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• W2170294911 abstract "Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is essential in DNA replication and repair. FEN-1 has specific endonuclease activity for repairing nicked double-stranded DNA substrates that have the 5′-end of the nick expanded into a single-stranded tail, and it is involved in processing Okazaki fragments during DNA replication. Magnesium is a cofactor required for nuclease activity. We used small-angle x-ray scattering to obtain global structural information pertinent to nuclease activity from FEN-1, the D181A mutant, the wild-type FEN-1·34-mer DNA flap complex, and the D181A·34-mer DNA flap complex. The D181A mutant, which has Asp-181 replaced by Ala, selectively binds to the flap structure, but has lost its cleaving activity. Asp-181 is thought to be involved in Mg2+binding at the active site (Shen, B., Nolan, J. P., Sklar, L. A., and Park, M. S. (1996) J. Biol. Chem. 271, 9173–9176). Our data indicate that FEN-1 and the D181A mutant each have a radius of gyration of ∼26 Å, and the effect of Mg2+ on the scattering from the proteins alone is insignificant. The 34-mer DNA fragment was constructed such that it readily forms a 5′-flap structure. The formation of the flap conformation of the DNA substrate was evident by both the extrapolatedI o scattering and radius of gyration and was supported by NMR spectrum and nuclease assays. In the absence of magnesium, the FEN-1·34-mer DNA flap complex has anR g value of ∼34 Å, whereas the D181A·34-mer DNA flap complex self-associates, suggesting that a significant protein conformational change occurs by addition of the flap DNA substrate and that Asp-181 is crucial for proper binding of the protein to the DNA substrate. A time course change in the scattering profiles arising from magnesium activation of the FEN-1·34-mer DNA flap complex is consistent with the protein completely releasing the DNA substrate after cleavage. Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is essential in DNA replication and repair. FEN-1 has specific endonuclease activity for repairing nicked double-stranded DNA substrates that have the 5′-end of the nick expanded into a single-stranded tail, and it is involved in processing Okazaki fragments during DNA replication. Magnesium is a cofactor required for nuclease activity. We used small-angle x-ray scattering to obtain global structural information pertinent to nuclease activity from FEN-1, the D181A mutant, the wild-type FEN-1·34-mer DNA flap complex, and the D181A·34-mer DNA flap complex. The D181A mutant, which has Asp-181 replaced by Ala, selectively binds to the flap structure, but has lost its cleaving activity. Asp-181 is thought to be involved in Mg2+binding at the active site (Shen, B., Nolan, J. P., Sklar, L. A., and Park, M. S. (1996) J. Biol. Chem. 271, 9173–9176). Our data indicate that FEN-1 and the D181A mutant each have a radius of gyration of ∼26 Å, and the effect of Mg2+ on the scattering from the proteins alone is insignificant. The 34-mer DNA fragment was constructed such that it readily forms a 5′-flap structure. The formation of the flap conformation of the DNA substrate was evident by both the extrapolatedI o scattering and radius of gyration and was supported by NMR spectrum and nuclease assays. In the absence of magnesium, the FEN-1·34-mer DNA flap complex has anR g value of ∼34 Å, whereas the D181A·34-mer DNA flap complex self-associates, suggesting that a significant protein conformational change occurs by addition of the flap DNA substrate and that Asp-181 is crucial for proper binding of the protein to the DNA substrate. A time course change in the scattering profiles arising from magnesium activation of the FEN-1·34-mer DNA flap complex is consistent with the protein completely releasing the DNA substrate after cleavage. The 5′-flap structure is a common DNA structural intermediate occurring during DNA replication, recombination, and repair (1Murante R.S. Huang L. Turch J.J. Bambara R.A. J. Biol. Chem. 1994; 269: 1191-1196Abstract Full Text PDF PubMed Google Scholar). In eukaryotic DNA replication, displacement of an upstream primer by an incoming polymerase can result in the formation of a 5′-flap structure (2Lyamichev V. Brow M.D. Dahlberg J.E. Science. 