Amino Acid Substitutions at Position 43 of NaeI Endonuclease

EVIDENCE FOR CHANGES IN NaeI STRUCTURE*

Kevin L. Carrick and Michael D. TopalDagger

From the Lineberger Comprehensive Cancer Center and the Department of Biochemistry & Biophysics, University of North Carolina Medical School, Chapel Hill, North Carolina 27599-7295

Received for publication, September 9, 2002, and in revised form, December 31, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NaeI endonuclease contains a 10-amino acid region with sequence similarity to the active site KXDG motif of DNA ligase except for leucine (Leu-43) in NaeI (43LXDG46). Changing Leu-43 to lysine abolishes the NaeI endonuclease activity and replaces it with topoisomerase and recombinase activities. Here we report the results of substituting Leu-43 with alanine, arginine, asparagine, glutamate, and histidine. Quantitating specific activities and DNA binding values for the mutant proteins determined the range of amino acids at position 43 that alter NaeI mechanism. Substituting alanine, asparagine, glutamate, and histidine for Leu-43 maintained endonuclease activity, but at a lower level. On the other hand, substituting positively charged arginine, like lysine at position 43, converted NaeI to a topoisomerase with no observable double-strand cleavage activity. The specific activities of NaeI-43K and NaeI-43R and their relative sensitivities to salt, the topoisomerase-inhibiting drug N-[4-(9-acridinylamino)-3-methoxyphenyl]methane-sulfonamide (amsacrine) and single-stranded DNA showed that the two activities are similar. The effect of placing a positive charge at position 43 on NaeI structure was determined by measuring (for NaeI and NaeI-43K) relative susceptibilities to proteolysis, UV, circular dichroism spectra, and temperature melting transitions. The results provide evidence that a positive charge at position 43 induces dramatic changes in NaeI structure that affect both the Endo and Topo domains of NaeI. The identification of four putative DNA ligase motifs in NaeI leads us to speculate that structural changes that superimpose these motifs on the ligase structure may account for the changes in activity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NaeI endonuclease (NaeI)1 is a prototype for the type IIe (enhancer) (1, 2) restriction endonucleases, so named (3) because they require interactions with an enhancing DNA sequence. One DNA sequence acts as enhancer to induce cleavage of the other sequence (4-7). In solution, NaeI protein is a 70-kDa homodimer (7) composed of two 317-amino acid polypeptides (8, 9) that recognize and cleave at the arrow, the DNA sequence 5'-GCCup-arrow GGC-3' using only Mg2+ as a cofactor. Two-site binding gives NaeI a specificity of DNA recognition ~104-fold better than single-site-binding proteins that recognize similar sized sequences (3). The two-site binding loops out intervening DNA sequences (5), hinting at more complicated functions than monofunctional DNA cleavage. Substituting lysine for leucine at position 43 (L43K) in NaeI endonuclease abolishes restriction endonuclease activity and in its place gives topoisomerase and recombinase activities (10). In addition, substitution L43K results in a preference for binding of single-stranded DNA and a sensitivity to salt and intercalative topoisomerase-inhibiting drugs, such as N-[4-(9-acridinylamino)-3-methoxyphenyl]methane-sulfonamide (amsacrine), which is lacking in restriction endonucleases but characteristic of topoisomerases (11, 12).

NaeI position 43 is located near the C terminus of alpha -helix H2, which is part of the central hydrophobic core of the Endo domain (Ref. 13 and Fig. 1). Unlike the structures of most restriction endonucleases, NaeI contains two separate domains, both of which bind DNA (14). The N-terminal, Endo domain contains the restriction endonuclease cleavage motif found in restriction enzymes as well as repair nucleases mutH, Vsr, and lambda  exonuclease (14-17) and transposases (18). The C-terminal, Topo domain contains a CAP motif also found in topoisomerases IA and II (19). Position 43 is positioned at the bottom edge of the Endo domain almost between the Endo and Topo domains and at the boundary separating the two NaeI monomers (Fig. 1) The two Leu-43 residues lie toward the center of the NaeI dimer ~15 Å apart.


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Fig. 1.   NaeI-DNA crystal structure (14). Wild-type (-L43) NaeI protein dimer is shown as red and gold ribbons. DNA is shown in green. Leucine at position 43 is shown as cyan ball and stick and oversized for clarity. Endo (N-terminal) and Topo (C-terminal) domains are indicated.

