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• W2012991166 abstract "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 ofNaeI-43K and NaeI-43R and their relative sensitivities to salt, the topoisomerase-inhibiting drugN-[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 onNaeI structure was determined by measuring (forNaeI 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. 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 ofNaeI-43K and NaeI-43R and their relative sensitivities to salt, the topoisomerase-inhibiting drugN-[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 onNaeI structure was determined by measuring (forNaeI 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. NaeI endonuclease (NaeI) 1The 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 is a prototype for the type IIe (enhancer) (1Oller A.R. Vanden Broek W. Conrad M. Topal M.D. Biochemistry. 1991; 30: 2543-2549Google Scholar, 2Reuter M. Kupper D. Pein C.-D. Petrusyte M. Siksnys V. Frey B. Krüger D. Anal. Biochem. 1993; 209: 232-237Google Scholar) restriction endonucleases, so named (3Yang C.C. Baxter B.K. Topal M.D. Biochemistry. 1994; 33: 14918-14925Google Scholar) because they require interactions with an enhancing DNA sequence. One DNA sequence acts as enhancer to induce cleavage of the other sequence (4Conrad M. Topal M.D. Proc. Nat. Acad. Sci. U. S. A. 1989; 86: 9707-9711Google Scholar, 5Topal M.D. Thresher R.J. Conrad M. Griffith J. Biochemistry. 1991; 30: 2006-2010Google Scholar, 6Yang C.C. Topal M.D. Biochemistry. 1992; 31: 9657-9664Google Scholar, 7Baxter B.K. Topal M.D. Biochemistry. 1993; 32: 8291-8298Google Scholar). In solution, NaeI protein is a 70-kDa homodimer (7Baxter B.K. Topal M.D. Biochemistry. 1993; 32: 8291-8298Google Scholar) composed of two 317-amino acid polypeptides (8Holtz J.K. Topal M.D. J. Biol. Chem. 1994; 269: 27286-27290Google Scholar, 9Taron C.H. Van Cott E.M. Wilson G.G. Moran L.S. Slatko B.E. Hornstra L.J. Benner J.S. Kucera R.B. Guthrie E.P. Gene (Amst.). 1995; 155: 19-25Google Scholar) that recognize and cleave at the arrow, the DNA sequence 5′-GCC↑GGC-3′ using only Mg2+ as a cofactor. Two-site binding givesNaeI a specificity of DNA recognition ∼104-fold better than single-site-binding proteins that recognize similar sized sequences (3Yang C.C. Baxter B.K. Topal M.D. Biochemistry. 1994; 33: 14918-14925Google Scholar). The two-site binding loops out intervening DNA sequences (5Topal M.D. Thresher R.J. Conrad M. Griffith J. Biochemistry. 1991; 30: 2006-2010Google Scholar), 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 (10Jo K. Topal M.D. Science. 1995; 267: 1817-1820Google Scholar). 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 asN-[4-(9-acridinylamino)-3-methoxyphenyl]methane-sulfonamide (amsacrine), which is lacking in restriction endonucleases but characteristic of topoisomerases (11Jo K. Topal M.D. Biochemistry. 1996; 35: 10014-10018Google Scholar, 12Jo K. Topal M.D. Nucleic Acids Res. 1996; 24: 4171-4175Google Scholar). NaeI position 43 is located near the C terminus of α-helix H2, which is part of the central hydrophobic core of the Endo domain (Ref. 13Huai Q. Colandene J.D. Chen Y. Luo F. Zhao Y. Topal M.D. Ke H. EMBO J. 2000; 19: 3110-3118Google Scholar and Fig. 1). Unlike the structures of most restriction endonucleases, NaeI contains two separate domains, both of which bind DNA (14Huai Q. Colandene J.D. Topal M.D. Ke H. Nat. Struct. Biol. 2001; 8: 665-669Google Scholar). The N-terminal, Endo domain contains the restriction endonuclease cleavage motif found in restriction enzymes as well as repair nucleases mutH, Vsr, and λ exonuclease (14Huai Q. Colandene J.D. Topal M.D. Ke H. Nat. Struct. Biol. 2001; 8: 665-669Google Scholar, 15Ban C. Yang W. EMBO J. 1998; 17: 1526-1534Google Scholar, 16Tsutakawa S.E. Muto T. Kawate T. Jingami H. Kunishima N. Ariyoshi M. Kohda D. Nakagawa M. Morikawa K. Mol. Cell. 1999; 3: 621-628Google Scholar, 17Kovall R.A. Matthews B.W. Curr. Opin. Chem. Biol. 1999; 3: 578-583Google Scholar) and transposases (18Hickman A.B. Li Y. Mathew S.V. May E.W. Craig N.L. Dyda F. Mol. Cell. 2000; 5: 1025-1034Google Scholar). The C-terminal, Topo domain contains a CAP motif also found in topoisomerases IA and II (19Berger J.M. Fass D. Wang J.C. Harrison S.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7876-7881Google Scholar). 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. 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. 20Shuman S. Schwer B. Mol. Microbiol. 1995; 17: 405-410Google Scholar). 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 ε-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 (10Jo K. Topal M.D. Science. 