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• W2043037006 abstract "Reconstitution of eukaryotic Okazaki fragment processing implicates both one- and two-nuclease pathways for processing flap intermediates. In most cases, FEN1 (flap endonuclease 1) is able to efficiently cleave short flaps as they form. However, flaps escaping cleavage bind replication protein A (RPA) inhibiting FEN1. The flaps must then be cleaved by Dna2 nuclease/helicase before FEN1 can act. Pif1 helicase aids creation of long flaps. The pathways were considered connected only in that the products of Dna2 cleavage are substrates for FEN1. However, results presented here show that Dna2, Pif1, and RPA, the unique proteins of the two-nuclease pathway from Saccharomyces cerevisiae, all stimulate FEN1 acting in the one-nuclease pathway. Stimulation is observed on RNA flaps representing the initial displacement and on short DNA flaps, subsequently displaced. Neither the RNA nor the short DNA flaps can bind the two-nuclease pathway proteins. Instead, direct interactions between FEN1 and the two-nuclease pathway proteins have been detected. These results suggest that the proteins are either part of a complex or interact successively with FEN1 because the level of stimulation would be similar either way. Proteins bound to FEN1 could be tethered to the flap base by the interaction of FEN1 with PCNA, potentially improving their availability when flaps become long. These findings also support a model in which cleavage by FEN1 alone is the preferred pathway, with the first opportunity to complete cleavage, and is stimulated by components of the backup pathway. Reconstitution of eukaryotic Okazaki fragment processing implicates both one- and two-nuclease pathways for processing flap intermediates. In most cases, FEN1 (flap endonuclease 1) is able to efficiently cleave short flaps as they form. However, flaps escaping cleavage bind replication protein A (RPA) inhibiting FEN1. The flaps must then be cleaved by Dna2 nuclease/helicase before FEN1 can act. Pif1 helicase aids creation of long flaps. The pathways were considered connected only in that the products of Dna2 cleavage are substrates for FEN1. However, results presented here show that Dna2, Pif1, and RPA, the unique proteins of the two-nuclease pathway from Saccharomyces cerevisiae, all stimulate FEN1 acting in the one-nuclease pathway. Stimulation is observed on RNA flaps representing the initial displacement and on short DNA flaps, subsequently displaced. Neither the RNA nor the short DNA flaps can bind the two-nuclease pathway proteins. Instead, direct interactions between FEN1 and the two-nuclease pathway proteins have been detected. These results suggest that the proteins are either part of a complex or interact successively with FEN1 because the level of stimulation would be similar either way. Proteins bound to FEN1 could be tethered to the flap base by the interaction of FEN1 with PCNA, potentially improving their availability when flaps become long. These findings also support a model in which cleavage by FEN1 alone is the preferred pathway, with the first opportunity to complete cleavage, and is stimulated by components of the backup pathway. Double-stranded DNA in eukaryotes is synthesized through elongation of leading and lagging strands copied using the original DNA as a template. The leading strand DNA is made in a continuous manner by DNA polymerase ϵ, which adds nucleotides in the same direction of the opening replication fork (1Kunkel T.A. Burgers P.M. Trends Cell Biol. 2008; 18: 521-527Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). The lagging strand, which grows in the opposite direction, must be made discontinuously, through the creation of short (∼150-nt) oligonucleotides known as Okazaki fragments (2Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman, New York1992: 113-195Google Scholar). This process begins with DNA polymerase α/primase (pol α), 2The abbreviation used is: ntnucleotide(s). which makes a mixed primer initiating with 10–12 nt of RNA, to which 10–20 nt of DNA is added (3Bambara 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). The primer is then lengthened by DNA polymerase δ (pol δ). After adding deoxynucleotides to make the bulk of the fragment, pol δ encounters the adjacent downstream Okazaki fragment. At this point, pol δ will displace the downstream fragment into a flap while continuing to synthesize DNA, a process called strand displacement synthesis (4Liu Y. Kao H.I. Bambara R.A. Annu. Rev. Biochem. 