The NaeI position 43 lies within a 10 amino acid region with similarity to the conserved, active-site KXDG motif for DNA ligases, RNA ligases, and RNA-capping enzymes, which together make up the nucleotidyl transferase superfamily (for discussions, see Ref. 20). Nucleotidyl transferase catalysis involves three steps. First, the ligase is activated by the formation of a covalent protein-AMP intermediate with the AMP linked to the epsilon -amino group of lysine by a phosphoramidate bond. The conserved lysine in the sequence KXDG forms the adenylated intermediate using the high energy cofactors ATP (generally found in eukaryotes, viruses, and Archaebacteria) and NAD+ (generally found in Eubacteria). Second, the AMP moiety is transferred from lysine to the 5'-phosphate at the nicked DNA. Finally, the DNA-free ends are joined in an enzyme-dependent reaction with loss of AMP. NaeI has leucine instead of the essential lysine at position 43 (43LXDG46). The topoisomerase activity of NaeI-43K is possibly the result of activating a cryptic ligase active site and thereby coupling restriction endonuclease cleavage with ligation. NaeI forms a covalent intermediate with a cleaved substrate (10), which may serve as the high energy intermediate needed for ligation, as is the case with the topoisomerases and recombinases. The amino acid that covalently links NaeI to its DNA substrate has not been identified. There is no similarity between the folds of NaeI (13, 14) and DNA ligase (21-24). Moreover, the KXDG-like motif in NaeI lies away from the endonuclease metal-binding site necessary for cleavage. Thus, the transformation to topoisomerase activity in L43K implies a conformational change in the ES complex that results in the KXDG-like motif lying closer to the phosphodiester scissile bond; the endonuclease fold may be altered to mimic aspects of the ligase fold found at the active sites of the DNA ligases.

To learn whether lysine at position 43 is unique in its ability to give NaeI topoisomerase activity, we substituted alanine, asparagine, glutamate, histidine, and arginine. We also substituted lysine for leucine at position 40. Alanine has a small, nonpolar, amino acid side chain. Asparagine has an uncharged, amide-bearing polar side chain. Like lysine, histidine, arginine, and glutamate all have charged, polar side chains depending on the pH. We report that alanine, asparagine, glutamate, and histidine at position 43 maintained NaeI wild-type endonuclease activity when substituted at position 43, but with significant decreases in DNA cleavage. Substitution with arginine, however, resulted in topoisomerase activity identical to that of NaeI-43K. NaeI and NaeI-43K susceptibility to protease, UV-circular dichroism (CD) spectra, and CD temperature melting transitions were determined. The results provide the first evidence that placing a positive charge at position 43 causes a dramatic change in NaeI folding. The finding of additional putative ligase motifs within the NaeI sequence offers a possible rationale for how the conformational changes may alter NaeI activity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Escherichia coli strain CAA1 (F-e14- (mcrA-) lacY1 or D (lac)6 SupE44 galK2 galT22 mcrA rfbD1 mcrBa hsd (rk- mk+) M·MspI+) and plasmid pNEB-786, containing the NaeIR gene, were obtained from New England Biolabs. Plasmid pMAL-C2 and amylose resin were purchased from New England Biolabs. Substrate pBR322 was purchased from Promega Corp. Amsacrine, a DNA-intercalating drug that inhibits topoisomerase activity was purchased from Topogen Inc. Oligodeoxyribonucleotides (oligonucleotides) were synthesized by the Nucleic Acid Core Facility at UNC. Cellulose phosphate, sp-Sepharose, and heparin resins were purchased from Sigma. Cognate (dTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCT) and noncognate (dTTTCTCGCCACGTTCGAAGAATTTCCCCGTCAAGCT) oligonucleotides were annealed to their complements to yield DNA fragments, which were gel purified.

NaeI Substitutions-- The NaeIR gene was subcloned into the expression vector pMAL-C2, and site-directed mutagenesis was performed using the method of Clackson et al. (25). Mutated NaeIR genes were sequenced to confirm the mutation and ensure that no secondary mutations were generated.

Purification of NaeI Mutants-- To express the fusion protein, pMAL-NaeI mutants (MBP·NaeI) were induced with 1 mM isopropyl-beta -D-thiogalactoside (IPTG). Cells were harvested by centrifugation and cell pellets resuspended in 4 volumes of Tris column buffer (10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 5% glycerol, 1.0 mM 2-mercaptoethanol, 50 mM NaCl) with 1 mM phenylmethylsulfonyl fluoride to inhibit serine proteases. The resuspended cell pellet was sonicated on ice for 1 min per 10 ml followed by centrifugation at 30,000 × g to remove cellular debris. The supernatant was subjected to amylose resin chromatography. The column was washed with column buffer containing 400 mM NaCl then equilibrated with column buffer containing 50 mM NaCl. Fusion protein was eluted in Tris column buffer containing 10 mM maltose. Maltose had no effect on NaeI and NaeI-43K activities.

