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

^*Istituto di Genetica Biochimica ed Evoluzionistica–Consiglio Nazionale delle Ricerche, I-27100 Pavia, Italy; ^§Department of Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, 8057 Zürich, Switzerland; and ^‡Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, 205 route de Narbonne, 31077 Toulouse Cedex, France

Edited by I. Robert Lehman, Stanford University School of Medicine, Stanford, CA, and approved September 25, 2001 (received for review April 19, 2001)

Abstract

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.

Footnotes

↵ † To whom reprint requests should be addressed at: Istituto di Genetica Biochimica ed Evoluzionistica–Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, I-27100 Pavia, Italy. E-mail: maga{at}igbe.pv.cnr.it.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

pol,

polymerase;

PCNA,

proliferating cell nuclear antigen;

RP-A,

replication protein A;

Fen 1,

flap endonuclease-1;

RF-C,

replication factor C;

ss,

single-stranded