Arginine Residues 47 and 70 of Human Flap Endonuclease-1 Are Involved in DNA Substrate Interactions and Cleavage Site Determination
2002; Elsevier BV; Volume: 277; Issue: 27 Linguagem: Inglês
10.1074/jbc.m111941200
ISSN1083-351X
AutoresJunzhuan Qiu, David N. Bimston, Arthur Partikian, Binghui Shen,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoFlap endonuclease-1 (FEN-1) is a critical enzyme for DNA replication and repair. Intensive studies have been carried out on its structure-specific nuclease activities and biological functions in yeast cells. However, its specific interactions with DNA substrates as an initial step of catalysis are not defined. An understanding of the ability of FEN-1 to recognize and bind a flap DNA substrate is critical for the elucidation of its molecular mechanism and for the explanation of possible pathological consequences resulting from its failure to bind DNA. Using human FEN-1 in this study, we identified two positively charged amino acid residues, Arg-47 and Arg-70 in human FEN-1, as candidates responsible for substrate binding. Mutation of the Arg-70 significantly reduced flap endonuclease activity and eliminated exonuclease activity. Mutation or protonation of Arg-47 shifted cleavage sites with flap substrate and significantly reduced the exonuclease activity. We revealed that these alterations are due to the defects in DNA-protein interactions. Although the effect of the single Arg-47 mutation on binding activities is not as severe as R70A, its double mutation with Asp-181 had a synergistic effect. Furthermore the possible interaction sites of these positively charged residues with DNA substrates were discussed based on FEN-1 cleavage patterns using different substrates. Finally data were provided to indicate that the observed negative effects of a high concentration of Mg2+ on enzymatic activity are probably due to the competition between the arginine residues and metal ions with DNA substrate since mutants were found to be less tolerant. Flap endonuclease-1 (FEN-1) is a critical enzyme for DNA replication and repair. Intensive studies have been carried out on its structure-specific nuclease activities and biological functions in yeast cells. However, its specific interactions with DNA substrates as an initial step of catalysis are not defined. An understanding of the ability of FEN-1 to recognize and bind a flap DNA substrate is critical for the elucidation of its molecular mechanism and for the explanation of possible pathological consequences resulting from its failure to bind DNA. Using human FEN-1 in this study, we identified two positively charged amino acid residues, Arg-47 and Arg-70 in human FEN-1, as candidates responsible for substrate binding. Mutation of the Arg-70 significantly reduced flap endonuclease activity and eliminated exonuclease activity. Mutation or protonation of Arg-47 shifted cleavage sites with flap substrate and significantly reduced the exonuclease activity. We revealed that these alterations are due to the defects in DNA-protein interactions. Although the effect of the single Arg-47 mutation on binding activities is not as severe as R70A, its double mutation with Asp-181 had a synergistic effect. Furthermore the possible interaction sites of these positively charged residues with DNA substrates were discussed based on FEN-1 cleavage patterns using different substrates. Finally data were provided to indicate that the observed negative effects of a high concentration of Mg2+ on enzymatic activity are probably due to the competition between the arginine residues and metal ions with DNA substrate since mutants were found to be less tolerant. flap endonuclease-1 proliferating cell nuclear antigen human FEN-1 position nucleotide(s) Flap endonuclease-1 (FEN-1)1 proteins possess a flap endonuclease activity as well as a 5′ to 3′ exonuclease activity (1Waga S. Stillman B. Nature. 