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• W2077146218 abstract "Despite its small size (27.6 kDa), the group I intron-encoded I-SceI endonuclease initiates intron homing by recognizing and specifically cleaving a large intronless DNA sequence. Here, we used gel shift assays and footprinting experiments to analyze the interaction between I-SceI and its target. I-SceI was found to bind to its substrate in monomeric form. Footprinting using DNase I, hydroxyl radical, phenanthroline copper complexes, UV/DH-MePyPs photosensitizer, and base-modifying reagents revealed the asymmetric nature of the interaction and provided a first glimpse into the architecture of the complex. The protein interacts in the minor and major grooves and distorts DNA at three distinct sites: one at the intron insertion site and the other two, respectively, downstream (−8, −9) and upstream (+9, +10) from this site. The protein appears to stabilize the DNA curved around it by bridging the minor groove on one face of the helix. The scissile phosphates would lie on the outside of the bend, facing in the same direction relative to the DNA helical axis, as expected for an endonuclease that generates 3′ overhangs. An internally consistent model is proposed in which the protein would take advantage of the concerted flexibility of the DNA sequence to induce a synergistic binding/kinking process, resulting in the correct positioning of the enzyme active site. Despite its small size (27.6 kDa), the group I intron-encoded I-SceI endonuclease initiates intron homing by recognizing and specifically cleaving a large intronless DNA sequence. Here, we used gel shift assays and footprinting experiments to analyze the interaction between I-SceI and its target. I-SceI was found to bind to its substrate in monomeric form. Footprinting using DNase I, hydroxyl radical, phenanthroline copper complexes, UV/DH-MePyPs photosensitizer, and base-modifying reagents revealed the asymmetric nature of the interaction and provided a first glimpse into the architecture of the complex. The protein interacts in the minor and major grooves and distorts DNA at three distinct sites: one at the intron insertion site and the other two, respectively, downstream (−8, −9) and upstream (+9, +10) from this site. The protein appears to stabilize the DNA curved around it by bridging the minor groove on one face of the helix. The scissile phosphates would lie on the outside of the bend, facing in the same direction relative to the DNA helical axis, as expected for an endonuclease that generates 3′ overhangs. An internally consistent model is proposed in which the protein would take advantage of the concerted flexibility of the DNA sequence to induce a synergistic binding/kinking process, resulting in the correct positioning of the enzyme active site. base pair(s) diethyl pyrocarbonate dimethyl sulfate I-SceI is a homing endonuclease encoded by the mobile group I intron of the large rRNA gene of Saccharomyces cerevisiae (1Dujon B. Gottarel G. Colleaux L. Betermier M. Jacquier A. d'Auriol L. Gallibert F. Quagliariello E. Slater E.C. Palnieri F. Saccone C. Kroon A.M. Achievement and Perspective of Mitochondrial Research II. Elsevier Sciences, Amsterdam1985: 215-225Google Scholar, 2Colleaux L. D'Auriol L. Betermier M. Cottarel G. Jacquier A. Galibert F. Dujon B. Cell. 1986; 44: 521-533Abstract Full Text PDF PubMed Scopus (199) Google Scholar). This family of enzymes mediates the propagation of the intron by cutting intronless genes at the site of intron insertion (reviewed in Ref. 3Mueller J.E. Bryk M. Loizos N. Belfort M. Linn S.M. Lloyd R.S. Roberts R.J. Nucleases. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 111-143Google Scholar). Like restriction enzymes, homing endonucleases cleave double-stranded DNA with high specificity in the presence of divalent metal ions. However, they differ from restriction endonucleases in their recognition properties and structures, as well as in their genomic location (4Belfort M. Roberts R. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (393) Google Scholar). In particular, whereas restriction enzymes have short recognition sequences (3–8 bp),1 homing endonucleases, despite their small size, recognize long DNA sequences (12–40 bp). They have been classified into four families on the basis of both their sequence motifs and DNA cleavage mechanism (3Mueller J.E. Bryk M. Loizos N. Belfort M. Linn S.M. Lloyd R.S. Roberts R.J. Nucleases. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 111-143Google Scholar). The protein I-SceI is a member of the largest class of homing enzymes (more than 130 proteins), characterized by the presence of either one or two conserved 12 amino acid residue sequence motifs (LAGLI-DADG motifs). Most of these proteins, like I-SceI, carry the motif in duplicate and are endonucleases. I-SceI has been purified as a monomeric globular protein of 235 amino acids (5Montheilet C. Perrin A. Thierry A. Colleaux L. Dujon B. Nucleic Acids Res. 1990; 18: 1407-1413Crossref PubMed Scopus (136) Google Scholar). Its endonuclease activity requires Mg2+ or Mn2+ but not Co2+, Ca2+, Cu2+, or Zn2+ to cleave DNA within its recognition sequence and leaves a 4-bp overhang presenting a 3′-hydroxyl terminus (5Montheilet C. Perrin A. Thierry A. Colleaux L. Dujon B. Nucleic Acids Res. 1990; 18: 1407-1413Crossref PubMed Scopus (136) Google Scholar, 6Colleaux L. D'Auriol L. Galibert F. Dujon B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6022-6026Crossref PubMed Scopus (225) Google Scholar). The enzyme displays a low turnover, probably because of its strong affinity for one of the products of the cleavage reaction (7Perrin A. Buckle M. Dujon B. EMBO J. 1993; 12: 2939-2947Crossref PubMed Scopus (110) Google Scholar).The interaction of homing endonucleases with their substrates raises an interesting question common to all the gene-regulatory proteins, namely: how can a small protein specifically recognize and modify a long DNA sequence? Understanding the molecular basis of such a mechanism is essential for elucidating many aspects of cellular control and is a prerequisite in any rational drug design program. It is clearly established that local and global DNA structural features that are highly sequence-dependent, play a primary role in the dynamics of protein-DNA recognition (8Steitz T.A. Quart. Rev. Biophys. 1990; 23: 205-280Crossref PubMed Scopus (460) Google Scholar). Sequence recognition would arise from the inherent sequence-dependent ability to adopt the conformation required for protein binding in the transient, biologically active complex. In an attempt to reveal intrinsic helical properties of the I-SceI nucleic acid target, we used this sequence as substrate in earlier studies analyzing the mechanisms of DNA chemical reactions and photosensitization processes (9Schaeffer F. Rimsky S. Spassky A. J. Mol. Biol. 1996; 260: 523-539Crossref PubMed Scopus (30) Google Scholar, 10Andreu Guillo L. Blais J. Vigny P. Spassky A. Photochem. Photobiol. 1995; 61: 331-335Crossref PubMed Scopus (6) Google Scholar, 11Andreu Guillo L. Beylot B. Vigny P. Spassky A. Photochem. Photobiol. 1996; 64: 349-355Crossref PubMed Scopus (18) Google Scholar,12Spassky A. Angelov D. Biochemistry. 1997; 36: 6571-6576Crossref PubMed Scopus (101) Google Scholar). We found evidence that the conformation of the helix deviates from the ideal B-form duplex along two segments of three and five base pairs located at a distance of approximately one helical turn, respectively, upstream and downtream from the site of junction of the two exons (9Schaeffer F. Rimsky S. Spassky A. J. Mol. Biol. 1996; 260: 523-539Crossref PubMed Scopus (30) Google Scholar). In the present study, we first performed DNase I footprinting and gel retardation assays to identify the complex formed between I-SceI and its target in the absence of a divalent metal ion. We then used chemical probing agents to characterize the conformation of DNA in the complex. I-SceI protein-DNA complex was thus submitted to the nucleolytic attack of the cuprous complexes of 1,10-phenanthroline (OP2Cu+ and Phe-OP2Cu+), which results from the abstraction of C-1′ hydrogen atom by a tetrahedral copper-oxo species bound within the minor groove (13Sigman D.S. Spassky A. Eckstein F. Lilley D.M.J. Nucleic Acids Molecular Biology. 3. Springer-Verlag, Berlin and Heidelberg1989: 13-27Google Scholar, 14Sigman D.S. Biochemistry. 1990; 29: 9098-9105Crossref Scopus (293) Google Scholar, 15Yoon C. Kuwabara M.D. Spassky A. Sigman D.S. Biochemistry. 