2004; 73: 589-615Crossref PubMed Scopus (306) Google Scholar, 5Rossi M.L. Purohit V. Brandt P.D. Bambara R.A. Chem. Rev. 2006; 106: 453-473Crossref PubMed Scopus (55) Google Scholar). These flaps are cleaved by specific endonucleases, creating a nick that will then be sealed by DNA ligase I. The ligation event finishes the production of the complete DNA strand. nucleotide(s). Two pathways have been suggested for flap processing. The first pathway, proposed by Burgers and co-workers (6Ayyagari 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, 7Garg P. Stith C.M. Sabouri N. Johansson E. Burgers P.M. Genes Dev. 2004; 18: 2764-2773Crossref PubMed Scopus (175) Google Scholar, 8Jin 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) based on reconstitutions in vitro, is referred to as the FEN1-only pathway, or “one-nuclease pathway.” As the name suggests, in this pathway, the only nuclease involved in flap cleavage is FEN1 (flap endonuclease 1). FEN1 is an endonuclease with a preferred substrate having a 5′-flap with a 1-nucleotide-long 3′-flap. After pol δ displaces a short flap of only a few nucleotides, FEN1 is able to bind to the 5′-end of the flap and track to its base, where it proceeds to cleave off the entire flap, leaving behind only a nick (4Liu Y. Kao H.I. Bambara R.A. Annu. Rev. Biochem. 2004; 73: 589-615Crossref PubMed Scopus (306) Google Scholar, 9Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (373) Google Scholar, 10Murante R.S. Rust L. Bambara R.A. J. Biol. Chem. 1995; 270: 30377-30383Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Reconstitutions of Okazaki fragment processing in vitro suggest that FEN1 cleaves most flaps when they are only a few nucleotides long. However, in these reactions, a small fraction of flaps escape FEN1 cleavage and achieve greater lengths (11Rossi M.L. Bambara R.A. J. Biol. Chem. 2006; 281: 26051-26061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 12Rossi 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). Once this occurs, the flaps can be bound by replication protein A (RPA), the eukaryotic single-stranded binding protein. RPA is able to stably bind flaps greater than 22 nt in length, and doing so has been shown to inhibit FEN1 cleavage (13Fanning E. Klimovich V. Nager A.R. Nucleic Acids Res. 2006; 34: 4126-4137Crossref PubMed Scopus (405) Google Scholar, 14Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar). Bae and Seo (15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) proposed a second pathway through which they suggested that flaps are cleaved in vivo. In the two-nuclease pathway, cleavage by the endonuclease Dna2 is promoted on RPA-coated flaps (14Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar). Dna2 exhibits both a directional 5′–3′ endonuclease activity and a 5′–3′ helicase activity that is specific for forked substrates (15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 16Bae 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). Like FEN1, Dna2 enters flaps from the 5′-end. However, whereas FEN1 cleaves a single time, Dna2 cleaves multiple times while tracking on the flap. Also, unlike FEN1, Dna2 is not able to cleave the flap entirely but leaves a terminal flap of 5–7 nt in length (17Kao 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). RPA is no longer able to bind these short flaps, allowing FEN1 to freely bind and cleave the remainder of the flap. Although the two-nuclease pathway was originally proposed to predominate, later reconstitutions suggest that its role is to complete processing of the small fraction of flaps that are missed by FEN1 (11Rossi M.L. Bambara R.A. J. Biol. Chem. 2006; 281: 26051-26061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 12Rossi 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, 15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). An additional protein, Pif1, has been implicated in lagging strand synthesis. Pif1 is a 5′–3′ helicase that has been shown to have an important role in telomere and mitochondrial DNA maintenance (18Boulé J.B. Zakian V.A. Nucleic Acids Res. 2006; 34: 4147-4153Crossref PubMed Scopus (104) Google Scholar). Interestingly, Pif1 has been shown to have a genetic interaction with Dna2; deletion of PIF1 rescues the lethality created by a Dna2 nuclease-deficient mutant in yeast (19Budd M.E. Reis C.C. Smith S. Myung K. Campbell J.L. Mol. Cell. Biol. 2006; 26: 2490-2500Crossref PubMed Scopus (167) Google Scholar). Previous work from our research group has also suggested that Pif1 plays a role in the two-nuclease pathway by aiding the creation of longer flaps during Okazaki fragment processing (12Rossi 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, 20Pike 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). Although originally formulated as two independent pathways, some evidence suggests that the one- and two-nuclease pathways have relevant interactions. Previous characterization of Dna2 suggests that it is able to stimulate FEN1 although indirectly. By cleaving long flaps bound by RPA, Dna2 