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• W2153298725 abstract "PI-SceI is an intein-encoded protein that belongs to the LAGLIDADG family of homing endonucleases. According to the crystal structure and mutational studies, this endonuclease consists of two domains, one responsible for protein splicing, the other for DNA cleavage, and both presumably for DNA binding. To define the DNA binding site of PI-SceI, photocross-linking was used to identify amino acid residues in contact with DNA. Sixty-three double-stranded oligodeoxynucleotides comprising the minimal recognition sequence and containing single 5-iodopyrimidine substitutions in almost all positions of the recognition sequence were synthesized and irradiated in the presence of PI-SceI with a helium/cadmium laser (325 nm). The best cross-linking yield (approximately 30%) was obtained with an oligodeoxynucleotide with a 5-iododeoxyuridine at position +9 in the bottom strand. The subsequent analysis showed that cross-linking had occurred with amino acid His-333, 6 amino acids after the second LAGLIDADG motif. With the H333A variant of PI-SceI or in the presence of excess unmodified oligodeoxynucleotide, no cross-linking was observed, indicating the specificity of the cross-linking reaction. Chemical modification of His residues in PI-SceI by diethylpyrocarbonate leads to a substantial reduction in the binding and cleavage activity of PI-SceI. This inactivation can be suppressed by substrate binding. This result further supports the finding that at least one His residue is in close contact to the DNA. Based on these and published results, conclusions are drawn regarding the DNA binding site of PI-SceI. PI-SceI is an intein-encoded protein that belongs to the LAGLIDADG family of homing endonucleases. According to the crystal structure and mutational studies, this endonuclease consists of two domains, one responsible for protein splicing, the other for DNA cleavage, and both presumably for DNA binding. To define the DNA binding site of PI-SceI, photocross-linking was used to identify amino acid residues in contact with DNA. Sixty-three double-stranded oligodeoxynucleotides comprising the minimal recognition sequence and containing single 5-iodopyrimidine substitutions in almost all positions of the recognition sequence were synthesized and irradiated in the presence of PI-SceI with a helium/cadmium laser (325 nm). The best cross-linking yield (approximately 30%) was obtained with an oligodeoxynucleotide with a 5-iododeoxyuridine at position +9 in the bottom strand. The subsequent analysis showed that cross-linking had occurred with amino acid His-333, 6 amino acids after the second LAGLIDADG motif. With the H333A variant of PI-SceI or in the presence of excess unmodified oligodeoxynucleotide, no cross-linking was observed, indicating the specificity of the cross-linking reaction. Chemical modification of His residues in PI-SceI by diethylpyrocarbonate leads to a substantial reduction in the binding and cleavage activity of PI-SceI. This inactivation can be suppressed by substrate binding. This result further supports the finding that at least one His residue is in close contact to the DNA. Based on these and published results, conclusions are drawn regarding the DNA binding site of PI-SceI. base pair(s) cross-link diethylpyrocarbonate double-stranded 5-iododeoxyuridine 5-iododeoxycytidine polyacrylamide gel electrophoresis Tris borate/EDTA Homing endonucleases are a fascinating new class of enzymes that cleave DNA with very high specificity within an extended recognition site and, thereby, in vivo initiate a double strand break repair that may lead to the insertion of the sequence coding for the homing endonuclease into an allele that it lacks (for reviews, see Refs. 1Belfort M. Roberts R.J. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (391) Google Scholar and 2Curcio M.J. Belfort M. Cell. 1996; 84: 9-12Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). They have been found in prokaryotes and eukaryotes as well as in archaebacteria and are encoded by introns or inteins (for review, see Ref. 3Lambowitz A.M. Belfort M. Annu. Rev. Biochem. 1993; 62: 567-622Crossref Scopus (532) Google Scholar). The largest group is characterized by the presence of one or two copies of a conserved dodecapeptide sequence, the LAGLIDADG motif (4Belfort M. Perlman P.S. J. Biol. Chem. 1995; 270: 30237-30240Crossref PubMed Scopus (195) Google Scholar). PI-SceI, a homing endonuclease from yeast, belongs to this group and occurs as an intein within the vacuolar H+-ATPase, from which it is spliced in an autocatalytic reaction (5Gimble F.S. Thorner J. J. Biol. Chem. 1993; 268: 21844-21853Abstract Full Text PDF PubMed Google Scholar). The mature protein recognizes an extraordinarily long DNA sequence of 35–45 bp,1 bends the DNA, and cleaves the substrate to produce a 4-bp 3′ overhang (5Gimble F.S. Thorner J. J. Biol. Chem. 1993; 268: 21844-21853Abstract Full Text PDF PubMed Google Scholar, 6Gimble F.S. Wang J. J. Mol. Biol. 1996; 263: 163-180Crossref PubMed Scopus (81) Google Scholar, 7Wende W. Grindl W. Christ F. Pingoud A. Pingoud V. Nucleic Acids Res. 1996; 24: 4123-4132Crossref PubMed Scopus (49) Google Scholar). The molecular details of DNA recognition and cleavage by PI-SceI are unclear, in spite of the fact that the crystal structure of PI-SceI is known (8Duan X. Gimble F.S. Quiocho F. Cell. 1997; 89: 555-564Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). According to the structure analysis and mutational studies (9Grindl W. Wende W. Pingoud V. Pingoud A. Nucleic Acids Res. 1998; 26: 1857-1862Crossref PubMed Scopus (34) Google Scholar), PI-SceI is composed of two domains, one responsible for protein splicing (domain I) and one for DNA cleavage (domain II), which are connected by two peptide segments. The structure of the elongated domain I consists almost entirely of β-sheets, whereas the compact domain II is an almost equal mixture of α-helices and β-strands. Domain II is built up from two substructures that are related by local 2-fold symmetry about an axis between the two LAGLIDADG sequences. The domain II of PI-SceI is structurally very similar to the homodimeric homing endonuclease I-CreI, which contains one LAGLIDADG motif per subunit and lacks the protein splicing domain. As shown by the crystal structure analysis (10Heath P.J. Stephens K.M. Monnat R. Stoddard B.L. Nat. Struct. Biol. 1997; 4: 468-476Crossref PubMed Scopus (116) Google Scholar), the LAGLIDADG motifs in I-CreI form part of the dimer interface while simultaneously positioning one of the conserved Asp residues adjacent to the scissile phosphates. These residues may function to coordinate Mg2+and thereby help to attack the DNA; substitution of these residues abolishes the endonuclease activity of I-CreI (11Seligman L.M. Stephens K.M. Savage J.H. Monnat R.J. Genetics. 1997; 147: 1653-1664Crossref PubMed Google Scholar). Mutation of the analogous residues Asp-218 and Asp-326 in PI-SceI also destroys the nucleolytic activity of this enzyme (12Gimble F.S. Stephens B.W. J. Biol. Chem. 1995; 270: 5849-5856Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), whereas substrate binding of the mutated PI-SceI is not affected, suggesting that these Asp residues are involved in catalysis. Similar results were obtained with I-SceII (13Henke R.M. Butow R.A. Perlman P.S. EMBO J. 1995; 14: 5094-5099Crossref PubMed Scopus (47) Google Scholar), I-DmoI (14Lykke-Andersen J. Garrett R.A. Kjems J. EMBO J. 