1990; 29: 2116-2121Crossref PubMed Scopus (31) Google Scholar, 9Schaeffer F. Rimsky S. Spassky A. J. Mol. Biol. 1996; 260: 523-539Crossref PubMed Scopus (30) Google Scholar) and to UVA/4′,5′-dihydro-7-methylpyrido [3,4-c]psoralen (DHMePyPs) photosensitization, which requires prior intercalation of the pyridopsoralen at selective 5′-TTA-3′ sites (10Andreu Guillo L. Blais J. Vigny P. Spassky A. Photochem. Photobiol. 1995; 61: 331-335Crossref PubMed Scopus (6) Google Scholar). We also employed chemical modification agents of base and sugar residues (for a review, see Ref. 16Nielsen P.E. J. Mol. Recogn. 1990; 3: 1-25Crossref PubMed Scopus (131) Google Scholar), diethyl pyrocarbonate (DEPC), which carboxylates purines at the N-7 atom, potassium permanganate (KMnO4), which oxidizes pyrimidine residues at the C5=C6double bond, dimethyl sulfate (DMS), which primarily methylates the N-7 of guanine residues and free hydroxyl radical, generated by Fe-EDTA reduction of hydrogen peroxide, which abstracts C-4′ hydrogen atoms from deoxyriboses of the DNA backbone. In the scheme that arises from present experiments, I-SceI appears to stabilize, in monomeric form, a constrained helical structure in which the minor groove is widened at the cleavage sites. The results are discussed in relation to previous reports on other endonucleases of the same family.EXPERIMENTAL PROCEDURESI-SceI Protein and DNA SubstratesI-SceI was purchased from Roche Molecular Biochemicals, aliquoted at 10 units/μl in phosphate buffer in the presence of 200 μg/ml bovine serum albumin, and conserved in 50% glycerol at −20 °C. Protein concentration was determined from the optical density of the bands on a Phast System minigel (12.5% acrylamide/5.5% SDS), using bovine serum albumin as internal standard (not shown). The solution used in the present study had a concentration of 0.72 × 10−12 mole per enzymatic unit.The 98-bp EcoRI-HindIII DNA fragment including the I-SceI recognition sequence (sequence shown in Fig.1A) was excised from its pUC19 vector supplied by B. Dujon (6Colleaux L. D'Auriol L. Galibert F. Dujon B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6022-6026Crossref PubMed Scopus (225) Google Scholar) and was purified by electrophoresis on a 15% preparative native polyacrylamide gel as described (50Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1123-1128Google Scholar). Concentration was measured by UV absorbance. The fragment was stored in 10 mm Tris, pH 7.5, 1 mm EDTA (TE). For only 5′ end-labeling, pUC19 plasmid vector was first digested with either the restriction enzymeEcoRI or HindIII, dephosphorylated with calf intestine alkaline phosphatase, 5′ end-labeled with T4 polynucleotide kinase in the presence of [γ-32P]ATP, and then digested with the second restriction enzyme before purification by 20% native polyacrylamide gel electrophoresis.Synthetic oligonucleotides used to form the 54- and 37-bp fragments (Fig. 1, B and C), were purchased from Genset (France). Purification, labeling, and annealing were carried out as previously described (12Spassky A. Angelov D. Biochemistry. 1997; 36: 6571-6576Crossref PubMed Scopus (101) Google Scholar).Gel Shift Analysis of I-SceI/DNA InteractionsThe conditions were derived from those described previously (17Fried M. Crothers D.M. Nucleic Acids Res. 1981; 9: 6505-6525Crossref PubMed Scopus (1683) Google Scholar,18Garner M.M. Revzin A. Nucleic Acids Res. 1981; 9: 3037-3060Crossref Scopus (1205) Google Scholar). I-SceI (10−9 to 10−7m) and 5′ 32P-labeled DNA (either 98, 54, or 37 bp DNA fragments) (10−10 to 10−8m) were preincubated separately for 2 min at 4 °C in the binding buffer (final concentration: 10 mm Tris-HCl, pH 8, 10 mm NaCl, 2.5 mm dithiothreitol and 20 μg/ml bovine serum albumin) and then mixed and incubated for 10 min at 20 °C. A 0.1 volume of loading buffer (50% glycerol/0.02% xylene cyanol) was added. 