1993; 260: 778-783Crossref PubMed Scopus (306) Google Scholar, 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 5′-flap intermediates are also formed during double-stranded break repair (4Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (373) Google Scholar, 5Roth D.B. Wilson J.H. Mol. Cell. Biol. 1986; 6: 4295-4303Crossref PubMed Scopus (416) Google Scholar), homologous recombination (6Pont K.G. Dawson R.J. Carroll D. EMBO J. 1993; 12: 23-24Crossref PubMed Scopus (23) Google Scholar), and excision repair (7Harrington J.J. Lieber M.R. Genes Dev. 1994; 8: 1344-1355Crossref PubMed Scopus (256) Google Scholar, 8Doetsch P.W. Trends Biochem. Sci. 1995; 20: 384-386Abstract Full Text PDF PubMed Scopus (32) Google Scholar, 9Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (666) Google Scholar, 10Kim K. Biade S. Matsumoto Y. J. Biol. Chem. 1998; 273: 8842-8848Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). In DNA repair and replication activities, structural recognition of the 5′-flap by specific DNA repair nucleases is essential. The importance of the DNA metabolic reactions, involving the structure-specific nucleases, is best illustrated by the human genetic disorder xeroderma pigmentosum (11Harrington J.J. Lieber M.R. J. Biol. Chem. 1995; 270: 4503-4508Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 12Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 13Vermeulen W. Jaeken J. Jaspers N.G. Bootsma D. Hoeijmakers J.H.J. Am. J. Hum. Genet. 1993; 53: 185-192PubMed Google Scholar). This disease, characterized by severe sensitivity to sunlight and a predisposition to skin cancer, results directly from defects in the nucleotide excision pathway. Mutation defects in the repair nucleases may be a point of breakdown in this DNA repair pathway. The design of a model flap DNA structure, similar to those conjectured to occur in the nucleotide excision pathway, has led to the discovery of human flap endonuclease-1 (FEN-1), 1The abbreviations used are: FEN-1, flap endonuclease-1; SAXS, small-angle x-ray scattering; R g, radius of gyration; R c, radius of gyration of cross-section; I o, forward scatter; P(r), vector distribution function; d max, maximum linear dimension. which structurally recognizes and cleaves the flap DNA structure (4Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (373) Google Scholar, 14Waga S. Bauer G. Stillman B. J. Biol. Chem. 1994; 269: 10923-10934Abstract Full Text PDF PubMed Google Scholar, 15Murray J.M. Tavassoli M. Al-harithy R. Sheldrick K.S. Lehmann A.R. Carr A.M. Watts F.Z. Mol. Cell. Biol. 1994; 14: 4878-4888Crossref PubMed Scopus (145) Google Scholar, 16Robins P. Pappin D.J.C. Wood R.D. Lindahl T. J. Biol. Chem. 1994; 269: 28535-28538Abstract Full Text PDF PubMed Google Scholar, 17Turchi J.J. Huang L. Murante R.S. Kim Y. Bambara R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9803-9807Crossref PubMed Scopus (169) Google Scholar). FEN-1, an ∼43-kDa Mg2+- or Mn2+-dependent enzyme, demonstrates both 5′-flap structure-specific endonuclease activity (1Murante R.S. Huang L. Turch J.J. Bambara R.A. J. Biol. Chem. 1994; 269: 1191-1196Abstract Full Text PDF PubMed Google Scholar, 4Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (373) Google Scholar, 7Harrington J.J. Lieber M.R. Genes Dev. 1994; 8: 1344-1355Crossref PubMed Scopus (256) Google Scholar) and nick-specific 5′ → 3′ exonuclease activity (4Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (373) Google Scholar,14Waga S. Bauer G. Stillman B. J. Biol. Chem. 1994; 269: 10923-10934Abstract Full Text PDF PubMed Google Scholar, 17Turchi J.J. Huang L. Murante R.S. Kim Y. Bambara R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9803-9807Crossref PubMed Scopus (169) Google Scholar, 18Turchi J.J. Bambara R.A. J. Biol. Chem. 1993; 268: 15136-15141Abstract Full Text PDF PubMed Google Scholar). The exonuclease activity of FEN-1 is similar to the function of the 5′ → 3′ exonuclease domain of Escherichia coli DNA polymerase I (16Robins P. Pappin D.J.C. Wood R.D. Lindahl T. J. Biol. Chem. 1994; 269: 28535-28538Abstract Full Text PDF PubMed