SMART MODE:

NORMAL GENOMIC

• Ossipov D, Pradeepkumar PI, Holmer M, Chattopadhyaya J
• Synthesis of [Ru(phen)(2)dppz](2+)-tethered oligo-DNA and studies on the metallointercalation mode into the DNA duplex.
• J Am Chem Soc. 2001; 123: 3551-62
• Display abstract

• To explore the binding properties of [Ru(phen)(2)dppz](2+) complex (phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine) in a sequence-specific manner in DNA duplex, it was tethered through the dppz ligand to a central position as well as both at the 3'- and 5'-ends of oligodeoxyribonucleotide (ODN). The middle [Ru(phen)(2)dppz](2+)-ODN tethered was resolved and isolated as four pure diastereomers, while the 3'- or 5'-[Ru(phen)(2)dppz](2+)-ODNs were inseparable on RP-HPLC. Thermal stability of the (Ru(2+)-ODN).DNA duplexes is found to increase considerably (DeltaT(m) = 12.8-23.4 degrees C), depending upon the site of the covalent attachment of the tethered [Ru(phen)(2)dppz](2+) complex, or the chirality of the [Ru(phen)(2)dppz](2+)-linker tethered at the middle of the ODN, compared to the unlabeled counterpart. Gross differences in CD between the [Ru(phen)(2)dppz](2+)-tethered and the native DNA duplexes showed that the global duplex conformation of the former has considerably altered from the B-type, but is still recognized by DNase I. The thermal melting studies, CD measurements, as well as DNase I digestion data, are interpreted as a result of intercalation of the dppz moiety, which is realized by threading of the Ru(phen)(2) complex part through the DNA duplex core. DNase I footprinting with four diastereomerically pure middle ([Ru(phen)(2)dppz](2+)-ODN).DNA duplexes furthermore showed that the tethered [Ru(phen)(2)dppz](2+)-linker chirality dictates the stereochemical accessibility of various phosphodiester moieties (around the intercalation site) toward the cleavage reaction by the enzyme. The diastereomerically pure ruthenium-modified duplexes, with the well-defined pi-stack, will be useful to explore stereochemistry-dependent energy- and electron-transfer chemistry to understand oxidative damage to the DNA double helix as well as the long-range energy- and electron-transfer processes with DNA as a reactant.

• Dou X, Limmer S, Kreutzer R
• DNA-binding of phenylalanyl-tRNA synthetase is accompanied by loop formation of the double-stranded DNA.
• J Mol Biol. 2001; 305: 451-8
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• The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus has previously been shown to bind DNA. We demonstrate that the "winged" helix-turn-helix motifs in the duplicate domains B5 are the relevant structural elements for this DNA-binding property. By altering particular amino acids in the "wing", the affinity of the FRS to DNA was significantly reduced. Based on experimental data, which indicate that the FRS prefers a certain DNA structure rather than a particular consensus sequence, we propose a novel loop model for the DNA-binding mode of the FRS. In our model we assume that two segments of the same DNA molecule are bound simultaneously by both B5 domains and are aligned in parallel, while the intervening DNA forms a loop. Due to the limited flexibility of the DNA, loop formation is only possible if the respective intervening DNA stretch exceeds a certain length. Several lines of evidence support this model. (1) We demonstrate by gel retardation assays that the DNA requires a minimal number of ca 80 base-pairs to be bound by the FRS. (2) In the presence of the FRS, DNA longer than ca 80 base-pairs has a significantly increased DNase I accessibility. This agrees well with its known preferential cleavage at positions where the minor grove is on the outside of looped-out DNA molecules. (3) The initial cleavage by DNase I of >80 bp long DNA occurs in the middle of the fragment. In a looped molecule this is the position with the highest accessibility to DNase I.The function of the FRS related to DNA binding is still unknown. Since the FRS exists in the nucleus of rapidly growing mammalian cells, and protein-induced DNA bending or looping contributes to several transcription, replication, and recombination systems in both prokaryotes and eukaryotes, it is likely that the FRS, in addition to its aminoacylation function, influences common cellular processes via DNA binding. Copyright 2001 Academic Press.

• Wojciak JM, Iwahara J, Clubb RT
• The Mu repressor-DNA complex contains an immobilized 'wing' within the minor groove.
• Nat Struct Biol. 2001; 8: 84-90
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• We have determined the solution structure of the complex between the 'winged-helix' enhancer binding domain of the Mu repressor protein and its cognate DNA site. The structure reveals an unusual use for the 'wing' which becomes immobilized upon DNA binding where it makes intermolecular hydrogen bond contacts deep within the minor groove. Although the wing is mobile in the absence of DNA, it partially negates the large entropic penalty associated with its burial by maintaining a small degree of structural order in the DNA-free state. Extensive contacts are also formed between the recognition helix and the DNA, which reads the major groove of a highly conserved region of the binding site through a single base-specific hydrogen bond and van der Waals contacts.

• Beylot B, Spassky A
• Chemical probing shows that the intron-encoded endonuclease I-SceI distorts DNA through binding in monomeric form to its homing site.
• J Biol Chem. 2001; 276: 25243-53
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• 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.

• Mucke M, Lurz R, Mackeldanz P, Behlke J, Kruger DH, Reuter M
• Imaging DNA loops induced by restriction endonuclease EcoRII. A single amino acid substitution uncouples target recognition from cooperative DNA interaction and cleavage.
• J Biol Chem. 2000; 275: 30631-7
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• EcoRII is a type IIE restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Transmission electron microscopy provided direct evidence that EcoRII mediates loop formation of linear DNA containing two EcoRII recognition sites. Specific DNA binding of EcoRII revealed a symmetrical DNase I footprint occupying 16-18 bases. Single amino acid replacement of Val(258) by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzyme-DNA complexes was significantly higher than that of the wild-type, we investigated if mutant V258N binds as a monomer to the substrate DNA. Analysis of the molecular mass of mutant V258N showed a high percentage of protein monomers in solution. The dissociation constant of mutant V258N confirmed a 350-fold decrease of the enzyme dimerization capability. We conclude that Val(258) is located in a region of EcoRII involved in homodimerization. This is the first report of a specific amino acid replacement in a restriction endonuclease leading to the loss of dimerization and DNA cleavage while retaining specific DNA binding.

• Horton NC, Perona JJ
• Crystallographic snapshots along a protein-induced DNA-bending pathway.
• Proc Natl Acad Sci U S A. 2000; 97: 5729-34
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• Two new high-resolution cocrystal structures of EcoRV endonuclease bound to DNA show that a large variation in DNA-bending angles is sampled in the ground state binary complex. Together with previous structures, these data reveal a contiguous series of protein conformational states delineating a specific trajectory for the induced-fit pathway. Rotation of the DNA-binding domains, together with movements of two symmetry-related helices binding in the minor groove, causes base unstacking at a key base-pair step and propagates structural changes that assemble the active sites. These structures suggest a complex mechanism for DNA bending that depends on forces generated by interacting protein segments, and on selective neutralization of phosphate charges along the inner face of the bent double helix.

• Goodsell DS
• The molecular perspective: DNA.
• Stem Cells. 2000; 18: 148-9

• Beniaminov AD, Miniat EE, Khomiakova EB, Dolinnaia NG, Ivanov VI
• [Cyclization of structures with shifted loops]
• Mol Biol (Mosk). 1999; 33: 471-5

• Bailly C, Echepare S, Gago F, Waring MJ
• Recognition elements that determine affinity and sequence-specific binding to DNA of 2QN, a biosynthetic bis-quinoline analogue of echinomycin.
• Anticancer Drug Des. 1999; 14: 291-303
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• Footprinting experiments with DNase I provide a starting-point for investigating the molecular basis of nucleotide sequence recognition by 2QN, a bis-quinoline derivative of the quinoxaline antibiotic echinomycin produced by directed biosynthesis in Streptomyces echinatus. Using tyrT DNA molecules variously substituted with inosine and/or 2,6-diaminopurine residues it is shown that the location of the 2-amino group of purine nucleotides in the minor groove of the double helix exerts a dominant influence in determining where the antibiotic will bind, as it does for echinomycin. However, newly created binding sites in DNA molecules substituted with diaminopurine (D), all located round TpD steps, bind 2QN with so much higher affinity than the canonical CpG steps that the latter fail completely to appear as footprints in D-substituted DNA; indeed CpG sequences appear in regions of enhanced susceptibility to nuclease cleavage as do CpI steps in doubly D + I-substituted DNA. Quantitative footprinting plots confirm that sequences surrounding TpD steps bind 2QN several hundred-fold more tightly than do CpG-containing sequences, with dissociation constants of the order of 25 nM. To test the hypothesis that differences in stacking interactions between the chromophores of the drug and the DNA base pairs could account for the differences in binding affinities, models of 2QN bound to two DNA hexamers containing either a central CpG or a central TpD step were built. Calculation of the molecular electrostatic potential (MEP) of 2QN in solution using a continuum method revealed a distinctive pattern that is considered relevant to DNA binding. When the MEPs calculated for the two DNA hexamers in the complexed state were compared, substantial differences were found in the major groove and in the space between the base pairs that is occupied by the chromophores of the drug upon binding. The modelling data support the notion that electrostatic stacking interactions underlie the considerably preferred binding of echinomycin and 2QN around TpD steps rather than CpG steps.

• Lebrun A, Lavery R
• Modeling DNA deformations induced by minor groove binding proteins.
• Biopolymers. 1999; 49: 341-53
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• Molecular modeling is used to demonstrate that the major structural deformations of DNA caused by four different minor groove binding proteins, TBP, SRY, LEF-1, and PurR, can all be mimicked by stretching the double helix between two 3'-phosphate groups flanking the binding region. This deformation reproduces the widening of the minor groove and the overall bending and unwinding of DNA caused by protein binding. It also reproduces the principal kinks associated with partially intercalated amino acid side chains, observed with such interactions. In addition, when protein binding involves a local transition to an A-like conformation, phosphate neutralization, via the formation of protein-DNA salt bridges, appears to favor the resulting deformation.

