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• W2094680135 abstract "Two pathways have been proposed for eukaryotic Okazaki fragment RNA primer removal. Results presented here provide evidence for an alternative pathway. Primer extension by DNA polymerase δ (pol δ) displaces the downstream fragment into an RNA-initiated flap. Most flaps are cleaved by flap endonuclease 1 (FEN1) while short, and the remaining nicks joined in the first pathway. A small fraction escapes immediate FEN1 cleavage and is further lengthened by Pif1 helicase. Long flaps are bound by replication protein A (RPA), which inhibits FEN1. In the second pathway, Dna2 nuclease cleaves an RPA-bound flap and displaces RPA, leaving a short flap for FEN1. Pif1 flap lengthening creates a requirement for Dna2. This relationship should not have evolved unless Pif1 had an important role in fragment processing. In this study, biochemical reconstitution experiments were used to gain insight into this role. Pif1 did not promote synthesis through GC-rich sequences, which impede strand displacement. Pif1 was also unable to open fold-back flaps that are immune to cleavage by either FEN1 or Dna2 and cannot be bound by RPA. However, Pif1 working with pol δ readily unwound a full-length Okazaki fragment initiated by a fold-back flap. Additionally, a fold-back in the template slowed pol δ synthesis, so that the fragment could be removed before ligation to the lagging strand. These results suggest an alternative pathway in which Pif1 removes Okazaki fragments initiated by fold-back flaps in vivo. Two pathways have been proposed for eukaryotic Okazaki fragment RNA primer removal. Results presented here provide evidence for an alternative pathway. Primer extension by DNA polymerase δ (pol δ) displaces the downstream fragment into an RNA-initiated flap. Most flaps are cleaved by flap endonuclease 1 (FEN1) while short, and the remaining nicks joined in the first pathway. A small fraction escapes immediate FEN1 cleavage and is further lengthened by Pif1 helicase. Long flaps are bound by replication protein A (RPA), which inhibits FEN1. In the second pathway, Dna2 nuclease cleaves an RPA-bound flap and displaces RPA, leaving a short flap for FEN1. Pif1 flap lengthening creates a requirement for Dna2. This relationship should not have evolved unless Pif1 had an important role in fragment processing. In this study, biochemical reconstitution experiments were used to gain insight into this role. Pif1 did not promote synthesis through GC-rich sequences, which impede strand displacement. Pif1 was also unable to open fold-back flaps that are immune to cleavage by either FEN1 or Dna2 and cannot be bound by RPA. However, Pif1 working with pol δ readily unwound a full-length Okazaki fragment initiated by a fold-back flap. Additionally, a fold-back in the template slowed pol δ synthesis, so that the fragment could be removed before ligation to the lagging strand. These results suggest an alternative pathway in which Pif1 removes Okazaki fragments initiated by fold-back flaps in vivo. During eukaryotic DNA replication, the lagging strand is synthesized in a series of segments, each ∼150 nucleotides (nt) 2The abbreviations used are: ntnucleotidepol αDNA polymerase α/primasePCNAproliferating cell nuclear antigenRFCreplication factor Cpol δDNA polymerase δLigIDNA ligase IFEN1flap endonuclease 1RPAreplication protein A. long, called Okazaki fragments (1.Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman & Co., New York1992: 113-195Google Scholar). An Okazaki fragment is initiated by DNA polymerase α/primase (pol α), which synthesizes a primer beginning with 10–12 nt of RNA followed by ∼20 nt of DNA (2.Bambara R.A. Murante R.S. Henricksen L.A. J. Biol. Chem. 1997; 272: 4647-4650Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). After primer synthesis, the sliding clamp proliferating cell nuclear antigen (PCNA) is loaded on the primer-template DNA by replication factor C (RFC). DNA polymerase δ (pol δ) then conjugates with PCNA and continues rapid and efficient extension of the primer. Upon reaching the downstream Okazaki fragment, pol δ displaces its 5′ end into a single-stranded flap that must be removed by nucleases (3.Liu Y. Kao H.I. Bambara R.A. Annu. Rev. Biochem. 