NaeI-- 43R was cloned into pNEB786 (to give pNEB786-NaeI-43R) and isolated from cells containing this plasmid. Cell extracts showed topoisomerase activity absent from extracts from cells containing pNEB786. Phosphocellulose, sp-Sepharose, and heparin columns were used for purification. Protein elution was achieved by NaCl gradients in column buffer. Fractions were assayed for topoisomerase activity. Peak fractions containing Topo activity were pooled and dialyzed with column buffer containing 50 mM NaCl after each column. Heparin fractions were dialyzed against 50 mM NaCl in column buffer for storage. NaeI-43R identity was confirmed by Western analysis using an affinity-purified antibody prepared against wild-type NaeI. The final purities of the proteins was estimated to be >90% based on optical density measurements of the protein resolved by SDS-PAGE by standard procedures.

Restriction Endonuclease Activity-- Restriction endonuclease activities relative to wild-type NaeI were determined from measurements of steady-state DNA cleavage rates. Protein concentrations were titrated while keeping reaction time (30 min) and DNA concentration (500 nM) constant. Reactions were prepared in 15-µl total volume to contain 10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and bovine serum albumin (0.1 mg/ml). Reactions were incubated at 37 °C for 30 min. Substrate was cognate DNA radiolabeled with [alpha -32P]ATP using T4 polynucleotide kinase. Reactions were stopped by addition of EDTA (40 nM) and glycerol (10%). Reaction products were separated on 8% polyacrylamide gels and analyzed using a Molecular Dynamics Storm 540 PhosphorImager. The cleaved and uncleaved gel bands were quantified using Imagequant 5.0 from Molecular Dynamics.

Gel Mobility Shift Assays-- Single-stranded oligonucleotides were radiolabeled and annealed to complementary oligonucleotides to give probe. Protein and probe were incubated in a 20-µl volume containing 10 mM Tris-HCl (pH 8.0), 10 mM CaCl2, 20 mM NaCl, 10% glycerol, and bovine serum albumin (0.1 mg/ml). Reactions were incubated at 25 °C for 20 min. Reaction products were analyzed by PAGE (6%). Apparent KD values (defined by the protein concentrations necessary to shift half the amount of probe) were determined with cognate probe. Nonspecific binding was tested using noncognate probe.

Assay of NaeI-43R Topoisomerase Activity-- Plasmid pBR322 (11.6 nM) and NaeI-43R (0.21 µM) were incubated at 37 °C for 30 min in 10 µl containing 10 mM Tris-HCl (pH 8.0), 20 mM NaCl (except for the assay of NaCl dependence), 5 mM MgCl2, bovine serum albumin (0.1 mg/ml), and 5 mM beta -mercaptoethanol. Its weak binding to DNA (12) necessitated the relatively high concentration of NaeI-43R. The DNA binding affinity was similar to that of NaeI-43K. Reactions were stopped by addition of SDS to 1%. Products were resolved on 1.0% agarose gels containing 0.5 µg/ml ethidium bromide in the gel and in the running buffer. Assays for the effects on NaeI-43R activity of single-stranded DNA, NaCl, and amsacrine varied the concentrations of these, as indicated.

Limited Proteolysis-- Trypsin (2.3 ng) was added to 7 µg of NaeI or NaeI-43K, and 1-µg aliquots were removed at the times indicated. Phenylmethylsulfonyl fluoride (1 mM) and 1.0% SDS were added to the aliquots, which were then heated to 100 °C for 10 min. Reaction products were resolved by SDS-PAGE (5% stacking and 15% resolving). The gel was stained with Coomassie Blue and photographed.

Size-exclusion Chromatography-- NaeI and NaeI-43K were sized relative to known molecular weight proteins using chromatography through Sephacryl S-200 resin (32-cm column) pre-equilibrated with phosphate column buffer (20 mM potassium phosphate (pH 7.4), 0.1 mM EDTA, 5% glycerol, 1.0 mM 2-mercaptoethanol, 50 mM NaCl). Protein (50 µl at 0.1 mg/ml) was loaded and eluted at 0.15 ml/min. Absorbance was monitored at 280 nm and the elution volume determined. The void volume was determined using blue dextran. Chromatography was performed with a Bio-Rad Biologic Chromatography System.