• Behrens C, Nielsen PE
• A combinatorial approach to new DNA minor groove binders.
• Comb Chem High Throughput Screen. 1998; 1: 127-34
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• A combinatorial approach towards new DNA minor groove binders capable of recognizing GC base pairs is reported. From a partly AT--biased library of 5832 different octapeptides of the type Py-Py-X1-X2-X3-Py-Py-gamma Abu synthesized following the one bead one compound methodology, two compounds containing the central peptide sequences Val-beta Ala-Tyr and Pip-beta Ala-Tyr were selected by a fluorescence--double stranded DNA probing assay using the sequence 5'-TTTGTTT-3' as the probe. The two hits were independently synthesized and binding to a recombinant pUC-19 EcoRl/Pvull DNA restriction fragment containing the sequence 5'-TTTGTTT-3' demonstrated. Binding constants to eight targets in the DNA fragment were estimated from quantitative DNAse I footprinting.

• Wemmer D
• Reading DNA.
• Nat Struct Biol. 1998; 5: 169-71
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• A new structure beings into sharp focus the structural basis for G-C base pair recognition by small ligands in the minor groove of DNA. At the same time the ability to distinguish specific sequences moves another major step forward through introduction of a new ligand modification.

• Horton NC, Perona JJ
• Role of protein-induced bending in the specificity of DNA recognition: crystal structure of EcoRV endonuclease complexed with d(AAAGAT) + d(ATCTT).
• J Mol Biol. 1998; 277: 779-87
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• The crystal structure of EcoRV endonuclease has been determined at 2. 1 A resolution complexed to two five-base-pair DNA duplexes each containing the cognate recognition half-site. The highly localized 50 degrees bend into the major groove seen at the center TA-step of the continuous GATATC site is preserved in this discontinuous DNA complex lacking the scissile phosphates. Thus, this crystal structure provides evidence that covalent constraints associated with a continuous target site are not essential to enzyme-induced DNA bending, even when these constraints are removed directly at the locus of the bend. The scissile phosphates are also absent in the crystal structure of EcoRV bound to the non-specific site TCGCGA, which shows a straight B-like conformation. We conclude that DNA bending by EcoRV is governed only by the sequence and is not influenced by the continuity of the phosphodiester backbone. Together with other data showing that cleavable non-cognate sites are bent, these results indicate that EcoRV bends non-cognate sites differing by one or two base-pairs from GATATC, but does not bend non-specific sites that are less similar. Structural and thermodynamic considerations suggest that the sequence-dependent energy cost of DNA bending is likely to play an important role in determining the specificity of EcoRV. This differential cost is manifested at the binding step for bent non-cognate sequences and at the catalytic step for unbent non-specific sequences.

• Cal S, Tan KL, McGregor A, Connolly BA
• Conversion of bovine pancreatic DNase I to a repair endonuclease with a high selectivity for abasic sites.
• EMBO J. 1998; 17: 7128-38
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• Bovine pancreatic deoxyribonuclease I (DNase I) is a nuclease of relatively low specificity which interacts with DNA in the minor groove. No contacts are made between the protein and the major groove of the nucleic acid. DNase I is structurally homologous to exonuclease III, a DNA-repair enzyme with multiple activities. One of the main differences between the two enzymes is the presence of an additional alpha-helix in exonuclease III, in a position suggestive of interaction with the major groove of DNA. Recombinant DNA techniques have been used to add 14 amino acids, comprising the 10 amino acids of the exonuclease III alpha-helix flanked by a glycine rich region, to DNase I. The polypeptide has been inserted after serine 174, an amino acid on the surface of DNase I corresponding to the location of the extra alpha-helix in exonuclease III. The recombinant protein, DNase-exohelix, has been purified and its catalytic activities towards DNA investigated. The recombinant protein demonstrated a high selectivity for endonucleolytic cleavage at abasic sites in DNA, a property of exonuclease III but not native DNase I. Thus the insertion of 14 amino acids at Ser174, converts DNase I to an exonuclease III-like enzyme with DNA-repair properties.

• Pan CQ, Ulmer JS, Herzka A, Lazarus RA
• Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition, catalysis, and metal ion dependence.
• Protein Sci. 1998; 7: 628-36
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• Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.

• Pan CQ, Lazarus RA
• Hyperactivity of human DNase I variants. Dependence on the number of positively charged residues and concentration, length, and environment of DNA.
• J Biol Chem. 1998; 273: 11701-8
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• Human DNase I, an enzyme used to treat cystic fibrosis patients, has been engineered to more effectively degrade double-stranded DNA to lower molecular weight forms by introducing positively charged amino acids at positions that can interact favorably with the proximal negatively charged phosphate groups of the DNA. A series of combination mutants having from one to six additional basic residues compared with the wild type has been constructed, expressed in human 293 cells, and characterized. The degree of hyperactivity for the mutants was highly dependent upon the conditions in various assays, including the concentration and length of the DNA substrate and the salt and divalent metal ion concentrations. The level of hyperactivity was inversely proportional to both DNA concentration and DNA length, consistent with the processive nicking mechanism for the hyperactive variants. Salt was inhibitory for wild type DNase I but actually enhanced the activity of the hyperactive variants. Under optimal conditions for wild type, variants with one additional positive charge possessed the highest activity, which was only severalfold greater than that for wild type. However, in the presence of low DNA concentrations and molecular weights, no Ca2+, and 150 mM NaCl, the variant with six engineered basic residues was most active, having >10,000-fold higher activity than the wild type enzyme. Therefore, any potential increase in potency for the hyperactive variants in vivo will be determined by the concentration, length, and environment of the DNA.

• Swaminathan K, Flynn P, Reece RJ, Marmorstein R
• Crystal structure of a PUT3-DNA complex reveals a novel mechanism for DNA recognition by a protein containing a Zn2Cys6 binuclear cluster.
• Nat Struct Biol. 1997; 4: 751-9
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• PUT3 is a member of a family of at least 79 fungal transcription factors that contain a six-cysteine, two-zinc domain called a 'Zn2Cys6 binuclear cluster'. We have determined the crystal structure of the DNA binding region from the PUT3 protein bound to its cognate DNA target. The structure reveals that the PUT3 homodimer is bound asymmetrically to the DNA site. This asymmetry orients a beta-strand from one protein subunit into the minor groove of the DNA resulting in a partial amino acid-base pair intercalation and extensive direct and water-mediated protein interactions with the minor groove of the DNA. These interactions facilitate a sequence dependent kink at the centre of the DNA site and specify the intervening base pairs separating two DNA half-sites that are contacted in the DNA major groove. A comparison with the GAL4-DNA and PPR1-DNA complexes shows how a family of related DNA binding proteins can use a diverse set of mechanisms to discriminate between the base pairs separating conserved DNA half-sites.

• Vaquero A, Portugal J
• Small ligands that neither bind to nor alter the structure of d(GA x TC)n sequences in DNA.
• FEBS Lett. 1997; 420: 156-60
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• Three minor-groove binding ligands have been used to study the characteristics of two d(GA x CT)n DNAs embedded in longer DNA fragments. The binding of mithramycin, netropsin or Thia-Net to these sequences has been studied using DNAse I footprinting. None of these ligands appeared to bind to d(GA x CT)5 nor to d(GA x CT)22 extensively, although with mithramycin some protected bonds were detected at the very edge of these sequences. In general, these small ligands did not enhance the DNAse I cleavage patterns at the alternating d(GA x CT)n flanking sequences located near DNA regions where the drug was bound. The d(GA x CT)n sequences could act as a rigid block in which it is not easy to propagate structural changes, whereas other sequences flanking the binding sites showed cleavage enhancements.

• Allemann RK, Egli M
• DNA recognition and bending.
• Chem Biol. 1997; 4: 643-50
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• DNA-binding proteins recognize their DNA targets not only through the formation of specific contacts with the nucleotide bases but also through inherent properties of the DNA sequence, including increased bendability and rigidity. Consideration of the properties of both the protein and the DNA is required before the sequence specificity and the observed DNA bend in DNA-protein complexes can be understood.

• Tateno M et al.
• DNA recognition by beta-sheets.
• Biopolymers. 1997; 44: 335-59
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• The modes of DNA recognition by beta-sheets are analyzed by using the known crystal and solution three-dimensional structures of DNA-protein complexes. Close fitting of the protein surface and the DNA surface determines the binding geometry. Interaction takes place so that essentially the N-to-C direction of the beta-strands either follows or crosses the DNA groove. Upon following the major groove a two-stranded antiparallel beta-sheet dives into the groove and contacts DNA bases with its convex side facing the DNA, while upon following the minor groove, it binds around the sugar-phosphate backbones, with its opposite concave side shielding the DNA. In order for the beta-strands crossing the minor groove to interact with the DNA, the dinucleotide steps need to almost totally helically untwist and roll around major groove. The beta-sheet, on the other hand, needs to adopt a concave curvature on the binding surface in the direction that follows the DNA minor groove, and a convex surface in the direction that bridges the sugar-phosphate backbones across the groove. The result is to produce a hyperbolic paraboloidal DNA-binding surface.

• Suzuki M, Yagi N
• An in-the-groove view of DNA structures in complexes with proteins.
• J Mol Biol. 1996; 255: 677-87
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• DNA structures bound to transcription factors are studied by using the crystal coordinates of complexes. Structural characteristics which are found at the sites where a protein secondary structure, an alpha-helix or a beta-sheet binds, can be understood in terms of fitting of the concave surface of the DNA major groove and the convex surface of the protein secondary structure; the former changes, becoming narrower or wider so that it fits the latter. An alpha-helix, independent of which groove it binds to, tends to produce a narrow major groove and a wide minor groove. When the major groove becomes narrower, the DNA helix axis bends around the major groove. Bending of DNA, which is overall structural change, is achieved by changes in local parameters (in particular, the roll parameter) so that an intermediate feature, the groove, fits the protein surface.