8–12% polyacrylamide gels (29:1 acrylamide/bisacrylamide) were prerun in 1× TBE (9 mm Tris-HCl, 8.8 mm boric acid, and 2 mm EDTA) for 2 h at 120 V. Samples were loaded at 50 V, and the gels were run at 120 V at 4 °C. Gels were dried and placed in phosphorimager cassettes. Screens were exposed for several hours and scanned using a Molecular Dynamics PhosphorImager with Image Quant software. The fraction of bound (free) DNA in each lane was calculated by dividing the area of bound (free) bands by the total area of bound and free bands. Each binding assay was performed in triplicate.Quantification of the Apparent Equilibrium Dissociation ConstantThe apparent equilibrium dissociation constant was derived from the Scatchard plot of the binding data in which the ratio of bound to free DNA concentration was plotted against bound DNA concentration using the Kaleidograph (Synergy software). The reciprocal of the negative slope of the linear plot gives the value of the apparentK d .DNA-Protein StoichiometryThe 5′ end-labeled DNA fragment (Fig. 1B,54bp) (8 × 10−10m) and I-SceI protein (0.01 units/μl) were incubated as described above, before loading onto a set of 8, 10, 12, and 15% polyacrylamide gels, alongside 10 μg of nondenatured protein molecular size standards (Sigma). Gels were stained with Coomassie Blue, destained, dried, and exposed to x-ray film. The relative mobility of each species including free DNA fragment (Rf) was calculated by dividing the distance of the corresponding band by that of the bromphenol blue tracking dye in the same lane. For each species, the plot of 100[log (100Rf)] against gel concentration was constructed. The negative slope or retardation coefficient (−K r) was then plotted as a function of the molecular mass for each protein standard, and this calibration line was used to determine the apparent molecular mass of the free DNA fragment and that of the protein I-SceI/DNA complex. The difference between these two values divided by the molecular mass of a protein monomer gives the number of protein monomers bound to the DNA (n).Complex Probing Using DNA Cleavage ReagentsUniquely 5′ end-labeled DNA fragments (Fig. 1, A,B, or C) were digested directly or after incubation with the I-SceI protein as described above. In each case, digestion was carried out under conditions such that the DNA molecule was broken only once.DNase I FootprintingDNase I footprinting was done essentially as previously described (51Spassky A. Busby S. Buc H. EMBO J. 1984; 3: 43-50Crossref PubMed Scopus (87) Google Scholar). Digestion was carried out at 23 °C using DNase I at a final concentration of 0.025 μg/ml for 15 or 45 s, depending on whether the DNA was free or I-SceI-bound.Hydroxyl Radical FootprintingA stock solution of iron(II)-EDTA was prepared immediately before use by mixing equal volumes of freshly prepared 0.4 mm(NH4)2Fe(SO4)2,6H2O and 0.8 mm EDTA. The footprinting reaction was initiated by placing iron(II)-EDTA solution (3 μl), 0.6% hydrogen peroxide (3 μl), and 20 mm sodium ascorbate (3 μl) on the inner wall of the 1.5-ml Eppendorf tube containing 21 μl of free or I-SceI-bound DNA, allowing the reagents to mix and then adding the cutting reagent to the sample solution. The reaction was allowed to run for 30 s and quenched by adding 3 μl of 1m thiourea.Phenanthroline Copper Complex FootprintingOrthophenanthroline-cuprous complex (OP2Cu+ or 5 Phe OP2Cu+) footprinting was carried out as previously described (29Spassky A. Sigman D.S. Biochemistry. 1985; 24: 8050-8056Crossref PubMed Scopus (169) Google Scholar, 30Thederahn T. Kuwabara M.D. Spassky A. Sigman D.S. Biochem. Biophys. Res. Commun. 1990; 168: 756-762Crossref PubMed Scopus (61) Google Scholar). One μl of a solution freshly prepared by diluting an ethanolic solution of 1,10-phenanthroline (1 mm) and an aqueous cupric sulfate solution (0.23 mm) was added to 10 μl of the appropriate free or I-SceI-bound DNA sample. Cleavage was initiated by the addition of 1 μl of 58 mm MPA (final concentration, 5.8 mm), and the mixture was incubated at 23 °C for either 30 s for OP2Cu+ or 2 min for 5 Phe OP2Cu+. Cleavage was quenched by the addition of 1 μl of 28 mm 2,9-dimethyl-orthophenanthroline (final concentration, 2.8 mm).Analysis of Cleaved FragmentsSpecific quenching of the footprinting reagent was followed by addition of a general stop solution to a final concentration of 1 mm EDTA, 0.3m sodium acetate, and 10 μg/ml tRNA. After phenol extraction, samples were ethanol-precipitated and lyophilized. The dried samples were resuspended in 10 μl of gel loading buffer and analyzed by denaturing gel electrophoresis in 15% (w/v) polyacrylamide containing 7 m urea. After electrophoresis, gels were dried on Whatman 3MM paper and exposed to x-ray film (X-OMAT) for documentation or to storage out of phosphor screens for quantification.Complex Probing Using UVA DHMePyPs PhotosensitizationUVA (365 nm) irradiation in the presence of DHMePyPs of the I-SceI protein-bound DNA fragment (98 bp) was performed exactly as previously described for free DNA (10Andreu Guillo L. Blais J. Vigny P. Spassky A. Photochem. Photobiol. 