not only removes the RPA block but also leaves short flaps that are more readily processed by FEN1 (21Stewart 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, 22Stewart 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). We therefore questioned here whether the two pathways truly operate independently (i.e. whether the protein components of the two-nuclease pathway performed no function until a flap grew to the length that would serve as a substrate for the two-nuclease pathway). In such a model, relative processing of substrates through either of the two pathways would depend solely on the fraction of flaps missed for cleavage by FEN1. Alternatively, the two pathways might be interactive, with components of one affecting the efficiency of the other. For example, the components of the two-nuclease pathway might interfere with FEN1 cleavage, helping to generate more substrate for their pathway. To test this concept, we measured the effect of the unique two-nuclease protein components on the ability of FEN1 to cleave substrates of the one-nuclease pathway. Surprising results showed that the two-nuclease proteins all stimulate FEN1 activity in a way that suppresses formation of the substrate for their own pathway. [α-32P]dCTP and [γ-32P]ATP were obtained from PerkinElmer Life Sciences. Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). T4 polynucleotide kinase and Escherichia coli DNA polymerase I Klenow were obtained from Roche Applied Science. Other reagents were of the best grade commercially available. Saccharomyces cerevisiae Rad27 (FEN1) was cloned into the T7 expression vector pET-24b (Novagen/EMD Biosciences, Madison, WI), expressed in E. coli BL21(DE3) codon plus strain (Stratagene), and purified as described previously (23Kao 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). S. cerevisiae Pif1 and Pif1 K264A were cloned into the pET-28b bacterial expression vector (Novagen/EMD Biosciences), expressed in the E. coli Rosetta strain (Novagen/EMD Biosciences), and purified as described previously (24Boulé J.B. Vega L.R. Zakian V.A. Nature. 2005; 438: 57-61Crossref PubMed Scopus (192) Google Scholar). S. cerevisiae RPA was overexpressed and purified from E. coli as previously described (25Sibenaller Z.A. Sorensen B.R. Wold M.S. Biochemistry. 1998; 37: 12496-12506Crossref PubMed Scopus (66) Google Scholar). S. cerevisiae Dna2 E675A was produced by site-directed mutagenesis as previously described. Wild-type Dna2 and Dna2 E675A were overexpressed and purified from baculovirus High Five cells as previously described (26Budd M.E. Choe W. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Oligonucleotides were used to design substrates that simulate Okazaki fragment processing intermediates. Downstream primers of 28, 32, and 56 nt in length were annealed at their 3′-ends to a 20-nt labeling template with a 5′-GCTA overhang and radiolabeled using [α-32P]dCTP and Klenow polymerase. For 5′ labeling, the downstream primer of 28 nt was labeled with [γ-32P]ATP using polynucleotide kinase. Radiolabeled primers were separated by electrophoresis on a 15%, 7 m urea polyacrylamide gel and then gel-purified. Substrates were then created by annealing primer components in annealing buffer (50 mm Tris-HCl, pH 8.0, 50 mm NaCl, 1 mm dithiothreitol), heating at 95 °C for 5 min, transferring to 70 °C, and slowly cooling to room temperature. The annealing ratio was 1:2:4 (downstream primer/template/upstream primer). Each downstream primer contains a region of 26 nt complementary to the template, resulting in 2-, 6-, and 30-nt unannealed 5′-flaps, respectively. The template itself is 51 nt in length. The upstream primer is 26 nt in length, containing a 25-nt region complementary to the template, leaving a 1-nt 3′-flap. These substrates were designed to allow measurement of cleavage by FEN1. Additionally, an RNA-DNA substrate was created utilizing the above procedure. However, for the RNA-DNA substrate, the 32-nt-long downstream primer contained 12 nt of RNA in the 5′-region. Annealing resulted in a substrate with a 6-nt 5′ RNA flap and 6 nt of RNA annealed to the template. Specific substrates used in each figure are indicated in the legends and pictured at the top of the figures. The location of the radiolabel on the downstream primer is indicated by an asterisk in the respective figures. For the fixed flap cleavage reactions, one or more of the proteins, FEN1, Pif1, Dna2, or RPA, were mixed together in reaction buffer containing 30 mm HEPES, 40 mm KCl, 4 mm MgCl2, 0.01% Nonidet P-40, 0.5% inositol, 0.1 mg/ml bovine serum albumin, 1 mm dithiothreitol, 0.5 mm ATP, and 5% glycerol. To start the reactions, 5 fmol of 3′-radiolabeled substrate was added for a total volume of 20 μl. Reactions were run for 10 min at 37 °C. Reactions were