©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Substitutions in Conserved Dodecapeptide Motifs That Uncouple the DNA Binding and DNA Cleavage Activities of PI-SceI Endonuclease (*)

(Received for publication, October 13, 1994; and in revised form, January 6, 1995)

Frederick S. Gimble (§) Brian W. Stephens

From the Center for Macromolecular Design, Institute of Biosciences and Technology and the Department of Biochemistry and Biophysics, Texas A & M University, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The PI-SceI endonuclease from yeast belongs to a protein family whose members contain two conserved dodecapeptide motifs within their primary sequences. The function of two acidic residues within these motifs, Asp and Asp, was examined by substituting alanine, asparagine, and glutamic acid residues at these positions. All of the purified mutant proteins bind to the PI-SceI recognition site with the same affinity and specificity as the wild-type enzyme. By contrast, substituting alanine or asparagine amino acids at the two positions completely eliminates strand cleavage of substrate DNA, whereas substitution with glutamic acid markedly reduces the cleavage activity. Experiments using nicked substrates demonstrate that the wild-type enzyme shows no strand preference during cleavage. These results are consistent with a model in which both acidic residues are part of a single catalytic center that cleaves both DNA strands. Furthermore, substrate binding by wild-type PI-SceI stimulates hydroxyl radical or hydroxide ion attack at the cleavage site while binding by the alanine-substituted proteins either stimulates this attack significantly less or protects the DNA at this position. These finding are discussed in terms of possible reaction mechanisms for PI-SceI-mediated endonucleolytic cleavage.


INTRODUCTION

The PI-SceI endonuclease (formerly named VDE) (^1)cleaves the yeast genome at a single location and initiates a gene conversion process that results in the transfer of the endonuclease gene to other yeast strains(1, 2) . The endonuclease is initially translated as part of a precursor protein and occurs as an internal protein sequence or ``intein'' that is flanked by two external protein sequences or ``exteins'' (for nomenclature information see(3) ). Through a process termed protein splicing, the PI-SceI intein excises itself from the precursor and joins together the two exteins to generate a second unrelated protein(4, 5, 6) . The excised PI-SceI protein belongs to a family of related DNA endonucleases that share common biochemical and structural properties (for reviews, see (7) and (8) ). These enzymes cleave DNA within their recognition sequences to leave 4-bp 3`-OH overhangs, require Mg ion as a co-factor, and, unlike the prokaryotic restriction endonucleases, recognize sites that are longer than 18 bp in length that contain no obvious dyad symmetry. Another distinguishing feature of these endonucleases are two conserved 12-residue peptide sequences that are spaced approximately 100 amino acids apart ((9-11), see Fig. 1). It is unlikely that these dodecapeptide motifs are responsible for mediating substrate specificity since each endonuclease recognizes and cleaves at a different site. Instead, the residues within these motifs may comprise part of the catalytic center.


Figure 1: Protein sequence alignment of the dodecapeptide motifs within related endonucleases (modified and updated from(8) ). Conserved amino acids are indicated by white on black lettering, and the number of intervening residues between the motifs is indicated. Amino acids are represented by the single-letter code. In the consensus sequences, specific amino acids are indicated if they are invariant or if they represent geq 50% of the total; hydrophobic residues are indicated by O (when geq65% of the total, regardless of specific amino acid content); acidic residues are indicated by B (geq90% of total), and X represents any residue. The I prefix indicates proteins involved in intron homing(40) ; the PI prefix denotes proteins generated from protein splicing events(3) . Proteins marked with an asterisk are generated by protein splicing, but have not been shown to be endonucleases. The arrows indicate the aspartic acid residues in PI-SceI that have been substituted by site-directed mutagenesis. Data base accession numbers for the sequences: Ctr VMA, M64984; PI-PspI, U00707, D29671; Mle recA, X73822; the remainder are given in (8) .