2004; 73: 589-615Crossref PubMed Scopus (306) Google Scholar, 4.Rossi M.L. Purohit V. Brandt P.D. Bambara R.A. Chem. Rev. 2006; 106: 453-473Crossref PubMed Scopus (55) Google Scholar). Cleavage of the flap produces a nick that DNA ligase I (LigI) will seal to complete the continuous DNA strand. nucleotide DNA polymerase α/primase proliferating cell nuclear antigen replication factor C DNA polymerase δ DNA ligase I flap endonuclease 1 replication protein A. Two pathways are proposed to process Okazaki flaps. In the first pathway, only one nuclease, flap endonuclease I (FEN1), is employed. In reconstitution studies, pol δ displaces short flaps, ∼1–5 nt long, that are efficiently cleaved by FEN1 to produce a nicked intermediate (5.Ayyagari R. Gomes X.V. Gordenin D.A. Burgers P.M. J. Biol. Chem. 2003; 278: 1618-1625Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 6.Garg P. Stith C.M. Sabouri N. Johansson E. Burgers P.M. Genes Dev. 2004; 18: 2764-2773Crossref PubMed Scopus (175) Google Scholar, 7.Jin Y.H. Ayyagari R. Resnick M.A. Gordenin D.A. Burgers P.M. J. Biol. Chem. 2003; 278: 1626-1633Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). FEN1 binds the 5′ end of the flap, tracks down the flap, and cleaves once at the base (8.Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (373) Google Scholar, 9.Murante R.S. Rust L. Bambara R.A. J. Biol. Chem. 1995; 270: 30377-30383Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). PCNA binds and stimulates both pol δ and FEN1, allowing for tight coordination between flap displacement and cleavage (10.Kao H.I. Bambara R.A. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 433-452Crossref PubMed Scopus (77) Google Scholar). This cooperation keeps flaps short, and the FEN1-only pathway has the potential to process virtually all flaps. However, reconstitutions have shown that some flaps can escape immediate cleavage and become long (11.Rossi M.L. Pike J.E. Wang W. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2008; 283: 27483-27493Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12.Rossi M.L. Bambara R.A. J. Biol. Chem. 2006; 281: 26051-26061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 13.Pike J.E. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2009; 284: 25170-25180Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). When flaps become ∼25–30 nt long, the eukaryotic single strand-binding protein replication protein A (RPA) can bind the flap stably (14.Fanning E. Klimovich V. Nager A.R. Nucleic Acids Res. 2006; 34: 4126-4137Crossref PubMed Scopus (405) Google Scholar). RPA binding inhibits FEN1 cleavage (15.Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar), necessitating the second pathway. This second, or two-nuclease, pathway was proposed to utilize Dna2 in addition to FEN1 to process long RPA-bound flaps (15.Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar). Dna2 displays both 5′–3′ helicase and endonuclease activities (16.Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 17.Budd M.E. Choe W. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 18.Bae S.H. Choi E. Lee K.H. Park J.S. Lee S.H. Seo Y.S. J. Biol. Chem. 1998; 273: 26880-26890Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Dna2, like FEN1, cleaves a 5′ flap structure by binding the 5′ end and tracking toward the base (19.Kao H.I. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 50840-50849Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). However, Dna2 cleaves multiple times before approaching the base, finally leaving a short flap of ∼5–10 nt (20.Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Dna2 is capable of cleaving an RPA-bound flap by displacing the RPA as it tracks (15.Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar, 21.Stewart J.A. Miller A.S. Campbell J.L. Bambara R.A. J. Biol. Chem. 2008; 283: 31356-31365Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Dna2 cleavage ultimately produces a short flap that RPA can no longer bind. FEN1 will then complete cleavage of the short flap, leaving a nick to be sealed by LigI. The importance of Dna2 cleavage is highlighted by the observation that Dna2 nuclease is essential in Saccharomyces cerevisiae (17.Budd M.E. Choe W. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 22.Lee K.H. Kim D.W. Bae S.H. Kim J.A. Ryu G.H. Kwon Y.N. Kim K.A. Koo H.S. Seo Y.S. Nucleic Acids Res. 2000; 28: 2873-2881Crossref PubMed Scopus (71) Google Scholar). In the absence of Dna2, it is likely that long RPA-bound flaps cannot be properly processed, leading to genomic instability and cell death. Genetic evidence suggests that Pif1 helicase influences the pathway chosen for flap processing by lengthening displaced flaps. Deletion of PIF1 rescues the lethality of dna2Δ in S. cerevisiae (23.Budd M.E. Reis C.C. Smith S. Myung K. Campbell J.L. Mol. Cell. Biol. 2006; 26: 2490-2500Crossref PubMed Scopus (167) Google Scholar, 24.Stith C.M. Sterling J. Resnick M.A. Gordenin D.A. Burgers P.M. J. Biol. Chem. 2008; 283: 34129-34140Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), suggesting that in the absence of Pif1, flaps do not become long enough to require cleavage by Dna2. Our biochemical studies support this conclusion (11.Rossi M.L. Pike J.E. Wang W. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2008; 283: 27483-27493Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 13.Pike J.E. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2009; 284: 25170-25180Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Using an Okazaki fragment processing reconstitution system, we showed that in the absence of Pif1 virtually all flaps remain too short for RPA to bind. When Pif1 is included, longer flaps are created, and their cleavage is inhibited by RPA (11.Rossi M.L. Pike J.E. Wang W. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2008; 283: 27483-27493Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Cleavage of these flaps and ligation of the nicked intermediates requires Dna2, demonstrating that Pif1 directs flaps to the two-nuclease pathway (13.Pike J.E. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2009; 284: 25170-25180Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Additionally, Pif1 stimulates strand displacement synthesis by pol δ, further supporting the hypothesis that Pif1 binds short flaps as they are displaced and lengthens them (13.Pike J.E. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2009; 284: 25170-25180Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The small portion of flaps lengthened by Pif1 implies that Pif1 binds flaps that escape immediate FEN1 cleavage. The precise role Pif1 plays in Okazaki fragment processing remains unknown. If virtually all flaps are capable of being processed by the FEN1-only pathway in the absence of Pif1 (11.Rossi M.L. Pike J.E. Wang W. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2008; 283: 27483-27493Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), then Pif1 activity at Okazaki fragments merely promotes inefficient energy use by requiring the action of Dna2. It is reasonable to assume that if Pif1 were not important for proper Okazaki fragment processing, evolution would have driven Pif1 to localize solely to telomeres and mitochondria, where it plays important roles in limiting telomere growth and maintaining mitochondrial DNA stability (25.Boulé J.B. Zakian V.A. Nucleic Acids Res. 2006; 34: 4147-4153Crossref PubMed Scopus (104) Google Scholar). Therefore, Pif1 likely also plays a biologically important role at Okazaki fragments. We considered the possibility that Pif1 is required for efficient synthesis and flap processing at specific sequences, such as regions of high GC content or having the potential to form fold-back flaps. pol δ does not displace through a sequence of high GC content as well as through a sequence of comparatively low GC content (12.Rossi M.L. Bambara R.A. J. Biol. Chem. 2006; 281: 26051-26061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), presumably because stable hydrogen bonding produces an energy barrier to strand separation. We considered that helicase activity of Pif1 might permit more rapid synthesis through such sequences. Pif1 is known to efficiently unwind G-quadruplexes (26.Ribeyre C. Lopes J. Boulé J.B. Piazza A. Guédin A. Zakian V.A. Mergny J.L. Nicolas A. PLoS Genet. 