1994; 369: 207-212Crossref PubMed Scopus (497) Google Scholar, 2Waga S. Bauer G. Stillman B. J. Biol. Chem. 1994; 269: 10923-10934Abstract Full Text PDF PubMed Google Scholar, 3Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (373) Google Scholar, 4Harrington J.J. Lieber M.R. Genes Dev. 1994; 8: 1344-1355Crossref PubMed Scopus (256) Google Scholar, 5Turchi J.J. Huang L. Murante R.S. Kim Y. Bambara R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8903-9807Crossref Scopus (169) Google Scholar). These nuclease activities allow FEN-1 proteins to remove the RNA primers during lagging-strand DNA synthesis and damaged DNA fragments in various DNA repair pathways (6Murray 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, 7Reagan M.S. Pittenberger C. Siede W. Friedberg E.C. J. Bacteriol. 1995; 177: 364-371Crossref PubMed Google Scholar, 8Sommers 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, 9Johnson R.E. Kovvali G.K. Prakash L. Prakash S. Science. 1995; 269: 238-240Crossref PubMed Scopus (195) Google Scholar, 10Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88 (263): 263Abstract Full Text Full Text PDF Scopus (394) Google Scholar, 11Yoon J.-H. Swiderski P. Kaplan B.E. Takao M. Yasui A. Shen B. Pfeifer G.P. Biochemistry. 1999; 39: 4909-4917Google Scholar). Besides its nuclease activities, it interacts with various proteins involved in DNA metabolic pathways including the Werner syndrome protein (12Brosh R.M., Jr. Kobbe C. Sommers J.A. Karmaker P. Opresko P.L. Piotrowski J. Dianova I. Dianov G., L. Bohr V.A. EMBO J. 2001; 20: 5791-5801Crossref PubMed Scopus (228) Google Scholar) and proliferating cell nuclear antigen (PCNA) (13Li X., Li, J. Harrington J.J. Lieber M.R. Burgers P.M.J. J. Biol. Chem. 1995; 270: 22109-22112Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 14Wu X., Li, J., Li, X. Hsieh C.-L. Burgers P.M.J. Lieber M.R. Nucleic Acids Res. 1996; 24: 2036-2043Crossref PubMed Scopus (198) Google Scholar). FEN-1 localizes into the nucleus in S phase during DNA synthesis as well as in response to DNA damage (15Qiu J., Li, X. Frank G. Shen B. J. Biol. Chem. 2001; 276: 4901-4908Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In yeast, loss of FEN-1 functions was determined to cause severely impaired phenotypes such as conditional lethality, dramatic increase of cellular sensitivity to alkylating agents,e.g. methyl methanesulfonate, and significant increase in the mutation rate. Interestingly a majority of mutations in the yFEN-1 mutant strains are large sequence duplications (10Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88 (263): 263Abstract Full Text Full Text PDF Scopus (394) Google Scholar). Duplication mutations in humans have been associated with disorders such as recessive retinitis pigmentosa, lethal junctional epidermolysis bullosa, familial hypertrophic cardiomyopathy, and cancer. FEN-1 is also a key enzyme for preventing expansion and contraction of bi- or trinucleotide repeats (16Gordenin D.A. Kunkel T.A. Resnick M.A. Nat. Genet. 1997; 16: 116-118Crossref PubMed Scopus (189) Google Scholar, 17Freudenreich C.H. Kantrow S.M. Zakian V.A. Science. 1998; 279: 853-856Crossref PubMed Scopus (364) Google Scholar, 18Spiro C. Pelletier R. Rolfsmeier M.L. Dixon M.J. Lahue R.S. Gupta G. Park M.S. Chen X. Mariappan S.V.S. McMurray C.T. Mol. Cell. 1999; 4: 1079-1085Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 19Tom S. Henricksen L.A. Bambara R.A. J. Biol. Chem. 2000; 275: 10498-10505Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). More recently we have demonstrated that FEN-1 nuclease plays an important role in limiting short sequence recombination for yeast genome stability (20Negritto M.C. Qiu J. Ratay D.O. Shen B. Bailis A.M. Mol. Cell. Biol. 2001; 21: 2349-2358Crossref PubMed Scopus (44) Google Scholar). All of these observations illustrate its critical roles in genome integrity. As an essential gene for DNA replication, FEN-1 defects resulting in a complete loss of function are unlikely to exist in the human population (21Kunkel T.A. Resnick M.A. Gordenin D.A. Cell. 1997; 88: 155-158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 22Ma X. Jin Q. Forsti A. Hemminki K. Kumar R. Int. J. Cancer. 