To examine whether the two dodecapeptide motifs are involved in the cleavage reaction and/or DNA binding, we used site-directed mutagenesis to substitute glutamic acid, asparagine, or alanine at the positions of two conserved aspartic acid residues within the motifs. Here, we show that all of the purified mutant proteins bind to DNA normally, but only the glutamic acid substituted enzymes cleave DNA. We conclude from these experiments that acidic amino acids are likely to be required at each of these two positions for cleavage to occur and that the two aspartic acid residues comprise a single catalytic center that cleaves both DNA strands. Previously, it has been reported that substitution of an analogous aspartic acid residue within the PI-TliI endonuclease, a member of the same protein family as PI-SceI, eliminates catalytic activity(12) . In this paper, we have significantly extended this analysis by examining the roles of both dodecapeptide motifs and by showing for the first time that the strand-scission and DNA binding activities can be effectively uncoupled for this class of enzymes.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis

Site-directed mutants of PI-SceI were created by cassette mutagenesis using plasmids pET23PI-Sce ESARC and pT7PI-Sce ESARC. Plasmid pT7PI-Sce ESARC is a derivative of pT7VDE(13) , a plasmid that expresses the PI-SceI intein from the T7 promoter in the absence of the flanking exteins. In pT7PI-Sce ESARC, polymerase chain reaction mutagenesis (14) was used to insert silent AvrII and RsrII sites near the borders of the N-terminal motif and EaeI and SnaBI sites at each end of the C-terminal motif. Plasmid pET23PI-Sce ESARC is a derivative of pET-23a (Novagen) that has the same PI-SceI gene as in pT7PI-Sce ESARC. The PI-SceI gene contained in these plasmids differs slightly from that in pT7VDE, since it expresses a version of the protein that contains an N-terminal rather than a C-terminal cysteine residue. This modified protein more closely resembles the PI-SceI produced in Saccharomyces cerevisiae(13, 15) and has identical activity to the enzyme that we characterized previously. (^2)To create site-directed mutations within the N-terminal motif, oligonucleotides MutIA (5`-CTAGGCCTATGGATTGGT(G/A)(A/C)(T/G)GGATTGTCG-3`) and MutIB (5`-GTCCGACAATCC(A/C)(T/G)(C/T)ACCAATCCATAGGC-3`) were annealed and inserted into the AvrII and RsrII sites of plasmid pET23PI-Sce ESARC. Mutant alleles were detected by DNA sequencing(16) . Similarly, oligonucleotides MutIIA (5`-GGCCGGTCTAATCGATTCT(G/A)(A/C)(T/G)GGCTAC-3`) and MutIIB (5`GTAGCC(A/C)(T/G)(C/T)AGAATCGATTAGACC-3`) were inserted into EaeI and SnaBI sites of plasmid pT7PI-Sce ESARC to create alleles that encoded mutations within the C-terminal motif.

Purification of PI-SceI

Preparations of PI-SceI were purified from Escherichia coli strain BL21 (DE3) containing plasmids pET23PI-Sce ESARC and pT7PI-Sce ESARC. Similar quantities of PI-SceI could be purified from strains that contained either plasmid. Plasmid derivatives of pT7PI-Sce ESARC were used to express the wild-type protein and Asp-substituted mutant proteins, and derivatives of plasmid pET23PI-Sce ESARC were used to express the Asp-substituted proteins. Conditions used to grow cells and purification protocols were as described previously (13) except that a 1-ml Resource S column (Pharmacia Biotech Inc.) was used instead of S-Sepharose resin during cation exchange chromatography. Purification was monitored by staining SDS-polyacrylamide gels with Coomassie Brilliant Blue(17) . The wild-type enzyme and each of the mutant derivatives exhibited the same purification behaviors, (^3)and each was purified greater than 95% as judged from stained SDS-polyacrylamide gels. Protein concentrations were determined using a corrected extinction coefficient of 4.68 times 10^4M cm that was determined by published procedures(18) .