Google Scholar) and identical to the activity in RNA primer removal that is necessary for in vitro mammalian DNA replication. In its endonuclease role, FEN-1 recognizes the phosphodiester backbone of a 5′-flap single strand and tracks down this arm to the cleavage site, the junction where the two strands of duplex DNA adjoin a single-stranded arm (1Murante R.S. Huang L. Turch J.J. Bambara R.A. J. Biol. Chem. 1994; 269: 1191-1196Abstract Full Text PDF PubMed Google Scholar, 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). FEN-1 does not cleave bubble substrates, single-stranded 3′-flaps, heterologous loops, or Holliday junctions, but acting as an exonuclease, FEN-1 will hydrolyze double-stranded DNA substrates containing a gap or 3′-overhang. FEN-1 endonuclease activity is independent of 5′-flap length, and endonuclease and exonuclease activities cleave both DNA and RNA without the need for accessory proteins (19Murante R.S. Rumbaugh J.A. Barnes C.J. Norton J.R. Bambara R.A. J. Biol. Chem. 1996; 271: 25888-25897Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). FEN-1 does, however, interact with other proteins at the replication fork, including a DNA helicase (20Budd M.E. Campbell J.E. Mol. Cell. Biol. 1997; 17: 2136-2142Crossref PubMed Scopus (193) Google Scholar), the proliferating cell nuclear antigen (21Li X. Li J. Harrington J. Lieber M.R. Burgers P.M. J. Biol. Chem. 1995; 270: 22109-22112Crossref PubMed Scopus (254) Google Scholar, 22Wu X.T. Li J. Li X.Y. Hsieh C.L. Burgers P.M.J. Lieber M.R. Nucleic Acids Res. 1996; 24: 2036-2043Crossref PubMed Scopus (198) Google Scholar, 23Chen J. Chen S. Saha P. Dutta A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11597-11602Crossref PubMed Scopus (116) Google Scholar, 24Jonsson Z.O. Hindges R. Hubscher U. EMBO J. 1998; 17: 2412-2425Crossref PubMed Scopus (236) Google Scholar), and possibly replication protein A (RPA) (25Biswas E.E. Zhu F.X. Biswas S.B. Biochemistry. 1997; 36: 5955-5962Crossref PubMed Scopus (38) Google Scholar). The biological significance of the FEN-1 gene (RAD27 inSaccharomyces cerevisiae and rad2 inSchizosaccharomyces pombe) is emphasized by genetic analysis in yeast. The yeast FEN-1 mutants display severely impaired phenotypes such as UV sensitivity, deficient chromosome segregation, conditional lethality, and accumulation in S phase (15Murray J.M. Tavassoli M. Al-harithy R. Sheldrick K.S. Lehmann A.R. Carr A.M. Watts F.Z. Mol. Cell. Biol. 1994; 14: 4878-4888Crossref PubMed Scopus (145) Google Scholar, 26Reagan M.S. Pittenger C. Siede W. Friedberg E.C. J. Bacteriol. 1995; 177: 365-371Crossref Google Scholar, 27Sommers C.H. Miller E.J. Dujon B. Prakash S. Prakash L. J. Biol. Chem. 1995; 270: 4193-4196Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 28Vallen E.A. Cross F.R. Mol. Cell. Biol. 1995; 15: 4291-4302Crossref PubMed Scopus (95) Google Scholar, 29Johnson R.E. Kovvali G.K. Prakash L. Prakash S. Science. 1995; 269: 238-240Crossref PubMed Scopus (195) Google Scholar). The yeastrad27 null mutant is a strong mutator, and the majority of mutations found are duplications. This is probably because unexcised flap strands in Okazaki fragments displaced by upstream DNA polymerization are subsequently annealed to the downstream complementary sequence. This part of the sequence will be duplicated in the next generation of DNA replication (30Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). FEN-1 activity requires afree 5′-end of the flap DNA strand. For instance, secondary structure formation of the single-stranded DNA into a hairpin structure is known to prevent the enzyme's function (31Gordenin D.A. Kunkel T.A. Resnick M.A. Nat. Genet. 1997; 16: 116-118Crossref PubMed Scopus (189) Google Scholar). At risk motif sequences such as trinucleotide repeats have a higher probability to form these structures. Indeed, the same FEN-1 null mutant displays length-dependent CTG tract destabilization and a marked increase in expansion frequency (32Freudenreich C.H. Kantrow S.M. Zakian V.A. Science. 1998; 279: 853-856Crossref PubMed Scopus (364) Google Scholar, 33Schweitzer J.K. Livingston D.M. Hum. Mol. Genet. 