2000; 88: 938-942Crossref PubMed Scopus (13) Google Scholar). A comprehensive structural and functional analysis of single amino acid residue mutations will assist in the formulation of models for biological and pathological roles of this enzyme. Initial domain analysis based on protein sequence comparison and biochemical assays displayed two major conserved motifs, N (N-terminal) and I (intermediate) motifs, which are essential for the nuclease activities of FEN-1 proteins (4Harrington J.J. Lieber M.R. Genes Dev. 1994; 8: 1344-1355Crossref PubMed Scopus (256) Google Scholar, 23Shen B. Qiu J. Hosfield D. Tainer J.A. TIBS. 1998; 23: 171-173Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). A third motif at the C-terminal end is involved in the interaction of FEN-1 proteins with PCNA (23Shen B. Qiu J. Hosfield D. Tainer J.A. TIBS. 1998; 23: 171-173Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Warbrick E. Lane D.P. Glover D.M. Cox L.S. Oncogene. 1997; 14: 2313-2321Crossref PubMed Scopus (136) Google Scholar, 25Frank G. Qiu J. Zheng L. Shen B. J. Biol. Chem. 2001; 276: 36295-36302Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Although we have recently demonstrated that stimulation of eukaryotic FEN-1 activities by PCNA is independent of its in vitrointeraction via a consensus PCNA binding region (25Frank G. Qiu J. Zheng L. Shen B. J. Biol. Chem. 2001; 276: 36295-36302Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), the interaction may be crucial for PCNA to recruit FEN-1 onto Okazaki fragment processing and DNA damage sites (24Warbrick E. Lane D.P. Glover D.M. Cox L.S. Oncogene. 1997; 14: 2313-2321Crossref PubMed Scopus (136) Google Scholar, 26Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (666) Google Scholar, 27Kim K. Biade S. Matsumoto Y. J. Biol. Chem. 1998; 273: 8842-8848Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 28Shibata Y. Nakamura T. J. Biol. Chem. 2002; 277: 746-754Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). An additional C-terminal motif exists in eukaryotic cells that is responsible for the localization of FEN-1 protein into the nucleus (15Qiu J., Li, X. Frank G. Shen B. J. Biol. Chem. 2001; 276: 4901-4908Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In recent years, we have performed site-directed mutagenesis on human FEN-1 nuclease coupled with biochemical and yeast genetic analysis. A number of amino acid residues, which are critical for biochemical and biological functions, have been revealed through this ongoing endeavor. The structural and functional relationship of these identified amino acid residues is summarized in TableI.Table ICritical amino acid residues identified in human FEN-1 via site-directed mutagenesis and biochemical analysisDesignated regionsBiochemical functionsResiduesRefs.N and ICatalytic centerAsp-34, Asp-86, Glu-158, Glu-160, Glu-178, Asp-179, Asp-181, Asp-23334Frank G. Qiu J. Somsouk M. Weng Y. Somsouk L. Nolan J. Shen B. J. Biol. Chem. 1998; 273: 33064-33072Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 38Shen B. Nolan J.P. Sklar L.A. Park M.S. J. Biol. Chem. 1996; 271: 9173-9176Abstract Full Text Full Text PDF PubMed Scopus (101) Google ScholarSubstrate bindingArg-47, Arg-70This studyIIIPCNA interactionLeu-340, Asp-341, Phe-343, Phe-34425Frank G. Qiu J. Zheng L. Shen B. J. Biol. Chem. 2001; 276: 36295-36302Abstract Full Text Full Text PDF PubMed Scopus (48) Google ScholarIVNuclear localizationLys-354, Arg-355, Lys-356, Lys-365, Lys-366, Lys-36715Qiu J., Li, X. Frank G. Shen B. J. Biol. Chem. 2001; 276: 4901-4908Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar Open table in a new tab Although relatively abundant information on the structural and functional relationship of some FEN-1 proteins is available, identification of structural elements for DNA substrate binding has been a difficult task without a three-dimensional structure of the protein-DNA complex. To identify amino acid residues directly involved in substrate binding, we constructed a three-dimensional molecular model for human FEN-1 based on the available homologous crystal structures (29Kim Y. Eom S.H. Wang J. Lee D.S. Suh S.W. Steitz T.A. Nature. 1995; 376: 612-616Crossref PubMed Scopus (329) Google Scholar, 30Mueser T.C. Nossal N.G. Hyde C.C. Cell. 1996; 85: 1101-1112Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 31Ceska T.A. Sayers J.R. Stier G. Suck D. Nature. 1996; 382: 90-93Crossref PubMed Scopus (165) Google Scholar, 32Hosfield D. Mol C.D. Shen B. Tainer J.A. Cell. 1998; 95: 135-146Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 33Hwang K.Y. Baek K. Kim H.