1997; 16: 3272-3281Crossref PubMed Scopus (34) Google Scholar), I-CeuI (15Turmel M. Otis C. Cote V. Lemieux C. Nucleic Acids Res. 1997; 25: 2610-2619Crossref PubMed Scopus (44) Google Scholar), I-PorI (14Lykke-Andersen J. Garrett R.A. Kjems J. EMBO J. 1997; 16: 3272-3281Crossref PubMed Scopus (34) Google Scholar), and PI-TliI (16Hodges R.A. Perler F.B. Noren C.J. Jack W.E. Nucleic Acids Res. 1992; 20: 6153-6157Crossref PubMed Scopus (106) Google Scholar), all members of the LAGLIDADG family of homing endonucleases. All homing endonucleases have long recognition sequences (15–45 bp) that can tolerate variation of the sequence, as shown for PI-SceI (6Gimble F.S. Wang J. J. Mol. Biol. 1996; 263: 163-180Crossref PubMed Scopus (81) Google Scholar) and other homing endonucleases, e.g.I-CreI (17Argast M.G. Stephens K.M. Emond M.J. Monnat R.J. J. Mol. Biol. 1998; 280: 345-353Crossref PubMed Scopus (100) Google Scholar), I-DmoI (18Ågaard C. Awayez M. Garrett R. Nucleic Acids Res. 1997; 25: 1523-1530Crossref PubMed Scopus (35) Google Scholar), I-PorI (19Lykke-Andersen J. Thi-Ngoc H.P. Garrett R.A. Nucleic Acids Res. 1994; 22: 4583-4590Crossref PubMed Scopus (29) Google Scholar), I-PpoI (17Argast M.G. Stephens K.M. Emond M.J. Monnat R.J. J. Mol. Biol. 1998; 280: 345-353Crossref PubMed Scopus (100) Google Scholar), and I-TevI (20Mueller J.E. Smith D. Bryk M. Belfort M. EMBO J. 1995; 14: 5724-5735Crossref PubMed Scopus (61) Google Scholar). Footprinting studies with PI-SceI (6Gimble F.S. Wang J. J. Mol. Biol. 1996; 263: 163-180Crossref PubMed Scopus (81) Google Scholar), I-DmoI (18Ågaard C. Awayez M. Garrett R. Nucleic Acids Res. 1997; 25: 1523-1530Crossref PubMed Scopus (35) Google Scholar), and I-TevII (21Loizos N. Silva G.H. Belfort M. J. Mol. Biol. 1996; 255: 412-424Crossref PubMed Scopus (26) Google Scholar) and their substrates show that they are involved in both major and minor groove interactions. After cleavage, PI-SceI (6Gimble F.S. Wang J. J. Mol. Biol. 1996; 263: 163-180Crossref PubMed Scopus (81) Google Scholar, 7Wende W. Grindl W. Christ F. Pingoud A. Pingoud V. Nucleic Acids Res. 1996; 24: 4123-4132Crossref PubMed Scopus (49) Google Scholar), I-SceI (22Perrin A. Buckle M. Dujon B. EMBO J. 1993; 12: 2939-2947Crossref PubMed Scopus (110) Google Scholar), F-SceII (23Jin Y. Binkowski G. Simon L.D. Norris D. J. Biol. Chem. 1997; 272: 7352-7359Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), I-TevI (24Mueller J.E. Smith D. Belfort M. Genes Dev. 1996; 10: 2158-2166Crossref PubMed Scopus (47) Google Scholar), and I-TevII (21Loizos N. Silva G.H. Belfort M. J. Mol. Biol. 1996; 255: 412-424Crossref PubMed Scopus (26) Google Scholar) remain bound to one of the two cleavage products that may be required for the subsequent recombination event which completes the homing reaction. The genetically engineered domain DI of PI-SceI binds specifically and with similar affinity as full-length PI-SceI to DNA containing the PI-SceI recognition site as well as to one of the two cleavage products (9Grindl W. Wende W. Pingoud V. Pingoud A. Nucleic Acids Res. 1998; 26: 1857-1862Crossref PubMed Scopus (34) Google Scholar), demonstrating that domain I is not only involved in protein splicing but also in DNA binding. In contrast, the genetically engineered domain II of PI-SceI is not able to bind DNA with strong affinity, suggesting that domain I is responsible for a decisive part of the contacts between PI-SceI and its substrate (9Grindl W. Wende W. Pingoud V. Pingoud A. Nucleic Acids Res. 1998; 26: 1857-1862Crossref PubMed Scopus (34) Google Scholar). A similar two-domain structure with a catalytic domain and a DNA binding domain has been proposed for I-TevI (25Derbyshire V. Kowalski J.C. Danserau J.T. Hauer C.R. Belfort M. J. Mol. Biol. 