Oligonucleotides

Six synthetic oligonucleotides (see Fig. 5, A-F) were used to construct substrates to analyze the binding and cleavage properties of PI-SceI. A 67-bp PI-Sce I binding site duplex was formed with gel-purified oligonucleotides A (5`-AGCTTTGACGCCATTATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGGCAGAAGTCTTGATGT-3`) and F (5`-CTAGACATCAAGACTTCTGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGATAATGGCGTCAA-3`). Individual oligonucleotides (8 pmol) were end-labeled with [-P]ATP (Amersham Corp.) and T4 polynucleotide kinase and purified on a small Sephadex G-50 (Pharmacia) column. Each of these labeled strands was annealed to a 5-fold molar excess of its unlabeled complement by heating to 90 °C for 2 min and by slow cooling. This creates two full-length substrates, each of which is labeled at a different end. A duplex substrate containing a ``nicked'' bottom strand was formed by annealing 5`-end-labeled oligonucleotide A with a 5-fold molar excess of the complementary oligonucleotides D (5`-CTAGACATCAAGACTTCTGCCATTTCATTACCTCTTTCTCCGCAC-3`) and E (5`-CCGACATAGATAATGGCGTCAA-3`). Similarly, oligonucleotides B (5`-AGCTTTGACGCCATTATCTATGTCGGGTGC-3`) and C (5`-GGAGAAAGAGGTAATGAAATGGCAGAAGTCTTGATGT-3`) were combined with 5`-end-labeled oligonucleotide F to create a substrate with a nicked top strand. To provide the nicked sites with 5`-phosphate and 3`-hydroxyl termini, oligonucleotides C and D were phosphorylated enzymatically with polynucleotide kinase prior to the annealing steps.


Figure 5: Cleavage activity assays of wild-type and mutant (D218A, D326A) PI-SceI proteins using full-length and nicked substrates. A, autoradiogram of a denaturing gel showing the cleavage products obtained following digestion of various synthetic duplex oligonucleotides with wild-type or mutant PI-SceI proteins. Cleavage reactions were assembled as described under ``Experimental Procedures'' and were incubated at 37 °C for 1 h. An equal volume of loading buffer was added to each reaction mixture, and the products were resolved by electrophoresis on a 15% denaturing acrylamide slab gel which was dried and exposed to x-ray film. The letters above each lane denote the oligonucleotides (shown in B) that were annealed to form the substrate for that reaction. An asterisk indicates which oligonucleotide was labeled at its 5`-end with P in each reaction. B, synthetic oligonucleotides (A-F) used to construct the full-length substrate and fragments used in this experiment. Cleavage sites on each strand are indicated by arrows.



Gel Retardation

The 67-bp (A + F) duplex (10,000 cpm, 0.25-0.5 nM) was incubated for 15 min at 25 °C with various amounts of purified PI-SceI protein in a total volume of 20 µl of gel binding buffer (25 mM Tris-HCl (pH 8.5), 100 mM KCl, 10% glycerol, 50 µg/ml bovine serum albumin, and 2.5 mM 2-mercaptoethanol) containing 1 µg (50 nM) of poly(dIbulletdC) (Sigma). The protein/DNA mixture was loaded onto a 7% nondenaturing polyacrylamide gel (acrylamide:bis, 19:1) and separated by electrophoresis in 0.5 times TBE (19) buffer at 350 V for 5 min and then at 200 V for 2-2.5 h at 4 °C. To derive apparent dissociation constants (K(d) values), dried gels were quantified with the use of a PhosphorImager system (Molecular Dynamics). Protein concentrations were varied over at least 4 orders of magnitude, where <5% and >85% of substrate was bound at the low and high ends of the binding curve, respectively. The binding curves were fitted to the equation f = K(d)/(K(d) + [P]) with the program KALEIDAGRAPH (Synergy Software, Reading, PA), where f is the fractional saturation of the DNA substrate, and [P] is the protein concentration. Curve fitting is preferred for estimating K(d) values over methods that require judging the half-maximal binding concentration because the entire data set is used in the calculation. Each binding assay was performed in duplicate for each of the protein samples. We estimate the standard errors for the dissociation constants to be approximately 50%.

DNase Footprinting

DNase I protection experiments were performed essentially as described(20) . Purified wild-type or mutant PI-SceI protein (5 nM total concentration) were incubated in 100 µl of assay buffer (100 mM KCl, 25 mM Tris-HCl (pH 8.5), 50 µg/ml bovine serum albumin (Life Technologies, Inc., Fraction V), 0.1 mg/ml sonicated salmon sperm DNA (Sigma), 2.5 mM 2-mercaptoethanol, 1 mM CaCl(2), and 5 mM MgCl(2)) with end-labeled 67-bp duplex substrate (25,000 cpm, 10-20 fmol) at 25 °C for 15 min. These protein/DNA mixtures were incubated for 5 min at 25 °C with DNase I (5 µl of a 3 µg/ml stock, Worthington enzymes). The DNA was precipitated with ethanol and resuspended in 95% formamide, 20 mM EDTA. The reaction products were resolved on 14% denaturing gels (7 M urea, 1 times TBE, acrylamide:bis, 19:1) which were subsequently exposed to x-ray film (Fuji RX).