-Y. Cho Y. Nat. Struct. Biol. 1998; 5: 707-713Crossref PubMed Scopus (149) Google Scholar). We have identified 10 amino acid residues including Arg-29, Arg-47, Arg-70, Arg-73, Lys-80, Lys-93, Lys-99, Arg-100, Arg-103, and Arg-104 as candidate residues that directly interact with DNA substrates. These residues are conserved in eukaryotes and are located on the surface of the molecule. We have performed site-directed mutagenesis on these residues. Indeed individual mutations of many of these residues affect biochemical activities. The first five residues are located in the N-terminal α-helix and β-sheet region near the active center of the enzyme, while the other five residues are exclusively in the loop region, which has been proposed to interact with the single-stranded flap of the DNA substrate (32Hosfield D. Mol C.D. Shen B. Tainer J.A. Cell. 1998; 95: 135-146Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 33Hwang K.Y. Baek K. Kim H.-Y. Cho Y. Nat. Struct. Biol. 1998; 5: 707-713Crossref PubMed Scopus (149) Google Scholar). In this report, we focused on the first five residues (Arg-29, Arg-47, Arg-70, Arg-73, and Lys-80) and determined that Arg-47 and Arg-70 are important for the interaction with the DNA substrates. Arg-70 might interact with the upstream double-stranded region of the DNA substrates, and Arg-47, possibly interacting with the upstream template region of DNA substrate, was revealed to play a role in determining cleavage sites via mutational analysis, biochemical assays, protonation, and competition experiments. All mutant proteins created for this study were prepared using the QuikChangeTM site-directed mutagenesis kit from Stratagene (La Jolla, CA). Mutagenic primers were synthesized at the City of Hope DNA/RNA/peptide synthesis core facility. Mutations and corresponding oligo sequences for primers are listed in TableII for clarity. Site-directed mutagenesis, overexpression, and purification of the wild type and mutant FEN-1 and Rad27 proteins were carried out based on our previously published procedures (15Qiu J., Li, X. Frank G. Shen B. J. Biol. Chem. 2001; 276: 4901-4908Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 34Frank G. Qiu J. Somsouk M. Weng Y. Somsouk L. Nolan J. Shen B. J. Biol. Chem. 1998; 273: 33064-33072Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 35Hosfield D.J. Frank G. Weng Y. Tainer J.A. Shen B. J. Biol. Chem. 1998; 273: 27154-27161Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 36Qiu J. Qian Y. Frank P. Wintersberger U. Shen B. Mol. Cell. Biol. 1999; 19: 8361-8371Crossref PubMed Scopus (137) Google Scholar). Mutagenesis reactions contain 50 ng of template pET28-derived plasmids harboring the wild type human FEN-1 sequence (37Nolan J.P. Shen B. Park M.S. Sklar L.A. Biochemistry. 1996; 35: 11668-11676Crossref PubMed Scopus (61) Google Scholar) so that the isolated plasmids containing a mutation can be directly expressed in Escherichia coli.Table IIDNA oligos for construction of DNA substrates and creation of FEN-1 mutantsF, forward; R, reverse; exo-, exonuclease. Bold sequences indicate codon changes. Open table in a new tab F, forward; R, reverse; exo-, exonuclease. Bold sequences indicate codon changes. The oligos used to formulate nuclease substrates are also listed in Table II. Protocols for DNA substrate preparation and nuclease activity assays were based on a previous publication (34Frank G. Qiu J. Somsouk M. Weng Y. Somsouk L. Nolan J. Shen B. J. Biol. Chem. 1998; 273: 33064-33072Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Briefly, Flap-G1 (labeled oligo of the flap substrate), Flap-ND (labeled oligo for single flap substrate and double flap substrate), or Exo-3PT (labeled oligo of exonuclease substrate) were individually phosphorylated at the 5′ end. This was done by incubating 40 pmol of the oligo with 10 μCi of [γ-32P]ATP and 1 μl (10 units/μl) of polynucleotide kinase at 37 °C for 60 min. Polynucleotide kinase was then inactivated by heating at 72 °C for 10 min. 80 pmol each of the Temp-3B and Prim-1G oligos were added to the labeled oligos, respectively. The samples were