1998; 7: 69-74Crossref PubMed Scopus (164) Google Scholar). Thus, FEN-1 is a key enzyme for maintaining genome integrity, and mutations in FEN-1 may give rise to a number of genetic diseases such as myotonic dystrophy, Huntington's disease, several ataxias, fragile X syndrome, and cancer (30Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 32Freudenreich C.H. Kantrow S.M. Zakian V.A. Science. 1998; 279: 853-856Crossref PubMed Scopus (364) Google Scholar). As the role of FEN-1 in DNA replication and repair is becoming more clear, it is important to structurally characterize this enzyme to better understand how it functions either as an exo- or endonuclease. To examine the structure-function relationship of FEN-1 in its nuclease capacity, we have studied the effect of magnesium on its conformation in aqueous solution, as observed by small-angle x-ray scattering (SAXS). Experiments were also done with a D181A mutant of FEN-1, which still selectively binds to the 5′-flap DNA structure, but has lost its catalytic ability (34Shen B. Nolan J.P. Sklar L.A. Park M.S. J. Biol. Chem. 1996; 271 (and references therein): 9173-9176Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 35Shen B. Nolan J.P. Sklar L.A. Park M.S. Nucleic Acids Res. 1997; 25: 3332-3338Crossref PubMed Scopus (95) Google Scholar). We show that no measurable structural change was evident in either FEN-1 or the D181A mutant due to the presence of Mg2+. A DNA fragment was constructed so that it readily adopts a 5′-flap conformation. When measurements of FEN-1 and the D181A mutant were performed in the presence of the 34-mer DNA flap fragment, the FEN-1·34-mer DNA flap complex was seen to be more compact than the D181A·34-mer DNA flap complex, which indicates that the wild-type FEN-1·34-mer DNA flap complex is in a cleavage-ready conformation. A time course scattering measurement showed that magnesium was able to activate the cleavage of the FEN-1·34-mer DNA flap complex, and the protein was found to completely release the remaining single- and double-stranded portions of the DNA products. Protein expression and purification were essentially carried out according to Nolan et al. (36Nolan J.P. Shen B. Sklar L.A. Park M.S. Biochemistry. 1996; 35: 11668-11676Crossref PubMed Scopus (61) Google Scholar). After FEN-1 was eluted from the column using elution buffer (20 mm Tris-HCl, pH 7.9, 0.5m NaCl, and 300 mm histidine), it was further dialyzed in Tris-HCl buffer, pH 7.9, containing 100 mmNaCl, 10 mm 2-mercaptoethanol, and 10% glycerol and then concentrated in a Centriprep-10 concentrator (Amicon, Inc.). Final protein concentrations used were ∼6 mg/ml in the SAXS study for both the wild-type and D181A mutant proteins, in which 10 mmMg2+ was either present or absent. Wild-type and D181A mutant protein concentrations were ∼4.7 mg/ml for the protein·DNA flap complex studies, and the protein and DNA had a 1:1 stoichiometric ratio. Final concentrations were determined by the Bradford assay (46Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using bovine serum albumin γ-globulin as a standard. Binding of the two proteins to the flap DNA substrate was confirmed by gel shift assay after labeling the 5′-end of the substrate with 32P. The purity of the concentrated protein samples was checked by SDS-polyacrylamide gel electrophoresis and gave single bands as illustrated in Fig. 1 a. The flap endonuclease activity of the proteins was assayed via a flow cytometry-based nuclease assay system (36Nolan J.P. Shen B. Sklar L.A. Park M.S. Biochemistry. 1996; 35: 11668-11676Crossref PubMed Scopus (61) Google Scholar) before and after each SAXS measurement. Activity was also observed by time-resolved SAXS measurements by activating the endonuclease with Mg2+ in the presence of the flap DNA substrate. An oligonucleotide (5′-CCCCCCATGCTACGTTTTCGTATACGTTTTCGTA-3′) was synthesized by the solid-phase phosphoramidite method using an Applied Biosystems synthesizer. The oligonucleotide was designed to form a 5′-flap substrate, which contains two Watson-Crick duplex arms folded by TTTT loops (37Blommers M.J.J. Haasnoot C.A.G. Hilbers C.W. van Boom J.H. van der Marel G.A. NATO Adv. Sci. Inst. Ser. Ser. E Appl. Sci. 1987; 133: 78-91Google Scholar) and a 10-base-long 