stopped by adding 20 μl of 2× termination dye (90% formamide (v/v), 10 mm EDTA, 0.01% bromphenol blue, and xylene cyanol), followed by heating at 95 °C for 5 min. Products were separated by electrophoresis on a 22.5%, 7 m urea polyacrylamide gel for 1 h and 40 min at 70 watts. The gel was placed on filter paper and vacuum-dried on a gel drier (Bio-Rad). Dried gels were exposed to a phosphor screen, which was scanned using a GE Healthcare PhosphorImager and analyzed with ImageQuant version 5.0 software. Binding efficiency of Pif1, RPA, or Dna2 to a substrate with a flap of either 2, 6, or 30 nt was assessed using EMSA. For clearer visualization, the 2-nt flap substrate was 5′-labeled in this experiment. Five fmol of substrate was incubated with increasing concentrations of the specific protein at either 50 or 500 fmol and incubated for 10 min at 37 °C in the same reaction buffer as described above but lacking ATP and MgCl2. The reactions were loaded on prerun, non-denaturing 12% polyacrylamide gels in 1× TBE. Gels were subjected to electrophoresis for 1.5 h at a constant 250 V. To measure the helicase activity of Pif1 or Dna2 or the strand melting activity of RPA, the respective protein was added to a reaction containing a flap substrate of either 2, 6, or 30 nt in the reaction buffer utilized for our cleavage reactions. For clearer visualization, the 2-nt flap substrate was 5′-labeled in this experiment. Five fmol of substrate was incubated with concentrations of protein of 50 or 500 fmol and incubated for 10 min at 37 °C. The reactions were terminated using a helicase dye consisting of 30% glycerol, 50 mm EDTA, 0.9% SDS, 0.125% bromphenol, and 0.125% xylene cyanol. The reactions were loaded on prerun, non-denaturing 12% polyacrylamide gels in 1× TBE. Gels were subjected to electrophoresis for 1.5 h at a constant 250 V. Purified S. cerevisiae FEN1 (1 ng) and 1 ng of a purified two-nuclease protein (S. cerevisiae Dna2, Pif1, or RPA) were allowed to bind together in a coupling buffer consisting of 25 mm HEPES (pH 7.5), 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, and 10% glycerol for 2 h at 4 °C (in a 1:1 ratio). When three proteins were bound to FEN1, 2 ng of the competing proteins were added first and allowed to bind for 2 h. Then 1 ng of the antibody-specific protein was added and incubated with the mixture for an additional 2 h. Antibody to either Dna2, prepared as previously described (27Budd M.E. Campbell J.L. Mol. Cell. Biol. 1997; 17: 2136-2142Crossref PubMed Scopus (193) Google Scholar), Pif1 (sc-48377 (Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), or RPA70 (kindly provided by Dr. Marc Wold) or control IgG (sc-2027, Santa Cruz Biotechnology, Inc.) were prebound to protein A-agarose for 1 h at room temperature and washed twice with phosphate-buffered saline. The bound proteins were then added to the washed protein A-agarose-antibody complex and incubated overnight at 4 °C with end-over mixing. The following day, the proteins were released from the protein A-agarose using elution buffer. The immunoprecipitates were separated on precast 4–20% gels (Bio-Rad). Western blot analysis was performed with anti-FEN1 polyclonal antibody (ab17993, Abcam). Cross-reactivity of mouse monoclonal Pif1 antibody with purified S. cerevisiae Pif1 was tested and confirmed before the immunoprecipitation. The amount of each protein used in each experiment is given in the appropriate figure legend. All enzyme assays were repeated at least in triplicate with a representative gel shown in each figure. Dna2 and RPA have been implicated in the two-nuclease pathway, important for the proper cleavage of long flaps formed during eukaryotic Okazaki fragment processing. Previous work has also shown that Pif1 helicase promotes the long flap pathway. Although the two-nuclease and FEN1-only pathways are generally represented as occurring separately from one another, we considered the possibility that the pathways have evolved to be interactive. Specifically, we set out to examine whether the components of the two-nuclease pathway influence the efficiency of the one-nuclease pathway. For our experiments, we created three substrates with flap lengths of 2, 6, or 30 nt. The 2-nt flap represents an intermediate formed during the beginning of strand displacement synthesis, which we would expect to have no affinity for Dna2, RPA, or Pif1. The 6-nt flap represents an intermediate considered at the borderline for detectable binding by the three two-nuclease pathway proteins. The 30-nt flap serves as a positive control to which all three proteins should be able to bind with maximum affinity. FEN1 is able to bind and cleave all of these flaps. By utilizing these three different lengths, we have been able to distinguish the influence of each protein on