1997; 265: 494-506Crossref PubMed Scopus (80) Google Scholar). The long recognition site of PI-SceI makes it an attractive model for studying the mechanism of DNA sequence recognition by proteins but makes it difficult to model the DNA into the structure of PI-SceI, in particular as it is known that both domains, which may be linked in a flexible manner, are involved in DNA binding (9Grindl W. Wende W. Pingoud V. Pingoud A. Nucleic Acids Res. 1998; 26: 1857-1862Crossref PubMed Scopus (34) Google Scholar, 26He Z. Crist M. Yen H. Duan X. Quiocho F.A. Gimble F.S. J. Biol. Chem. 1998; 273: 4607-4615Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 27Pingoud V. Grindl W. Wende W. Thole H. Pingoud A. Biochemistry. 1998; 37: 8233-8243Crossref PubMed Scopus (13) Google Scholar). In the study presented here, we tried to identify residues of PI-SceI in close contact to the recognition site by a photocross-linking technique using 5-iodine-substituted pyrimidines (5-IdU and 5-IdC). We show that PI-SceI can be cross-linked via His-333 to a 5-Iododeoxyuridine (5-IdU) residue located in position +9 of the bottom strand, i.e. the right half of the PI-SceI recognition sequence. Substitution of the cross-linked amino acid His-333 by Ala results in a PI-SceI mutant that is not significantly impaired in its ability to bind and cleave DNA. However, no photocross-linking could be observed with the H333A mutant, demonstrating that the region around His-333 is in close contact with the DNA. With this information and knowing where the active site is located, it is possible to present a model that describes the approximate location of the DNA binding site in the structure of PI-SceI. His6-tagged PI-SceI was expressed in Escherichia coli and purified as described by Wendeet al. (7Wende W. Grindl W. Christ F. Pingoud A. Pingoud V. Nucleic Acids Res. 1996; 24: 4123-4132Crossref PubMed Scopus (49) Google Scholar). Oligodeoxynucleotides were chemically synthesized by automated β-cyanoethylphosphoramidite DNA synthesis using 5-IdU-β-cyanoethylphosphoramidites (Glen Research) on a Cyclone plus DNA synthesizer (Millipore) or obtained by INTERACTIVA. To reduce possible deiodination of the 5-IdU and 5-IdC, the final deprotection step was carried out at ambient temperature for 24 h, as suggested by the manufacturer. We used either the minimal full-length recognition sequence (cf. Fig. 1) or the right-half cleavage product comprising the upper strand 5′-GGAGAAAGAGGTAATGAAATGGCAGAAGTCT-3′ (31-mer) and the lower strand 5′-GATCAGACTTCTGCCATTTCATTACCTCTTTCTCCGCAC-3′ (39-mer). Some oligodeoxynucleotides were labeled at their 5′-terminus using T4 polynucleotide kinase and [γ-32P]ATP. For analytical scale photocross-linking, approximately 10 μmPI-SceI was preincubated with 10 μmradioactively labeled ds oligodeoxynucleotide monosubstituted with 5-IdU or 5-IdC at various positions of the top and bottom strand of either the full-length recognition site or the right-half cleavage product (cf. Fig. 1) in buffer P (10 mmTris/HCl, pH 8.5, 100 mm KCl, 2.5 mm EDTA) for 30 min at ambient temperature in a volume of 50 μl. Photocross-linking was carried out with a 40 milliwatt helium/cadmium laser emitting at 325 nm (Laser 2000). The total irradiation time was usually 2 h; in kinetic experiments, 0–3 h. Samples of 2.5 μl were withdrawn before and after cross-linking and analyzed on a 15% (w/v) SDS-polyacrylamide gel. Gels were silver-stained and dried, and radioactive bands were visualized by autoradiography with intensifying screens or by using an imager. For preparative isolation of the cross-linked PI-SceI/oligodeoxynucleotide complex (see below), the analytical scale was increased 10-fold. PI-SceI (20 μm) was digested under limiting conditions in 50 mm Tris/HCl, pH 8.0 with trypsin at a substrate:protease ratio of 500:1 (w/w) at ambient temperature, similar to that described recently (27Pingoud V. Grindl W. Wende W. Thole H. Pingoud A. Biochemistry. 