1995; 267: 1817-1820Google Scholar), 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 linksNaeI to its DNA substrate has not been identified. There is no similarity between the folds of NaeI (13Huai Q. Colandene J.D. Chen Y. Luo F. Zhao Y. Topal M.D. Ke H. EMBO J. 2000; 19: 3110-3118Google Scholar, 14Huai Q. Colandene J.D. Topal M.D. Ke H. Nat. Struct. Biol. 2001; 8: 665-669Google Scholar) and DNA ligase (21Subramanya H.S. Doherty A.J. Ashford S.R. Wigley D.B. Cell. 1996; 85: 607-615Google Scholar, 22Singleton M.R. Håkansson K. Timson D.J. Wigley D.B. Structure. 1999; 7: 35-42Google Scholar, 23Lee J.Y. Chang C. Song H.K. Moon J. Yang J.K. Kim H.-K. Kwon S.-T. Suh S.W. EMBO J. 2000; 19: 1119-1129Google Scholar, 24Odell M. Sriskanda V. Shuman S. Nikolov D.B. Mol. Cell. 2000; 6: 1183-1193Google Scholar). Moreover, the KXDG-like motif inNaeI 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 giveNaeI 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 maintainedNaeI 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 andNaeI-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. 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 theNaeIR 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. The NaeIR gene was subcloned into the expression vector pMAL-C2, and site-directed mutagenesis was performed using the method of Clackson et al. (25Clackson T. Gussow D. Jones P.T. Quirke P. Taylor G.R. PCR-A Practical Approach. Oxford University Press, New York1991: 187-214Google Scholar). MutatedNaeIR genes were sequenced to confirm the mutation and ensure that no secondary mutations were generated. To express the fusion protein, pMAL-NaeI mutants (MBP·NaeI) were induced with 1 mmisopropyl-β-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 mm2-mercaptoethanol, 50 mm NaCl) with 1 mmphenylmethylsulfonyl 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 onNaeI and NaeI-43K activities. 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 mmNaCl 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 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 [α-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. 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 mmCaCl2, 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%). ApparentK D 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. 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 mmβ-mercaptoethanol. Its weak binding to DNA (12Jo K. Topal M.D. Nucleic Acids Res. 1996; 24: 4171-4175Google Scholar) 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. Trypsin (2.3 ng) was added to 7 μg ofNaeI 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. NaeI andNaeI-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 mmNaCl). 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. UV-CD spectra were measured using anApplied Photophysics PiStar-180 spectrometer. NaeI andNaeI-43K were extensively dialyzed into 10 mmphosphate, 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 andNaeI-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. 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. WhenNaeI-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 forNaeI-43K and NaeI-43R.Table IEffects of amino acid substitutions for Leu-40 and Leu-43 on endonuclease activityMutant1-aAll mutants were expressed as fusions with MBP.Sp. activityWild typeapp K DWild type(×1000)%nm1-bApparent K D is abbreviated app K D.%Wt30 ± 31-cErrors are variation from average of two determinations. Errors for normalized % values are based on propagation of errors.10010.0 ± 0.2100L40K0.8 ± 0.053 ± 11250 ± 750.8 ± 0.6L43A3.7 ± 0.0112 ± 110 ± 0.2100 ± 4L43E0.1 ± 0.10.3 ± 0.4225 ± 54.4 ± 0.6L43H1.5 ± 0.055 ± 110 ± 0.2100 ± 4L43N0.5 ± 0.32 ± 1125 ± 28.0 ± 0.3L43KND1-dND, not detectable. L43K and L43R showed no detectable endonuclease activity when expressed either as protein fusions or unfused. Unfused they demonstrated DNA relaxation activities.L43RND1-a All mutants were expressed as fusions with MBP.1-b Apparent K D is abbreviated app K D.1-c Errors are variation from average of two determinations. Errors for normalized % values are based on propagation of errors.1-d ND, not detectable. L43K and L43R showed no detectable endonuclease activity when expressed either as protein fusions or unfused. Unfused they demonstrated DNA relaxation activities. Open table in a new tab The apparent K Dvalues 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 apparentK D 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 K D values. NaeI-43K has topoisomerase activity rather than endonuclease activity, which is sensitive to salt, amsacrine, and ssDNA (10Jo K. Topal M.D. Science. 1995; 267: 1817-1820Google Scholar, 11Jo K. Topal M.D. Biochemistry. 