PI-SceI Cleavage Analysis

The cleavage of a plasmid substrate by wild-type and mutant PI-SceI enzymes was assayed as described previously(13) . Purified PI-SceI (0.2 µM total concentration) was incubated with XmnI-linearized plasmid pBSVDEX (5.5 nM) (13) in 15 µl of cleavage buffer (100 mM KCl, 25 mM Tris-HCl (pH 8.5), 2.5 mM 2-mercaptoethanol) that contained 2.5 mM MgCl(2) or 2.5 mM MnCl(2). The DNA substrate was digested for 1.5 h at 37 °C, and the reactions were terminated by the addition of 5 µl of stop buffer (10 mM EDTA, 5 mM Tris-HCl (pH 7.5), 0.2% SDS, 2.5% Ficoll). Aliquots of each reaction were separated by electrophoresis in 1 times TBE buffer on a 0.9% agarose gel, which was stained with ethidium bromide and photographed. Cleavage assays of 5`-end-labeled full-length and nicked duplexes (5 nM total concentration) by wild-type and mutant PI-SceI (20 nM) were performed in cleavage buffer (25 µl) containing 2.5 mM MgCl(2). Cleavage of each single-stranded, P-labeled oligonucleotide was also carried out with wild-type PI-SceI. The reaction mixtures were incubated at 37 °C for 1 h and were stopped by the addition of an equal volume 95% formamide, 20 mM EDTA, 0.2% SDS. Following heating for 4 min at 90 °C, the products were resolved by electrophoresis on a 15% denaturing acrylamide slab gel (8 M urea, 1 times TBE) which was dried and exposed to x-ray film.

Hydroxyl Radical Footprinting

Hydroxyl radical footprinting was performed according to published procedures(21) . To observe protected regions of the substrate, protein-DNA complexes were isolated from gels following DNA modification by the Fenton reaction. Protein-DNA complexes were formed by mixing PI-SceI protein (90 nM final concentration) and 0.5 pmol (14 nM, 1 times 10^6 cpm) of end-labeled 67-bp (A + F) duplex together for 20 min at 25 °C in a total volume of 35 µl in 100 mM KCl, 25 mM Tris-Cl (pH 8.5), and 2.5 mM 2-mercaptoethanol. The samples were adjusted to 1.5 mM (NH(4))Fe(SO(4))(2)bullet6H(2)0 (Aldrich), 3.0 mM EDTA (Life Technologies, Inc.), 0.23% H(2)O(2) (J. T. Baker), and 7.7 mML-ascorbic acid (Sigma), which are the components of the Fenton reaction. After 3 min of incubation at 25 °C, the reaction was quenched with 5 µl of 0.1 M thiourea (Sigma) and 10 µl of loading buffer (50% glycerol, 0.25% bromphenol blue, 0.25% xylene cyanol). The samples were loaded onto a 7% nondenaturing polyacrylamide gel and separated by electrophoresis as described under the ``Gel Retardation'' section. The protein-DNA complexes were located by autoradiography and were isolated by electroelution. Following extraction with phenol and precipitation of the DNA, the pellets were resuspended into 4 µl of 95% formamide and 20 mM EDTA and separated by electrophoresis on a 12% denaturing gel (7 M urea, 1 times TBE). The gel was dried and the amount of radioactivity in each band was quantified using a PhosphorImager.