1995; 61: 331-335Crossref PubMed Scopus (6) Google Scholar). One μl of an ethanolic psoralen solution (10−5m) was added to 19 μl of the appropriate labeled DNA sample in binding buffer. After 10 min of incubation at room temperature in the dark, sample-containing droplets were irradiated on ice at 365 nm using an HPW 125 Philips mercury lamp at a fluence of 25 J/m2/s, as determined by a VLX 365 radiometer. After irradiation, psoralen and protein were extracted with chloroform/isoamyl alcohol/phenol followed by G50-Sephadex column chromatography. The DNA was then ethanol-precipitated and treated as above.Complex Probing Using Nucleobase Modifications (52McCarthy J.G. Williams L.D. Rich A. Biochemistry. 1990; 29: 6071-6081Crossref PubMed Scopus (56) Google Scholar)Whereas quite unreactive toward double-stranded adenine and guanine residues, diethyl pyrocarbonate can carbethoxylate the N-7 atom of purines of distorted structures, with a strong preference for adenines, thus destabilizing the imidazole ring and creating a piperidine-sensitive site. Similarly, because potassium permanganate oxidizes the C5=C6 double bond of pyrimidines (T≫C) from above or below the plane of the base, these residues are susceptible to attack by KMnO4 only if the stacking interaction is disrupted.Thymidines Using Potassium Permanganate (KMnO4)One microliter of freshly prepared 0.1m KMnO4 was added to 5 μl of the appropriate labeled DNA sample. The reaction was stopped after 4 min at 23 °C by addition of 2 μl of β-mercaptoethanol. After phenol extraction, samples were ethanol-precipitated, washed and dried.Adenines Using Diethyl Pyrocarbonate (DEPC)One microliter of freshly prepared 3% DEPC was added to 10 μl of the appropriate labeled DNA sample. The reaction mixture was incubated for various time (30 s; 2.5 and 10 min.) at 30 °C and then stopped by addition of 10 μl of 50 mm imidazole. After phenol extraction, samples were ethanol-precipitated, washed, and dried.Guanines Using DimethylsulfateThe DNA fragment (Fig. 1), uniquely 5′ 32P-end-labeled on the top or on the bottom strand, was methylated either directly or after incubation with I-SceI protein (see above), by adding dimethyl sulfate directly to the reaction mixture. The concentration of methylating agent, reaction temperature, and incubation time were determined so as to obtain in each case 1 N-7-MeG lesion per strand.Processing of Modified DNAThe pellets of modified DNA samples were resuspended in 100 μl of 1 m piperidine at 95 °C for 30 min. Piperidine was removed by extensive lyophilization. The dried samples were resuspended in 10 μl of gel loading buffer (as described in ref. 50Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1123-1128Google Scholar) and analyzed by denaturing gel electrophoresis in 15% (w/v) polyacrylamide containing 7 murea. After electrophoresis, gels were dried on Whatman 3MM paper and exposed to x-ray film (X-OMAT) for documentation or to storage out of phosphor screens for quantification.Quantification of ResultsThe autoradiograms were scanned by using a PhosphorImager and Image Quant software (Molecular Dynamics). Measurements and normalization were carried out exactly as previously described (12Spassky A. Angelov D. Biochemistry. 1997; 36: 6571-6576Crossref PubMed Scopus (101) Google Scholar).DISCUSSIONIn the cellular context, the endonuclease I-SceI discriminates its target site among ∼107 bp (40Thierry A. Perrin A. Boyer J. Fairhead C. Dujon B. Frey B. Schmidt G. Nucleic Acids Res. 1991; 19: 189-190Crossref PubMed Scopus (43) Google Scholar). Present results establish that, as with other proteins of the same family, the absence of divalent metal ions eliminates cleavage but not sequence-specific DNA binding. Consistent with the general tendency of the two-motif LAGLIDADG homing endonucleases (4Belfort M. Roberts R. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (393) Google Scholar), I-SceI was found to bind to its substrate in monomeric form. Furthermore, the difference in the length of the downstream and upstream exonic sequences involved in protein-DNA binding as well as the position of the footprinting protection and cleavage maxima clearly reveal that the downstream part of the recognition sequence is primarily involved in I-SceI binding. Experimental results provide also the evidence that I-SceI binding is accompanied by DNA distortion. In the model of the complex that arises from the present findings (summarized in Fig. 10 and 11), the binding of the protein appears to distort its bound substrate to widen the minor groove at the cleavage site and make the scissile phosphates accessible to the enzyme active site. A sequential binding-kinking model is suggested in which the first step of the protein binding would be facilitated by the helical features of the sequence located at one helical turn downstream from the intron insertion site. The high tendency of this region to unwind is apparent from the OP2Cu+hypersensitivity at positions 10 to 15 and from the strikingly high sensitivity of position G7 to DMS methylation (Fig. 8), observed to characterize guanines positioned in open DNA regions (41). This intrinsic unwinding must facilitate protein binding deep in the minor groove resulting in kinking the double helix at the base step A9A10, 5′ to the protein side chain minor groove intercalation (42Werner M.H. Gronenborn A.M. Clore M.G. Science. 1996; 271: 778-784Crossref PubMed Scopus (254) Google Scholar) and easily propagating helical distortion. The induced helical distortion would position the sugar-phosphate backbone of residues 2 and 5 on the top strand and −1 to +3 on the bottom strand in register to be contacted by the protein from the outside of the minor groove. This would result in the induction of a new constraint that deforms the helical area encompassing the cleavage sites. Note that the experiments using the variant substrate with the substitution G/C to A/T at +7 support this direct relationship. The protein would therefore be positioned closer to the first steps of the next minor groove opening on the same side of the helix, i.e. positions −5, −6 on the top strand and −7 on the bottom strand giving rise in turn to an upstream distortion identified by the Phe-OPCu hypersensitivity at positions −8, −9, and the unstacking of the bases A−6 and T−7. Thus, by bridging the minor groove opening on the same face of the helix, the protein would induce DNA to curve around it, the major groove being directly accessible to the binding surfaces of the protein on either side of the center of the homing site. The protein would thus stabilize the natural tendency of the helix to bend, predicted by a theoretical calculation using the program proposed by De Santiset al. (43De Santis P. Palleschi A. Savino M. Scipion A. Biochemistry. 1990; 29: 9269-9273Crossref PubMed Scopus (120) Google Scholar) according to which the DNA primary sequence would induce a global curvature to the helix with a maximum distortion angle at the step A4G5. 2F. Schaeffer, personal communication. Figure 10Summary of the chemical probing data.Upper, helical accessibility and protection from the inside and the outside of the minor groove. Between the sequences of the two DNA strands: filled and open horizontal rectangles identify, respectively, OP2Cu+hyperreactive and protected areas; the filled vertical rectangle at the intron insertion site represents the intercalated pyridopsoralen molecule. The filled vertical rectanglecrossing two filled horizontal rectangles represent the two orthogonal phenanthroline planes (OP) fitting the geometry of the minor groove from positions 1 to 4: one OP is deeply intercalated between base pairs and the other OP is close to the wall of the minor groove of one or the other strand (Schaeffer et al., Ref. 9Schaeffer F. Rimsky S. Spassky A. J. Mol. Biol. 1996; 260: 523-539Crossref PubMed Scopus (30) Google Scholar).Arrows indicate Phe OP2Cu+ hypersensitive sites. On each side of the strands, the first lane indicates hydroxyl radical protection (open rectangles) and enhancement (arrows), and