1998; 37: 8233-8243Crossref PubMed Scopus (13) Google Scholar). After 2 h of incubation, the reaction was terminated by the addition of 5 mm phenylmethylsulfonyl fluoride. The cross-linking reaction with nicked PI-SceI (10 μm) was performed in the presence of radioactively labeled ds oligodeoxynucleotide T + 9 (10 μm, right-half cleavage product) for 2 h in buffer P. 5-μl aliquots withdrawn before and after irradiation were analyzed by electrophoresis on a 15% (w/v) SDS-polyacrylamide gel with subsequent silver-staining and autoradiography. Sequencing of the cross-linked C-terminal tryptic fragment was performed as described recently (27Pingoud V. Grindl W. Wende W. Thole H. Pingoud A. Biochemistry. 1998; 37: 8233-8243Crossref PubMed Scopus (13) Google Scholar). The cross-linked PI-SceI·oligodeoxynucleotide T + 9 (right-half cleavage product) complex was purified from unreacted PI-SceI by anion exchange chromatography on a Mono Q column (HR5/5, Amersham Pharmacia Biotech). After irradiation, the reaction mixture was incubated in the presence of 2 m urea for 5 min at 60 °C and directly applied to the column. The elution buffers used were A, 50 mm Tris/HCl, pH 8.0 and B, 50 mm Tris/HCl, pH 8.0 with 1 m NaCl. The gradient applied was 0–80% B in 40 min. The flow-rate was 1.0 ml/min. The elution was monitored by measuring the absorbance at 260 nm. Fractions of 1 ml were collected, and aliquots were analyzed by electrophoresis on a 15% (w/v) SDS-polyacrylamide gel. The purified cross-linked complex of PI-SceI with oligodeoxynucleotide T + 9 obtained by anion-exchange chromatography was concentrated and washed in a Centricon 50 tube with 50 mm Tris/HCl, pH 8.0. An aliquot (5 pmol) was radioactively labeled with [γ-32P]ATP in the presence of T4 polynucleotide kinase. Free [γ-32P]ATP was removed using a NAP5 column (Amersham Pharmacia Biotech). The radioactively labeled PI-SceI·oligodeoxynucleotide T + 9 complex was digested in 50 mm Tris/HCl, pH 8.0, 1 mm CaCl2by various proteases in a reaction volume of 50 μl. Trypsin and chymotrypsin were added to give a final concentration of 2, 5, 10, 20, 40, 80, and 200 μg/ml. Proteinase K and subtilisin were added to give a final concentration of 2 μg/ml and 20 μg/ml. The digestions were performed for 16 h at 37 °C. The reactions were terminated by precipitation with 0.1 volume of 1 m sodium acetate, pH 6.8, and 2 volumes of ethanol. The samples were dissolved in 20 μl of 6 m urea, 0.025% (w/v) bromphenol blue, and 0.025% xylene cyanole and subjected to electrophoresis on a 12% (w/v) polyacrylamide gel containing 0.5 × TBE and 2 m urea after a pre-run for 30 min with a cathode buffer containing 1 mmthioglycolic acid. The gel was dried, and radioactive bands were visualized by autoradiography. 5 nmol of PI-SceI were cross-linked with an equimolar amount of ds oligodeoxynucleotide T + 9 (right-half cleavage product) by irradiation for 2 h at 325 nm. The DNA in the reaction mixture was subsequently radioactively labeled with [γ-32P]ATP and T4 polynucleotide kinase. The buffer was adjusted to 50 mmTris/HCl, pH 8.0, 1 mm CaCl2, and 40 μg/ml chymotrypsin was added. The digestion was performed for 2 h at 37 °C. To test the progress of chymotryptic proteolysis, an aliquot of the reaction mixture was precipitated with ethanol and analyzed by electrophoresis on a 12% (w/v) polyacrylamide gel containing TBE (0.5× concentration) and 2 m urea. After the digestion was complete, the whole sample was ethanol-precipitated, redissolved in 50 μl of 50 mm Tris/HCl, pH 8.0 and 2 m urea, and applied onto a Mono Q column. For elution, the following buffers were used: Buffer A, 50 mm Tris/HCl, pH 8.0; buffer B, 50 mm Tris/HCl, pH 8.0, and 1 m NaCl. The gradient applied was 0–80% B in 40 min. The flow rate was 1 ml/min. Fractions of 1 ml were collected, ethanol-precipitated, and redissolved in 6m urea, 0.025% (w/v) bromphenol blue, and 0.025% (w/v) xylene cyanole. 