2009; 5: e1000475Crossref PubMed Scopus (274) Google Scholar), consistent with an ability to destabilize structures that might produce barriers to primer extension. Observations in vivo support this interpretation, as Pif1 is important in maintaining genomic stability at loci likely to form G-quadruplexes (26.Ribeyre C. Lopes J. Boulé J.B. Piazza A. Guédin A. Zakian V.A. Mergny J.L. Nicolas A. PLoS Genet. 2009; 5: e1000475Crossref PubMed Scopus (274) Google Scholar). We also anticipated that Pif1 would be needed for replication of sequences that have the potential to form stable fold-back flaps. Neither FEN1 nor Dna2 can cleave such flaps (20.Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). If the fold-back is relatively weak and initiated with a 5′ single-stranded tail, Dna2 helicase activity can unwind the fold-back and allow cleavage of the flap. However, if no tail is present or the structure is very stable, Dna2 will not be able to enter to affect cleavage. Additionally, RPA strand melting activity can unwind weak structure in flaps and permit Dna2 cleavage, but RPA is unable to unwind flaps with strong secondary structure (21.Stewart J.A. Miller A.S. Campbell J.L. Bambara R.A. J. Biol. Chem. 2008; 283: 31356-31365Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 27.Bartos J.D. Willmott L.J. Binz S.K. Wold M.S. Bambara R.A. J. Biol. Chem. 2008; 283: 21758-21768Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Thus, flaps that form strong fold-backs, likely to occur in certain sequences such as triplet repeat regions, cannot be processed by either pathway. We considered that Pif1 might unwind such flaps and permit FEN1 cleavage, Dna2 cleavage, or RPA binding. In this study, we examined potential biologically important roles for Pif1 in Okazaki fragment processing. We first examined possible stimulation of synthesis through a GC-rich sequence. Next, we asked whether Pif1 is capable of unwinding a fold-back flap and allowing FEN1 or Dna2 to cleave or RPA to bind. Finally, we used a reconstitution system to examine the effect of Pif1 on strand displacement synthesis through an Okazaki fragment initiated with a stable fold-back flap. Our results provide evidence for an alternative Okazaki fragment-processing pathway, in which Pif1 promotes removal of an entire fragment initiated by a fold-back flap from the template DNA. Radioactive nucleotides [γ-32P]ATP and [α-32P]dCTP were obtained from PerkinElmer Life Sciences. Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA) or Midland Certified Reagents Co. (Midland, TX). The primers and their sequences are listed in Table 1. Streptavidin, Escherichia coli DNA polymerase I Klenow fragment, and polynucleotide kinase were obtained from Roche Applied Science. Other reagents were the best grade commercially available.TABLE 1Oligonucleotide sequencesa Underline indicates a nucleotide with a 3′-phosphate.b Boldface indicates fold-back region.c Underline and italics indicates a nucleotide with a 5′-phosphate.d Templates T1, T2, T3, and T4 are biotinylated at both the 5′ and 3′ ends. a Underline indicates a nucleotide with a 3′-phosphate. b Boldface indicates fold-back region. c Underline and italics indicates a nucleotide with a 5′-phosphate. d Templates T1, T2, T3, and T4 are biotinylated at both the 5′ and 3′ ends. S. cerevisiae pol δ (28.Burgers P.M. Gerik K.J. J. Biol. Chem. 1998; 273: 19756-19762Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) and LigI (5.Ayyagari R. Gomes X.V. Gordenin D.A. Burgers P.M. J. Biol. Chem. 2003; 278: 1618-1625Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) were overexpressed in S. cerevisiae and purified as described previously. S. cerevisiae PCNA (12.Rossi M.L. Bambara R.A. J. Biol. Chem. 2006; 281: 26051-26061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), RFC (29.Gerik K.J. Gary S.L. Burgers P.M. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), FEN1 (30.Kao H.I. Henricksen L.A. Liu Y. Bambara R.A. J. Biol. Chem. 2002; 277: 14379-14389Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), RPA (31.Sibenaller Z.A. Sorensen B.R. Wold M.S. Biochemistry. 1998; 37: 12496-12506Crossref PubMed Scopus (66) Google Scholar), and Pif1 and helicase-deficient Pif1 K264A (11.Rossi M.L. Pike J.E. Wang W. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2008; 283: 27483-27493Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) were overexpressed in E. coli and purified as described previously. PCNA and FEN1 recombinant proteins had C-terminal His6 tags. Pif1 recombinant protein had an N-terminal His6 tag. S. cerevisiae Dna2 was overexpressed and purified from baculovirus High Five cells as described previously (17.Budd M.E. Choe W. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Substrates composed of the oligonucleotides listed in Table 1 were designed to simulate intermediates of Okazaki fragment processing. The 5′ ends of primers U2, U3, and I1 were radiolabeled with [γ-32P]ATP using polynucleotide kinase. Primers D1, D4, D5, D6, D7, D8, D9, D10, D11, or D12 were annealed at the 3′ end to a 20-nt labeling template with a 5′-GCTA overhang and radiolabeled with [α-32P]dCTP using Klenow polymerase. The radiolabeled primers were separated by running the reactions on a 15%, 7 m urea polyacrylamide gel. The radiolabeled products were then gel-purified. To anneal substrates, component oligonucleotides were mixed in annealing buffer (50 mm Tris-HCl, pH 8.0, 50 mm NaCl, 1 mm dithiothreitol), heated at 95 °C for 5 min, adjusted to 70 °C, and slowly cooled to room temperature. When the upstream primer was labeled, the oligonucleotides were annealed at a 1:2:4 ratio of upstream primer to template to downstream primer. When the downstream primer was labeled, the oligonucleotides were annealed at a 1:2:4 ratio of downstream primer to template to upstream primer. When the internal primer was labeled, the oligonucleotides were annealed at a 1:2:4:4 ratio of internal primer to template to upstream primer to downstream primer. Four sets of substrates were used in the following experiments. The first set consisted of 10 fixed flap configurations that were designed to examine Pif1 helicase activity and Pif1 stimulation of FEN1 cleavage, Dna2 cleavage, and RPA binding of fixed fold-back flaps with certain structural elements. The first fixed flap substrate had a 30-nt unstructured control flap and consisted of a 71-nt upstream primer (U1) and a 70-nt downstream primer (D1) annealed to a 110-nt template (T1). This will be referred to as the 30-nt flap substrate in the text. The next three fixed flap substrates had an 18-, 15-, or 12-nt fold-back flap with a 12-nt 5′ tail and a 6-nt gap between the fold-back and the downstream annealed region. These consisted of a 71-nt upstream primer (U1) and a 100-, 94-, or 88-nt downstream primer (D4, D5, or D6, respectively) annealed to a 110-nt template (T1). In the text, these will be referred to as the 18-, 15-, and 12-nt fold-back flap substrates, respectively. The next three flap substrates were identical to the fold-back flap substrates but lacked the 6-nt gap between the fold-back and the downstream annealed region. These consisted of a 71-nt upstream primer (U1) and a 94-, 88-, or 82-nt downstream primer (D7, D8, or D9, respectively) annealed to a 110-nt template (T1). In the text, these will be referred to as the 18-, 15-, and 12-nt fold-back –G flap substrates, respectively. The final three flap substrates were identical to the fold-back flap substrates but lacked both the 12-nt 5′ tail and the 6-nt gap between the fold-back and downstream annealed region. These consisted of a 71-nt upstream primer (U1) and an 82-, 76-, or 70-nt downstream primer (D10, D11, or D12, respectively) annealed to a 110-nt template (T1). In the text, these will be referred to as the 18-, 15-, and 12-nt fold-back –G–T flap substrates, respectively. The second set of substrates was designed to examine the effect of Pif1 on pol δ strand displacement synthesis. Both substrates in this set were identical in structure but different in sequence. The first substrate consisted of a 44-nt upstream primer (U2) and a 60-nt downstream primer (D2) annealed to a 110-nt template (T1), leaving a 6-nt gap between the upstream and downstream primers. This substrate has been used in previous reconstitution experiments (11.Rossi M.L. Pike J.E. Wang W. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2008; 283: 27483-27493Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 13.Pike J.E. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2009; 284: 25170-25180Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and will be referred to as the standard-44 substrate in the text. The second substrate consisted of the same 44-nt upstream primer (U2) and a different 60-nt downstream primer (D3) annealed to a different 110-nt template (T2), also leaving a 6-nt gap between the upstream and downstream primers. This substrate will be referred to as the GC-44 substrate in the text. The downstream annealed region of the GC-44 substrate was identical in nucleotide composition to that of the standard-44 substrate but different in sequence. The first 12 nts of the GC-44 downstream annealed region were 75% GC as opposed to 50% in the standard-44 substrate. The third single-member substrate set was designed to examine strand displacement synthesis through, and cleavage and ligation of, an Okazaki fragment initiated by a pre-created 18-nt fold-back flap. The substrate consisted of a 25-nt upstream primer (U3), a 100-nt internal primer (I1), and a 30-nt downstream primer (D13) annealed to a 110-nt template (T1), forming a 2-nt gap between the upstream and internal primers and a nick between the internal and downstream primers. The internal primer approximated a full-length Okazaki fragment, and the upstream and downstream primers represent the adjacent fragments. This substrate will be referred to as the internal 18-nt fold-back substrate. The final single-member substrate set was designed to examine synthesis through a fold-back in the template DNA. The substrate consisted of a 44-nt upstream primer (U2) annealed to the 3′ end of a 110-nt template (T3). The template has an 18-nt fold-back 10 nt downstream of the upstream primer. This substrate will be referred to as the template fold-back substrate. The standard-44 substrate lacking the downstream primer was used as an unstructured control. Five fmol of radiolabeled biotinylated substrate were first incubated on ice with 500 fmol of streptavidin for 20 min. Streptavidin complexes with biotin on the template ends, blocking the substrate ends and requiring that RFC loads PCNA. For simplicity, the blocked ends are not depicted in the figures. Streptavidin-conjugated substrate was then incubated with various combinations and amounts of pol δ, PCNA, RFC, FEN1, Dna2, RPA, LigI, and Pif1 for 10 min at 30 °C in 20 μl of reconstitution buffer (50 mm Tris-HCl, pH 7.5, 2 mm dithiothreitol, 25 μg/ml bovine serum albumin, 50 μm dNTPs, 1 mm ATP, 4 mm MgCl2, and 75 mm NaCl). Reactions were stopped with 20 μl of 2× termination dye (90% formamide (v/v), 10 mm EDTA, 0.01% bromphenol blue, and 0.01% xylene cyanole), followed by heating for 5 min at 95 °C. Reaction products were separated by electrophoresis on a 22.5%, 7 m urea polyacrylamide gel for 1 h and 30 min at 80 watts. The gel was dried and exposed to a phosphor screen, which was scanned with a GE Healthcare PhosphorImager and analyzed using ImageQuant version 1.2 software. For the kinetic experiment shown in Fig. 5, the reactions were initiated in a total of 120 μl of reconstitution buffer and at given time points (0, 0.5, 1, 2.5, 5, and 10 min), a 20-μl sample was removed from each reaction, added to 20 μl of 2× termination dye, and heated for 5 min at 95 °C. Products were then separated by electrophoresis and analyzed as described above. For the strand displacement-coupled cleavage assay shown in Fig. 4A, reactions were run and analyzed