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• W2096549833 abstract "Okazaki fragments are initiated by short RNA/DNA primers, which are displaced into flap intermediates for processing. Flap endonuclease 1 (FEN1) and Dna2 are responsible for flap cleavage. Replication protein A (RPA)-bound flaps inhibit cleavage by FEN1 but stimulate Dna2, requiring that Dna2 cleaves prior to FEN1. Upon cleavage, Dna2 leaves a short flap, which is then cut by FEN1 forming a nick for ligation. Both enzymes require a flap with a free 5′-end for tracking to the cleavage sites. Previously, we demonstrated that FEN1 disengages the tracking mechanism of Dna2 to remove it from the flap. To determine why the disengagement mechanism evolved, we measured FEN1 dissociation of Dna2 on short RNA and DNA flaps, which occur during flap processing. Dna2 tracked onto these flaps but could not cleave, presenting a block to FEN1 entry. However, FEN1 disengaged these nonproductively bound Dna2 molecules, proceeding on to conduct proper cleavage. These results clarify the importance of disengagement. Additional results showed that flap substrate recognition and tracking by FEN1, as occur during fragment processing, are required for effective displacement of the flap-bound Dna2. Dna2 was recently shown to dissociate flap-bound RPA, independent of cleavage. Using a nuclease-defective Dna2 mutant, we reconstituted the sequential dissociation reactions in the proposed RPA/Dna2/FEN1 pathway showing that, even without cutting, Dna2 enables FEN1 to cleave RPA-coated flaps. In summary, RPA, Dna2, and FEN1 have evolved highly coordinated binding properties enabling one protein to succeed the next for proper and efficient Okazaki flap processing. Okazaki fragments are initiated by short RNA/DNA primers, which are displaced into flap intermediates for processing. Flap endonuclease 1 (FEN1) and Dna2 are responsible for flap cleavage. Replication protein A (RPA)-bound flaps inhibit cleavage by FEN1 but stimulate Dna2, requiring that Dna2 cleaves prior to FEN1. Upon cleavage, Dna2 leaves a short flap, which is then cut by FEN1 forming a nick for ligation. Both enzymes require a flap with a free 5′-end for tracking to the cleavage sites. Previously, we demonstrated that FEN1 disengages the tracking mechanism of Dna2 to remove it from the flap. To determine why the disengagement mechanism evolved, we measured FEN1 dissociation of Dna2 on short RNA and DNA flaps, which occur during flap processing. Dna2 tracked onto these flaps but could not cleave, presenting a block to FEN1 entry. However, FEN1 disengaged these nonproductively bound Dna2 molecules, proceeding on to conduct proper cleavage. These results clarify the importance of disengagement. Additional results showed that flap substrate recognition and tracking by FEN1, as occur during fragment processing, are required for effective displacement of the flap-bound Dna2. Dna2 was recently shown to dissociate flap-bound RPA, independent of cleavage. Using a nuclease-defective Dna2 mutant, we reconstituted the sequential dissociation reactions in the proposed RPA/Dna2/FEN1 pathway showing that, even without cutting, Dna2 enables FEN1 to cleave RPA-coated flaps. In summary, RPA, Dna2, and FEN1 have evolved highly coordinated binding properties enabling one protein to succeed the next for proper and efficient Okazaki flap processing. During eukaryotic DNA replication, synthesis of the leading strand occurs in continuous fashion in the direction of DNA unwinding. In contrast, the lagging strand is replicated in a discontinuous fashion via short Okazaki fragments. Each Okazaki fragment is between 100 and 150 nucleotides (nt) 3The abbreviations used are: nt, nucleotide; pol, polymerase; FEN1, flap endonuclease 1; RPA, replication protein A; ss, single-stranded; SPR, surface plasmon resonance. in length. In Saccharomyces cerevisiae, ∼100,000 fragments are created per replication cycle. These fragments are initiated by the DNA polymerase (pol) α-primase complex, which synthesizes 10–12 nt of RNA followed by 20–30 nt of DNA (1Rossi M.L. Purohit V. Brandt