5′-flap single strand (see Fig.1 b). It was purified by eluting the material through a POROS R2/H reverse-phase chromatography column, followed by eluting through a POROS HQ/M anion-exchange chromatography column if necessary, equipped with a Bio-Cad Workstation 700E (PerSeptive Biosystems). The purity was analyzed using a Tris borate/EDTA-urea gel (Novex) and found to be >98% pure. NMR spectroscopic analysis was used to establish the formation of the flap structure as shown in Fig. 1 b. Electrophoretic mobility shift and flap endonuclease assays were used to determine the suitability of the newly designed substrate for structural analysis. SAXS data were reduced as described elsewhere (38Heidorn D.B. Trewhella J. Biochemistry. 1988; 27: 909-915Crossref PubMed Scopus (283) Google Scholar, 39Blechner S.L. Olah G.A. Strynadka N.C.J. Hodges R.S. Trewhella J. Biochemistry. 1992; 31: 11326-11334Crossref PubMed Scopus (33) Google Scholar) to giveI(Q) versus Q, whereI(Q) is the scattered x-ray intensity andQ is the amplitude of the scattering vector. For elastic scattering processes, Q is equal to 4πsinθ/λ, where θ is half the scattering angle and λ is the wavelength of the incident and scattered x-rays. Guinier (40Guinier A. Ann. Rev. Physiol. (Paris). 1939; 12: 161-237Google Scholar) and the indirect Fourier transform (41Moore P. J. Appl. Crystallogr. 1980; 13: 168-175Crossref Google Scholar) analyses were used to calculate R g, forward scatter (I o), and the vector distribution function (P(r)). Aggregation was ascertained fromI o, which was expected to be proportional to the molecular mass (39Blechner S.L. Olah G.A. Strynadka N.C.J. Hodges R.S. Trewhella J. Biochemistry. 1992; 31: 11326-11334Crossref PubMed Scopus (33) Google Scholar). Lysozyme was used as a standard for scalingI o with the implicit assumption that lysozyme has the same mean scattering density as FEN-1 and the D181A mutant. Interparticle interference contribution to the scattering at the concentrations used was assumed to be negligible since preliminary measurements from the samples at concentrations between 0.5 and 6 mg/ml scaled linearly and gave the same R g values.P(r) is the frequency of all interatomic vectors within the scattering particle weighted by the product of their scattering powers. The zeroth and second moments ofP(r) are equal to I o andR g, respectively, and the maximum linear dimension of the scattering particle (d max) was determined from the corresponding value at which the P(r) function goes to zero. SAXS measurements were done using the instrument described elsewhere (38Heidorn D.B. Trewhella J. Biochemistry. 1988; 27: 909-915Crossref PubMed Scopus (283) Google Scholar) at the Los Alamos National Laboratory. The instrument configuration used nickel-filtered 1.542 x-rays produced from a 1.5-kilowatt sealed tube copper target source and was line-focused by a single mirror giving a full width at a half-maximum of 0.74 mm and a full height of 26 mm as measured at the detector. A 4-inch-long position-sensitive linear detector (TEC Model 210Q) was placed 64 cm from the sample. Measurements spanned a Q range of 0.015–0.27 Å−1. The net scattering from the samples was calculated by subtracting a normalized buffer spectrum measured in the same sample cell. Time course SAXS measurements, whereby data were recorded for 30 min at subsequent intervals, was used to track endonuclease activation after addition of magnesium to protein/DNA substrate samples. Typically, measurements at these protein concentrations require ∼6 h for sufficient statistics; however, 30-min scans were good enough for observing changes inI o. SAXS from at least two separate sample preparations were measured for all measurements. Measurements were made at 10 °C. P(r) analysis of the data collected included a deconvolution of the slit geometric contribution to the scattering. Omission of this correction results in a systematic ∼0.3-Å smallerR g for FEN-1, well within the statistics of the data measured in this report (0.5–1.0 Å; 1 S.D.). Guinier analysis of the data collected was not deconvoluted for instrument geometry. Magnesium is an essential cofactor for many enzymes involved in DNA