Essential amino acids for substrate binding and catalysis of human flap endonuclease 1.

Human flap endonuclease 1 (FEN-1) is a member of the structure-specific endonuclease family and is involved in DNA repair. Eight restrictively conserved amino acids in FEN-1 have been converted individually to an alanine to elucidate their roles in specific DNA substrate binding and catalysis. Flap endonuclease activity of the wild type and mutant enzymes was measured by kinetic flow cytometry. Mutants D34A, D86A, and D181A lost their cleavage activity completely but retained substrate binding ability, as measured by their ability to inhibit the wild type enzyme in a competition assay. This indicates that these amino acids contribute to integrity of the enzyme active site. Loss of both binding and cleavage competency for the flap substrate by mutants E156A, G231A, and D233A suggests that these amino acids are involved in substrate binding. Mutants R103A and D179A retained wild type-like enzyme activity.

Human flap endonuclease 1 (FEN-1) is a member of the structure-specific endonuclease family and is involved in DNA repair. Eight restrictively conserved amino acids in FEN-1 have been converted individually to an alanine to elucidate their roles in specific DNA substrate binding and catalysis. Flap endonuclease activity of the wild type and mutant enzymes was measured by kinetic flow cytometry. Mutants D34A, D86A, and D181A lost their cleavage activity completely but retained substrate binding ability, as measured by their ability to inhibit the wild type enzyme in a competition assay. This indicates that these amino acids contribute to integrity of the enzyme active site. Loss of both binding and cleavage competency for the flap substrate by mutants E156A, G231A, and D233A suggests that these amino acids are involved in substrate binding. Mutants R103A and D179A retained wild type-like enzyme activity.
Human flap endonuclease 1, a structure-specific endonuclease, recognizes a specific 5Ј flap DNA structure, the junction where the two strands of duplex DNA adjoin a single-stranded arm (Lyamichev et al., 1993). 1 FEN-1 2 binds to the arm with a free 5Ј end, slides down to the double strand-single strand junction of the substrate, and then cleaves the single-stranded arm (Murante et al., 1995). This in vitro activity suggests an in vivo function of the enzyme in DNA damage repair pathways (Harrington and Lieber, 1994b;Murray et al., 1994;Sommers et al., 1995;Reagan et al., 1995;Johnson et al., 1995). Mouse FEN-1 and the yeast FEN-1 homologs, Saccharomyces cerevisiae YKL510 and Schizosaccharomyces pombe rad2 have been identified (Jacquier et al., 1992;Harrington and Lieber, 1994a;Murray et al., 1994). Sequence comparison of FEN-1 with XPG family proteins revealed that two regions of the protein sequences are conserved (Carr et al., 1993;MacInnes et al., 1993;Scherly et al., 1993). The in vitro activity of the flap endonuclease has been found in the human XPG nuclease (Cloud et al., 1995). This indicates that members of the XPG family including human and mouse XPG and the yeast counterparts, S. cerevisiae RAD2 and S. pombe rad13, and the FEN-1 family are conserved not only in sequence but also in function.
Previously known 5Ј exonuclease domains of several prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I and Thermus aquaticus (Taq) DNA polymerase have now been shown to posses a structure-specific endonuclease activity that cleaves the 5Ј flap structure (Lyamichev et al., 1993). When four bacteriophage exonuclease sequences have been compared with the above exo/endonuclease domains of the DNA polymerases, five conserved regions of these two groups of nucleases were identified (Gutman and Minton, 1993). Interestingly, the sequence of human flap endonuclease has some similarities to the conserved exo/endonuclease domain of E. coli DNA polymerase I (Robins et al., 1994). Taken together, this indicates that the structure and function of this 5Ј structurespecific nuclease activity are conserved among bacteria, yeasts, and mammals.
In this report, we compared sequences of the two conserved regions of 8 eukaryotic FEN-1/XPG family members, 6 exo/ endonuclease domains of prokaryotic DNA polymerases, and 4 bacteriophage exonucleases. We have identified eight amino acids that are restrictively conserved throughout these 18 endo/exonucleases (Fig. 1). We predict that these amino acids might be essential for substrate binding or catalysis. To test this hypothesis, we have converted these amino acids individually to alanine, expressed the mutant genes in E. coli, and purified the mutant FEN-1 proteins to near homogeneity. The mutants enzymes were characterized for their ability to recognize and cleave the flap DNA structure by a recently developed assay employing kinetic flow cytometry. 1 This approach allows us to identify critical amino acids involved in DNA substrate binding and catalysis.