P.D. Bambara R.A. Chem. Rev. 2006; 106: 453-473Crossref PubMed Scopus (55) Google Scholar, 2Burgers P.M. J. Biol. Chem. 2009; 284: 4041-4045Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). The pol α-primase complex is then displaced by the clamp loader, replication factor C. The toroidal sliding DNA clamp, proliferating cell nuclear antigen, and DNA pol δ are then loaded onto the DNA. pol δ then synthesizes DNA until it encounters the downstream Okazaki fragment. The downstream RNA/DNA primer is then displaced into a flap intermediate by pol δ. The flap must then be removed and the fragments joined to avoid genome instability (3Gordenin D.A. Kunkel T.A. Resnick M.A. Nat. Genet. 1997; 16: 116-118Crossref PubMed Scopus (189) Google Scholar, 4Kunkel T.A. Resnick M.A. Gordenin D.A. Cell. 1997; 88: 155-158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Removal of the primer is proposed to occur by at least two parallel-acting pathways in S. cerevisiae (2Burgers P.M. J. Biol. Chem. 2009; 284: 4041-4045Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). In one pathway, flap endonuclease 1 (FEN1) cleaves the flap intermediate to create a nicked product for ligation (5Ayyagari 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, 6Garg P. Stith C.M. Sabouri N. Johansson E. Burgers P.M. Genes Dev. 2004; 18: 2764-2773Crossref PubMed Scopus (175) Google Scholar). FEN1 is a structure-specific, single-stranded nuclease that recognizes and cleaves at the base of a flap structure on both DNA and RNA (7Liu Y. Kao H.I. Bambara R.A. Annu. Rev. Biochem. 2004; 73: 589-615Crossref PubMed Scopus (306) Google Scholar). The FEN1-only model suggests that strand displacement synthesis by pol δ produces short flaps, which are successively cleaved by FEN1 until the primer has been removed. DNA ligase I then joins the resultant nicked product. Another model of primer removal involves both FEN1 and the nuclease/helicase Dna2 (8Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar). Dna2 possesses both single-stranded DNA (ssDNA) nuclease and 5′ to 3′ ATP-dependent helicase activities (9Bae 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, 10Budd M.E. Campbell J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7642-7646Crossref PubMed Scopus (151) Google Scholar). It is functionally conserved from yeast to humans (11Liu Q. Choe W. Campbell J.L. J. Biol. Chem. 2000; 275: 1615-1624Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 12Kang H.Y. Choi E. Bae S.H. Lee K.H. Gim B.S. Kim H.D. Park C. MacNeill S.A. Seo Y.S. Genetics. 2000; 155: 1055-1067Crossref PubMed Google Scholar, 13Kim J.H. Kim H.D. Ryu G.H. Kim D.H. Hurwitz J. Seo Y.S. Nucleic Acids Res. 2006; 34: 1854-1864Crossref PubMed Scopus (52) Google Scholar, 14Masuda-Sasa T. Imamura O. Campbell J.L. Nucleic Acids Res. 2006; 34: 1865-1875Crossref PubMed Scopus (85) Google Scholar). Originally identified in a screen for DNA replication mutants, S. cerevisiae Dna2 has also been shown to play a role in telomere processing and DNA repair (15Budd M.E. Reis C.C. Smith S. Myung K. Campbell J.L. Mol. Cell. Biol. 2006; 26: 2490-2500Crossref PubMed Scopus (167) Google Scholar, 16Choe W. Budd M. Imamura O. Hoopes L. Campbell J.L. Mol. Cell. Biol. 2002; 22: 4202-4217Crossref PubMed Scopus (65) Google Scholar, 17Masuda-Sasa T. Polaczek P. Peng X.P. Chen L. Campbell J.L. J. Biol. Chem. 2008; 283: 24359-24373Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 18Budd M.E. Tong A.H. Polaczek P. Peng X. Boone C. Campbell J.L. PLoS Genet. 2005; 1: e61Crossref PubMed Scopus (0) Google Scholar, 19Weitao T. Budd M. Hoopes L.L. Campbell J.L. J. Biol. Chem. 2003; 278: 22513-22522Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 20Vernon M. Lobachev K. Petes T.D. Genetics. 