Circular Dichroism-- UV-CD spectra were measured using an Applied Photophysics PiStar-180 spectrometer. NaeI and NaeI-43K were extensively dialyzed into 10 mM phosphate, pH 7.0 (buffer conditions that showed similar specific activities to that measured using the above assay conditions). CD spectra were measured for NaeI, NaeI-43K, and buffer between wavelengths 185 nm and 260 nm at 25 °C. Buffer spectrum was subtracted from NaeI and NaeI-43K spectra. The concentrations of both NaeI and NaeI-43K were 0.1 mg/ml as determined from absorbance measurements at 280 nm. CD measurements were also made at a wavelength of 208 nm while increasing the temperature from 10 to 90 °C.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activity of NaeI Mutants-- All of the mutants were expressed as fusions with maltose-binding protein. The effects of substitutions at position 43 were determined. DNA specific activities for each mutant were determined from the slope of the line defined by amount cleaved per amount of protein (Fig. 2). The plots of DNA cleavage versus amount of protein were reproducible and linear over the entire range of protein concentration (Fig. 2 and Table I). Substitutions with alanine, asparagine, glutamate, and histidine retained endonuclease activity. The cleavage patterns of the fusion protein mutants were identical to that of wild-type NaeI fused to maltose-binding protein (MBP·NaeI). The specific activities of the mutants, however, were reduced compared with that of MBP·NaeI. When NaeI-43K and -43R were expressed as protein fusions with maltose-binding protein neither restriction endonuclease activity nor topoisomerase activity was detected. NaeI, expressed from this vector, showed no topoisomerase activity. Therefore, NaeI-43K and -43R were expressed in E. coli from pNEB786 where they both demonstrated topoisomerase activities. Therefore, all of the mutants were studied as fusion proteins (MBP·NaeI-43A, -43H, -43E, -43N), except for NaeI-43K and NaeI-43R.


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Fig. 2.   Increase in restriction endonuclease DNA cleavage activity with increase in MBP·NaeI mutant protein concentrations. A, MBP·NaeI-43A was incubated with 500 nM DNA substrate for 30 min. Reaction products were separated by 8% PAGE and visualized by phosphorimager. B, plots of % DNA substrate cleaved versus nanomolar NaeI mutant protein for position 43 substitutions, except for NaeI-43K and -43R, which do not have detectable activities as fusion proteins and have DNA relaxation activities, but no restriction enzyme activities when fused to MBP. Wild type (black-diamond ), 43A (), 43H (black-triangle), 43N (black-square), 43E ().

                              
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Table I
Effects of amino acid substitutions for Leu-40 and Leu-43 on endonuclease activity

Relative DNA Binding-- The apparent KD values were determined for NaeI mutant interactions with DNA from the amount of protein necessary to shift half of the cognate DNA probe during PAGE (Fig. 3 and Table I). DNA-binding results are the average of two determinations. The apparent KD values varied significantly from wild type only for MBP·NaeI-43N, -43E, and -40K, which gave values of 125 ± 2, 225 ± 5, and 1250 ± 75 (nM), respectively. The gel-shift results show a small amount of density in the wells probably due to a small amount of aggregated protein that binds DNA. The small amount of protein in the wells is counted in the determination of relative KD values.


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Fig. 3.   Gel mobility-shift assay of NaeI-43A. Upper panel, PAGE (6%) analysis of complexes formed from incubation of increasing concentration of NaeI-43A with radiolabeled DNA probe. Bands were visualized by phosphorimager and quantitated by Imagequant software. Lower panel, plot of percent DNA shifted versus increasing NaeI-43A protein concentration. Apparent KD is defined as the protein concentration required to shift half of the starting amount of DNA probe.

Comparison of NaeI-43R and NaeI-43K Activities-- NaeI-43K has topoisomerase activity rather than endonuclease activity, which is sensitive to salt, amsacrine, and ssDNA (10-12). Incubating similar concentrations of NaeI-43R and NaeI-43K with pBR322 resulted in almost identical banding ladders characteristic of topoisomerase activity (Fig. 4A). The differences between the ladders in Fig. 4A can be attributed to small differences in specific activities between the two protein preparations.


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Fig. 4.   Topoisomerase activity of NaeI-43R. A, NaeI-43K and NaeI-43R proteins (0.21 µM) were incubated with pBR322 (11.6 nM) for 30 min. at 37 °C. The products were resolved by 1% agarose gel electrophoresis. The products were visualized by ethidium bromide staining. B, reaction products from above reactions were analyzed as above but gel contained ethidium bromide (0.5 µg/ml). Commercially available human Topo II was used in a control reaction.