• Jones SJ, Worrall AF, Connolly BA
• Site-directed mutagenesis of the catalytic residues of bovine pancreatic deoxyribonuclease I.
• J Mol Biol. 1996; 264: 1154-63
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• Bovine pancreatic deoxyribonuclease I (DNase I) is a well characterised endonuclease which cleaves double-stranded DNA to yield 5' phosphorylated polynucleotides. Co-crystal structures of DNase I with two different oligonucleotides have revealed the presence of several residues (R9, E78, H134, D168, D212 and H252) close to the scissile phosphate. The roles that these amino acids play in the catalytic mechanism have been investigated using site-directed mutagenesis. The following variants were used: R9A, E78T, H134Q, D168S, D212S and H252Q. The kinetics of all six mutants with both DNA and a small chromophoric substrate, thymidine-3',5'-di(p-nitrophenyl)-phosphate, were studied. Only R9A and E78T showed any significant turnover of the two substrates. D168S, H134Q, D212S and H252Q showed vanishingly low activities towards DNA and no detectable activity with thymidine-3',5'-di(p-nitrophenyl)-phosphate. These results demonstrate that H134, D168, D212 and H252 play a critical role in the catalytic mechanism. It is suggested that H134 and H252 (which are hydrogen-bonded to E78 and D212, respectively) provided general acid and general base catalysis. DNase I also requires Mg2+ and E39 has been identified as a ligand for this metal ion. We propose that D168 serves as a ligand for a second Mg2+, and thus DNase I, uses a two metal-ion hydrolytic mechanism. Both magnesium ions are used to supply electrophilic catalysis. Role assignment is based on the mutagenesis results, structural information, homologies between DNase I from different species and a comparison with exonuclease III. However, it is still not feasible to unequivocally assign a particular catalytic role to each amino acid/metal ion.

• Long EC, Eason PD, Liang Q
• Synthetic metallopeptides as probes of protein-DNA interactions.
• Met Ions Biol Syst. 1996; 33: 427-52

• Suzuki M, Loakes D, Yagi N
• DNA conformation and its changes upon binding transcription factors.
• Adv Biophys. 1996; 32: 53-72

• Timsit Y, Moras D
• Cruciform structures and functions.
• Q Rev Biophys. 1996; 29: 279-307

• Brukner I, Sanchez R, Suck D, Pongor S
• Sequence-dependent bending propensity of DNA as revealed by DNase I: parameters for trinucleotides.
• EMBO J. 1995; 14: 1812-8
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• Structural parameters characterizing the bending propensity of trinucleotides were deduced from DNase I digestion data using simple probabilistic models. In contrast to dinucleotide-based models of DNA bending and/or bendability, the trinucleotide parameters are in good agreement with X-ray crystallographic data on bent DNA. This improvement may be due to the fact that the trinucleotide model incorporates more sequence context information than do dinucleotide-based descriptions.

• Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK
• Structure of Bam HI endonuclease bound to DNA: partial folding and unfolding on DNA binding.
• Science. 1995; 269: 656-63
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• The crystal structure of restriction endonuclease Bam HI complexed to DNA has been determined at 2.2 angstrom resolution. The DNA binds in the cleft and retains a B-DNA type of conformation. The enzyme, however, undergoes a series of conformational changes, including rotation of subunits and folding of disordered regions. The most striking conformational change is the unraveling of carboxyl-terminal alpha helices to form partially disordered "arms." The arm from one subunit fits into the minor groove while the arm from the symmetry related subunit follows the DNA sugar-phosphate backbone. Recognition of DNA base pairs occurs primarily in the major groove, with a few interactions occurring in the minor groove. Tightly bound water molecules play an equally important role as side chain and main chain atoms in the recognition of base pairs. The complex also provides new insights into the mechanism by which the enzyme catalyzes the hydrolysis of DNA phosphodiester groups.

• Duckett DR, Panis MJ, Lilley DM
• Binding of the junction-resolving enzyme bacteriophage T7 endonuclease I to DNA: separation of binding and catalysis by mutation.
• J Mol Biol. 1995; 246: 95-107
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• Bacteriophage T7 endonuclease I is a resolving enzyme that selectively cleaves four-way DNA junctions, and related branched species. We have isolated mutants of this protein that retain full structural selectivity of binding to four-way junctions, but which are completely inactive as nucleases. This is consistent with a divisibility of structure-selective binding and catalysis. The mutations that inactivate endonuclease I as a nuclease are clustered into the second quarter of the primary sequence, a region that displays some sequence similarity with the related junction-resolving enzyme endonuclease VII from bacteriophage T4. This suggests that these residues may form the active site of these enzymes. The configuration of the helical arms of the junction bound by mutant endonuclease I has been investigated by gel electrophoretic methods. We find that the junction is bound in the presence or absence of magnesium ions, and that the global structure of the bound form is apparently identical with or without cations. The patterns of mobilities suggest that the structure of the junction becomes perturbed by the binding of the protein.

• Werner MH et al.
• The solution structure of the human ETS1-DNA complex reveals a novel mode of binding and true side chain intercalation.
• Cell. 1995; 83: 761-71
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• The solution structure of a 24.4 kDa specific complex of the DNA-binding domain (DBD) of the human ETS1 (hETS1) oncoprotein with a 17-mer DNA has been solved by NMR. The interaction of the hETS1 DBD with DNA reveals a surprising twist on the general features of helix-turn-helix (HTH)-DNA interactions. Major groove recognition involves the C-terminal two thirds of the HTH recognition helix, while minor groove recognition occurs via true intercalation of the side chain of Trp-28, which extends from the minor to the major groove. This results in a sharp kink of approximately 60 degrees and a widening of the minor groove over one-half turn of the DNA. The orientation of the HTH element of the hETS1 DBD with respect to the major groove is significantly rotated relative to other HTH proteins. These observations establish the ETS family of DNA-binding proteins as a distinct family of HTH proteins.

• Mannherz HG, Peitsch MC, Zanotti S, Paddenberg R, Polzar B
• A new function for an old enzyme: the role of DNase I in apoptosis.
• Curr Top Microbiol Immunol. 1995; 198: 161-74

• Wolf E, Brukner I, Suck D
• Mutational analysis of DNase I-DNA interactions: design, expression and characterization of a DNase I loop insertion mutant with altered sequence selectivity.
• Protein Eng. 1995; 8: 283-91
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• A mutant of bovine pancreatic DNase I containing two additional residues in a loop next to C173 has been expressed in Escherichia coli, purified and characterized biochemically. Modelling studies suggest that the inserted arginine and glutamate side chains of the modified loop sequence C173-R-E-G-T-V176 could contact the bases 3' to the cleaved bond in the major groove of a bound DNA, and that up to 10 bp could interact with the enzyme and potentially influence its cutting rate. The loop insertion mutant has an 800-fold lower specific activity than wild-type and shows overall cleavage characteristics similar to bovine pancreatic DNase I. Compared with the wild-type enzyme, the mutant shows a strongly enhanced preference for cutting the inverted repeat: (formula: see text) or close variants thereof. Unexpectedly for a minor groove binding protein, the preferred cutting sites in opposite strands are staggered by 1 bp in the 5' direction, causing the cleavage of a TA and a TT step, respectively. This finding demonstrates that the sequence context is relatively more important for the cutting frequency than the nature of the dinucleotide step of the cleaved bond, and clearly shows that base recognition is involved in determining the sequence selectivity of the mutant. The importance of the sequence 5' to the cleaved bond for the cutting rate suggests that the additional major groove contacts may require a distortion of the DNA associated with a higher energy barrier, resulting in an increased selectivity for flexible DNA sequences and a lower overall activity of the mutant enzyme.

• Abu-Daya A, Brown PM, Fox KR
• DNA sequence preferences of several AT-selective minor groove binding ligands.
• Nucleic Acids Res. 1995; 23: 3385-92
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• We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments containing different arrangements of AT base pairs by DNase I footprinting. For fragments which contain multiple blocks of (A/T)4 quantitative DNase I footprinting reveals that AATT and AAAA are much better binding sites than TTAA and TATA. Hoechst 33258 shows that greatest discrimination between these sites with a 50-fold difference in affinity between AATT and TATA. Alone amongst these ligands, Hoechst 33258 binds to AATT better than AAAA. These differences in binding to the various AT-tracts are interpreted in terms of variations in DNA minor groove width and suggest that TpA steps within an AT-tract decrease the affinity of these ligands. The behaviour of each site also depends on the flanking sequences; adjacent pyrimidine-purine steps cause a decrease in affinity. The precise ranking order for the various binding sites is not the same for each ligand.

• Bailly C, Mollegaard NE, Nielsen PE, Waring MJ
• The influence of the 2-amino group of guanine on DNA conformation. Uranyl and DNase I probing of inosine/diaminopurine substituted DNA.
• EMBO J. 1995; 14: 2121-31
• Display abstract

• The conformation of the DNA helix is supposed to be a critical element in site-specific recognition by ligands both large and small. Groove width is one important measure of the conformation which varies with the local nucleotide composition, perhaps because of the presence of a purine 2-amino group on G.C base pairs. We have probed DNA with G-->inosine (I) and/or A-->diaminopurine (DAP) substitutions to see whether the location of the purine 2-amino group can indeed affect the minor groove width. At acid pH, the reactivity towards uranyl nitrate is modulated in substituted DNA quite differently from natural DNA, consistent with a marked narrowing of the minor groove at sites of G-->I substitution and widening at sites of A-->DAP replacement. The latter exerts the dominant effect. The expected changes in conformation are equally evident in the patterns of susceptibility to DNase I cleavage, but not to hydroxyl radical attack. Nuclease cleavage is maximal in normal and substituted DNA at regions of inferred moderate groove width which are generally little affected by the nucleotide substitutions. Consistent with models of sequence-dependent cutting by DNase I we find that the presence of a purine 2-amino group on the base pair three places upstream of the cutting site has a profound influence on the rate of reaction.

• Ladbury JE
• Counting the calories to stay in the groove.
• Structure. 1995; 3: 635-9
• Display abstract

• High-sensitivity microcalorimetry is beginning to make an impact on the determination of thermodynamic parameters associated with protein-DNA interactions and the understanding of the relationship of these data to structural details of complex formation.

• Hanes SD, Brent R
• Backward bicoid?
• Structure. 1994; 2: 894-894

• Nekludova L, Pabo CO
• Distinctive DNA conformation with enlarged major groove is found in Zn-finger-DNA and other protein-DNA complexes.
• Proc Natl Acad Sci U S A. 1994; 91: 6948-52
• Display abstract

• We have analyzed DNA conformations in a series of protein-DNA complexes, and we find that a distinctive conformation--with an enlarged major groove--occurs in a number of different complexes. During this analysis, we also developed a simplified model of DNA structure that illustrates the relative position of (i) the base pairs, (ii) the phosphate backbone, and (iii) the double-helical axis. This model highlights the key structural features of each duplex, facilitating the analysis and comparison of structures that are distinct from canonical A-DNA or B-DNA. Comparing DNA conformations in this way revealed that an otherwise unrelated set of protein-DNA complexes have interesting structural similarities, including an enlarged major groove. We refer to this class of structures as Beg-DNA (where eg means enlarged groove). Since related features occur in such a diverse set of protein-DNA complexes, we suggest that this conformation may have a significant role in protein-DNA recognition.