2008; 179: 237-247Crossref PubMed Scopus (25) Google Scholar, 21Tomita K. Kibe T. Kang H.Y. Seo Y.S. Uritani M. Ushimaru T. Ueno M. Mol. Cell. Biol. 2004; 24: 9557-9567Crossref PubMed Scopus (60) Google Scholar). Recently, it was identified as a major nuclease for resection of double strand breaks, in both S. cerevisiae and Xenopus laevis (22Liao S. Toczylowski T. Yan H. Nucleic Acids Res. 2008; 36: 6091-6100Crossref PubMed Scopus (57) Google Scholar, 23Zhu Z. Chung W.H. Shim E.Y. Lee S.E. Ira G. Cell. 2008; 134: 981-994Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar). In S. cerevisiae, Dna2 was shown to physically interact with FEN1 (24Budd M.E. Campbell J.L. Mol. Cell. Biol. 1997; 17: 2136-2142Crossref PubMed Scopus (193) Google Scholar). Also, the overexpression of FEN1 rescued the temperature-sensitive phenotype of the dna2-1 nuclease-impaired mutant, and overexpression of Dna2 rescued the temperature-sensitive rad27Δ (FEN1-null) strain. Moreover, Dna2 interacts with the single-stranded binding protein, replication protein A (RPA), which is involved in both DNA replication and repair (25Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1188) Google Scholar). RPA stimulates flap cleavage by Dna2, while repressing cleavage by FEN1. Based on these findings, Seo and co-workers proposed that pol δ displaces flaps that become long enough to be coated by RPA (8Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar). Once RPA is bound, Dna2 cleavage is required, because FEN1 is inhibited. After cleavage by Dna2, the shortened flap is then free of RPA but must be further processed because Dna2, unlike FEN1, cannot cut at the base of the flap. Instead it leaves a short flap of ∼5 nt (8Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar, 26Kao 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), which is removed by FEN1 to create a nick for ligation. The FEN1-only model is consistent with results obtained from the reconstitution of Okazaki fragment processing with S. cerevisiae proteins. These results showed that the coordination between pol δ and FEN1 is highly efficient, resulting in mostly mononucleotide cleavage products and the production of nicked replication intermediates for ligation (6Garg P. Stith C.M. Sabouri N. Johansson E. Burgers P.M. Genes Dev. 2004; 18: 2764-2773Crossref PubMed Scopus (175) Google Scholar). Later, however, we showed that although mostly short flaps were created during strand displacement by pol δ, a minor subset of longer flaps arose (27Rossi M.L. Bambara R.A. J. Biol. Chem. 2006; 281: 26051-26061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). This subset reached a length at which RPA could stably bind, suggesting a role for Dna2 in processing at least some flaps. Relevant to this issue, Budd et al. (15Budd M.E. Reis C.C. Smith S. Myung K. Campbell J.L. Mol. Cell. Biol. 2006; 26: 2490-2500Crossref PubMed Scopus (167) Google Scholar) showed that the elimination of Pif1, a 5′ to 3′ helicase, rescued the lethality of the dna2Δ strain in S. cerevisiae. Cell growth was even more robust when both Pif1 and Pol32, the nonessential subunits of pol δ, were simultaneously deleted in the dna2Δ strain. Significantly, the pol δ mutant lacking Pol32 exhibits decreased strand displacement activity (28Burgers P.M. Gerik K.J. J. Biol. Chem. 1998; 273: 19756-19762Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Furthermore, we have recently shown that the addition of Pif1 in reconstituted Okazaki fragment processing augmented the subset of longer flaps that escaped FEN1 cleavage and were bound by RPA (29Rossi 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). These results suggest that Pif1 aids pol δ strand displacement in creating long flap substrates that require Dna2 nuclease function. Although the FEN1-only pathway is likely to be the dominant mechanism of flap removal, employment of both pathways appears to be critical to process and join all Okazaki fragments. A characteristic feature of both FEN1 and Dna2 is that they must enter a free flap 5′-end for substrate cleavage. If a double-stranded region or a streptavidin-biotin conjugate is used to block the 5′-end of the flap, then cleavage is inhibited (30Kao 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, 31Murante R.S. Rust L. Bambara R.A. J. Biol. Chem. 1995; 270: 30377-30383Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Because tracking is required for cleavage, we previously