EXPERIMENTAL PROCEDURES
Materials -Restriction enzymes, EcoRI, BamHI, and NcoI, and 100 mg/ml bovine serum albumin solution were obtained from New England Biolabs. Sequenase Version 2.0 DNA sequencing kit was from United States Biochemical Corp. Expression vectors and the host E. coli strains were from Novagen (Madison, WI). All electrophoresis gels and reagents were obtained from Novex (San Diego, CA). Chelating Sepharose fast flow resin was from Pharmacia Biotech Inc. Protein assay kit was from Bio-Rad. All buffers and salts were molecular biology grade from Fisher Scientific. Streptavidin-coated polystyrene microspheres (6.2-m diameter) were from Spherotech (Libertyville, IL). Fluorescein isothiocyanate-labeled standard microspheres were from Flow Cytometry Standards Corp (San Juan, PR). Sodium fluorescein was from Sigma. Oligonucleotide substrates were synthesized on an Applied Biosystems (ABI) DNA synthesizer, using nucleotide or BioTEG CPG support and nucleotide or fluorescein phosphoramidites (Glen Research, Sterling, VA).
Site-directed Mutagenesis- Table I shows the sequences of the oligonucleotides used to construct the eight site-directed mutants used in this study. The altered nucleotides are underlined. Nucleotide substitutions were incorporated at the desired locations in the cloned FEN-1 in pBluescript vector by using Chameleon Double-stranded Site-directed Mutagenesis Kit from Stratagene. The mutations were confirmed by double-stranded DNA sequencing using U. S. Biochemical Corp. Sequenase Version 2.0 kit.
Mutant Enzyme Expression and Purification-The pET-FCH plasmid has been made to overexpress the wild type human FEN-1 protein as described previously. 1 All of the mutated sequences described above were subcloned into pET-FCH with NcoI and BamHI sites. The resultant plasmids were transformed into the E. coli host strain BL21(DE3) for protein expression. Enzyme expression procedures for most of the mutant proteins were essentially the same as for the wild type. For some mutant proteins with low solubility in LB medium, a sorbitol/ betaine medium was used according to Blackwell and Horgan (1991), and the expression temperature was shifted to 25°C in order to enhance solubility.
All of the purification steps were carried out either on ice or in a 4°C cold room. Bacterial lysates were made by sonicating in buffer S (50 mM Tris, pH 8.0, 50 mM sorbitol). The extract then was centrifuged at 30,000 ϫ g for 1 h. The recombinant proteins were purified by passing the supernatant over a Ni 2ϩ -Sepharose column under nondenaturing conditions. The column was washed extensively with buffer A (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 5 mM imidazole). Buffer A containing 60 mM imidazole was used to remove weakly bound proteins. The FEN-1 mutant proteins were eluted with buffer A containing 1.0 M imidazole. The eluted proteins were diluted 10-fold with phosphate-buffered saline (50 mM sodium phosphate, pH 7.0, 100 mM NaCl). The diluted proteins were dialyzed against 3 liters of phosphate-buffered saline for 12 h with 3 changes and then concentrated with polyethylene glycol powder (molecular mass ϭ 15-20 kDa). The purity of the protein was analyzed on 10 -20% gradient SDS-polyacrylamide gel electrophoresis. The protein was quantitated using a Bio-Rad detergent-compatible protein assay based on the Lowry assay. Enzyme concentrations were calculated based on the protein mass measurements and an estimated molecular mass of 42.5 kDa.
Flow Cytometric Nuclease Activity Assay-The flap substrate labeling, immobilization of the flap substrate to the microsphere, and calibration were carried out according to the protocol described recently. 1 In a typical reaction, carried out at room temperature, 5-7 nM immobilized flap substrate was mixed in R buffer (50 mM Tris, pH 8.0, 10 mM MgCl 2 , 100 g/ml bovine serum albumin) in total volume of 500 l. The substrate alone was analyzed ϳ8 s to establish a base line. The sample tube was removed from the tube holder, approximately 50 nM enzyme was added, the tube was vortexed, and sample was reintroduced to the instrument. The first time point was acquired 8 -9 s after mixing.