as described above. For fixed fold-back flap cleavage assays, 5 fmol of radiolabeled substrate were incubated with either FEN1 or Dna2 and various amounts of Pif1 for 10 min at 30 °C in 20 μl of reaction buffer (same as reconstitution buffer described above but without dNTPs). Reactions were stopped, separated by electrophoresis, and analyzed as described above. Five fmol of radiolabeled substrate were incubated with RPA and various amounts of Pif1 for 10 min at 30 °C in 20 μl of reaction buffer. Reaction samples were loaded onto a 12% native polyacrylamide gel, and products were separated by electrophoresis for 2 h at 250 V. The gel was dried, scanned, and analyzed as described above. Five fmol of radiolabeled substrate were incubated with either various amounts of Pif1 or pol δ, PCNA, and RFC with increasing amounts of Pif1 for 10 min at 30 °C in 20 μl of reaction buffer. Reactions were stopped by adding 4 μl of 6× helicase dye (50 mm EDTA, 0.9% SDS, 30% glycerol, 0.125% bromphenol blue, and 0.125% xylene cyanole). Reactions to be boiled were further incubated for 5 min at 95 °C. Reaction samples were loaded onto a 12% native polyacrylamide gel, and products were separated by electrophoresis for 2 h at 250 V. The gel was dried, scanned, and analyzed as described above. The amount of each protein used in each experiment is given in the corresponding figure legend. All experiments were performed at least in triplicate, and a representative gel is shown in the corresponding figure. Our goal in this study was to determine whether there are specific substrate structures on which Pif1 promotes Okazaki fragment processing. One possibility was that Pif1 stimulation of synthesis is necessary for rapid and efficient synthesis through GC-rich sequences, as these sequences inhibit strand displacement by pol δ (12.Rossi M.L. Bambara R.A. J. Biol. Chem. 2006; 281: 26051-26061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). We therefore examined Pif1 stimulation of synthesis using a reconstitution substrate with a relatively GC-rich region at the 5′ end of the downstream primer, the GC-44 substrate (Fig. 1). The standard-44 substrate served as a control. The first 12 nt of the downstream primer of the GC-44 substrate were 75% G or C, and they were 50% G or C in the standard-44 substrate. As expected, Pif1 stimulated full-length synthesis with the standard-44 substrate (Fig. 1, lanes 6–8). Stimulation of synthesis by FEN1 was greatly reduced on the GC-44 substrate (Fig. 1, lane 13 compared with lane 5), as observed previously (12.Rossi M.L. Bambara R.A. J. Biol. Chem. 2006; 281: 26051-26061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Interestingly, Pif1 did not stimulate full-length synthesis with the GC-44 substrate (Fig. 1, lanes 14–16). This suggests that Pif1 does not stimulate synthesis through sequences of stable structure even though it has been shown to unwind stable G-rich structures, such as G-quadruplexes (26.Ribeyre C. Lopes J. Boulé J.B. Piazza A. Guédin A. Zakian V.A. Mergny J.L. Nicolas A. PLoS Genet. 2009; 5: e1000475Crossref PubMed Scopus (274) Google Scholar). Another possibility was that Pif1 activity is required for proper processing of fold-back flaps. Triplet repeats, especially CTG repeats, are particularly prone to form such structures (20.Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 32.Ryu G.H. Tanaka H. Kim D.H. Kim J.H. Bae S.H. Kwon Y.N. Rhee J.S. MacNeill S.A. Seo Y.S. Nucleic Acids Res. 2004; 32: 4205-4216Crossref PubMed Scopus (49) Google Scholar). Fold-back flaps present a challenge to the flap-processing pathways. Neither FEN1 nor Dna2 can cleave a fold-back (20.Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Additionally, RPA does not bind double-stranded DNA. Therefore, fold-back flaps are inert to all of the components of either flap-processing pathway. As a 5′–3′ helicase, Pif1 would appe" @default.
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• W2094680135 title "An Alternative Pathway for Okazaki Fragment Processing" @default.
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