incubated at 70 °C for 5 min followed by slow cooling to 25 °C, allowing the oligos to slowly anneal and form the flap endonuclease and exonuclease substrates as shown in Fig. 2, B and C. Substrates were precipitated at −20 °C overnight after adding 20 μl of 3 m NaOAc and 1 ml of 100% ethanol. Substrates were collected by centrifugation, washed once with 70% ethanol, and resuspended in sterile water. Reactions were carried out with the amount of hFEN-1 protein as indicated and 500 fmol of flap or exonuclease substrate with reaction buffer containing 50 mm Tris (pH 8.0) 10 mmMgCl2, and 100 μg/ml bovine serum albumin. Each reaction was then brought to a total volume of 13 μl with water. All reactions were incubated at 30 °C for 15 min and terminated by adding an equal volume of stop solution (95% formamide, 20 mm EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). An aliquot of each reaction was then run on a 12% denaturing polyacrylamide gel at 1900 V for 1 h. The gel was dried at 70 °C for 50 min and then visualized by autoradiography. FEN-1 cleavage kinetics were performed using various concentrations of DNA substrates (31.25–500 nm) and constant amounts of FEN-1 (92 nm) following the procedures described in Hosfield et al. (35Hosfield D.J. Frank G. Weng Y. Tainer J.A. Shen B. J. Biol. Chem. 1998; 273: 27154-27161Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Briefly, reactions were initiated by combining standard reaction buffer, substrate, and enzyme in that order. Samples were mixed and incubated for 10 min. The products and substrate were separated by denaturing gel electrophoresis. The initial velocity was calculated by measuring products and substrate intensity on the gel by using the IP Lab Gel program and by using the equation ν = {I 1/(I 0+0.5I 1)t} × [substrate], where t = time in seconds, I 1 = product intensity, and I 0 = final substrate concentration. The substrate concentration was expressed in nm. V max and K m values were calculated by directly fitting the data to the Michaelis-Menten equation, and k cat andk cat/K m were then derived. The experiment was based on a constant amount of wild type protein with various concentrations of each mutant protein. Three mutants were used for this analysis, including D181A, R47A/D181A, and R70A/D181A. A plasmid overexpressing the D181A mutant gene (D181A/pET) was constructed previously (38Shen B. Nolan J.P. Sklar L.A. Park M.S. J. Biol. Chem. 1996; 271: 9173-9176Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Based on this plasmid, two other mutants, R47A/D181A and R70A/D181A, were constructed, overexpressed, and purified. The competition assay was conducted with standard reaction buffer and32P-labeled substrates as described above. The amount of wild type hFEN-1 and mutant proteins are as specified in the Fig. 3legend. After a 15-min incubation at 30 °C, the reaction was stopped with stop solution. The products and substrate were separated by 10% denaturing PAGE to assess the substrate binding capacity of the mutants. This assay was conducted using immobilized substrates on beads and based on a method modified from Gomes and Burgers (39Gomes X.V. Burgers P.M. EMBO J. 2000; 19: 3811-3821Crossref PubMed Scopus (126) Google Scholar). Briefly, 1 nm Temp-1G with biotin at the 5′ end (5′-Biotin-GGACTCTGCCTCAAGACGGTAGTCAACGTG-3′) (Integrated DNA Technologies, Inc., Coralville, IA) was annealed to 1.5 nm Prim-3B (5′-CACGTTGACTACCGTC-3′) and 1.5 nm Flap-G1 (5′-GATGTCAAGCAGTCCTAACTTTGAGGCAGAGTCC-3′) for flap substrate or Exo-3PT (5′-TTGAGGCAGAGTCC-3′) for exonuclease substrate. The annealed flap endo- or exonuclease substrate was immobilized to the NeutrAvidinTM beads in buffer A (10 mm Tris-HCl, pH 7.5, 1 mm EDTA, and 1m NaCl) by incubation for 2–3 h at room temperature. The unbound substrate was washed off with buffer A. The bead-bound substrate was resuspended in buffer C (30 mm HEPES-NaOH, pH 7.5, 125 mm NaCl, 0.2 mg/ml bovine serum albumin, and 1 mm dithiothreitol). The binding assay was performed on ice for 20 min in a 16-μl reaction volume in buffer C with addition of 8 μl of substrate-bound beads and the amount of protein as indicated in the Fig. 4 legend. The beads were then washed twice with prechilled buffer B (30 mm HEPES-NaOH, pH 7.5, 5% glycerol, 0.1 mg/ml bovine serum albumin, 1 mm dithiothreitol, 0.01% Nonidet P-40, 0.1 mm EDTA, and 125 mm NaCl). 