The internal clock of the FACScalibur's Macintosh acquisition computer was used to track the time parameter. The data obtained were analyzed using IDLYK, a flow cytometry data analysis program developed at Los Alamos National Laboratory. Time data were collected at 1024-channel resolution (500 ms/channel) and rebinned at 64-channel resolution (8 s/channel) for display. The time value for a given data point was the midpoint of the time window measured. For clarity, only every fifth data point is displayed.
Competition of Wild Type Enzyme with Inactive Mutant Proteins -To test the binding activity of the catalytically inactive mutants, the mutant enzymes (150 nM) were preincubated with the immobilized flap substrate in R buffer at room temperature for 15 min. The reactions were started by adding 50 nM wild type enzyme, and flap DNA cleavage was measured as described above.

RESULTS
The amino acid sequences of eight proteins from the XPG/ FEN-1 family in mammals and yeast have been compared using the computer program DNA Star. This analysis revealed conserved regions in the N-terminal (29 -114 for hFEN-1) and in the middle part (129 -260 for hFEN-1) of the protein amino acid sequence as described by Murray et al. (1994). Within these two regions (218 total amino acids for hFEN-1), 39 amino acids are identical. Moreover, exonuclease domains of prokaryotic DNA polymerases have been shown to have flap endonuclease activity (Lyamichev et al., 1993). When we extended this comparison to 6 exonuclease domains of the prokaryotic DNA polymerases, including E. coli DNA polymerase I and Taq DNA polymerase and 4 bacteriophage exonucleases (Lopez et al., 1989;Beck et al., 1989;Hollingsworth and Nossal, 1991), we found that 11 amino acids are similar and 8 of them are identical among these 18 enzymes (Fig. 1, A and B).
Each of these 8 charged or hydrophilic amino acids was converted to alanine by site-directed mutagenesis. The mutant proteins were overexpressed to a level similar to that of the wild type enzyme (Fig. 2A). The solubility of the mutant proteins differed although most of mutant proteins were comparable to the recombinant wild type protein. The most insoluble mutant proteins (G231A and D233A) were solubilized in vivo by employing the sorbitol/betaine medium according to Blackwell and Horgan (1991). The His-6-tagged FEN-1 mutant proteins were purified from the soluble part of the total crude E. coli cell extract using a nickel-Sepharose column as shown in Fig. 2B. The purified mutant proteins migrated as wild type protein in a 10 -20% SDS-polyacrylamide gel electrophoresis  (Gutman and Minton, 1993). Middle, human FEN-1 (Murray et al., 1994) representing mouse FEN-1 (Harrington and Lieber, 1994a), S. cerevisiae YKL510 (Jacquier et al., 1992) and S. pombe rad2 (Murray et al., 1994). Bottom, human XPG (MacInnes et al., 1993;Scherly et al., 1993) conservative domains to FEN-1 representing the XPG family members: mouse XPG (Shiomi et al., 1994), S. cerevisiae RAD2, and S. pombe rad13 (Carr et al., 1993).
Flap endonuclease activity of the wild type and mutant proteins was screened by a flow cytometry-based assay system which has been described recently. 1 Six of the eight mutant proteins (D34A, D86A, E158A, D181A, G231A, D233A) exhibited no flap endonuclease activity when the assay was carried out at a protein concentration of 50 nM (Fig. 3) or at 150 nM even after 30 min of incubation (data not shown). Conversion of an arginine at position 103 or aspartate 179 to an alanine did not affect the flap endonuclease activity compared to the wild type enzyme.