flap cleavage by FEN1 and whether that influence depends on binding of each protein to the flap. Because our experimental protocol utilized fixed flap substrates and because Dna2 and Pif1 are helicases, the potential existed that the helicase functions of these proteins would disrupt the substrates. The concern was that the helicases would partially displace the downstream primer, creating a gap. This would alter the double flap configuration favored by FEN1, decreasing FEN1 cleavage activity and masking any stimulatory effects. This is not an issue in vivo because elongation of the upstream primer by pol δ would continuously renew the favored double flap structure. To ensure that we did not experience interference, we chose a buffer system in which the helicase functions would cause minimal disruption of our substrates. Dna2 was found to stimulate FEN1 cleavage on long flaps. It has been proposed that the ability of Dna2 to cleave long 5′-flaps, terminating in a minimum flap length of about 6 nt, promotes FEN1 to cleave the remainder of the flap more efficiently. The action of Dna2 would remove regions of secondary structure in the flap that might interfere with tracking of FEN1. Moreover, Dna2 would also reduce the flap length so that RPA could not bind to interfere with FEN1 tracking. However, we were interested in determining whether Dna2 is able to directly stimulate FEN1 on the short flaps cleaved in the one-nuclease pathway. To measure whether Dna2 influences FEN1 activity, we first performed a cleavage assay. For this experiment, amounts of Dna2 ranging from 0 to 50 fmol were added to reactions containing FEN1 and varying flap length substrates. We observed a dose-dependent stimulation of FEN1 cleavage by Dna2 (Fig. 1A). Interestingly, in addition to measuring stimulation on the 30-nt flap substrate, to which Dna2 is expected to bind avidly, we also measured significant stimulation on the 6- and 2-nt flaps, implying that the ability of Dna2 to stimulate FEN1 is independent of Dna2 binding to the substrate. After adding 50 fmol of Dna2 to the reaction in the presence of ATP, we measured a stimulation of FEN1 cleavage of 12.5-fold on the 2-nt flap substrate, 44.2-fold on the 6-nt flap substrate, and 15.9-fold on the 30-nt flap substrate. Additionally, this experiment was performed in the absence of ATP. This was done to determine whether the helicase function of Dna2 is responsible for its ability to stimulate FEN1. The outcome was similar, showing that Dna2 is able to stimulate FEN1 cleavage even in the absence of helicase function. To confirm the expected binding characteristics of Dna2 to the different length flap substrates, we performed an electrophoretic mobility shift assay (EMSA) utilizing all three substrates (Fig. 1B). For these EMSA measurements, we used protein levels of 50 and 500 fmol. Whereas 50 fmol of Dna2 represents the highest amount used in our cleavage assays, to observe high efficiency binding of the protein for an EMSA, a 10-fold increase in protein (i.e. 500 fmol) was needed. In the EMSA reactions, we utilized a buffer lacking both ATP and Mg2+ to prevent unwinding or, in the case of Dna2, cleavage. At 50 fmol of Dna2, we detected no binding to the 2- and 6-nt flap substrates, and a small amount of Dna2 bound to the 30-nt flap substrate. At 500 fmol, there was little binding to the 2-nt flap substrate. More binding was observed on the 6-nt flap substrate, with the most binding occurring on the 30-nt flap substrate in the presence of 500 fmol of Dna2. This is consistent with the binding properties expected from Dna2. Finally, we performed a helicase assay utilizing the nuclease-deficient mutant of Dna2. Although the absence of ATP negated the helicase activity of Dna2 in the cleavage assay, the helicase assay was performed in conditions that allowed for maximal helicase activity by the nuclease-deficient Dna2 mutant. Although Dna2 did not show stable binding to the short flap substrates in our EMSA study, we envisioned that there could be transient binding of Dna2 to these substrates, allowing for some helicase activity. However, at 50 fmol of Dna2, the maximum amount used in our cleavage assay, we saw only minimal unwinding and only on the 30-nt flap (Fig. 2). Even at 500 fmol of Dna2, unwinding of the downstream primer could only be observed on the 30-nt flap, suggesting that robust helicase activity by Dna2 requires a longer single-stranded DNA region for it to track and unwind the substrate. This confirms that although Dna2 can stimulate FEN1 cleavage on short flaps, it cannot perform its helicase or endonuclease functions. Pif1 has previously been shown to promote utilization of the two-nuclease pathway in reconstitution reactions. Because it appears to be a two-nuclease pathway component, we