12 μl of 2× protein sample loading buffer was then added. The reaction mixture was boiled for 5 min to release the FEN-1 protein from the beads. After a 2-min centrifugation at 14,000 rpm, the supernatant was loaded onto the 10% SDS-polyacrylamide gel. The proteins were blotted onto a nitrocellulose membrane (Schleicher & Schuell) using a Trans-Blot SD semidry electrophoretic transfer cell from Bio-Rad. The blot was probed with polyclonal antibodies raised in rabbits against hFEN-1 (36Qiu J. Qian Y. Frank P. Wintersberger U. Shen B. Mol. Cell. Biol. 1999; 19: 8361-8371Crossref PubMed Scopus (137) Google Scholar). A specific FEN-1 protein was detected using the Pierce SuperSignal West Pico chemiluminescence kit and a protocol recommended by the manufacturer. Since a three-dimensional crystal structure of human FEN-1 has not been solved, we constructed a molecular model of hFEN-1 using the archaebacterial structures. Based on that model of hFEN-1 (Fig. 1), five residues including Arg-29, Arg-47, Arg-70, Arg-73, and Arg-80 were visualized on the surface of the molecule near the catalytic center. To experimentally test the roles of these amino acid residues in substrate binding and nuclease activity, we created the following non-conservative amino acid substitution mutants: R29A, R47A, R70A, R73A, R80A, R47A/R70A, and R70A/R73A. The mutant proteins were overexpressed and purified to photographic homogeneity as shown in Fig.2 A. These proteins were then assayed for their FEN-1 activities. Fig. 2, B andC, shows that only R47A or R70A among the five single mutants had a significant effect on FEN-1 enzyme activities. Subsequently R47A, R70A, R73A (as a control since it is so close to Arg-70), R47A/R70A, and R70A/R73A were included for further investigations. The R47A and R70A mutants had a more severe impact on exonuclease rather than on flap endonuclease activity. R70A significantly reduced exonuclease activity while retaining most of its flap endonuclease activity (Table III). In contrast, the R47A mutation maintained both activities but shifted the endonuclease cleavage site from nucleotide position 19 (Pos19) to 21 (Pos21) on the flap strand and the exonuclease cleavage site from Pos1, Pos2, and Pos3 to Pos2 and Pos3. Double mutants R47A/R70A and R70A/R73A failed to show a synergistic effect on nuclease activities. In fact, the combined mutational effect of R47A and R70A as well as R70A and R73A was additive (Fig. 2, B and C).Table IIIKinetic parameters of wild type and mutant FEN-1 proteinsProteinsEnzyme activity K m k cat k cat/K m nm (1/min) × 10 −2 (1/nm·min) × 10 −4wtFEN-145.23.57.7R47A76.33.14.1R70AEndo-101.22.62.6R73A47.53.67.6R47A/R70A111.82.52.2R70A/R73A106.72.72.5wtFEN-156.22.54.4R47A74.71.62.1R70AExo-ND3-aND, the relevant parameters were unmeasurable due to the impaired enzyme activity.NDNDR73A55.62.64.7R47A/R70ANDNDNDR70A/R73ANDNDNDwt, wild type; Endo-, endonuclease; Exo-, exonuclease.3-a ND, the relevant parameters were unmeasurable due to the impaired enzyme activity. Open table in a new tab wt, wild type; Endo-, endonuclease; Exo-, exonuclease. To further explore the roles of Arg-47 and Arg-70 in substrate DNA binding, we performed kinetic analysis of the wild type and mutant hFEN-1 enzymes. Conventional steady-state kinetic analyses were carried out using various concentrations of radioactively labeled substrates, a constant amount of enzyme, and gel electrophoresis. We then measured cleavage activity with both the flap endo- and exonuclease DNA substrates. The results were analyzed using Michaelis-Menten kinetics to derive K m ,k cat, andk cat/K m values as listed in Table III. While the k cat values for flap endonuclease activity were largely not affected, K m values for the R70A, R47A/R70A, and R70A/R73A mutants were doubled. The second order rate constants (k cat/K m ) for these three mutants were significantly reduced (3-fold) with the endonuclease activity, suggesting that residue Arg-70 is critical for DNA substrate binding. Interestingly the R47A/R70A