• Feng JA, Johnson RC, Dickerson RE
• Hin recombinase bound to DNA: the origin of specificity in major and minor groove interactions.
• Science. 1994; 263: 348-55
• Display abstract

• The structure of the 52-amino acid DNA-binding domain of the prokaryotic Hin recombinase, complexed with a DNA recombination half-site, has been solved by x-ray crystallography at 2.3 angstrom resolution. The Hin domain consists of a three-alpha-helix bundle, with the carboxyl-terminal helix inserted into the major groove of DNA, and two flanking extended polypeptide chains that contact bases in the minor groove. The overall structure displays features resembling both a prototypical bacterial helix-turn-helix and the eukaryotic homeodomain, and in many respects is an intermediate between these two DNA-binding motifs. In addition, a new structural motif is seen: the six-amino acid carboxyl-terminal peptide of the Hin domain runs along the minor groove at the edge of the recombination site, with the peptide backbone facing the floor of the groove and side chains extending away toward the exterior. The x-ray structure provides an almost complete explanation for DNA mutant binding studies in the Hin system and for DNA specificity observed in the Hin-related family of DNA invertases.

• Stonehouse TJ, Fox KR
• DNase I footprinting of triple helix formation at polypurine tracts by acridine-linked oligopyrimidines: stringency, structural changes and interaction with minor groove binding ligands.
• Biochim Biophys Acta. 1994; 1218: 322-30
• Display abstract

• We have investigated the binding of short (10 base) acridine-linked triplex-forming oligonucleotides to the target sequence A6G6.C6T6 by DNase I footprinting. Specific binding is detected at low pH (< 6.0) for 5'-Acr-T5C5 and 5'-Acr-5BrU5(5Me)C5. The sequence T5C5, lacking the acridine modification, binds less strongly, though specific binding is still evident. 5'-Acr-T5C5 produces footprints at slightly lower concentrations than 5'-Acr-5BrU5(5Me)C5. All three oligonucleotides produce enhanced DNase I digestion at the 3'-end of the target purine strand, suggesting that there is a DNA structural change at the triplex-duplex boundary. Target sequences AnG4A and TAC3Tn, containing one and two triplex mismatches, show no interaction with the acridine-free oligonucleotide, but bind the acridine-linked oligonucleotides. In these secondary binding modes the third strand is positioned so that the mismatches are located at the 3'-end of the oligonucleotide. Mithramycin and distamycin, binding in the minor groove to GC- and AT-rich sequences respectively, abolish triple helix formation.

• Herrera JE, Chaires JB
• Characterization of preferred deoxyribonuclease I cleavage sites.
• J Mol Biol. 1994; 236: 405-11
• Display abstract

• The preferred DNase I cleavage sites within the 160 bp tyrT DNA fragment were identified by studying the initial rate of cleavage of individual bonds. The results show that there is no correlation between the rate of cleavage and the identity of the dinucleotide sequence that is cleaved. Examination of the sequences surrounding the seven most rapidly cleaved bonds suggests that an A-T base-pair is preferred at the position three bases to the 5' side of the cleavage site. Preferential cleavage at such sites is consistent with predictions based on the recently obtained high resolution structure of a DNase I-octanucleotide complex. A statistical analysis of 54 additional preferred DNase I cleavage sites, using sequence data taken from published literature, confirms that DNase I exhibits a local sequence preference in addition to its relatively well characterized global structural specificity. Our analysis indicates preferential cleavage at the sequences 5'ATYAT--ATVN, where -- indicates the cleavage site, the notation AT indicates a preference for an A-T base-pair, and V indicates not-T. Comparative kinetic studies of the digestion of three deoxyoctanucleotides by DNase I quantitatively support the sequence preference inferred from the sequence analysis. Poor DNase I cleavage sites were also examined, and found to be characterized by the sequence motif 5'GCRR--TTY. Notably, poor cleavage sites characteristically contain G or C at position -3. While DNase I certainly does not cleave with an absolute sequence specificity, our studies reveal a distinct sequence preference in DNase I cleavage that has heretofore been unappreciated and uncharacterized.

• Mirkin SM, Frank-Kamenetskii MD
• H-DNA and related structures.
• Annu Rev Biophys Biomol Struct. 1994; 23: 541-76

• Mohan S, Yathindra N
• A study of the interaction of DAPI with DNA containing AT and non-AT sequences--molecular specificity of minor groove binding drugs.
• J Biomol Struct Dyn. 1994; 11: 849-67
• Display abstract

• The binding specificity of DAPI to DNA has been probed by analysing its interactions with DNA octamers consisting of different base sequences, which include adenine, guanine, 2-amino adenine and inosine, using molecular mechanics methods. Presence of AT and non-AT base pairs in the immediate vicinity of the binding site, containing AT and non-AT base pairs is also investigated. Results show that DAPI most prefers to bind to homopolymer of AT, and least to the duplex containing alternating GC bases. DAPI interacts with homopolymeric duplexes in two possible orientations related by 180 degrees with nearly same affinity. Affinity of DAPI towards DNA comprising the modified bases, inosine and 2-amino adenine, is in between these extremities. The binding affinity is reduced to some extent by the occurrence of non AT bases flanking the four base paired binding region. An interesting revelation is that one can visualise DAPI to form a hydrogen bond with O2 of cytosine indicating that the 2-amino group of purines does not per se sterically preclude DAPI from residing in the minor groove of B-DNA helix. On the other hand, repulsive nature of electrostatic interactions that prevail at the minor groove consequent to the presence of these sequences contribute decisively in preventing further diffusion of the drug. Thus, electrostatics, rather than hydrogen bonding to bases, seemingly play an important role in determining the specificity of interaction. The retention of drug binders in the minor groove and therefore recognition, is governed by the combined effect of these various forces.

• Leblanc B, Moss T
• DNase I footprinting.
• Methods Mol Biol. 1994; 30: 1-10

• Sandaltzopoulos R, Becker PB
• Solid phase DNase I footprinting: quick and versatile.
• Nucleic Acids Res. 1994; 22: 1511-2

• Winkler FK et al.
• The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.
• EMBO J. 1993; 12: 1781-95
• Display abstract

• The crystal structure of EcoRV endonuclease has been determined at 2.5 A resolution and that of its complexes with the cognate DNA decamer GGGATATCCC (recognition sequence underlined) and the non-cognate DNA octamer CGAGCTCG at 3.0 A resolution. Two octamer duplexes of the non-cognate DNA, stacked end-to-end, are bound to the dimeric enzyme in B-DNA-like conformations. The protein--DNA interactions of this complex are prototypic for non-specific DNA binding. In contrast, only one cognate decamer duplex is bound and deviates considerably from canonical B-form DNA. Most notably, a kink of approximately 50 degrees is observed at the central TA step with a concomitant compression of the major groove. Base-specific hydrogen bonds between the enzyme and the recognition base pairs occur exclusively in the major groove. These interactions appear highly co-operative as they are all made through one short surface loop comprising residues 182-186. Numerous contacts with the sugar phosphate backbone extending beyond the recognition sequence are observed in both types of complex. However, the total surface area buried on complex formation is > 1800 A2 larger in the case of cognate DNA binding. Two acidic side chains, Asp74 and Asp90, are close to the reactive phosphodiester group in the cognate complex and most probably provide oxygen ligands for binding the essential cofactor Mg2+. An important role is also indicated for Lys92, which together with the two acidic functions appears to be conserved in the otherwise unrelated structure of EcoRI endonuclease. The structural results give new insight into the physical basis of the remarkable sequence specificity of this enzyme.

• Boutonnet N, Hui X, Zakrzewska K
• Looking into the grooves of DNA.
• Biopolymers. 1993; 33: 479-90
• Display abstract

• The present study is aimed at understanding the effects of DNA sequence, local conformation, and curvature on groove geometry. Energy-optimized structures are obtained by Jumna methodology; groove geometry is analyzed by a recently developed technique that allows an accurate and continuous measurement of width and depth. The mechanics of groove deformations is also studied and analyzed in terms of helicoidal parameters.

• Reitz M, Knitza R, Lanz E
• Increasing DNase I activity after exposure of isolated DNA to halothane.
• Arzneimittelforschung. 1993; 43: 92-4
• Display abstract

• DNA was exposed to halothane (CAS 151-67-7) in a cell-free system. After exposure the DNA was used as substrate for DNase I from bovine pancreas. The DNase I activity increased after halothane exposure of the substrate depending on time and doses. Drugs are able to influence the DNA conformation. Conformational changes in the DNA can enhance the DNase I cleavage rate. Therefore, it is possible that halothane exposure induces changes in DNA conformation demonstrable by an increased DNase I activity. The results suggest a mechanism by which halothane may contribute to chromosomal defects and disturbances of DNA metabolism in cells.

• Chandler SP, Fox KR
• Triple helix formation at A8XA8.T8YT8.
• FEBS Lett. 1993; 332: 189-92
• Display abstract

• We have examined the formation of DNA triple helices between the oligonucleotides T8XT8 (X = A,C,G,T) and DNA fragments containing the target sequences A8XA8.T8YT8 (X = T,C,G; Y = A,G,C), by DNase I footprinting. We find that A8GA8.T8CT8 yields a footprint with T8CT8 and shows a weaker interaction with T17 and T8GT8. A8CA8.T8GT8 yields a footprint with T17, and shows weaker interaction with T8CT8. A8TA8.T8AT8 yields a footprint with T8GT8 and shows weaker interaction with T17. Each of the successful complexes is characterised by enhanced DNase I cleavage at the 3' end of the purine strand of the target, as well as protection at the 5' end. We have been unable to from triplexes with third strands of the type A8XA8.

• Kochanek S, Renz D, Doerfler W
• Differences in the accessibility of methylated and unmethylated DNA to DNase I.
• Nucleic Acids Res. 1993; 21: 5843-5
• Display abstract

• DNase I binds in the minor groove of DNA and is used as an enzymatic tool to investigate the interaction of proteins with DNA. Here we show that the major groove located 5-methyldeoxycytidine can enhance or inhibit the cleavage rates of DNA by DNase I. This effect may be caused in part by changes in DNA structure affecting the accessibility of the minor groove of DNA to DNase I.