50-μl aliquots of the fractions were analyzed by electrophoresis on a 12% (w/v) polyacrylamide, 0.5 × TBE, 2m urea gel. For further purification of the cross-linked peptide/oligodeoxynucleotide adduct, the peak fractions from the Mono Q column were combined and subjected to preparative electrophoresis under the same conditions as used for the electrophoretic analysis. Radioactive bands were visualized by an imager. The cross-linked peptide/oligodeoxynucleotide adduct was extracted from the gel and eluted into 0.5 ml of 10 mm ammonium bicarbonate, pH 8.8, by shaking for 2.5 h at 37 °C. This solution was lyophilized, and the cross-linked peptide/oligodeoxynucleotide adduct was redissolved in 50 μl of 10 mm ammonium bicarbonate, pH 8.8, was applied to a 4-ml column of Sephadex G25 (Amersham Pharmacia Biotech), eluted with the same buffer, and lyophilized. The recovery was 400 pmols. For sequencing, the cross-linked peptide/oligodeoxynucleotide complex was solubilized with 200 μl of H2O and centrifuged using a ProSorb cartridge (Applied Biosystems). The membrane was cut out, washed with 5% (v/v) methanol for 5 min, and dried. The peptide was sequenced on a pulsed liquid phase sequenator Model 477A (Applied Biosystems) with a 120A on-line high performance liquid chromatography system according to Tholeet al. (28Thole H.H. Maschler I. Jungblut P.W. Eur. J. Biochem. 1995; 231: 510-516Crossref PubMed Google Scholar). 50 pmols of the material were amenable to sequencing. Site-directed mutagenesis of the PI-SceI gene was performed by a polymerase chain reaction-based technique (29Ito W. Ishiguro H. Kurosawa Y. Gene. 1991; 102: 67-70Crossref PubMed Scopus (261) Google Scholar) using the primer 5′-GCTATGTTACTGATGAGGCCGGCATCAAAGCAACAATAAAG-3′ to introduce the desired mutation. The sequence of the mutated gene was confirmed by sequencing. The purification of the PI-SceI mutant H333A was carried out as described for wild type PI-SceI (7Wende W. Grindl W. Christ F. Pingoud A. Pingoud V. Nucleic Acids Res. 1996; 24: 4123-4132Crossref PubMed Scopus (49) Google Scholar). Binding and cleavage experiments were performed as described by Pingoudet al. (27Pingoud V. Grindl W. Wende W. Thole H. Pingoud A. Biochemistry. 1998; 37: 8233-8243Crossref PubMed Scopus (13) Google Scholar), and bending assays were performed as described by Wende et al. (7Wende W. Grindl W. Christ F. Pingoud A. Pingoud V. Nucleic Acids Res. 1996; 24: 4123-4132Crossref PubMed Scopus (49) Google Scholar). Photocross-linking experiments with the H333A variant and ds oligodeoxynucleotide T + 9 were carried out as described above for wild type PI-SceI. A 311-bp substrate carrying the PI-SceI cleavage site in the center and the 5-IdU modification in position +9 of the lower strand was generated by ligating two polymerase chain reaction products. The left half, a 187-bp-long DNA fragment with the cleavage site and the modification was produced with the primers 5′-GCGTCGGATCCAGGTCAAAGAGTTTTGG-3′ and 5′-AGACTTCTGCCATTTCATTACCCTCXTTCTCCGCAC-3′ (X, 5-IdU) and a 311-bp DNA fragment as template in the presence of [α-32P]dATP (7Wende W. Grindl W. Christ F. Pingoud A. Pingoud V. Nucleic Acids Res. 1996; 24: 4123-4132Crossref PubMed Scopus (49) Google Scholar). For the generation of the right half, a 124-bp DNA fragment, we used the primers 5′-GATGGAATTCCCAGAGTTATATC-3′ and 5′-GCGTCGGATCCAAGCTTCTCTGGCTGC-3′ and the