RESULTS

Substitution of Conserved Aspartic Acid Residues within PI-SceI Does Not Affect Substrate Binding or Specificity

Alignment of the dodecapeptide motifs from several endonucleases reveals that there is substantial variation at most positions (Fig. 1). The weakly conserved positions are typically occupied by hydrophobic amino acids, whereas the most highly conserved positions are usually glycine or acidic residues. For example, each of the two motifs usually contains an aspartic acid residue at the ninth position. A motif found in many restriction enzymes (Pro-Asp-Xaa-Glu/Asp-Xaa-Lys,(22, 23) ), including EcoRI, EcoRV, and FokI, resembles the dodecapeptide motif to some extent in that it contains acidic amino acids at two positions, but it displays little other similarity (Fig. 1). In the crystal structures of the EcoRV and EcoRI endonucleases bound to their recognition sequences, these motifs are situated near the scissile phosphodiester bonds(24) . The BamHI and PvuII endonucleases also contain similarly arranged acidic residues(25, 26) . Alteration of the acidic amino acids by mutation inactivates these enzymes, but does not affect their ability to bind to their recognition site(23, 27, 28, 29, 30) . To investigate the functional roles of the two aspartic acid residues (Asp and Asp) within PI-SceI, we used site-directed mutagenesis to generate mutant proteins in which each acidic residue had been changed to glutamic acid, asparagine, or alanine.

We overproduced and purified wild-type PI-SceI and the D218A, D218N, D218E, D326A, D326N, D326E mutants in order to assay their enzymatic and DNA binding properties in vitro. Each of the mutant proteins exhibited the same purification behavior as the wild-type protein. Gel shift experiments were performed to examine substrate binding by using a 67-bp duplex oligonucleotide that contains the PI-SceI cleavage/recognition site(1, 13) . In the absence of Mg ion, a required co-factor for cleavage, the wild-type protein and each of the mutant proteins binds to the labeled 67-bp probe and yields a single protein-DNA complex (Fig. 2A). Gel shift assays were used to measure apparent equilibrium dissociation constants for each of the proteins (Table 1). These values (average K(d) 2.4 nM) are essentially the same for the mutant and wild-type endonucleases.


Figure 2: Gel shift analysis of wild-type and mutant PI-SceI proteins. A, sequence of the synthetic 67-bp duplex used as substrate. Oligonucleotides containing the wild-type recognition sequence were synthesized as described under ``Experimental Procedures.'' CS indicates the cleavage sites on both strands. The arrows mark the boundaries of a DNase I footprint (Fig. 3). B, gel shift analysis in the absence of Mg ion. Approximately 5-10 fmol of 5`-end-labeled 67-bp duplex substrate were incubated with wild-type or mutant PI-SceI protein (13 nM) in gel binding buffer (25 mM Tris-HCl (pH 8.5), 100 mM KCl, 10% glycerol, 50 µg/ml bovine serum albumin, and 2.5 mM 2-mercaptoethanol) that did not contain Mg ion. The unbound and bound DNA species were separated by electrophoresis on a 7% nondenaturing gel and migrated to the positions labeled Free and Complex, respectively. The proteins used are indicated above each lane. The designations top and bottom refer to which strand shown in A is end-labeled in the DNA duplex. C, gel shift analysis was performed as in B except that the gel binding buffer contained 2.5 mM MgCl(2). The two cleavage products migrate to the positions labeled F1 and F2. These gels have been overexposed in order to show the low levels of cleavage product. The faint bands seen in the control reactions that co-migrate with the complex are due to spillover between lanes.






Figure 3: DNase I protection analysis of wild-type and mutant PI-SceI proteins. A, footprint of the PI-SceI cleavage/recognition site. Purified wild-type or mutant PI-SceI protein (5 nM total concentration) were incubated with end-labeled 67-bp duplex substrate (25,000 cpm, 10-20 fmol) as described under ``Experimental Procedures.'' Digestion with DNase I (5 µl of a 3 µg/ml stock) was performed for 5 min at 25 °C. The reaction products were resolved on 14% denaturing gels (7 M urea, 1 times TBE, acrylamide:bisacrylamide, 19:1) which were subsequently dried and exposed to x-ray film. Lanes are designated as in Fig. 2; the lane marked A + G contains products from a Maxam-Gilbert A + G reaction(19) . CS indicates the cleavage site; brackets represent protected areas. B, schematic of DNase I footprint (indicated by brackets) relative to the cleavage site. These data are representative of the results from two different experiments, which were indistinguishable.