metabolism such as DNA polymerases, nucleases, and ligases. This divalent metal, commonly ligated by acidic amino acid residues in nucleases, attacks water molecules to produce a nucleophile that can break phosphodiester bonds (34Shen B. Nolan J.P. Sklar L.A. Park M.S. J. Biol. Chem. 1996; 271 (and references therein): 9173-9176Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Chelating of the metal ions out of human FEN-1 can inactivate the enzyme completely. It has been hypothesized that activation of the enzyme by adding Mg2+to the reaction requires conformational changes in the enzyme before it can cleave the DNA substrate (36Nolan J.P. Shen B. Sklar L.A. Park M.S. Biochemistry. 1996; 35: 11668-11676Crossref PubMed Scopus (61) Google Scholar). To test this hypothesis, we performed small-angle x-ray scattering from wild-type FEN-1, the D181A mutant, and their complex with DNA substrate to determine their global structure and possible conformational changes upon addition of the Mg2+ cofactor. To perform SAXS experiments, it was necessary to develop an experimental approach to produce a large quantity of flap DNA substrate. Unfortunately, the conventional approach, which utilizes annealing of three independent oligonucleotides, was inadequate for this purpose due to its low yield. To overcome this problem, we designed a single oligonucleotide that has a high propensity to form the flap DNA structure, as shown in Fig.1 b (see “Experimental Procedures”). We used NMR spectroscopy and gel mobility shift and flap endonuclease assays to determine that the newly designed oligonucleotide forms a predicted flap DNA substrate. NMR spectroscopy showed the presence of a double-stranded region registered by A=T and G≡C base pairs and showed the presence of a TTTT loop, and wild-type FEN-1 was able to bind to DNA and yielded a correct cleavage product with an expected size of the released flap strand (10 bases) (data not shown). Based upon all of these results, we concluded that the new flap DNA substrate could be used for our further study described. Fig. 2 shows Guinier plots calculated for FEN-1 and the D181A mutant as well as for FEN-1·34-mer DNA flap and D181A·34-mer DNA flap complexes. Each sample gives a Guinier region that can be fit with a straight line with reduced χ2below 1 (Table I). Molecular masses calculated for the protein samples from I o and using the lysozyme standard were within 10% of the expected value of 43,416 Da. In addition, there was no significant upturn at low Q in the scattering profiles, except for the D181A·34-mer DNA flap complex. The D181A·34-mer DNA flap complex sample had the same concentration as the FEN-1·34-mer DNA flap complex and gave an extrapolated I o value consistently ∼10% larger than that found for the FEN-1·34-mer DNA flap complex. This increase in I o indicates slight aggregation of the D181A·34-mer DNA flap complex samples. The R g andd max parameters in Table I were calculated from combinations of two to four scattering measurements using different sample preparations. Guinier plots of log(I·Q)versus Q 2 showed a linearQ region (0.03–0.08 Å−1) with a negative slope, from which the radius of gyration of cross-section (R c) could be calculated. A linear region in such a plot suggests that the proteins have an elongated shape, at least in one dimension. The R c values are also tabulated in Table I.Table IX-ray scattering data from FEN-1 and D181ASampleConcGuinier analysisP(r) analysisR gR cR gd maxmg/mlÅÅFEN-1−Mg2+5.826.4 ± 0.412.7 ± 0.726.8 ± 0.782+Mg2+5.926.6 ± 0.912.6 ± 0.926.2 ± 0.484D181−Mg2+626.4 ± 0.512.9 ± 1.026.1 ± 0.586+Mg2+625.9 ± 0.612.5 ± 0.726.1 ± 0.78834-mer DNA−Mg2+220.4 ± 0.510.1 ± 2.020.2 ± 0.460+Mg2+221.8 ± 0.69.3 ± 2.021.4 ± 0.370FEN-1 · DNA (1:1), −Mg2+4.734.4 ± 0.913.1 ± 0.434.4 ± 0.7104D181A · DNA (1:1), −Mg2+4.740.6 ± 0.913.9 ± 0.943.9 ± 0.8138R g and R c were calculated from the scattering data using Guinier analysis. R g andd max were calculated usingP(r) analysis. FEN-1 and the D181A mutant alone give approximately the same R g,R c, and d max values in both the absence and presence of Mg2+, suggesting that Mg2+either induces no conformational change or induces a localized or