unmodified 311-bp substrate as template, again in the presence of [α-32P]dATP. Both DNA fragments were annealed with the 311-bp DNA by incubation at 95 °C for 10 min and, after cooling to 37 °C, ligated with T4 DNA ligase (AGS). The 32P-labeled ligation product was purified by electrophoresis on a 10% (w/v) polyacrylamide gel. The 311-bp substrate modified in position T + 9 of the recognition site was incubated with 50 nmPI-SceI and irradiated for 1.5 h at 325 nm as described above. Analytical gel shift experiments were performed in the absence and presence of the 56-bp competitor oligodeoxynucleotide F (7Wende W. Grindl W. Christ F. Pingoud A. Pingoud V. Nucleic Acids Res. 1996; 24: 4123-4132Crossref PubMed Scopus (49) Google Scholar). Preparative gel shift experiments of the PI-SceI/311-bp T + 9 complex were carried out before and after photocross-linking. Bands corresponding to the upper and lower complex were excised, eluted in 50 mm Tris/HCl, pH 8.0, precipitated, and analyzed on a 6% (w/v) polyacrylamide gel containing 1% (w/v) SDS. PI-SceI was dialyzed against a buffer consisting of 30 mm inorganic sodium phosphate, pH 6.4, 150 mm NaCl, and 20 mmdithiothreitol. PI-SceI (6 μm) in the absence and presence of equimolar amounts of a 62-bp substrate (oligodeoxynucleotide G (7Wende W. Grindl W. Christ F. Pingoud A. Pingoud V. Nucleic Acids Res. 1996; 24: 4123-4132Crossref PubMed Scopus (49) Google Scholar)) was treated with DEPC at final concentrations of 0.25, 0.5, 1, 2.5, 5, and 10 mm for 30 min at ambient temperature. To remove the ethoxyformyl residue from the histidine residues, an aliquot of the DEPC-treated mixture was acidified by NaH2PO4 to reach a pH value of 6.25. Hydroxylamine was added to a final concentration of 250 mm, and the reaction mixture was incubated for 16 h at 4 °C. Circular dichroism spectra of 17 μmPI-SceI before and after incubation with DEPC (3 mm) were measured in a buffer consisting of 30 mm inorganic sodium phosphate, pH 6.4, and 150 mm NaCl on a JASCO J-710 spectrophotometer at ambient temperature. For electrophoretic mobility shift assays, PI-SceI was mixed with 10,000 cpm of32P-labeled 311-mer polymerase chain reaction product containing the PI-SceI recognition sequence in a total volume of 10 μl of binding buffer (10 mm Tris/HCl, pH 7.5, 50 mm NaCl, 1 mm EDTA, 0.05% (w/v) nonfat dry milk, 5% (v/v) glycerol, 10 mm dithiothreitol, 0.1 μg of poly(dI-dC)) (27Pingoud V. Grindl W. Wende W. Thole H. Pingoud A. Biochemistry. 1998; 37: 8233-8243Crossref PubMed Scopus (13) Google Scholar). After electrophoresis, the gels were dried, and bands were visualized and quantified in an imager. To obtain detailed topological information about specific contacts between PI-SceI and its DNA substrate, we performed photocross-linking experiments using either 5-IdU or 5-IdC monosubstituted ds oligodeoxynucleotides comprising the recognition sequence for PI-SceI (Fig. 1). Photocross-linking of halogenated pyrimidines has been used successfully to identify contacts in DNA- and RNA-protein complexes (30Blatter E.E. Ebright Y.W. Ebright R.H. Nature. 1992; 359: 650-652Crossref PubMed Scopus (71) Google Scholar, 31Meisenheimer K.M. Meisenheimer P.L. Willis M.C. Koch T.H. Nucleic Acids Res. 1996; 24: 981-982Crossref PubMed Scopus (34) Google Scholar, 32Wang Y. Adzuma K. Biochemistry. 1996; 35: 3563-3571Crossref PubMed Scopus (48) Google Scholar, 33Malkov V.A. Biswas I. Camerini-Otero R.D. Hsieh P. J. Biol. Chem. 1997; 272: 23811-23817Crossref PubMed Scopus (91) Google Scholar, 34Jenkins T.M. Esposito D. Engelman A. Craigie R. EMBO J. 1997; 16: 6849-6859Crossref PubMed Scopus (214) Google Scholar). 5-IdU is an almost perfect analogue of thymidine (35Willis M.C. Hicke B.J. Uhlenbeck O.C. Cech T.R. Koch T.H. Science. 