Nicking versus relaxation by NaeI-43R was assayed to determine the relative amounts of nicked versus covalently closed, fully relaxed products produced (Fig. 4B). About half the final products were the latter. NaeI-43K results in a similar amount of nicked versus relaxed DNA (26).

The effects of varying salt concentration on the relaxation reaction were determined (Fig. 5). The optimum NaCl concentration was below 30 mM. At 210 mM salt, relaxation was completely inhibited. The NaeI-43R mutation also made NaeI sensitive to the intercalative drug amsacrine (Fig. 5B). Inhibition of NaeI-43R Topo activity by amsacrine was apparent at a concentration of 5 µM with near total inhibition at 10 µM. Single-stranded DNAs, containing the NaeI cognate or noncognate sites, were tested for their ability to inhibit NaeI-43R activity. Nearly complete inhibition of NaeI-43R occurred with 10 nM ssDNA with or without cognate recognition sequence (Fig. 5, C and D). The results are identical to those for NaeI-43K.


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Fig. 5.   Effects of NaCl (A), amsacrine (B), and single-stranded (ss) DNA (C and D) on the topoisomerase activity of NaeI-43R. Reaction conditions were as defined in the Fig. 4 legend and under "Experimental Procedures." Reaction products were resolved by 1% agarose gel and stained with ethidium bromide.

Effect of L43K Substitution on NaeI Structure-- To determine the effect of a positive charge at position 43 on NaeI structure, NaeI and NaeI-43K conformations were probed using size-exclusion chromatography, limited proteolysis, and circular dichroism. NaeI-43K elutes by size-exclusion chromatography at the same volume as NaeI, indicating that it is a dimer of about 70 kDa (Fig. 6). Ultracentrifugation (27), gel filtration (7), and crystallization (13, 14) show that the preferred structure of NaeI in solution is a dimer. Trypsin was used to probe the domain structure of NaeI-43K relative to NaeI. The sites accessible to trypsin cleavage were clearly different between NaeI-43K and NaeI (Fig. 7). NaeI showed stable domains at molecular sizes of 16 and 19 kDa. NaeI-43K showed no stable domains and a significantly different initial banding pattern from NaeI at 5-min digestion with trypsin (Fig. 7). Additionally, NaeI was more resistant to trypsin cleavage than NaeI-43K.


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Fig. 6.   Determination of the native molecular weight of NaeI-43K by size-exclusion chromatography. A, chromatograph for NaeI on Sephacryl S-200 (see "Experimental Procedures"). Ultracentrifugation shows NaeI in solution to be a mixture of higher order complexes and dimer (27). Thus, the first peak is assigned to the higher order complexes of NaeI, which eluted at the void volume (Vo). The two V-shaped dips were probably caused by bubbles. The elution volume (Ve) for the NaeI dimer is defined as the volume at which the peak maximum eluted. B, (Ve)/(Vo) is plotted against the log of the molecular weight for: 1) sweet potato beta -amylase (200 kDa), 2) yeast alcohol dehydrogenase (150 kDa), 3) bovine albumin (66 kDa), 4) bovine carbonic anhydrase (29 kDa), and 5) horse cytochrome c (12.4 kDa). NaeI (70 kDa) is a dimer (7, 13).


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Fig. 7.   Limited proteolysis of NaeI and NaeI-43K. Products of incubation of substrate protein (7 µg) with trypsin (2.3 ng) SDS-PAGE (15%) for the time intervals indicated. Gel was stained with Coomassie Blue. Stable Endo and Topo domains of NaeI are indicated.

The protease digestion experiments implied conformational differences between NaeI and NaeI-43K. To confirm this, we measured the UV CD spectra of both proteins. CD measurements showed distinct differences between the CD spectra of NaeI and NaeI-43K (Fig. 8A). Most notably, the minima between 205 and 230 nm were larger for NaeI-43K, with a distinct difference at the alpha -helical characteristic wavelength of 222 nm. The CD curves were reproducible and overlapped above lambda  of 250 nm, which indicated no significant concentration differences. CD was also used to monitor the temperature melting profiles of the two proteins to determine their relative Tm values. The thermal transition point was determined at lambda  of 208 nm, which is within the wavelength area where the CD values are most sensitive to protein structure (28). NaeI and NaeI-43K gave well defined melting points of 56 ± 0.3 °C and 59 ± 0.3 °C, respectively (Fig. 8B). The melting point values are the averages of two determinations.