tested whether a bound nuclease-defective mutant of Dna2, E675A, inhibited FEN1 cleavage (32Stewart J.A. Campbell J.L. Bambara R.A. J. Biol. Chem. 2006; 281: 38565-38572Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). We were surprised to find that cleavage was not inhibited and discovered that FEN1 disengages the tracking mechanism of Dna2 to allow dissociation. FEN1 also dissociated Dna2 from RNA flaps, which Dna2 cannot cleave. Furthermore, we recently demonstrated the ability of Dna2 to dissociate flap-bound RPA (33Stewart 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). These findings suggest a sequential dissociation of RPA by Dna2 followed by the dissociation of Dna2 by FEN1. Here we are investigating the significance of the FEN1 disengagement of Dna2 on relevant substrates of the proposed RPA/Dna2/FEN1 pathway. Dna2 binds but cannot cleave RNA (32Stewart J.A. Campbell J.L. Bambara R.A. J. Biol. Chem. 2006; 281: 38565-38572Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 34Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Additionally, cleavage by Dna2 produces short ∼5-nt flaps, which Dna2 cannot cleave. In this study, we analyzed these substrates for Dna2 binding and FEN1 dissociation of flap-bound Dna2. We also probed the role of tracking and flap structure for disengagement of Dna2 by FEN1. Finally, we tested the proposed sequential dissociation reactions by reconstituting the RPA/Dna2/FEN1 pathway with the nuclease-defective Dna2 E675A. Materials-Synthetic oligonucleotides, including ones with biotin modifications, were synthesized by Integrated DNA Technologies. Radioactive [α-32P]dCTP and [γ-32P]ATP were acquired from PerkinElmer Life Sciences. Both the polynucleotide kinase and the Klenow fragment of Escherichia coli DNA polymerase I, used for labeling, were purchased from Roche Applied Sciences. All other reagents were the best available commercial grade. Oligonucleotides-Primers used in this study are listed in Table 1. 32P was incorporated at either the 5′- or 3′-end of the downstream primers for visualization as described (32Stewart J.A. Campbell J.L. Bambara R.A. J. Biol. Chem. 2006; 281: 38565-38572Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). For 5′-end labeling [γ-32P]ATP was incorporated using polynucleotide kinase, and for 3′-end labeling [α-32P]dCTP was added by the Klenow fragment. Substrates were then PAGE-purified and resuspended in 1× TE. Radiolabeled primers were then annealed in a 1:2:4 ratio of downstream primer to template to upstream primer to create a flap substrate. Substrates containing RNA included Protector RNase (Roche Applied Science) during substrate purification and annealing to prevent degradation.TABLE 1OligonucleotidesPrimerLength (nt)SequenceDownstreamaNucleotides shown in boldface type are biotinylated.,bRNA segment is shown in italic type.,cUnderlined nucleotide indicates the last annealed nucleotide. (5′-3′)D123GCC GU C CAC CCG U CC ACC CGA CGD228GCC GTC GTT TTA CAA CGA CGT GAC TGG GD353TTC ACG CCT GTT AGT TAA TTC ACT GGC CGT CGT TTT ACA ACG ACG TGA CTG GGD476GTA CCG AGC TCG AAT TCG CCC GTT TCA CGC CTG TTA GTT AAT TCA CTG GCC GTC GTT TTA CAA CGA CGT GAC TGG GD576GTA CCG AGC TCG AAT TCG CCC GTT TCA CGC CTG TTA GTT AAT TCA CTG GCC GTC GTT TTA CAA CGA CGT GAC TGG GD650GTA CCG AGC TCG AAT TCG CCC GTT TCA CGC CTG TTA GTT AAT TCA CTG GCD746CAC TGG CCG TCG TTT TAC GGA CCC GTC CAC CCG ACG CCA CCT CCT GUpstream (5′-3′)U126CGA CCG TGC CAG CCT AAA TTT CAA GAU226CGC CAG GGT TTT CCC AGT CAC GAC CAU326CGA CCG TGC CAG CCT AAA TTT CAA TATemplate (3′-5′)T144GCT GGC ACG GTC GGA TTT AAA GTT CGG TGG GCA GGT GGG CTG CGT249GCG GTC CCA AAA GGG TCA GTG CTG GGC AAA ATG TTG CTG CAC TGA CCC GT351GCT GGC ACG GTC GGA TTT AAA GTT AGG GCA GGT GGG CTG CGG TGG AGG ACGa Nucleotides shown in boldface type are biotinylated.b RNA segment is shown in italic type.c Underlined nucleotide indicates the last annealed nucleotide. Open table in a new tab Protein Purification-Wild-type and E675A Dna2 proteins from S. cerevisiae were overexpressed in baculovirus High Five cells and purified as described (35Budd M.E. Choe W. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Dna2 E675A was created using site-directed mutagenesis as described (35Budd M.E. Choe W. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). S. cerevisiae FEN1 (36Kao 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) and RPA (37Henricksen L.A. Wold M.S. J. Biol. Chem. 1994; 269: 24203-24208Abstract Full Text PDF PubMed Google Scholar) were overexpressed in E. coli and then purified as described. Surface Plasmon Resonance-Association and dissociation of wild-type Dna2 with a single-stranded segment of DNA was analyzed using a Reichert SR7000 dual channel instrument (Reichert Inc., Depew, NY). A mixture of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide/N-hydroxysuccinimide was used to activate the dithiol carboxyl surface of the sensor chip as described (38Subramanian A. Irudayaraj J. Ryan T. Biosens. Bioelectron. 2006; 21: 998-1006Crossref PubMed Scopus (248) Google Scholar). Approximately 900 micro-refractive index units of Dna2 were then immobilized over one channel, whereas the other served as a control to detect nonspecific binding, refractive index changes, and instrument drift. Following Dna2 immobilization, the chip was inactivated by flowing 1 m ethanolamine, pH 8.5, over both chambers. The running buffer then consisted of 30 mm HEPES, pH 7.5, 0.1 mg/ml bovine serum albumin, 40 mm KCl, 2 mm CaCl2, 50 μm ATP, and 0.05% Tween 20. For association, ssDNA (D4) was run over the immobilized Dna2 at a flow rate of 50 μl/min for 3 min. For dissociation, the reaction buffer only was run over the chip for 3 min at the same flow rate of 50 μl/min. After each run the chip was regenerated for 2 min with 1 m NaCl in the running buffer to remove the bound DNA. Regeneration was verified by a return to the base line established prior to each run. The resulting data were then analyzed using Scrubber 2 software (Biologic Software Pty. Ltd.). Gel Shift Assay-Reactions contained 5 fmol of radiolabeled substrate and various amounts of Dna2 and/or FEN1, as indicated. The reaction buffer contained 50 mm Tris-HCl, pH 8.0, 2 mm dithiothreitol, 30 mm NaCl, 0.1 mg/ml bovine serum albumin, 5% glycerol, and 50 μm ATP. Dna2 was pre-bound to the substrate for 5 min at room temperature prior to the addition of FEN1, which was then incubated with the reaction for 5 min at room temperature. When streptavidin was added, it was incubated with the substrate for 10 min prior to the addition of protein, unless otherwise indicated. Reactions were then loaded onto a 5% polyacrylamide gel and subjected to electrophoresis at 150 V for 30–40 min. Nuclease Assays-Samples contained 5 fmol of radiolabeled substrate and various amounts of protein, as stated in the figure legends. The reaction buffer contained 50 mm Tris-HCl, pH 8.0, 2 mm dithiothreitol, 30 mm NaCl, 0.1 mg/ml bovine serum albumin, 5% glycerol, 50 μm ATP, and 2 mm MgCl2. In Fig. 1A, Dna2 was bound to the radiolabeled substrate for 5 min at room temperature. Unlabeled substrate (1 pmol) was then added at time 0. At each time point, MgCl2 was added, to a final concentration of 2 mm, to initiate the reaction. Reactions were then incubated at 37 °C for 10 min. In Fig. 5, RPA, Dna2 E675A, and FEN1 were mixed followed by the addition of the flap substrate. The reactions were then incubated at 37 °C for 10 min. After incubation, all reactions were then stopped by the addition of 2× termination dye, consisting of 90% formamide (v/v), 10 mm EDTA, 0.01% bromphenol blue, and 0.01% xylene cyanole. Reactions were then incubated at 95 °C for 5 min and loaded onto a 15% polyacrylamide gel, containing 7 m urea and subjected to electrophoresis at 80 watts for 1–1.5 h.FIGURE 5Dna2 E675A overcomes RPA inhibition of FEN1. RPA (200 fmol), FEN1 (0.25 fmol), and Dna2 E675A (10, 20, 100, and 200 fmol) were mixed followed by the addition of a 21-nt flap substrate (D7:U3:T3) (lanes 8–11). Denaturing PAGE was then used to separate the products. Lane 1 is the substrate alone control. Lanes 2–4 are substrate with Dna2 E675A (200 fmol), substrate with FEN1 (0.25 fmol), and substrate with RPA (200 fmol), respectively. Lanes 5–7 are substrate with Dna2 E675A (200 fmol) and FEN1 (0.25 fmol), substrate with RPA (200 fmol) and FEN1 (0.25 fmol), and substrate with Dna2 E675A (200 fmol) and RPA (200 fmol), respectively. B, graphical analysis of A. Each bar of the graph represents the conditions shown in the corresponding lane in A. The bars are an average of four independent experiments, and error bars represent the S.D. The substrate is depicted above the gel in A, and the asterisk indicates the site of the 3′-32P radiolabel.