1996; 35: 10014-10018Google Scholar, 12Jo K. Topal M.D. Nucleic Acids Res. 1996; 24: 4171-4175Google Scholar). Incubating similar concentrations ofNaeI-43R and NaeI-43K with pBR322 resulted in almost identical banding ladders characteristic of topoisomerase activity (Fig. 4 A). The differences between the ladders in Fig. 4 A can be attributed to small differences in specific activities between the two protein preparations. Nicking versus relaxation by NaeI-43R was assayed to determine the relative amounts of nicked versuscovalently closed, fully relaxed products produced (Fig.4 B). About half the final products were the latter.NaeI-43K results in a similar amount of nickedversus relaxed DNA (26Jo K. Topal M.D. Nucleic Acids Res. 1998; 26: 2380-2384Google Scholar). The effects of varying salt concentration on the relaxation reaction were determined (Fig. 5). The optimum NaCl concentration was below 30 mm. At 210 mmsalt, relaxation was completely inhibited. The NaeI-43R mutation also made NaeI sensitive to the intercalative drug amsacrine (Fig. 5 B). 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 inhibitNaeI-43R activity. Nearly complete inhibition ofNaeI-43R occurred with 10 nm ssDNA with or without cognate recognition sequence (Fig. 5, C andD). The results are identical to those forNaeI-43K. 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 (27Colandene J.D. Topal M.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3531-3536Google Scholar), gel filtration (7Baxter B.K. Topal M.D. Biochemistry. 1993; 32: 8291-8298Google Scholar), and crystallization (13Huai Q. Colandene J.D. Chen Y. Luo F. Zhao Y. Topal M.D. Ke H. EMBO J. 2000; 19: 3110-3118Google Scholar,14Huai Q. Colandene J.D. Topal M.D. Ke H. Nat. Struct. Biol. 2001; 8: 665-669Google Scholar) show that the preferred structure of NaeI in solution is a dimer. Trypsin was used to probe the domain structure ofNaeI-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.Figure 7Limited 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.View Large Image Figure ViewerDownload (PPT) 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 andNaeI-43K (Fig. 8 A). Most notably, the minima between 205 and 230 nm were larger forNaeI-43K, with a distinct difference at the α-helical characteristic wavelength of 222 nm. The CD curves were reproducible and overlapped above λ 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 relativeTm values. The thermal transition point was determined at λ of 208 nm, which is within the wavelength area where the CD values are most sensitive to protein structure (28Provencher S.W. Glockner J. Biochemistry. 1981; 20: 33-37Google Scholar).NaeI and NaeI-43K gave well defined melting points of 56 ± 0.3 °C and 59 ± 0.3 °C, respectively (Fig. 8 B). The melting point values are the averages of two determinations. 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 (10Jo K. Topal M.D. Science. 1995; 267: 1817-1820Google Scholar). 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 (20Shuman S. Schwer B. Mol. Microbiol. 1995; 17: 405-410Google Scholar, 21Subramanya H.S. Doherty A.J. Ashford S.R. Wigley D.B. Cell. 1996; 85: 607-615Google Scholar, 22Singleton M.R. Håkansson K. Timson D.J. Wigley D.B. Structure. 1999; 7: 35-42Google Scholar, 23Lee J.Y. Chang C. Song H.K. Moon J. Yang J.K. Kim H.-K. Kwon S.-T. Suh S.W. EMBO J. 2000; 19: 1119-1129Google Scholar, 24Odell M. Sriskanda V. Shuman S. Nikolov D.B. Mol. Cell. 2000; 6: 1183-1193Google Scholar,29Sriskanda V. Schwer B. Ho C.K. Shuman S. Nucleic Acids Res. 1999; 27: 3953-3963Google Scholar). 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 (30Dayhoff M.O. Schwartz R.M. Orcott B.L. Dayhoff M.O. Atlas of Protein Sequence and Structure. 5, Suppl. 3. National Biomedical Research Foundation, Washington, D. C.1978: 345-352Google Scholar) as the corresponding amino acid in NaeI are shown inbold and underlined. Substituting lysine for leucine at position 43 abolishes NaeI endonuclease activity and replaces it with topoisomerase and recombinase activities (10Jo K. Topal M.D. Science. 1995; 267: 1817-1820Google Scholar). 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 (TableI). 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 forNaeI 8-fold (27Colandene J.D. Topal M.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3531-3536Google Scholar) because of the strong DNA binding of the Topo domain (6Yang C.C. Topal M.D. Biochemistry. 1992; 31: 9657-9664Google Scholar, 14Huai Q. Colandene J.D. Topal M.D. Ke H. Nat. Struct. Biol. 2001; 8: 665-669Google Scholar, 27Colandene J.D. Topal M.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3531-3536Google Scholar). 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 (27Colandene J.D. Topal M.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3531-3536Google Scholar). No activity is recovered from NaeI expressed as a C-terminal fusion protein. The apparent loss of activity wh" @default.
• W2012991166 created "2016-06-24" @default.
• W2012991166 creator A5015396575 @default.
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