; DNA; DNA - metabolism; DNA Ligases - metabolism; DNA Polymerase III - metabolism; DNA Primers; DNA Replication; DNA-Binding Proteins - metabolism; Endodeoxyribonucleases - metabolism; Enzymes; Fens; flap endonuclease 1; Flap Endonucleases; Gels; Hybridity; In Vitro Techniques; Kinetics; Models, Biological; Nucleotides; pol^d protein; Proliferating Cell Nuclear Antigen - metabolism; Replication Protein A
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.251193198

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.