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Gel Analysis-At least two independent experiments were performed for each figure, and representative gels are shown. After electrophoresis, the gels were placed on filter paper and dried on a gel dryer (Bio-Rad) with vacuum (Savant). Dried gels were then exposed to a phosphor screen, visualized by phosphorimaging (GE Healthcare), and analyzed using ImageQuantMac, version 1.2. Calculation of Dissociation Rates-Data points in Fig. 1B are an average of five independent experiments and were fit using nonlinear least squares regression of either the single exponential decay Equation 1, y=a·exp(-b·x)Eq. 1 or the double exponential decay Equation 2, y=a·exp(-b·x)+c·exp(-d·x)Eq. 2 where y is the relative cleavage; a and c are the amplitudes of each dissociation curve, and b and d are the rates of dissociation for each curve. In Fig. 1C, data were fit using the Scrubber 2 software (Biologics Software Pty. Ltd.). Dna2 Dissociates Slowly from a Flap Substrate-Previously, we observed that FEN1 disengages Dna2 from a flap substrate to gain access for cleavage (32Stewart J.A. Campbell J.L. Bambara R.A. J. Biol. Chem. 2006; 281: 38565-38572Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). To understand the details of FEN1-promoted disengagement of Dna2, we used a DNA competition assay and surface plasmon resonance (SPR) to assess Dna2 dissociation (Fig. 1). We reasoned that if Dna2 dissociates slowly from flap substrates the disengagement reaction is likely to have evolved to facilitate rapid joining of Okazaki fragments. However, if spontaneous dissociation of Dna2 were rapid, the disengagement process has another purpose. To assess the rate of Dna2 dissociation from the flap, we incubated Dna2 with a radiolabeled 53-nt double flap substrate. After binding, an excess of unlabeled flap substrate was added to the reaction followed by the addition of MgCl2 at the indicated time points (Fig. 1A). Reactions were then incubated for 10 min to allow Dna2 cleavage. Because MgCl2 is required for Dna2 cleavage, the cleavage rate was proportional to the amount of Dna2 still bound to the labeled substrate at each time point. These results were compared with a control in which the labeled and excess unlabeled substrates were incubated prior to the addition of Dna2 (Fig. 1A, lane 12). A graph was then generated, and points were fit to an exponential decay curve to determine the dissociation rate (see “Experimental Procedures”) (Fig. 1B). Initially, we fit the curve to a single exponential decay equation, which showed a half-time of about 25 min (Fig. 1B, gray line). Based on the shape of the curve, we then utilized the double exponential decay equation and found a better fit (Fig. 1B, black line). This suggests two dissociation phases, an initial rapid dissociation followed by a much slower one. Based on the small amplitude (∼20% of the relative cleavage) and the short time frame (∼1 min), we believe that nonspecific binding or a weak binding mode accounts for the initial dissociation phase of Dna2. The second phase would account for the majority of Dna2 binding. Dna2 bound in this manner dissociates slowly from the DNA, with a half-time of about 40 min. These data show that binding of Dna2 to the substrate is quite stable. To further assess the binding and dissociation rates of Dna2 to DNA, we performed SPR. Dna2 was immobilized onto a chip, and various amounts of ssDNA were allowed to flow over the chip while association was measured (Fig. 1C). This was followed by a