the second lanerepresents the influence of protein binding on the DNase I cleavage frequency (same symbols as in Fig. 2). Lower, base residue modification and interference. The adenines and thymines reactive, respectively, to DEPC and KMnO4 are indicated byblack-filled squares (strongly reactive), gray-filled squares (moderately reactive), and open squares (weakly reactive). The guanines protected against DMS methylation are indicated by black-filled circles (strongly protected),gray-filled circles (moderately protected), and open circles (weakly protected). Black arrows indicate the methylated guanines responsible for strong binding interference.View Large Image Figure ViewerDownload (PPT)Figure 11Superimposition of the chemical probing data on the helical representation of the I-SceI DNA homing site (from −13 to +17).Blue ribbon areasindicate the regions of the backbone (green ribbon) protected from hydroxyl radical attack, and blue dashesindicate the minor groove area protected from OP2Cu+ cleavage.Red rectanglesshow the adenines and thymines that strongly (filled rectangles), moderately (hatched rectangles), or weakly (blank rectangles) react with DEPC and KMnO4, respectively. Red dashesindicate the binding domain of the tetrahedral coordination complex OP2Cu+; red arrowsshow the phosphodiester bonds hypersensitive to OP2Cu+attack; and red asterisksthe phosphodiester bonds hypersensitive to hydroxyl radical attack. Black arrowsshow the phosphodiester bonds hypersensitive to Phe OP2Cu+ attack. The filled vertical rectangleidentifies the highly favored pyridopsoralen intercalation base step. ISindicates the intron insertion site. Bases are numbered from −1 and +1 extending, respectively, upstream (on the left) and downstream (on theright) from this site. The model was constructed using the program Insight II (Molecular Simulations, version 98.0).View Large Image Figure ViewerDownload (PPT)The stabilization of a distorted DNA double helix appears to be a common requirement for the homing endonucleases. From recent reports of the high-resolution crystal structures of PI-SceI (44Duan X. Gimble F.S. Quiocho F.A. Cell. 1997; 89: 555-564Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), I-DmoI (45Silva G.H. Dalgaard J.Z. Belfort M. Van Roey P. J. Mol. Biol. 1999; 286: 1123-1136Crossref PubMed Scopus (86) Google Scholar), and I-CreI (46Heath P.J. Stephens K.M. Monnat R.J. Stoddard B.L. Nat. Struct. Biol. 1997; 4: 468-476Crossref PubMed Scopus (116) Google Scholar) and of the proteins I-CreI or I-PpoI complexed to their DNA target (47Jurica M. Monnat R. Stoddard B. Mol. Cell. 1998; 2: 469-476Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 48Flick K.E. Jurica M.S. Monnat R.J. Stoddard B.L.,. Nature. 1998; 394: 96-101Crossref PubMed Scopus (190) Google Scholar), it appears that these relatively small homing endonucleases utilize the same principle to recognize and cleave their long DNA targets. They form extended folds that allow them to form long interfaces across lengthy DNA homing sites and display preformed binding motifs, consisting of antiparallel β-ribbons, making extended contacts with the DNA. However, the different conserved motifs that characterize each family give each of them a specific interface structure. In particular, the proteins of the LAGLIDADG family share a domain fold characterized by the topology αββαββα. This gives rise to an unusual β-ribbon helical interface whose architecture displays an extensive curvature complementary to the DNA major groove in the cleavage sites region and further facilitates the recognition of extended DNA sequences (49Philipps S.E.V. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 671-701Crossref PubMed Scopus (47) Google Scholar). Nevertheless, the proteins of the LAGLIDADG family differ greatly in the relative shap" @default.
• W2077146218 created "2016-06-24" @default.
• W2077146218 creator A5005907388 @default.
• W2077146218 creator A5080989699 @default.
• W2077146218 date "2001-01-01" @default.
• W2077146218 modified "2023-10-14" @default.
• W2077146218 title "Chemical Probing Shows That the Intron-encoded Endonuclease I-SceI Distorts DNA through Binding in Monomeric Form to Its Homing Site" @default.
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