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Fig. 8.   CD measurements of NaeI and NaeI-43K. A, UV-CD spectra for NaeI (---) and NaeI-43K (---) at 25 °C. B, CD measurements at lambda  of 208 nm (chosen because it lies within the wavelength region where the CD shows maximum dependence on protein structure, Refs. 28 and 31) for NaeI (---)and NaeI-43K (---) as a function of temperature from 10 to 90 °C. Protein concentrations were 0.1 mg/ml, as determined by absorbance at 280 nm.

Visual Inspection of the NaeI Amino Acid Sequence-- Initial inspection of the NaeI sequence led to the discovery of a 10 amino acid sequence in NaeI with similarity to motif I of DNA ligase, the KXDG motif (10). Visual inspection, taking into consideration the secondary structures of the DNA ligase motifs, identified three additional regions of NaeI protein sequence (Fig. 9) with similarity to three additional motifs that define the DNA ligase enzyme family (20-24, 29). Fig. 9 shows the four ligase motifs for several of the DNA ligases and for NaeI. The NaeI amino acid sequence regions are shown at the top of Fig. 9 in bold. Amino acids in the DNA ligase motifs in the same exchange group (30) as the corresponding amino acid in NaeI are shown in bold and underlined.


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Fig. 9.   Nucleotidyl transferase motifs in the NaeI protein sequence. Five collinear sequence elements, designated nucleotidyl transferase motifs I, III, IIIa, IV, and V, are conserved in ATP-dependent DNA ligases (ATP) and NAD-dependent DNA ligases (NAD) (20-24, 29). The amino acid sequences shown aligned with NaeI-43K (top, bold) include: the ATP-dependent ligases human ligase I (Hu 1), human ligase 3 (Hu 3), Schizosaccharomyces pombe (Spo), Saccharomyces cerevisiae (Sce), vaccinia virus (Vac), Chlorella virus (ChV), and bacteriophage T7 (T7); and the NAD-dependent ligases Escherichia coli (Eco), Bacillus stearothermophilus (Bst) and Thermus thermophilus (Tth). The number of amino acids separating the motifs is indicated by (-n-). Amino acids in the ligase motifs that are in the same exchange group (30) as the corresponding NaeI amino acid are shown in bold and underlined. NaeI was compared with the DNA ligases by visual inspection of amino acid sequences keeping in mind the secondary structures of the individual ligase motifs (21-24).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NaeI Position 43-- Substituting lysine for leucine at position 43 abolishes NaeI endonuclease activity and replaces it with topoisomerase and recombinase activities (10). Alanine, arginine, asparagine, glutamate, and histidine were substituted at position 43 and lysine at position 40 and the activities of the respective N-terminal MBP fusion proteins quantitated to test the chemical characteristics that lead to topoisomerase activity. All substitutions, except arginine and lysine substituted at position 43, retained sequence-specific endonuclease activity, albeit at lower levels (Table I). Substitution of alanine at position 43 retained the most cleavage activity, whereas inserting lysine at position 40 and the negatively charged glutamate at position 43 reduced cleavage ~33- and 100-fold, respectively. Alanine and histidine substitutions at position 43 retained wild-type levels of DNA binding. Substituting lysine for leucine at position 40 and glutamate for leucine at position 43, however, reduced DNA binding 125-fold and 23-fold, respectively. Deletion of the entire Endo domain only reduces DNA binding for NaeI 8-fold (27) because of the strong DNA binding of the Topo domain (6, 14, 27). Thus, placing a charge within the core hydrophobic region of the Endo domain appears to have effects that reach beyond the local environment of the Endo domain.

NaeI-43K lacks activity when expressed either as an N-terminal or as a C-terminal MBP fusion protein (not shown). NaeI-43R fused at its N terminus to MBP also lacks activity. The effect of fusion at the C terminus was not determined. When expressed without MBP, on the other hand, both mutant proteins gave identical topoisomerase activities. NaeI has endonuclease activity either when expressed alone or as an N-terminal MBP fusion protein (27). No activity is recovered from NaeI expressed as a C-terminal fusion protein. The apparent loss of activity when fused at the C terminus can be rationalized from the crystal structure of the NaeI-DNA complex (14): The C terminus of each NaeI monomer lies over the DNA binding pocket. A bulky protein fused in this position would block access of DNA to the NaeI Topo domain. On the other hand, the NaeI N terminus lies on the outside of NaeI away from the binding faces of the protein (14). Thus, the loss of activity when MBP is fused to the N terminus of NaeI-43K and -43R, but not when fused to the N terminus of NaeI, suggests an altered structure for NaeI-43K and -43R.