• Fletcher MC, Fox KR
• Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting.
• Nucleic Acids Res. 1993; 21: 1339-44
• Display abstract

• We have investigated the kinetics of dissociation of actinomycin D from DNA by a variation of the footprinting technique. Complexes of actinomycin with a radiolabelled DNA fragment (tyrT) were dissociated by addition of a large excess of unlabelled calf thymus DNA and the mixture subjected to DNase I footprinting at subsequent intervals. The rates at which the footprints disappeared varied between the different binding sites. The dissociation was temperature dependent with average time constants of 30 s, 10 mins and 2 hours at temperatures of 37 degrees C, 20 degrees C and 4 degrees C respectively. The dissociation from a DNA fragment containing the synthetic insert T9GCA9 was significantly faster, with a half-life of about 1 min at 20 degrees C. In contrast, the dissociation of distamycin was too fast to measure (< 5 s) even at 4 degrees C.

• Weston S, Suck D
• X-ray structures of two single-residue mutants of DNase I: H134Q and Y76A.
• Protein Eng. 1993; 6: 349-57
• Display abstract

• The structures of the single-residue mutants H134Q and Y76A of bovine pancreatic DNase I have been determined and refined including data to 2.3 and 2.4 A resolution respectively, by X-ray crystallography. H134 is an essential catalytic residue, while Y76 contributes to the binding of DNA by providing a large van der Waals contact area that stabilizes the wide minor groove seen in DNase I-DNA complexes. The mutant proteins, which show strongly reduced activities of 0.001% (H134Q) and 0.3% (Y76A), were expressed in E. coli and both crystallize in space-group C2 with almost identical unit cells. The crystal packing scheme is different from that found in wild type crystals grown under very similar conditions, presumably due to the absence of the carbohydrate moiety. In both mutants the conformation of the protein is nearly identical to that of the wild type enzyme and changes are confined to surface loops involved in packing. The disruption of the hydrogen bonds between H134, E78 and Y76 in both mutants leads to an increased mobility and positional shifts in the DNA-binding loop, mainly around residue Y76. This in turn may further reduce DNA-binding affinity and, thus, contribute to the low activity. In contrast, symmetry contacts involving residues 97-108 lead to a stabilization of the flexible loop compared to wild type DNase I.

• Weston SA, Lahm A, Suck D
• X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution.
• J Mol Biol. 1992; 226: 1237-56
• Display abstract

• The crystal structure of a complex between DNase I and the self-complementary octamer duplex d(GGTATACC)2 has been solved using the molecular replacement method and refined to a crystallographic R-factor of 18.8% for all data between 6.0 and 2.3 A resolution. In contrast to the structure of the DNase I-d(GCGATCGC)2 complex solved previously, the DNA remains uncleaved in the crystal. The general architecture of the two complexes is highly similar. DNase I binds in the minor groove of a right-handed DNA duplex, and to the phosphate backbones on either side over five base-pairs, resulting in a widening of the minor groove and a concurrent bend of the DNA away from the bound enzyme. There is very little change in the structure of the DNase I on binding the substrate. Many other features of the interaction are conserved in the two complexes, in particular the stacking of a deoxyribose group of the DNA onto the side-chain of a tyrosine residue (Y76), which affects the DNA conformation and the binding of an arginine side-chain in the minor groove. Although the structures of the DNA molecules appear at first sight rather similar, detailed analysis reveals some differences that may explain the relative resistance of the d(GGTATACC)2 duplex to cleavage by DNase I: whilst some backbone parameters are characteristic of a B-conformation, the spatial orientation of the base-pairs in the d(GGTATACC)2 duplex is close to that generally observed in A-DNA. These results further support the hypothesis that the minor-groove width and depth and the intrinsic flexibility of DNA are the most important parameters affecting the interaction. The disposition of residues around the scissile phosphate group suggests that two histidine residues, H134 and H252, are involved in catalysis.

• Goodisman J, Dabrowiak JC
• Structural changes and enhancements in DNase I footprinting experiments.
• Biochemistry. 1992; 31: 1058-64
• Display abstract

• In footprinting experiments, an increase in DNA cleavage with addition of ligand to a system may be due to a ligand-induced structural change. Ligand binding also enhances cleavage by displacing the cleavage agent from ligand-binding sites, thus increasing its concentration elsewhere. The theory and characteristics of this mass-action enhancement are given, and it is shown how it may be recognized. Results of DNase I footprinting of small oligomers, with actinomycin D as ligand, are analyzed to reveal which enhancements are due to mass action, and which can reasonably be ascribed to structural changes. Patterns in the footprinting plots from our experiments on actinomycin D binding to a 139-base-pair DNA fragment (with DNase I as a probe) are studied in the same way. The likely origins of these patterns are discussed, as are enhancements occurring with other probes commonly used in footprinting experiments.

• Bickler SW, Heinrich MC, Bagby GC
• Magnesium-dependent thermostability of DNase I.
• Biotechniques. 1992; 13: 64-6
• Display abstract

• DNase I can be used to remove contaminating DNA from RNA samples. Heat treatment has been recommended as a method to inactivate DNase I enzymatic activity, thereby allowing subsequent reverse transcription and PCR amplification of DNase I treated RNA. We have found that inactivation of DNase I by heat treatment is strongly dependent on Mg2+ concentration. In addition, deoxyribonucleolytic activity of "inactivated" enzyme may be restored by changes in Mg2+ concentration following heat treatment. Caution should be exercised when using heat treatment alone as a method of DNase I inactivation.

• Goodisman J, Rehfuss R, Ward B, Dabrowiak JC
• Site-specific binding constants for actinomycin D on DNA determined from footprinting studies.
• Biochemistry. 1992; 31: 1046-58
• Display abstract

• We report site-specific binding constants for the intercalating anticancer drug actinomycin D (Act-D), binding to a 139-base-pair restriction fragment from pBR 322 DNA. The binding constants are derived from analysis of footprinting experiments, in which the radiolabeled 139-mer is cleaved using DNase I, the cleavage products undergo gel electrophoresis, and, from the gel autoradiogram, spot intensities, proportional to amounts of cleaved fragments, are measured. A bound drug prevents DNase I from cleaving at approximately 7 bonds, leading to decreased amounts of corresponding fragments. With the radiolabel on the 3' end of the noncoding strand (A-label), we measured relative amounts of 54 cleavage products at 25 Act-D concentrations. For cleavage of the 139-mer with the label on the 3' end of the coding strand (G-label), relative amounts of 43 cleavage products at 11 Act-D concentrations were measured. These measurements give information about approximately 120 base pairs of the restriction fragment (approximately 12 turns of the DNA helix); in this region, 14 strong and weak Act-D binding sites were identified. The model used to interpret the footprinting plots is derived in detail. Binding constants for 14 sites on the fragment are obtained simultaneously. It is important to take into account the effect of drug binding at its various sites on the local concentration of probe elsewhere. It is also necessary to include in the model weak as well as strong Act-D sites on the carrier DNA which is present, since the carrier DNA controls the free-drug concentration. As expected, the strongest sites are those with the sequence (all sequences are 5'----3') GC, with TGCT having the highest binding constant, 6.4 x 10(6) M-1. Sites having the sequence GC preceded by G are weak binding sites, having binding constants approximately 1 order of magnitude lower than those of the strong sites. Also, the non-GC-containing sequences CCG and CCC bind Act-D with a binding constant comparable to those of the weak GGC sites. The analysis may reveal drug-induced structural changes on the DNA, which are discussed in terms of the mechanism of Act-D binding.

• Yonezawa A, Kuwahara J, Fujii N, Sugiura Y
• Binding of tachyplesin I to DNA revealed by footprinting analysis: significant contribution of secondary structure to DNA binding and implication for biological action.
• Biochemistry. 1992; 31: 2998-3004
• Display abstract

• In view of the cationic amphipathic structure of tachyplesin I and antiparallel beta-sheet as a general DNA binding motif, DNA binding of the antimicrobial peptide has been examined. Several footprinting-like techniques using DNase I protection, dimethyl sulfate protection, and bleomycin- (BLM-) induced DNA cleavage were applied in this study. Some distinct footprints with DNase I are detected, and also the sequence-specific cleavage mode of the BLM-Fe(II) complex clearly is altered in the presence of tachyplesin I. In addition, methylation of the N-7 residue of guanine situated in the DNA major groove is not entirely inhibited (or activated) by tachyplesin I. The results suggest that tachyplesin I interacts with the minor groove of DNA duplex. Disappearance of the footprints by dithiothreitol-treated tachyplesin I and Ala-tachyplesin strongly suggests a significant contribution of secondary structure containing an antiparallel beta-sheet to the DNA binding of tachyplesin I. This is the first report on DNA interaction with a small peptide which contains a unique antiparallel beta-sheet structure. The mechanism for antimicrobial action of tachyplesin I has also been inferred.

• Cons BM, Fox KR
• Effects of the antitumor antibiotic mithramycin on the structure of repetitive DNA regions adjacent to its GC-rich binding site.
• Biochemistry. 1991; 30: 6314-21
• Display abstract

• Regions of An.Tn, (GA)n.(TC)n, and (GT)n.(AC)n have been cloned into the SmaI (CCC/GGG) site of plasmid pUC19. HindIII-EcoRI restriction fragments containing these inserts have been used as substrates for footprinting experiments using DNase I, DNase II, and micrococcal nuclease as probes. These present good mithramycin binding sites (GGG) flanking repetitive regions to which the drug does not bind. In each case, mithramycin footprints are observed at the CCC/GGG sites, which are not affected by the nature of the surrounding sequences. Some weaker binding is detected at TCGA and ACCA sites and at regions of alternating GA. No binding is found to regions of alternating GT. An.Tn inserts (n = 23 or 69) are normally resistant to cleavage by all these probes; in the presence of mithramycin, a dramatic increase in DNase I cleavage is observed throughout the entire insert and is indicative of an alteration in DNA structure. Similar changes are seen with DNase II and micrococcal nuclease. These changes cannot be explained by invoking changes in the ratio of free substrate to cleavage agent. In contrast, cleavage of (GA)n.(CT)n and (GT)n.(AC)n inserts is not affected by drug binding. The results are consistent with a model in which mithramycin causes dramatic changes in the width of the DNA minor groove, generating a structure which has some properties of A-DNA, and suggest that this can be propagated into surrounding DNA regions in a sequence-dependent manner. The structural alterations with An.Tn are highly cooperative and can be transmitted over at least three turns of the DNA helix.