dissociation phase with only buffer flowing over the chip. A second surface in which Dna2 was not immobilized served as a reference. When we attempted to fit the curves, they did not fit a simple 1:1 binding model, suggesting a complex interaction between Dna2 and the DNA. Although we were unable to simultaneously fit both the association and dissociation rates, we could independently fit the dissociation rate using the Scrubber 2 software. Because the curves appeared strikingly similar to those in Fig. 1B, we fit the data 30 s into the dissociation phase. By doing so, we were able to bypass the initial dissociation phase and fit a 1:1 binding model for the second dissociation phase. Again, these curves suggest a slow rate of dissociation, with a half-time of ∼50 min. Both the excess substrate and SPR dissociation measurements clearly indicate that the half-time for dissociation of Dna2 is in the range of ½ to 1 h. These findings are consistent with the conclusion that, because Dna2 binding to the flap is stable, FEN1 has evolved the ability to disengage Dna2 to efficiently gain access to the flap base for cleavage. Dna2 Binds, but Does Not Cleave, Short RNA and DNA Flaps-Previously, we hypothesized that FEN1 evolved to remove Dna2 molecules that are unproductively bound to the flap. The need for disengagement is envisioned to arise at two stages of Okazaki fragment processing. Each Okazaki fragment is initiated by a short segment of RNA, 10–12 nt in length (1Rossi M.L. Purohit V. Brandt P.D. Bambara R.A. Chem. Rev. 2006; 106: 453-473Crossref PubMed Scopus (55) Google Scholar). The first stage requiring disengagement would occur during initial strand displacement by pol δ, when RNA flaps begin to emerge. Although FEN1 can readily cleave short RNA flap intermediates, RNA is not a substrate for the nuclease activity of Dna2 (34Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). A bound, inactive Dna2 molecule could block progressive FEN1 cleavage. We previously showed that Dna2 bound but did not cleave a 30-nt RNA flap and that Dna2 was dissociated by FEN1 (32Stewart J.A. Campbell J.L. Bambara R.A. J. Biol. Chem. 2006; 281: 38565-38572Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Here we employed a substrate with 5 nt of RNA on the flap and an additional 8 nts of RNA in the annealed portion of the labeled primer. This substrate simulates the initial partial displacement of the RNA primer by pol δ. The substrate was used to test Dna2 cleavage and binding. Consistent with previous findings, Dna2 was unable to cleave the RNA flap (Fig. 2A). By way of a control, we measured robust Dna2 cleavage activity on a 30-nt DNA flap substrate. We then tested the ability of Dna2 to bind the 5-nt RNA flap. Dna2 was incubated with the substrate, and the reactions were then analyzed by gel shift (Fig. 2B). The labeled substrate band shifted upon the addition of Dna2 to indicate formation of a higher molecular weight complex. Next, we assessed FEN1 dissociation of Dna2 on the 5-nt RNA flap substrate (Fig. 2C). Dna2 was pre-bound to the flap. FEN1 was then added with the Dna2-bound substrate. The reactions were then analyzed by gel shift to separate the products and determine which protein remained bound to the substrate. Because Dna2 is three times the size of FEN1, the bound complexes of these proteins with the labeled substrate are easily distinguished (Fig. 2C, lanes 2 and 7). With increasing amounts of FEN1, the bands were shifted from a Dna2-bound substrate to a FEN1-bound substrate (Fig. 2C, lanes 3–6). This shift is indicative of the removal of Dna2 from the 5-nt RNA flap by FEN1. These results suggest that FEN1 disengagement of flap-bound Dna2 would promote FEN1 cleavage on initially displaced fl" @default.
• W2096549833 created "2016-06-24" @default.
• W2096549833 creator A5000378293 @default.
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• W2096549833 title "Significance of the Dissociation of Dna2 by Flap Endonuclease 1 to Okazaki Fragment Processing in Saccharomyces cerevisiae" @default.
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