DNase I protection experiments were used to establish whether the mutant proteins bound to the recognition sequence with the same binding pattern as wild-type PI-SceI. Fig. 3shows that the wild-type protein uniformly protects a 35-bp region on each strand from DNase I cleavage. This region overlaps the cleavage site and is nearly coincident with a 30-bp region that was determined previously to be the minimum region required for PI-SceI-mediated cleavage(13) . Under the conditions used in this assay, cleavage products are not observed because of the slow rate of cleavage by the enzyme. Each of the mutant proteins yields a protection pattern that is identical to that of the wild-type protein (Fig. 3A). The amount of protection produced by the D218E and D326A proteins at the concentration used is approximately 2-fold to 3-fold less than that of wild-type PI-SceI even though the equilibrium dissociation constants determined by gel-shift analysis are very similar. This difference may be due to the fact that the DNase I experiments are performed in solution, whereas the gel-shift experiments require passage of the protein-DNA complex through the gel matrix.

Substitution of Asp and Asp with Non-acidic Amino Acids Eliminates PI-SceI Endonucleolytic Activity

The ability of the six mutant PI-SceI proteins to cleave DNA was first assayed by performing gel-shift experiments that included the essential Mg ion co-factor in the binding buffer. Incubation of the wild-type protein with the end-labeled 67-bp substrate yields DNA cleavage fragments that migrate faster than the uncut substrate (labeled F1 and F2 in Fig. 2C). Surprisingly, the amount of the larger cleavage product (F1 in Fig. 2C, bottom strand) appears to be significantly less than that of the smaller product (F2 in Fig. 2C, top strand). This occurs because most of the larger product binds with high affinity to the enzyme following cleavage and co-migrates with the bound, uncleaved substrate. (^4)In contrast to these results, only the D326E mutant protein generates cleavage products, even though each of the mutant proteins binds to the substrate to yield protein-DNA complexes. The amount of cleavage by the D326E mutant protein after 1 h is at least an order of magnitude lower than that generated by the wild-type enzyme.

Similar results are observed when the wild-type and mutant enzymes are used to cleave a linearized plasmid substrate that contains a single copy of the PI-SceI cleavage/recognition site (Fig. 4). The wild-type enzyme cleaves the substrate nearly completely, the D326E mutant protein generates at least 6-fold less cleavage product and the other mutants yield no products. Previously, we have shown that replacing the Mg ion in the reaction buffer with Mn ion causes PI-SceI to cut DNA at non-cognate sites and to cleave the cognate site at faster rates (1) . When Mn ion is used in these experiments, the D218E mutant enzyme functions to cleave the substrate (Fig. 4). Furthermore, the amount of cleavage by the D326E protein in the presence of Mn ion is greater than in the presence of Mg ion. Although we have not mapped the cleavage sites created by the glutamic acid mutants at the nucleotide level, it is likely that they are the same as or similar to those created by the wild-type enzyme as judged by the size of the cleavage products.


Figure 4: Cleavage activity assays of wild-type and mutant PI-SceI proteins using a linearized plasmid substrate. Cleavage of plasmid pBSVDEX (13) linearized with XmnI was performed as described under ``Experimental Procedures.'' Approximately 10 units of wild-type PI-SceI were used where one unit is sufficient to cleave 100 ng of linearized substrate in 1 h at 37 °C. Aliquots of each reaction were separated by electrophoresis in 1 times TBE buffer on a 0.9% agarose gel, which was stained with ethidium bromide and photographed. PI-SceI-mediated cleavage generates 2.6- and 1.1-kilobase products. The reactions included either 2.5 mM MgCl(2) or 2.5 mM MnCl(2) as indicated above the gel.



The alanine- and asparagine-substituted mutant proteins lack double-strand cleavage activity, but it is possible that they are able to cleave single strands of the substrate (i.e. ``nick'' the DNA). We tested for this activity by incubating the wild-type enzyme and the two alanine mutants with end-labeled substrates and by analyzing the cleavage products on denaturing gels. The wild-type protein cleaves the full-length substrate and generates the two expected cleavage products (Fig. 5). Unlike the experiment shown in Fig. 2C, equal amounts of both cleavage products are observed here because the denaturing gel system being used prevents one of the products from remaining bound to the protein. In contrast to these results, the two alanine mutants do not produce either cleavage product, indicating that both proteins are incapable of nicking the DNA on either strand. By using nicked substrates (see ``Experimental Procedures''), we also examined whether these mutants cleave the second strand if the first strand is already cut. The wild-type enzyme effectively cleaves the second strand of both nicked substrates, whereas neither alanine mutant cleaves either nicked DNA substrate (Fig. 5). In sum, these results demonstrate that the alanine mutant proteins are unable to cleave either strand of the substrate.