• Veaute X, Fuchs RP
• Polymorphism in N-2-acetylaminofluorene induced DNA structure as revealed by DNase I footprinting.
• Nucleic Acids Res. 1991; 19: 5603-6
• Display abstract

• In this paper, we have constructed double stranded helices (60-mers) containing a single N-2-acetylaminofluorene (-AAF) adduct covalently bound to one of the three guanine residues of the Narl site (G1G2CG3CC). This sequence was identified as a strong frameshift mutation hot spot for many carcinogens that bind to the C8 position of guanine. Using DNase I as a probe for DNA conformation we show i) that the average size of the helix deformation extends over 3 to 5 base pairs in both directions from the adduct site, and ii) that there is a strong polymorphism in the adduct induced DNA conformation. The present study supports the idea that adducts induce specific sequence dependent local conformational changes in DNA that are differentially recognized and processed by the enzymatic machineries that lead to repair or mutagenesis.

• Bishop KD, Borer PN, Huang YQ, Lane MJ
• Actinomycin D induced DNase I hypersensitivity and asymmetric structure transmission in a DNA hexadecamer.
• Nucleic Acids Res. 1991; 19: 871-5
• Display abstract

• DNase I cleavage rates and nmr chemical shifts are shown to change for DNA sequences distal to an intercalated actinomycin D molecule in a duplex hexadecamer upon drug binding. Both sets of observations suggest that the source of these changes is a DNA-mediated structural response. The nmr results imply the response is transmitted preferentially in a 5'-to-3' direction from the drug binding site. An inequivalent response of the two strands to a ligand-induced conformational change immediately suggests a mechanism for distinguishing the sense and antisense strands of DNA.

• Murchie AI, Carter WA, Portugal J, Lilley DM
• The tertiary structure of the four-way DNA junction affords protection against DNase I cleavage.
• Nucleic Acids Res. 1990; 18: 2599-606
• Display abstract

• The accessibility of phosphodiester bonds in the DNA of four-way helical junctions has been probed with the nuclease DNase I. Regions of protection were observed on all four strands of the junctions, that tended to be longer on the strands that are exchanged between the coaxially stacked pairs of helices. The protected regions on the continuous strands of the stacked helices were not located exactly at the junction, but were displaced towards the 3' side of the strand. This is the region of backbone that becomes located in the major groove of the opposed helix in the non-crossed, right-handed structure for the junction, and might therefore be predicted to be protected against cleavage by an enzyme. However, the major grooves of the structure remain accessible to the much smaller probe dimethyl sulphate.

• Fox KR, Sansom CE, Stevens MF
• Footprinting studies on the sequence-selective binding of pentamidine to DNA.
• FEBS Lett. 1990; 266: 150-4
• Display abstract

• The sequence-selective binding of pentamidine, an antimicrobial aromatic diamidine, has been investigated by footprinting studies on two different DNA fragments using DNase I, micrococcal nuclease and hydroxyl radical as probes. Each probe reveals drug-induced protection from cleavage in AT-rich regions. The best binding sites consist of at least 5 consecutive AT base pairs. Three or less AT pairs do not constitute a pentamidine binding site.

• Mendoza R, Markovits J, Jaffrezou JP, Muzard G, Le Pecq JB
• DNase I susceptibility of bent DNA and its alteration by ditercalinium and distamycin.
• Biochemistry. 1990; 29: 5035-43
• Display abstract

• The bending of kinetoplast DNA from Crithidia fasciculata is thought to be related to the periodic distribution of AA or TT cluster sequences. The sensitivity to DNase I of the two strands of this DNA was analyzed at nucleotide resolution by sequencing gel electrophoresis. The effect on the DNase I cleavage pattern of two drugs, ditercalinium and distamycin, that are able to remove bending was analyzed. The same analysis was done on a pBR 322 DNA fragment of random sequence as a control. The periodic distribution of the AA or TT clusters in the bent DNA fragment was first analyzed by computing the autocorrelation function of the AA or TT clusters in the bent DNA fragment. It is shown that the AT tracts are on average 10.5 base pairs apart. This value is almost identical with that of the B-DNA helix pitch in solution [10.5 (Wang, 1979); 10.6 +/- 0.1 (Rhodes & Klug, 1980)]. To reveal the periodic pattern of DNase I cleavage on this bent DNA, alone or in presence of drugs, the cross correlation between the different bands obtained from DNAse I cleavage and the presence of AA or TT sequences was computed. This shows that GC and mixed sequences are the most sensitive regions. These data also suggest that there is a periodic fluctuation in the width of the minor groove in the bent fragment. Ditercalinium and distamycin alter the DNase I cutting pattern of the bent DNA fragment but in an inverse fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

• Brukner I, Jurukovski V, Savic A
• Sequence-dependent structural variations of DNA revealed by DNase I.
• Nucleic Acids Res. 1990; 18: 891-4
• Display abstract

• Two global helix parameters important for DNA-DNase I interaction are the geometry of the minor groove and the DNA stiffness that resists bending toward major groove. Thus, local averaging of P-O3' bonds cutting frequencies (InP) reflects global helix parameters revealed by DNase I. Using the approximation that locally averaged InP values depend only on the type of the dinucleotide steps involved in the region of interaction, we calculated the collective contribution (sigma Dd) for ten different dinucleotide steps. Our results suggest that, at the first approximation, global varying helix parameters revealed by DNase I, might be predicted from sequence. Obtained sigma Dd function can be used as a sequence-dependent measure of protein-induced DNA flexure in the direction towards the major groove, which is usually connected to widening of the minor groove. In the course of analysis of Mg2+ and Mn2+ dependent DNase I digestions, no significant difference was found, in spite of the supposed differences in enzyme activity. These results suggest that if the second Mn2(+)-dependent active site exists, its activity is lower than that of the first one.

• Brotherton TW, Zenk DW
• Bovine pancreatic DNase I binds very tightly to DNA fragments and may be mistaken for putative endogenous nuclear proteins covalently bound to DNA.
• Biochem Biophys Res Commun. 1990; 166: 443-8
• Display abstract

• Using published methods for the isolation of nuclear proteins tightly bound to DNA, and resistant to removal by SDS or 16-BAC detergent and urea, several new protein bands in the region of 55 kd and 62 kd on SDS gel and 43 kd and 70 kd on 16--BAC gel electrophoresis were identified in extracts of avian erythroid nuclei. These bands were radiolabelled by subjecting the DNA--protein complexes to nick--translation in the presence of [32P]--dCTP, followed by prolonged digestion with excess bovine DNase I. Amino acid sequence analysis shows that these bands contain DNase I. These results indicate that DNase I can form stable complexes with DNA, and suggest that DNase I--DNA complexes may be mistakenly identified as nuclear proteins covalently bound to DNA.

• Hogan ME, Roberson MW, Austin RH
• DNA flexibility variation may dominate DNase I cleavage.
• Proc Natl Acad Sci U S A. 1989; 86: 9273-7
• Display abstract

• In a previous experimental study, we proposed that the bending and torsional stiffness of DNA display a systematic sequence dependence. Subsequently, we developed an elastic strain model to quantify the sequence dependence of the bending and torsional rigidity in terms of nearest neighbor interactions and used that model to analyze the sequence dependence of the 434 repressor binding to its operator. The analysis presented here shows that, in the absence of significant local variation of DNA secondary structure, DNase I cleavage is strongly correlated with local variation in the bending flexibility as calculated from our elastic strain model and that the agreement is also quantitatively significant. It is proposed that analysis using elastic strain models will provide a preliminary set of biochemical and chemical tools to explore the relation between DNA flexibility and the binding of other proteins.

• Ramesh N, Brahmachari SK
• Structural alteration from non-B to B-form could reflect DNase I hypersensitivity.
• J Biomol Struct Dyn. 1989; 6: 899-906
• Display abstract

• Preferential cleavage of active genes by DNase I has been correlated with a structurally altered conformation of DNA at the hypersensitive site in chromatin. To have a better understanding of the structural requirements for gene activation as probed by DNase I action, digestability by DNase I of synthetic polynucleotides having the ability to adopt B and non-B conformation (like Z-form) was studied which indicated a marked higher digestability of the B-form of DNA. Left handed Z form present within a natural sequence in supercoiled plasmid also showed marked resistance towards DNase I digestion. We show that alternating purine-pyrimidine sequences adopting Z-conformation exhibit DNAse I foot printing even in a protein free system. The logical deductions from the results indicate that 1) altered structure like Z-DNA is not a favourable substrate for DNase I, 2) both the ends of the alternating purine-pyrimidine insert showed hypersensitivity, 3) B-form with a minor groove of 12-13 A is a more favourable substrate for DNase I than an altered structure, 4) any structure of DNA deviating largely from B form with a capacity to flip over to the B-form are potential targets for the DNase I enzymic probes in naked DNA.

• Portugal J
• A model for the ability of drugs to induce enhanced DNase I cleavage.
• FEBS Lett. 1989; 251: 8-12
• Display abstract

• A common property of sequence-selective DNA-binding drugs lies in their ability to induce an enhanced DNase I cleavage in regions surrounding their binding sites. A hypothetical model to explain the enhancements induced by drug binding to the minor-groove of DNA is presented. It involves the participation of three different single models: a mass action effect produced by the enzyme redistribution after drug binding; changes in the minor groove width size; and interactions between the enzyme and the drug, so increasing the cleavage in places located close to the binding site. The model is tested by using statistical data analysis. The hypothetical model might explain the experimental results better than any of the single models alone, but these models also appear to render significant results.

• Lu M, Guo Q, Seeman NC, Kallenbach NR
• DNase I cleavage of branched DNA molecules.
• J Biol Chem. 1989; 264: 20851-4
• Display abstract

• We report here a potentially useful signature of branched DNA structures. The base 5' to the branch and the five bases flanking the 3' side of the branch site are protected from cleavage by DNase I in both three- and four-arm branched DNA molecules. Our procedure is to measure the cleavage profile for each 5' -labeled strand in a control duplex and compare this with that of the same strand in a branched structure under conditions yielding less than one cut per strand. The resulting cleavage pattern in an immobile four-arm junction is roughly 2-fold symmetric, consistent with the pattern of Fe(II).EDTA-induced cleavage that has been observed previously. In the three-arm junction, the DNase I cleavage pattern is asymmetric, indicating lack of 3-fold symmetry. A variable pattern of protection occurs to the 5' side of the branch in some strands only for both three- and four-arm junctions, extending 2-4 residues 5' to the branch.