NaeI-43R Has Topoisomerase Activity-- NaeI-43R and NaeI-43K were found to be similar with respect to specific activity, NaCl dependence, amsacrine inhibition, and ssDNA inhibition. Amsacrine and ssDNA have no affect on NaeI, but inhibited the topoisomerase activities of NaeI-43K and NaeI-43R, demonstrating the equivalency of the L43K and L43R substitutions. To probe whether introducing a positive charge into the NaeI hydrophobic core could explain the switch to topoisomerase activity, lysine was substituted for leucine at position 40 in NaeI. Leucine 40 lies in the same alpha -helix, H2, as leucine 43 and is also part of the hydrophobic core of NaeI. Substitution of lysine for Leu-40 of NaeI decreased NaeI endonuclease activity, relative to wild type, but did not produce topoisomerase activity (Table I). The decrease in binding caused by L40K was similar to that seen when a negative charge (on glutamate) was inserted at position 43. The result implies that the topoisomerase activities in NaeI-43K and NaeI-43R depend on chemistry that involves more than the disruption caused by the introduction of a positive charge into the hydrophobic core. It is also possible, however, that a positive charge placed at position 40 causes a different disruption to folding than when placed at position 43.

Positive Charge at Position 43-- The addition of a positive charge in the form of lysine or arginine is sufficient to convert NaeI from a restriction endonuclease to a topoisomerase. NaeI position 43 is located near the interface between the two subunits that compose the homodimer (Fig. 1). The addition of a positive charge could interfere with NaeI dimerization, but size-exclusion chromatography demonstrated that NaeI-43K is a dimer. Limited proteolysis of NaeI shows two distinct and stable domains as products (Ref. 27 and Fig. 7). Limited proteolysis of NaeI-43K showed a very different initial pattern of digestion products (Fig. 7), indicating that in NaeI-43K versus NaeI, different arginines and lysines are accessible to trypsin. It is clear from the proteolysis results that NaeI-43K is less stable to trypsin than NaeI. At 90 min, a large portion of full-length NaeI remained undigested, whereas the full-length NaeI-43K was almost completely digested in 60 min and by 90 min showed no domain remaining resistant to trypsin. The most resistant band from digestion of NaeI-43K with trypsin was at 17 kDa. Peptide sequencing showed that this product and the 17-kDa product of NaeI (27) come from the Endo domain. Thus, the L43K substitution, which lies in the Endo domain, has significant effect on trypsin digestion of both the Topo and Endo domains. This implies significant refolding of the protein.

The protein UV-CD spectrum is determined by the protein conformation (28). The UV-CD spectra of NaeI and NaeI-43K indicate significant structural differences (Fig. 8A). The CD spectrum for NaeI-43K has the classical minima at 208 and 222 nm indicative of significant alpha -helical structure (31), whereas in NaeI they are not as prominent. The CD spectra were analyzed using the computer program Continll (28). NaeI-43K showed higher alpha -helical content than wild-type NaeI: 40 versus 15% alpha -helix, 33 versus 42% beta -sheet, 17 versus 24% turn, and 10 versus 20% unordered, respectively. The errors associated with these values are all better than ± 1% as determined from the best-fit results. CD analysis of protein structure is not very accurate. Here, the CD results underestimate the amount of NaeI alpha -helix; the crystal structure of NaeI shows that ~30% of the protein is alpha -helical. We assume that the alpha -helical content of NaeI-43K was underestimated to the same extent. Thus, by CD analysis, NaeI-43K has an altered structure relative to wild-type NaeI.

The presence of the positive charges in NaeI-43K and NaeI-43R could destabilize the proteins. To measure relative stability, CD measurements at lambda  of 208 nm as a function of increasing temperature were used to monitor NaeI and NaeI-43K temperature denaturation (Tm) profiles (Fig. 8B). NaeI-43K was more heat-stable. The 3 °C increase in Tm value for NaeI-43K relative to NaeI was surprising considering the location of position 43 in the hydrophobic core. L43K substitution causes a significant change in NaeI structure. The change may move lysine 43 out of the hydrophobic core so that it can participate in electrostatic interactions that help stabilize NaeI-43K. We speculate that the change in structure is required for the switch from endonuclease to topoisomerase activity.