Hydroxyl Radical Protection Experiments Reveal That the Mutant and Wild-type PI-SceI Proteins Interact with the Cleavage Site Differently

We used hydroxyl radical protection experiments to probe the specific contacts made by the wild-type and mutant enzymes. The hydroxyl radical provides high resolution data, because it is small, highly reactive, and it cleaves at every nucleotide with almost equal efficiency. In the presence of the wild-type protein or the two alanine mutants, areas of strong and weak protection occur on either side of the cleavage site spanning a 35-bp region (Fig. 6) that is nearly identical to the region protected from DNase I (Fig. 3). Fig. 6shows clearly that in the regions that flank the cleavage site, the same nucleotide positions are protected by the wild-type and mutant proteins. The region that is most strongly protected is located on the right arm of the cleavage/recognition site (positions +6 through +21). We have shown in other experiments that this heavily protected region binds to PI-SceI with an affinity that is similar to that of the entire substrate and likely contains the most important binding contacts. (^5)A smaller, weakly protected region is observed on the other side of the cleavage site. In general, the cleavage site and the other points of contact are aligned along one face of the DNA duplex.


Figure 6: Hydroxyl radical protection analysis. A, footprint of the PI-SceI cleavage recognition site. Hydroxyl radical reactions were performed as described under ``Experimental Procedures,'' and the modified DNA products were separated by electrophoresis on a 12% denaturing gel (7 M urea, 1 times TBE). Lane AG, products from a Maxam-Gilbert DNA sequencing reaction; lane F, free duplex treated with [Fe(II)EDTA]



Surprisingly, the protection patterns near the cleavage site are very different for the wild-type and mutant proteins. In the presence of wild-type PI-SceI, some positions near the cleavage site are hypersensitive to hydroxyl radical cleavage. On the top strand, the cytosine adjacent to the cleavage site (C) is hypersensitive to cleavage, and nucleotides G, G and G are also hypersensitive, but to a lesser degree. On the bottom strand, the two cytosines that border the cleavage strand (C and C) are equally hypersensitive. No hypersensitivity is observed if either hydrogen peroxide or [Fe(II)EDTA]


DISCUSSION

We have constructed six variants of the PI-SceI endonuclease whose behaviors suggest that the enzyme uses a single catalytic center to effect strand cleavage. Substitutions were made for two aspartic acid residues that occur in two dodecapeptide motifs that define a family of site-specific DNA endonucleases and RNA maturases (7, 8) . We found that substitution of either aspartic acid residue with asparagine or alanine effectively uncouples the DNA binding and strand scission activities and results in mutant proteins that bind to the substrate DNA with the same affinity as the wild-type protein, but fail to cleave it. Substitution with glutamic acid results in proteins with decreased cleavage activity, suggesting that the acidic nature of the side chain plays a critical role in the cleavage mechanism. Taken together, our results provide strong evidence that the dodecapeptide motifs contain the active site residues for the cleavage reaction.

Two models for PI-SceI-mediated cleavage are possible, either the enzyme contains a single active site that cuts both strands or two active sites, each of which cuts one DNA strand. If there are two sites and each is comprised of the amino acids within a single dodecapeptide motif, it might be expected that disabling one of them would result in an enzyme that is still able to nick the DNA on one strand. However, we show here that introducing mutations at either of the two conserved aspartic acid residues prevents the enzyme from cleaving the duplex DNA on either strand. It could still be argued, however, that each single mutation disrupts two active sites by globally perturbing the protein conformation. This is unlikely because 1) the behaviors of the mutant proteins during purification are indistinguishable from that of wild-type PI-SceI enzyme, and 2) the binding affinities of the mutant and wild-type enzymes for the substrate DNA are the same. Alternatively, nicking activity may not be observed because the double-strand cleavage reaction involves two sequential single-strand cleavage steps that occur in a fixed order. If the first step is prevented by mutation, the second step cannot occur. This possibility was tested in the case of the FokI restriction endonuclease by using a nicked substrate that was identical in structure to a duplex substrate where the first strand had already been cleaved(23) . When we performed similar experiments, the PI-SceI mutant enzymes, like FokI(23) , failed to cleave the nicked substrates. These data are consistent with a single active site model.