• Kimura K, Morinaga T, Miyata N, Kawanishi G
• Nucleotide sequence of SN-07 chromophore binding site.
• J Antibiot (Tokyo). 1989; 42: 1838-43
• Display abstract

• DNase I footprinting was used to investigate binding sites for SN-07 chromophore on DNA fragments prepared from pBR322. Six sites were protected on about 150 base pair DNA fragments by SN-07 chromophore, but not by related anthracycline antibiotics from DNase I digestion. All the protected sites contained the dinucleotide sequence 5'-GC-3', but no other regularities could be discerned. A drug-induced conformational change of DNA was suggested by enhancement of DNase I sensitivity between the protected sites. These results support covalent interaction of the carbinolamine function of SN-07 chromophore to 2-amino group of guanine residues.

• Kuwahara J, Sugiura Y
• Sequence-specific recognition and cleavage of DNA by metallobleomycin: minor groove binding and possible interaction mode.
• Proc Natl Acad Sci U S A. 1988; 85: 2459-63
• Display abstract

• The DNase I cleavage-inhibition analysis shows binding sites of approximately 2 or 3 base pairs--in particular, 5' N-G-C sequences--for the green-colored CoIII and fully oxidized FeIII complexes of bleomycin. The apparent binding constant of the bleomycin-CoIII complex is quite similar for glucosylated and nonglucosylated phage T4 DNAs, whereas poly[d(I-C)] clearly gives a smaller binding constant than does poly[d(G-C)]. In contrast to the covalent attachment of the guanine N-7 with aflatoxin B1, the modification of the guanine 2-amino group with anthramycin remarkable inhibits the DNA cleavages at 5' G-C and 5' G-T sites by the FeIII and CoIII complex systems of bleomycin. These results strongly indicate that metallobleomycin binds in the minor groove of B-DNA and that the 2-amino group of guanine adjacent to the 5' side of the cleaved pyrimidine base is one key element of the specific 5' G-C or G-T recognition by the bleomycin-metal complex. A possible binding mode of metallobleomycin in the DNA helix has been proposed by computer-constructed model building.

• Ward B, Dabrowiak JC
• Stability of DNase I in footprinting experiments.
• Nucleic Acids Res. 1988; 16: 8724-8724

• Lobanenkov VV
• [Occurrence of DNA structures which differ from the canonic B-form in sites of highly specific interaction with chromatin proteins]
• Mol Biol (Mosk). 1988; 22: 77-85
• Display abstract

• According to the three-dimensional structure of DNase I and the mechanism of its action on linear double-stranded DNA, helix regions in conformations considerably different from the canonical B-form should be resistant to endonucleolysis. A number of DNA sequences specifically bound by nonhistone factors within 5'-flanking regions of the chicken beta A-globin, beta H-globin and c-myc genes are shown to contain short DNase I-resistant DNA domains. Several examples of the occurrence of such DNase I-resistant domains within the sites for high-specific recognition by different proteins are given. The role of the DNA structural polymorphism in site-specific interaction with protein factors is discussed.

• Fox KR
• Footprinting studies on the interactions of nogalamycin, arugomycin, decilorubicin and viriplanin with DNA.
• Anticancer Drug Des. 1988; 3: 157-68
• Display abstract

• DNase I footprinting studies employing several DNA fragments have confirmed that nogalamycin binds preferentially to regions of DNA containing alternating purines and pyrimidines. Arugomycin and viriplanin A, related compounds which contain additional sugar residues at both ends of the molecule, produce similar patterns of nuclease protection though at higher drug concentrations. The pattern induced by decilorubicin, which has charged groups at both ends of the aglycone, differs in many details and this analogue appears to display a modified DNA sequence selectivity. The results have been confirmed by similar studies using DNase II. All four compounds increase the susceptibility of certain adenine residues to modification by diethylpyrocarbonate. The results suggest an intercalative mode of binding for these nogalamycin analogues, and reveals an increased complexity in compounds which can bind to DNA by this mechanism.

• Rachkus IA, Kanopkaite SI
• [Effect of various factors on the methylation of DNAse I]
• Biokhimiia. 1988; 53: 278-82
• Display abstract

• The effects of NaCl, EDTA and tRNA on methylation and enzymatic properties of deoxyribonuclease I (DNase I) were investigated. The methylation was carried out by S-methylmethionine (vitamin U) in the phosphate-citric buffer pH 4.0. It was found that 0.5 M NaCl decreases by about 30% the incorporation of CH3-groups into the DNase, whereas 1.5 M NaCl-by 47%. A similar, although a less pronounced effect, was exerted by tRNA within the concentration range of 1.36-34.7 microM. On the contrast, EDTA (0.01-0.05 M) stimulated the incorporation of CH3-groups by 15 and 30%, respectively. The functional properties of methylated DNase I in the presence of EDTA remained unaffected. The enzyme methylation in the presence of NaCl or tRNA caused deceleration of the 3H-DNA hydrolysis (by 15-30%) only within the first 20 min of the reaction.

• Suck D, Lahm A, Oefner C
• Structure refined to 2A of a nicked DNA octanucleotide complex with DNase I.
• Nature. 1988; 332: 464-8
• Display abstract

• The cutting rates of bovine pancreatic deoxyribonuclease I (DNase I) vary along a given DNA sequence, indicating that the enzyme recognizes sequence-dependent structural variations of the DNA double-helix. In an attempt to define the helical parameters determining this sequence-dependence, we have co-crystallized a complex of DNase I with a self-complementary octanucleotide and refined the crystal structure at 2 A resolution. This structure confirms the basic features of an early model, namely that an exposed loop of DNase I binds in the minor groove of B-type DNA and that interactions do occur with the backbone of both strands. Nicked octamer duplexes that have lost a dinucleotide from the 3'-end of one strand are hydrogen-bonded across a two-fold axis in the crystal to form a quasi-continuous double helix of 14 base pairs. The DNA 14-mer has a B-type conformation and shows substantial distortion of both local and overall helix parameters, induced mainly by the tight interaction of Y73 and R38 in the unusually wide minor groove. Directly coupled to the widening of the groove by approximately 3A is a 21.5 degree bend of the DNA away from the bound enzyme towards the major groove, suggesting that both DNA stiffness and groove width are important in determining the sequence-dependence of the enzyme cutting rate. A second cut of the DNA which is induced by diffusion of Mn2+ into the co-crystals suggests that there are two active sites in DNase I separated by more than 15A.

• Davis SJ, Burgoyne LA
• The DNase I generated disomal series is coherent to 16N. Implications for coiling models of chromatin structure.
• FEBS Lett. 1987; 226: 88-90
• Display abstract

• The disomal series generated by the digestion of chromatin with DNase I has been followed to its highest orders using Klenow end-labelling and field-inversion gel electrophoresis to maximise the resolution of large DNA fragments. The series is coherent to the 16N level and as such is incompatible with the most structurally acceptable coiling models. We propose that this is evidence for the general unsuitability of coiling models and is support for the existence of simple 'back-to-back' double-stranded structures within chromatin.

• Price CM, Cech TR
• Telomeric DNA-protein interactions of Oxytricha macronuclear DNA.
• Genes Dev. 1987; 1: 783-93
• Display abstract

• Telomeres of Oxytricha macronuclear chromatin exist as discrete nonnucleosomal DNA-protein complexes, each of which encompasses the terminal 100-150 bp of a macronuclear DNA molecule. We have used chemical and nuclease footprinting to examine the internal structure of these telomeric complexes. Remarkably salt-stable DNA-protein interactions result in methylation-protection of specific guanine residues in the 3'-terminal T4G4T4G4 tail. The methylation pattern seen in vivo and in isolated macronuclei is reconstituted in vitro when purified 55-kD and 43-kD telomere proteins are added to purified macronuclear DNA. Very different interactions are observed between protein and DNA within the region approximately 45-135 bp from the 5' terminus. The DNase I cleavage pattern indicates that this DNA lies on the outside surface of protein but is not part of a nucleosome. Our data suggest that the telomeric complexes have two structural domains characterized by their dissimilar DNA-protein interactions. We propose that functionally equivalent telomeres from other organisms could be accommodated in a similar telomeric chromatin structure.

• Ward B, Rehfuss R, Dabrowiak JC
• Quantitative footprinting analysis of the netropsin-DNA interaction.
• J Biomol Struct Dyn. 1987; 4: 685-95
• Display abstract

• The results of a series of quantitative footprinting experiments of the netropsin-DNA interaction as studied using two different DNA cleaving probes, the enzyme DNase I and a cationic manganese porphyrin complex, are described. Plots of the relative change in oligonucleotide concentration as a function of drug concentration, covering approximately 110 base pairs of a DNA restriction fragment, revealed netropsin induced changes in the cleavage rates of both probes. These appeared as inhibitions for the binding sites, enhancements where no binding took place, and enhancement/inhibitions for the weak binding sites. Determination of the concentration of drug necessary to reduce the amount of a particular oligomer to half of its initial value allowed a ranking of the affinities of the various binding sites on the fragment. In addition to uncovering the location of a number of overlapping netropsin binding sites, the data allowed additional insight on the manner in which both probes alter their DNA cleavage rates in the drug-footprinting experiment.

• Fish EL, Vournakis JN
• Properties of DNase I digestion of the deoxyoligonucleotide: 5'd(ATCGTACGAT)2(3').
• Nucleic Acids Res. 1987; 15: 9417-28
• Display abstract

• Deoxyribonuclease I digestion of the deoxyoligodecamer 5'd(ATCGTACGAT)2(3') has been examined in detail to study the kinetic and structural properties of this enzyme substrate system in solution. In addition, these studies have defined, in general, those DNase I conditions to be used in future drug-DNA footprinting experiments. Special attention has been taken of those properties of DNase I that are critical for quantitation of ligand binding to small DNA fragments, and that aid in designing oligomers to be used in footprinting experiments. Enzyme activity was observed at all phosphodiester bonds in the decamer studied with varying affinity, except for the first four bonds at the 5' end of the oligomer. The DNA substrate concentration is always in excess, in order to achieve conditions of no more than one DNase I cleavage per DNA molecule. Reactions were controlled so that 65% or more of the initial amount of decamer substrate remained after DNase I digestion. It was observed that the rate of enzyme reactivity decreases with digestion time and is sensitive to the experimental conditions.