1993; 262: 1255-1257Crossref PubMed Scopus (174) Google Scholar) and particularly useful for the study of DNA-binding proteins using oligodeoxynucleotides with a T → 5-IdU substitution in defined positions. It is a photoactivated zero-length cross-linker that has the advantage that cross-linking to regions of a protein not involved in DNA binding is minimized and that irradiation with long wavelength UV light (325 nm) does not lead to the excitation of other nucleic acid and protein chromophores. Mechanistic studies of the 5-IdU chromophore relevant to its use in nucleoprotein photocross-linking have been performed by Norris et al. (36Norris C.L. Meisenheimer P.L. Koch T.H. J. Am. Chem. Soc. 1996; 118: 5796-5803Crossref Scopus (46) Google Scholar). The site-specific 5-IdU- or 5-IdC-mediated photocross-linking method does not depend on information regarding the structure of the protein or the structure of the protein·DNA complex. Efficient cross-linking requires the close proximity of the modified base and a reactive amino acid. Preferential targets are Phe, Tyr, Trp, His, and Met residues (35Willis M.C. Hicke B.J. Uhlenbeck O.C. Cech T.R. Koch T.H. Science. 1993; 262: 1255-1257Crossref PubMed Scopus (174) Google Scholar, 37Wong D.L. Pavlovich J.G. Reich N.O. Nucleic Acids Res. 1998; 26: 645-649Crossref PubMed Scopus (26) Google Scholar). In addition, the cross-link yield depends on a suitable orientation of the reacting groups. With aromatic amino acid side chains as acceptor, the yield of the cross-linking reaction is significantly enhanced when a π-π stacking interaction between the base analog and an aromatic amino acid residue is possible (36Norris C.L. Meisenheimer P.L. Koch T.H. J. Am. Chem. Soc. 1996; 118: 5796-5803Crossref Scopus (46) Google Scholar). We used synthetic oligodeoxynucleotides comprising either the minimal full-length recognition sequence of 36 bp (Fig. 1) or containing the right-half cleavage product of the PI-SceI recognition sequence required for specific binding by PI-SceI (7Wende W. Grindl W. Christ F. Pingoud A. Pingoud V. Nucleic Acids Res. 1996; 24: 4123-4132Crossref PubMed Scopus (49) Google Scholar). These oligonucleotides were substituted with a single 5-IdU moiety that substituted for T, A, or G, or with 5-IdC, which substituted for C, as shown in Fig. 1. It was important to ensure that the presence of such a substitution in the PI-SceI recognition sequence does not interfere with the binding to PI-SceI. We therefore compared the binding of PI-SceI to modified and unmodified DNA in gel shift experiments and found that they are bound by PI-SceI with the same apparent K D of 5–10 nm as the wild type sequence (data not shown). For analytical cross-linking experiments designed to find out which position produces the best cross-link yield, PI-SceI was incubated with the different mono-substituted ds oligodeoxynucleotides for 30 min at ambient temperature at a PI-SceI to DNA ratio of 1:1. The photocross-linking reactions were carrried out by irradiation at 325 nm with a helium/cadmium laser for 2 h at ambient temperature. Among the various positions modified by 5-IdU or 5-IdC in the recognition site, only three positions gave rise to a substantial amount of cross-linked PI-SceI (Fig. 1). Oligodeoxynucleotides with thymine in position +9 of the bottom strand and, to a somewhat smaller extent, guanine in position +4 and adenine in position +5 of the upper strand when substituted by 5-IdU, were efficiently cross-linked to PI-SceI. The absence of s" @default.
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• W2153298725 title "Photocross-linking of the Homing Endonuclease PI-SceI to Its Recognition Sequence" @default.
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