assessed the ability of Pif1 to stimulate FEN1 on short flaps. As with Dna2, we first performed a cleavage assay, now utilizing FEN1 and Pif1. Varying amounts of Pif1, ranging from 0 to 50 fmol, were added to reactions containing FEN1. Results revealed a dose-dependent stimulation of FEN1 cleavage by Pif1 (Fig. 3A). As with Dna2, stimulation of FEN1 occurred not only for cleavage of the 30-nt flap substrate but also with the 6- and 2-nt flap substrates. This implies that the ability of Pif1 to stimulate FEN1 is not dependent on Pif1 binding to the flap. The addition of 50 fmol of Pif1 to the reaction resulted in a stimulation of FEN1 cleavage by 16.6-fold on the 2-nt flap substrate, 10.5-fold on the 6-nt flap substrate, and 9.4-fold on the 30-nt flap substrate. A similar experiment was also performed in the absence of ATP to determine whether the helicase function of Pif1 is in any way responsible for its ability to stimulate FEN1. We observed that, like Dna2, Pif1 is able to stimulate FEN1 cleavage even in the absence of ATP. A Pif1 helicase-deficient mutant was also utilized to further confirm that the stimulation of FEN1 by Pif1 does not require its helicase function (data not shown). We expected that Pif1 would exhibit weak binding to a 2- or 6-nt flap but would bind avidly to a 30-nt flap. To confirm, we performed an EMSA utilizing all three substrates in the presence of Pif1 (Fig. 3B). We observed a very low level of binding on the 2-nt flap, even at 500 fmol of protein. As expected, the amount of binding increased on the 6-nt flap, although the amount of Pif1 bound was still low. On the 30-nt flap, binding was evident even at 50 fmol of Pif1 and even more efficient at 500 fmol of Pif1. This result is consistent with our expectation that Pif1 would bind poorly to the short flap substrates, if at all, but readily to the 30-nt flap substrate. In order to determine how the helicase of Pif1 is functioning on the different length flaps utilized in our previous assays, we performed a helicase assay. In addition to characterizing the helicase ability of Pif1, we also sought to confirm that Pif1 was not disrupting our substrates by removing the downstream primer. At 50 fmol of Pif1, the maximum amount used in our cleavage assay, we saw minimal unwinding of the downstream primer and only on the 30-nt flap (Fig. 2). This level of Pif1 was chosen intentionally with the expectation that it would not disrupt the substrate. At the higher concentration of 500 fmol, however, Pif1 displayed helicase activity, most effective on the 30-nt flap substrate, with some unwinding on the 6-nt flap. This confirms what we expected; there is no helicase activity of Pif1 directly on the 2-nt flap and minimal activity on the 6-nt flap, and Pif1 is able to freely interact with the 30-nt flap. This pattern of unwinding is consistent with our hypothesis that Pif1, on short flaps, is at first working to stimulate FEN1 cleavage. However, after a critical flap length is reached, Pif1 is able to bind to the flap, allowing its helicase function to act. This allows Pif1 to lengthen the flap, thereby biasing the processing of the flap toward the two-nuclease pathway. RPA is a key component of the Okazaki fragment maturation pathway, responsible for switching processing from the one-nuclease pathway to the two-nuclease pathway via binding of the flaps. To better understand these actions of RPA, we measured regulation of FEN1 by RPA on the same substrates that revealed stimulation of FEN1 by Dna2 and Pif1. Amounts of RPA ranging from 0 to 100 fmol were added to reactions containing FEN1. This resulted in a dose-dependent stimulation of FEN1 cleavage by RPA (Fig. 4A). On the 2- and 6-nt flap substrates, to which RPA cannot stably bind, FEN1 cleavage was increased by 9.3- and 8.3-fold, respectively, at 100 fmol of RPA. For the 30-nt flap substrate, we included two additional, higher amounts of RPA, 200 and 300 fmol. This allowed us to measure the expected inhibition of FEN1 by RPA on long flaps. At lower levels, RPA was indeed able to stimulate FEN1 cleavage; there was a 2-fold stimulation of FEN1 cleavage at 100 fmol of RPA. However, at 300 fmol of RPA, at which we expected stable coating of the flap, FEN1 cleavage was inhibited to 25% of its basal level. As" @default.
• W2043037006 created "2016-06-24" @default.
• W2043037006 creator A5000378293 @default.
• W2043037006 creator A5005315226 @default.
• W2043037006 creator A5021657789 @default.
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• W2043037006 date "2010-09-01" @default.
• W2043037006 modified "2023-10-17" @default.
• W2043037006 title "Components of the Secondary Pathway Stimulate the Primary Pathway of Eukaryotic Okazaki Fragment Processing" @default.
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