How might PI-SceI effect cleavage of its substrate using a single active site? Examining the similarities between PI-SceI and the FokI enzyme, which is a type IIS restriction endonuclease(31) , may provide an answer. Both enzymes recognize asymmetric sequences, are monomers in solution and require Mg co-factor for cleavage, but not for binding(13, 32) . More importantly, each of two aspartic acid residues within FokI are required for DNA cleavage activity, but not for DNA binding(23) . These two aspartic acids may be part of a single active site within FokI which cleaves both strands(23) . Following cleavage of the first strand, a conformational change in the enzyme or the DNA substrate could be required to allow cleavage of the second strand to proceed(23) . A mechanism of sequential strand scission could also by used by the PI-SceI enzyme. However, even if the reaction mechanisms are similar for FokI and PI-SceI, their strategies for recognizing and binding to DNA are likely to be different. The FokI endonuclease can be divided into a DNA binding domain and a catalytic domain(33) , but there is no evidence that the same is true for PI-SceI. In addition, the recognition sequence for FokI is 5 bp long, whereas the minimum sequence required for PI-SceI-mediated cleavage is in excess of 30 bp.

The exact role of the two aspartic acid residues in PI-SceI during catalytic cleavage is unclear, but it may be similar to the function of the conserved acidic residues that are found in the EcoRI, EcoRV, FokI, BamHI, and PvuII restriction endonucleases(22, 23, 25, 26) . Substantial structural and mechanistic information exists in the case of the EcoRI and EcoRV proteins that suggests a function for these acidic residues. In these proteins, the two acidic residues as well as a conserved lysine are positioned almost identically with respect to the scissile phosphodiester bond(34) . In one model, the acidic residues are thought to bind to the Mg ion at the active site which is believed to help stabilize the negative charges on the pentavalent transition state following nucleophilic attack by an activated water molecule. Although the sequences of the dodecapeptide motifs within PI-SceI are dissimilar from the motifs that occur in these restriction endonucleases, the acidic residues may play similar roles in chelating a Mg ion. However, even if the two aspartic acid residues have a similar function in PI-SceI, they are not equivalent since the Asp and Asp mutant proteins behave differently in the cleavage assay and during hydroxyl radical footprinting. Little is known about the function of the conserved hydrophobic and glycine residues within the dodecapeptide motifs. The singular ability of glycine amino acids to adopt conformations not available to other residues may be critical for orienting the aspartic acid side chains during catalysis.

The hypersensitivity to hydroxyl radical attack that occurs upon binding of wild-type PI-SceI to its substrate was unexpected. This hypersensitivity is markedly reduced when the alanine mutants bind to the substrate; in fact, the D326A protein significantly protects this region. The hypersensitivity is clearly the result of hydroxyl radical or hydroxide ion attack and not due to the normal PI-SceI-mediated cleavage reaction, since this effect is only observed if all of the components of the Fenton reaction are present. Two models can be used to explain the source of the observed hypersensitivity. First, if the aspartic acid residues normally chelate Mg ion, they may also be able to bind Fe ion, whose ionic radius is only slightly larger (0.66 Å for Mgversus 0.74 Å for Fe(35) ), or the [Fe(II)EDTA]


FOOTNOTES

*
This work was supported by National Institutes of Health Research Grant GM50815 (to F. S. G.). and by the Institute of Biosciences and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Center for Macromolecular Design, Institute of Biosciences and Technology, Texas A & M University, 2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-677-7605; Fax: 713-677-7641; fgimble{at}ibt.tamu.edu.

(^1)
The abbreviations used are: VDE, VMA1-derived endonuclease; bp, base pair; TBE, Tris borate-EDTA.

(^2)
F. S. Gimble, unpublished results.

(^3)
F. S. Gimble and B. W. Stephens, unpublished results.

(^4)
F. S. Gimble, manuscript in preparation.

(^5)
F. S. Gimble, manuscript in preparation.

(^6)
F. S. Gimble, unpublished results.

(^7)
F. S. Gimble, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Richard Sinden, Jim Hu, and Timothy Palzkill for critical comments on the manuscript.


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