double mutant again revealed an additive effect on kinetic parameters. The effects of these mutations to the exonuclease activity were so severe that their kinetic parameters were not measurable any more. The K m of the R47A exonuclease activity was slightly increased, while thek cat and second order rate constant were both decreased. Since R47A and R70A of hFEN-1 retained partial flap endonuclease activity, they could not be directly used for the competition assays. However, our previous studies indicated that the D181A hFEN-1 mutant had a wild type or even enhanced binding capability but lost cleavage activity, and it could compete for substrates with the wild type enzyme and suppress wild type enzyme activity (38Shen B. Nolan J.P. Sklar L.A. Park M.S. J. Biol. Chem. 1996; 271: 9173-9176Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The competition assay utilized in our current study has been established based on these observations. Knowing the biochemical characteristics of the D181A mutant, we created two double mutants D181A/R47A and D181A/R70A to confirm the roles of the Arg-47 and Arg-70 residues in DNA binding. Null nuclease activities of these mutants were verified before they were used in competition assays. Circular dichroism spectra of the wild type, single mutants, and double mutants were taken and compared to confirm that no significant structural alterations took place due to these mutations (data not shown). Then if Arg-47 and Arg-70 are involved in DNA binding, the double mutant proteins, R47A/D181A and R70A/D181A, should demonstrate reduced DNA substrate binding ability in contrast to the D181A mutant. The competition assays were performed based on a constant amount of wild type hFEN-1 protein and increasing amounts of mutant proteins (D181A, R47A/D181A, or R70A/D181A). The result indicates that the double mutants did in fact have a significantly reduced ability to suppress the enzyme activity of the wild type hFEN-1 protein (Fig.3, A and B). R47A/D181A had even more severe effects on substrate binding. To further examine the roles of Arg-47 and Arg-70 in substrate binding, we performed DNA-protein binding assays. This assay was conducted using biotinylated DNA substrates immobilized onto avidin-coated beads. After FEN-1 proteins were loaded onto the beads with the DNA substrates, the remaining unbound proteins were removed by extensively washing the beads. Bound protein was then dissociated by boiling and subsequently detected by Western blotting using anti-hFEN-1 antibody. The result of this experiment is shown in Fig. 4,A and B, with the flap DNA and exonuclease substrates. It is clear that R47A and R70A mutations produced significantly lower binding affinity to both of the substrates. The above data allow us to propose that the two amino acids, Arg-47 and Arg-70, are involved in DNA binding of FEN-1 protein. One further question is which parts of the DNA substrate interact with these two amino acids. We designed an experiment using five different substrates. Some of the substrates lack a part of the configuration of the normal double flap substrate. The names and different structural elements of the substrates are indicated in Fig.5. Analysis of various responses of the wild type protein and R47A and R70A mutants to the different substrates was expected to delineate the specific site of the normal substrate that interacts with these two residues. Fig. 5 Ashows the cleavage results of three FEN-1 proteins based on normal flap, pseudo Y, and 5′ overhanging substrates. The result with the normal flap substrate was consistent with those described for Fig.1 B. That is, R70A mutant had a reduction of enzyme cleavage capability but had no change of cleavage sites in comparison to the wild type; however, mutation R47A changed both cleavage efficiency and sites (a shift from Pos19 to Pos21 and larger products). With pseudo Y substrate missing the upstream primer, the enzyme activities of the wild type and the R47A and R70A mutants were reduced, particularly at the Pos21 cleavage site. With a 5′ overhanging substrate, the enzyme activities of the three FEN-1 proteins were all further reduced, and the cleavage products wer
Referência(s)