Changes in Structure and Activity-- It is reasonable to assume that a significant refolding of NaeI is necessary to convert its activity from endonuclease to that of topoisomerase and recombinase. If this is true, then the significant global conformational changes demonstrated here may be the result of this refolding. How the changes in protein folding lead to the changes in protein activity is unknown. We can speculate about a possible mechanism, however, based on sequence homologies found by visual comparisons of the NaeI protein sequence with the sequences of the DNA ligases (Fig. 9). There are five conserved nucleotidyl transferase motifs that define the ligase/capping enzyme superfamily (20-24, 29). The five collinear sequence elements, designated nucleotidyl transferase motifs I, III, IIIa, IV, and V, are conserved in ATP-dependent DNA ligases, mRNA-capping enzymes, and NAD-dependent DNA ligases. Four of five sequence motifs were detected in NaeI by visual inspection (Fig. 9). The NaeI sequences are collinear with the conserved nucleotidyl transferase motifs, and the sequence distances between the NaeI sequences are approximately within the ranges predicted from that between the corresponding nucleotidyl transferase motifs. The motifs are spread over both domains of NaeI, and the NaeI structure cannot be superimposed on the DNA ligase structure. It is interesting to speculate that the refolding of NaeI caused by the placement of a positive charge at position 43 causes at least some of these motifs to superimpose. Active site residues in NaeI bind divalent metal, which stabilizes the DNA binding and the pentavalent transition state. DNA ligase motif III contains two conserved residues: one is conserved among all the DNA ligases, the other is conserved among the ATP ligases (Fig. 9). The two conserved amino acids in motif III contribute to the third (nick-joining) step of DNA ligation. For example, in Chlorella virus DNA ligase, Asp-65 and Glu-67 enhance the rate of step 3 phosphodiester formation by 20- and 1000-fold, respectively (32). Asp-65 overlaps with NaeI Asp-86, an essential metal ion-binding amino acid in NaeI (Fig. 9), which enhances DNA cleavage by 50-fold.2 Mutations of Glu-88 in NaeI have not been studied because this position is not conserved among restriction enzymes. Study of the effects on topoisomerase activity of mutation of NaeI-43K amino acids with similarity to the ligase motifs shown in Fig. 9 are in progress. The presence of additional ligase motifs in NaeI would imply homology with the DNA ligase family and support the notion that NaeI topoisomerase activity arises through linkage of DNA cleavage and ligation activities through the NaeI-DNA covalent intermediate (10).

Comparison with Nucleotidyl Transferases-- The nucleotidyl transferase KXDG-like motif in NaeI-43K appears to provide another example of the motif retaining some activity after substitution of the active site KXDG lysine with arginine but not when substituted with other amino acids. Two nucleotidyl transferases that manifest similar behavior are vaccinia virus RNA-capping enzyme and T4 RNA ligase (33, 34). Active-site lysine to arginine substitution in vaccinia virus RNA-capping enzyme gives low overall activity, whereas other mutations give no activity (33). The equivalent substitution in T4 RNA ligase gives intermediate levels of activity in all three steps of nucleotidyl transfer (34), whereas asparagine substitution gives no activity in any of the three steps (34).

NaeI-43K and NaeI-43R can catalyze many cycles of DNA relaxation in the absence of ATP and NAD+ (10). Therefore, they do not require adenylation to relax DNA. Rather they form a covalent enzyme-DNA complex (10). The energy from the covalent complex is used for ligation, replacing the need for an enzyme-adenylate intermediate. This suggests that NaeI-43K and -43R require only the third step of the nucleotidyl transferase reaction, strand closure, for topoisomerase activity. Studies of vaccinia virus RNA-capping enzyme (33), T4 RNA ligase (34), and Chlorella virus DNA ligase (35) show that the KXDG lysine contributes to strand closure. In T4 RNA ligase, substitution of the active-site lysine with asparagine inactivates the strand-closure step (34). Lysine is not absolutely required for this step by RNA-capping enzyme and DNA ligase but contributes 16-fold to the rate of closure (35). Thus, the importance of the active site lysine for strand closure may vary with the protein being considered. In NaeI, arginine and lysine substitutions at position 43 contribute a positive charge that alters the structure of NaeI and gives it the ability to catalyze strand closure of newly cleaved substrate. The result of this cleavage and ligation, when linked by the enzyme-DNA covalent intermediate, is relaxation of supercoiled DNA.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM52123.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295. Tel.: 919-966-8208; Fax: 919-966-3015; E-mail: mdtopal@ med.unc.edu.

Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M209192200

2 K. Carrick and M. Topal, unpublished results.

    ABBREVIATIONS

The abbreviations used are: NaeI, NaeI endonuclease; CAP, catabolite-activating protein; ES, enzyme-substrate complex; CD, circular dichroism; oligonucleotides, oligodeoxyribonucleotides; Topo, topoisomerase; MBP, maltose-binding protein; ssDNA, single-stranded DNA; amsacrine, N-[4-(9-acridinylamino)-3-methoxyphenyl]methane-sulfonamide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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