Mutants with no catalytic activity are expected to have a defect in substrate binding, cleavage, or both. To examine whether an individual inactive mutant was capable of substrate binding, we performed a competition assay with the wild type enzyme. For these experiments, the inactive mutant enzymes were preincubated with the substrate at room temperature for 15 min to allow them to bind to the substrate followed by the addition of wild type enzyme and measurement of cleavage. If an inactive mutant is capable of binding to the flap substrate, it will compete with and reduce the apparent activity of the wild type enzyme. Fig. 4 shows that of the six inactive mutants, three (D34A Ͻ D86A Ͻ D181A) partially inhibit the wild type FEN-1 activity at a concentration three times higher than wild type FEN-1. By increasing the concentration of mutant enzyme, the wild type activity can be completely inhibited (data not shown) although the concentration required for complete inhibition by these three mutants are different. On the other hand, the other three catalytically inactive mutants E158A, G231A, and D233A did not inhibit the wild type enzyme activity at all under the same experimental conditions. The results are summarized in Table II. DISCUSSION Isolation and characterization of homologous genes from different organisms provide valuable information on structurally conserved regions that are likely to be important for protein function or for macromolecular interactions. Flap endonuclease 1 genes from human, mouse, and two yeast species constitute a highly conserved family. The encoded proteins have homologous regions with the XPG family (Carr et al., 1993;MacInnes et al., 1993;Scherly et al., 1993;Murray et al., 1994). When the comparison of these protein sequences was extended to the prokaryotic and viral proteins that exhibit exonuclease/endonuclease activities, the number of absolutely conserved amino acids is reduced to a few. In order to evaluate the roles of these evolutionarily conserved amino acids in catalysis, we changed these amino acids to alanine. FEN-1 enzyme activity was assayed with a recently developed flow cytometer-based nuclease assay. Substrate binding by catalytically inactive mutants was measured by competition against the wild-type enzyme. Table  II summarizes the ability of eight mutant enzymes to catalyze DNA cleavage and compete with the wild type enzyme.
These data indicate that mutation of Asp-34, Asp-86, and Each of five inactive mutants was preincubated with substrate for 15 min in room temperature. Then a base line was obtained, wild type enzyme was added, and reaction progress was measured.
Asp-181 lead the enzyme to lose catalytic activity completely but retain the binding ability to the flap substrate which is on the same order as wild type substrate binding. Therefore, we propose that these amino acids are required for the catalytic activity of the enzyme. Three other amino acids, Glu-158, Gly-231, and Asp-233, are important for binding of DNA substrate because their replacement by alanine leads not only to the loss of cleavage activity, but also to the loss of the ability to compete with the wild type enzyme. This indicates that the affinity of these mutants for the flap DNA substrate has been reduced several orders of magnitude relative to the wild type enzyme, and that the K D for substrate binding must be much greater than 150 nM, the concentration of mutant enzyme tested here. Mutation of Arg-103 and Asp-179 did not change activity of the enzyme. Interestingly, the amino acids constituting the active site of the enzyme (Asp-34, Asp-86, Asp-181) are all aspartate. The amino acids with a carboxyl group have been identified as components of an active site in several nucleases involved in DNA repair or recombination. The amino acids constituting the active center of the human DNA repair enzyme HAP1 have been assigned to the conserved Asp-90, Asp-219, Asp-308, and Glu-96 (Barzilay et al., 1995). Saito et al. (1995) recently identified Asp-7, Glu-66, Asp-138, and Asp-141 as the catalytic center for the RuvC Holliday junction resolvase.
In most hydrolytic nucleases for which detailed structural information is available, the active site residues are required either to interact directly with a water molecule which attacks the scissile phosphate of the DNA backbone or to precisely orient two metal ions with respect to the phosphodiester backbone and the attacking water molecule (Suck and Oefner, 1986;Suck et al., 1988;Beeze and Steitz, 1991;Derbyshire et al., 1991;Volbeda et al., 1991;Weber et al., 1991;Steitz and Steitz, 1993). The requirement to bind metals or to abstract protons from a water molecule to generate a nucleophile which attacks the P-O3Ј bond limits the choice of amino acid residues which can function effectively in the active site of such nucleases. Histidine residues have been implicated previously in metal binding or in the activation of water molecules, while residues with carboxylic acid side groups are commonly associated with divalent cation binding (Saenger, 1991). We recently observed a Mg 2ϩ -induced conformational change of the wild type FEN-1 enzyme studied by small angle x-ray scattering and Fourier transform infrared spectroscopy, and this conformational change did not occur in the mutant D181A FEN-1 enzyme. 3 In summary, this work has combined site-directed mutagenesis and a flow cytometric nuclease assay system to study a novel structure-specific endonuclease. We have found amino acids essential for substrate binding and catalysis. The information provided here should be useful for further studies on structure and function of this enzyme and other nucleases involved in DNA repair.
Ϫ Ϫ a Relative flap DNA cleavage activity tested at a single concentration (50 nM) of mutant or wild type enzyme.
b Degree of inhibition of wild type enzyme (50 nM) by inactive mutant enzyme (150 nM). c ND, not determined.