Okazaki fragment processing: Modulation of the strand displacement activity of DNA polymerase 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 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 delta coupled to DNA synthesis. Our results suggested that pol delta 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 delta, 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 delta is modulated by the concerted action of PCNA, RP-A and Fen 1. Okazaki fragment processing: Modulation of the strand displacement activity of DNA polymerase by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1 Giovanni Maga*†, Giuseppe Villani‡, Vanessa Tillement‡, Manuel Stucki§, Giada A. Locatelli*, Isabelle Frouin*, Silvio Spadari*, and Ulrich Hübscher‡ *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) DNA polymerase (pol) is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol 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 coupled to DNA synthesis. Our results suggested that pol 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 , 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 is modulated by the concerted action of PCNA, RP-A and Fen 1. D polymerase (pol) is the major pol involved in chromosomal replication in eukaryotes (reviewed in ref. 1). It is essential for both leading and lagging strand synthesis in the in vitro reconstituted SV40 replication system (2) and in eukaryotic cells. The current view of DNA replication in eukaryotes predicts that pol primase synthesizes the first RNA DNA primer on the leading strand and at each Okazaki fragment on the lagging strand. Then replication factor C (RF-C) binds to the 3 -OH end of the nascent DNA strand and loads proliferating cell nuclear antigen (PCNA), thereby displacing pol (3). The displacement of pol occurs after 30 nt (4), likely by RF-C action (5). Next, pol is attracted and this event is called DNA polymerase switch. Beside its role in DNA replication, pol has been shown to be involved in DNA repair processes such as long patch base excision repair, nucleotide excision repair, and mismatch repair (reviewed in refs. 6 and 7). Pol is a heteromultimeric enzyme composed of one major catalytic subunit of 125 kDa and three small accessory subunits (8); by itself pol is a poorly processive enzyme due to its unstable interaction with the DNA substrate, but a physical interaction with the processivity factor PCNA leads to a catalytically competent processive holoenzyme (9). The consequence of such an interaction is a marked increase in the processivity (10). Pol has intrinsic DNA polymerization and 3 3 5 exonucleolytic activities. In addition, a limited strand displacement activity has been described for the pol PCNA RF-C holoenzyme on a gapped M13 DNA template (11). Intrinsic strand-displacement activity of pols has been inversely correlated to their tendency to perform template slippage during replication (12). However, in the case of pol , it is not known whether the observed strand displacement activity depends on PCNA RF-C or it is an intrinsic property of the enzyme. The endonuclease flap endonuclease-1 (Fen 1) has been shown to interact with PCNA (13, 14) and has been proposed to work in a coordinated fashion with pol , replication protein A (RP-A), and DNA ligase 1 in Okazaki fragment maturation (15). In the current model, RNase H is proposed to cut and degrade the initiator RNA of the primer DNA, leaving a single ribonucleotide at the RNA–DNA junction that is subsequently removed by the 5 3 3 exonuclease activity of Fen 1 (16–19). In mammalian cells, pol , RP-A, PCNA, RF-C, RNase H, Fen 1, and DNA ligase I are necessary and sufficient to reconstitute lagging strand synthesis in vitro (20). Fen 1 contains a 5 3 3 exonuclease activity and a structure-specific endonuclease activity that cleaves the 5 unannealed singlestranded (ss)-DNA or RNA at the duplex junction (21, 22). In addition, mammalian RNase H can cleave 5 of the last ribonucleotide of ssRNA–DNA hybrid molecules (23). These findings suggest that Okazaki fragment maturation is likely to require formation of a 5 tail before the action of Fen 1 and or RNase H (24). Recently, it has been shown that the helicase endonuclease Dna2 in yeast cells interacts with Fen 1 and can substitute for RNase H, leading to a novel model for Okazaki fragment processing, involving the sequential action of Fen 1 and Dna2 enzymes for the removal of primer RNA and DNA (15, 22). Interestingly, in this model the action of pol is not only critical for the extension of the newly synthesized Okazaki fragment, but also for the displacement of the RNA segment of the preexisting downstream Okazaki fragment, thus creating an intermediate flap structure that is the target for the subsequent concerted action of Dna2 and Fen 1. This process could also have the advantage of removing entirely the RNA–DNA hybrid 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. †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: [email protected]. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact. 14298–14303 PNAS December 4, 2001 vol. 98 no. 25 www.pnas.org cgi doi 10.1073 pnas.251193198 fragment synthesized by pol primase, potentially containing nucleotide misincorporations due to the lack of a proofreading exonuclease activity of pol primase, and substituting it with a more accurate copy synthesized by pol . In this paper we have measured the strand displacement by pol coupled to DNA synthesis, using a single-stranded minicircular template with an annealed primer. We found that pol can synthesize through a double-stranded DNA. PCNA and RP-A can control the strand displacement activity of pol by either enhancing the processivity (PCNA) or by limiting the size of the displaced fragment down to 20–30 nt (RP-A). In addition, Fen 1 can exactly process the displaced fragment in the presence of RP-A into a ligatable product. Based on these results, we propose a model in which the concerted action of RP-A, PCNA, and Fen 1 can modulate the strand displacement activity of pol . Materials and Methods Chemicals. [ -32P]dCTP (3,000 Ci mmol; 1 Ci 37 GBq) and [ -32P]ATP (3,000 Ci mmol) were from Amersham Pharmacia. Unlabeled dNTPs and poly(dA) oligo(dT)10:1 were from Amersham Pharmacia. All other reagents were of analytical grade and purchased from Merck or Fluka. T4 DNA kinase and T4 DNA ligase were from New England Biolabs Construction of the Minicircle Template-Primer. The sequence of the 72-mer template was: 5 -CTTCTAGTTGTGAATTCGGCACTGGCCGTCGTATGCTCTTGGTTGTAGGATCCCAGCACATTGAAGGATGCA-3 . Bold letters indicated the sequence annealed to the 17-mer primer. The 72-mer primer was converted into a single-stranded minicircle as described by using a 39-mer scaffold oligonucleotide (5 -CAGTGCCGAATTCACAACTAGAAGTGCATCCTTCAATGT-3 ) complementary to the first 5 -end 24 bases and the last 3 -end 15 bases of the 72-mer primer (25). The 72-mer primer was first phosphorylated by using T4 polynucleotide kinase followed by annealing with the 39-mer bridging oligonucleotide that brought together the ends of the 72-mer primer for subsequent ligation using T4 DNA ligase. The 72-mer primer minicircle was then purified from a 10% polyacrylamide 7 M urea gel. After elution from the gel and ethanol precipitation, its concentration was determined spectrophotometrically. The annealing mixture, in a final volume of 10 l, contained 20 mM Tris HCl (pH 7.5) and 150 mM NaCl, 0.5 pmol of minicircle, and 0.8 pmol of linear 17-mer DNA primer [5 -d(CATACGACGGCCAGTGC)-3 ] or the RNA DNA 27-mer hybrid [5 -r(CAACCAAGAG)d(CATACGACGGCCAGTGC)-3 ]. The mixture was heated to 98°C for 3 min and then left to slowly cool down to room temperature. When indicated, the 17-mer primer was 5 -end-labeled before annealing with [ -32P]ATP and polynucleotide kinase, according to the manufacturer’s protocol (New England Biolabs). Enzymes and Proteins. Calf thymus pol and pol were purified as described (26). The pol used in this study was 2,200 units ml (0.08 mg/ml 27,500 units/mg), and pol was 580 units ml. 1 unit of pol activity corresponds to the incorporation of 1 nmol of total dTMP into acid-precipitable material for 60 min at 37°C in a standard assay containing 0.5 g (nucleotides) of poly(dA) oligo(dT)10:1 and 20 M dTTP. Recombinant human wt PCNA was prepared as described (27). Recombinant human RP-A and Fen 1 were isolated as described (28, 29). Mutants Fen 1 D86A and Fen 1 C (lacking amino acid 360–380), were prepared as described (29). Human RF-C was purified from HeLa cells nuclei according to ref. 30. Enzymatic Assays. Pol activity was assayed on poly(dA) oligo(dT) as described (30, 31). For studies with the singly primed minicircular d17:d72 oligodeoxynucleotide as template, a final volume of 10 l contained 50 mM Tris HCl (pH 7.6), 0.25 mg ml BSA, 1 mM DTT, 6 mM MgCl2, 12 nM (3 -OH ends) of primed minicircular DNA template, 1 M [ -32P]dCTP (3,000 Ci mmol), and 50 M unlabeled dATP, dGTP, and dTTP. When the minicircular DNA template was used with a 5 -32Plabeled d17 primer, the reaction conditions were the same as above, except that 50 M of all four unlabeled dNTPs were added. For single-turnover experiments, 600 nM (3 -OH ends) of poly(dA) oligo(dT)10:1 was added as the trapping agent. Enzymes and proteins were added as indicated in the figure legends. All reactions were incubated for 15 min at 37°C, unless otherwise stated in the figure legends, and samples were mixed with denaturing gel loading buffer (95% vol/vol Formamide 10 mM EDTA 0.25 mg/ml bromophenol blue 0.25 mg/ml xylene cyanol), heated at 95°C for 5 min, and then subjected to electrophoresis on a 7 M Urea 14% polyacrylamide gel. Quantification of the reaction products on the gel was performed by using a Molecular Dynamics PhosphoImager and IMAGE QUANT software. Kinetic Parameters Calculations. The kburst (kb) and ksteady-state (kss) values for time-dependent nucleotide incorporation by pol on the minicircular template were determined according to the exponential equation: products A 1 e kbt ksst, where A is the burst amplitude and t is time.