global conformational change that is not measurable within the precision of these measurements. The 34-mer DNA in the presence of Mg2+ aggregates as shown by a 16.6% increase inI p, therefore, the larger R g andd max values observed relative to the case without Mg2+ are not necessarily attributable only to a Mg2+-induced conformational change in the DNA substrate. The D181A · 34-mer DNA flap complex has a consistently ∼10% larger I o relative to the FEN-1 · 34-mer DNA flap complex, suggesting that slight aggregation occurs in the mutant · DNA substrate complex. Thus, the scattering data show that the D181A mutant possibly binds and interacts differently with the DNA fragment compared with wild-type FEN-1. Open table in a new tab R g and R c were calculated from the scattering data using Guinier analysis. R g andd max were calculated usingP(r) analysis. FEN-1 and the D181A mutant alone give approximately the same R g,R c, and d max values in both the absence and presence of Mg2+, suggesting that Mg2+either induces no conformational change or induces a localized or global conformational change that is not measurable within the precision of these measurements. The 34-mer DNA in the presence of Mg2+ aggregates as shown by a 16.6% increase inI p, therefore, the larger R g andd max values observed relative to the case without Mg2+ are not necessarily attributable only to a Mg2+-induced conformational change in the DNA substrate. The D181A · 34-mer DNA flap complex has a consistently ∼10% larger I o relative to the FEN-1 · 34-mer DNA flap complex, suggesting that slight aggregation occurs in the mutant · DNA substrate complex. Thus, the scattering data show that the D181A mutant possibly binds and interacts differently with the DNA fragment compared with wild-type FEN-1. Fig. 3 shows the R gvalues calculated from the P(r) analysis of the individual measurements for the proteins alone in the presence and absence of magnesium. Also, a comparison of theP(r) functions is given in Fig.4. The P(r) functions have a single peak at ∼27 Å and are fairly symmetric, suggesting that the proteins are globular (ellipsoidal). Modeling the data with one ellipsoid using a Monte Carlo modeling method described elsewhere (42Mecklenburg S.L. Donohoe R.D. Olah G.A. J. Mol. Biol. 1997; 270: 739-750Crossref PubMed Scopus (35) Google Scholar) gave dimensions for FEN-1 of a = 13.6 ± 0.2 Å, b = 32.4 ± 1.0 Å, andc = 45.1 ± 1.0 Å for the best fit. The scattering profile generated from this model also gave anR c value consistent with the R cvalue determined from the Guinier plots. Within the statistics ofR g measurements, it is evident that magnesium has no effect on FEN-1 or the D181A mutant in the absence of DNA. The fact that Mg2+-induced conformational changes was not observed is probably because the Mg2+ binding causes a localized instead of global conformational effect or the induced global conformational changes are quite small.Figure 4P(r) functions for FEN-1 (a) and the D181A mutant (b) in the absence of DNA. Black is in the absence of Mg2+, and gray is in its presence. TheP(r) functions are fairly symmetric, indicating that the two proteins are globular (ellipsoid shape) in solution and are not affected by the presence of Mg2+ or by replacement of Asp-181 by alanine. Single ellipsoid modeling against FEN-1 showed it to have dimensions of a = 13.6 ± 0.2 Å,b = 32.4 ± 1.0 Å, and c = 45.1 ± 1.0 Å.View Large Image Figure ViewerDownload (PPT) Next, we examine the effect of Mg2+ on the DNA substrate by both scattering and modeling. Scattering profiles andP(r) functions from the free 34-mer DNA fragment in the absence and presence of Mg2+ are shown in Fig.5. In the absence of Mg2+, the P(r) function showed a peak at ∼16 Å and decreased approximately linearly out to ∼60 Å. A simple two-cylinder model was constructed based on the 5′-flap DNA structure shown in Fig.1" @default.
• W2170294911 created "2016-06-24" @default.
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• W2170294911 title "Structural Changes Measured by X-ray Scattering from Human Flap Endonuclease-1 Complexed with Mg2+ and Flap DNA Substrate" @default.
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