• Hurley LH, Needham-VanDevanter DR, Lee CS
• Demonstration of the asymmetric effect of CC-1065 on local DNA structure using a site-directed adduct in a 117-base-pair fragment from M13mp1.
• Proc Natl Acad Sci U S A. 1987; 84: 6412-6
• Display abstract

• Using DNase I and Alu I endonuclease analysis of a site-directed CC-1065-[N3-adenine]DNA adduct in a 117-base-pair fragment from M13mp1 DNA, we have demonstrated that CC-1065 produces an asymmetric effect on DNA conformation that extends more than one helix turn to the 5' side of the covalently modified adenine. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis, which is believed to mediate its cytotoxic effects through covalent binding to DNA. Previous studies have demonstrated that CC-1065 binds covalently to N3 of adenine and lies within the minor groove of DNA spanning a 4-base-pair sequence to the 5' side of the modified adenine. DNase I footprinting of this site-directed CC-1065-DNA adduct on the noncovalently modified strand shows that inhibition of cleavage occurs over a 12-base region, which is bordered on the 3' side by a site of 2-fold enhancement of cleavage. On the covalently modified strand a much less pronounced inhibition/enhancement pattern of cleavage occurs as far as 11 bases to the 5' side of the covalently modified adenine. While Hae III is able to cleave the DNA on both strands on the 3' side of the covalently modified adenine, Alu I is only able to cleave the covalently modified strand on the 5' side of the adduct. By taking into account the recently published structure of DNase I, we are able to interpret these results and develop a model for the effect of CC-1065 on local DNA structure. In this model, we propose selective drug-induced distortion of the covalently modified strand as a consequence of the alkylation of adenine by CC-1065.

• Fox KR, Waring MJ, Brown JR, Neidle S
• DNA sequence preferences for the anti-cancer drug mitoxanthrone and related anthraquinones revealed by DNase I footprinting.
• FEBS Lett. 1986; 202: 289-94
• Display abstract

• The interaction has been studied of several anthraquinone-based intercalating drugs, including the anti-cancer agent mitoxantrone, with defined sites of DNA. A 160 base pair DNA sequence from tyrT was employed for footprinting with DNase I. The anthraquinones had aminoalkylamino substituents in various positions of the ring system. Inhibition of enzymatic cutting of the DNA was observed at various positions on the sequence, mostly around some of the pyrimidine-3',5'-purine sites. Enhancements to cutting were observed clustered around AT-rich regions. The compounds showed differences in detailed footprinting behaviour, which have been related to differences in their mode of interaction with DNA as found in earlier computer modelling studies.

• Oefner C, Suck D
• Crystallographic refinement and structure of DNase I at 2 A resolution.
• J Mol Biol. 1986; 192: 605-32
• Display abstract

• The structure of bovine pancreatic deoxyribonuclease I (DNase I) has been refined at 2 A resolution using the restrained parameter, reciprocal least-squares procedure of Hendrickson and Konnert. The conventional R-factor for 16,104 reflections with I greater than or equal to 3 sigma (I) from 6.0 to 2.0 A resolution is 0.157. Bond lengths and angles of the refined structure are close to ideal values with root-mean-square (r.m.s.) deviations of 0.023 A and 1.4 degrees, respectively. The r.m.s. deviation of short non-bonded contacts from the sum of van der Waals' radii is 0.18 A. The orientation of side-chains shows a clear trimodal distribution of chi 1-angles at -60 degrees, 180 degrees, 60 degrees (in the order of preference) corresponding to staggered conformations. The chemically determined sequence was corrected at four positions, the major correction being an insertion of the tripeptide Ile-Val-Arg between Arg27 and Arg28. Extended hydrophobic regions in between, and on either side of, the two central six-stranded beta-pleated sheets are mainly responsible for the low average isotropic temperature factor of 11.9 A2 for the 2033 protein atoms. Besides the flexible loop region between Gly97 and Gly102 (Glu99 and Ser100 are disordered) and the carbohydrate side-chain, which both extend into a large solvent channel, only the exposed loop Arg70 to Lys74 shows elevated thermal mobility. The longest of the eight helices in DNase I, together representing 26% of the structure, has a 22 degree kink and consists of two alpha-helical segments (residues 136 to 144 and 145 to 155) separated by a 3(10)-helical turn. DNase I fragments 1 to 120 and 121 to 257 can be superimposed by an approximate 2-fold axis (r.m.s. deviation 1.49 A for 61 equivalent C alpha positions), suggesting that the enzyme might be the result of gene duplication. The two Ca2+ bound to DNase I under crystallization conditions are important for its structural integrity by stabilizing the surface loop Asp198 to Thr204 and limiting the region of high thermal mobility in the flexible loop to residues Gly97 to Gly102. The N-linked carbohydrate side-chain attached to Asn18 is of the high-mannose type with a branching point at the mannose residue in position 3.(ABSTRACT TRUNCATED AT 400 WORDS)

• Bromley SD, Ward BW, Dabrowiak JC
• Cationic porphyrins as probes of DNA structure.
• Nucleic Acids Res. 1986; 14: 9133-48
• Display abstract

• The DNA binding specificity of a group of cationic manganese porphyrin complexes has been examined using DNase I footprinting methodology and by observing the sites of porphyrin-induced DNA strand scission in the presence of potassium superoxide. The compounds, which possess systematic changes in total charge, its distribution on the periphery on the macrocycle and ligand shape, bind in the minor groove of AT rich regions of DNA. While changes in total charge and charge arrangement do not significantly influence specificity, a shape change which blocks close ligand contact with the minor groove relaxes the original AT specificity causing the compound to cleave at both AT and GC sites. The observed changes in binding sequence specificity were interpreted in terms of electrostatic and steric factors associated with both the compounds and DNA.

• Kabsch W, Mannherz HG, Suck D
• Three-dimensional structure of the complex of actin and DNase I at 4.5 A resolution.
• EMBO J. 1985; 4: 2113-8
• Display abstract

• The shape of an actin subunit has been derived from an improved 6 A map of the complex of rabbit skeletal muscle actin and bovine pancreatic DNase I obtained by X-ray crystallographic methods. The three-dimensional structure of DNase I determined independently at 2.5 A resolution was compared with the DNase I electron density in the actin:DNase map. The two structures are very similar at 6 A resolution thus leading to an unambiguous identification of actin as well as DNase I electron density. Furthermore the correct hand of the actin structure is determined from the DNase I atomic structure. The resolution of the actin structure was extended to 4.5 A by using a single heavy-atom derivative and the knowledge of the atomic coordinates of DNase I. The dimensions of an actin subunit are 67 A X 40 A X 37 A. It consists of a small and a large domain, the small domain containing the N terminus. Actin is an alpha,beta-protein with a beta-pleated sheet in each domain. These sheets are surrounded by several alpha-helices, comprising at least 40% of the structure. The phosphate peak of the adenine nucleotide is located between the two domains. The complex of actin and DNase I as found in solution (i.e., the actin:DNase I contacts which do not depend on crystal packing) was deduced from a comparison of monoclinic with orthorhombic crystals. Residues 44-46, 51, 52, 60-62 of DNase I are close to a loop region in the small domain of actin. At a distance of approximately 15 A there is a second contact in the large domain in which Glu13 of DNase I is involved. A possible binding region for myosin is discussed.

• Suck D, Oefner C, Kabsch W
• Three-dimensional structure of bovine pancreatic DNase I at 2.5 A resolution.
• EMBO J. 1984; 3: 2423-30
• Display abstract

• The three-dimensional structure of bovine pancreatic deoxyribonuclease I (DNase I) has been determined at 2.5 A resolution by X-ray diffraction from single crystals. An atomic model was fitted into the electron density using a graphics display system. DNase I is an alpha, beta-protein with two 6-stranded beta-pleated sheets packed against each other forming the core of a 'sandwich'-type structure. The two predominantly anti-parallel beta-sheets are flanked by three longer alpha-helices and extensive loop regions. The carbohydrate side chain attached to Asn 18 is protruding by approximately 15 A from the otherwise compact molecule of approximate dimensions 45 A X 40 A. The binding site of CA2+-deoxythymidine-3',5'-biphosphate (Ca-pdTp) has been determined by difference Fourier techniques confirming biochemical results that the active centre is close to His 131. Ca-pdTp binds at the surface of the enzyme between the two beta-pleated sheets and seems to interact with several charged amino acid side chains. Active site geometry and folding pattern of DNase I are quite different from staphylococcal nuclease, the only other Ca2+-dependent deoxyribonuclease whose structure is known at high resolution. The electron density map indicates that two Ca2+ ions are bound to the enzyme under crystallization conditions.

• Fox KR, Waring MJ
• DNA structural variations produced by actinomycin and distamycin as revealed by DNAase I footprinting.
• Nucleic Acids Res. 1984; 12: 9271-85
• Display abstract

• The technique of DNAase I footprinting has been used to investigate preferred binding sites for actinomycin D and distamycin on a 160-base-pair DNA fragment from E. coli containing the tyr T promoter sequence. Only sites containing the dinucleotide step GpC are protected by binding of actinomycin, and all such sites are protected. Distamycin recognizes four major regions rich in A + T residues. Both antibiotics induce enhanced rates of cleavage at certain regions flanking their binding sites. These effects are not restricted to any particular base sequence since they are produced in runs of A and T by actinomycin and in GC-rich sequences by distamycin. The observed increases in susceptibility to nuclease attack are attributed to DNA structural variations induced in the vicinity of the ligand binding site, most probably involving changes in the width of the helical minor groove.

• Suck D, Kabsch W, Mannherz HG
• Three-dimensional structure of the complex of skeletal muscle actin and bovine pancreatic DNAse I at 6-A resolution.
• Proc Natl Acad Sci U S A. 1981; 78: 4319-23
• Display abstract

• The structure of rabbit skeletal muscle actin complexed with bovine pancreatic DNase I has been determined by x-ray crystallographic methods at 6-A resolution. The analysis was based on a new orthorhombic crystal form, space group P212121, with one complex in the asymmetric unit. Six isomorphous heavy-atom derivatives yielding an overall figure of merit of 0.72 have been used to calculate the electron-density map. Molecular models for actin and DNase I derived from this map have dimensions 67 X 40 X 37 A and 50 X 50 X 40 A, respectively. The actin molecule is elongated and consists of a larger and a smaller domain, each containing density regions resembling a central beta-pleated sheet surrounded by alpha-helices. The highest electron-density peak is found in the cleft between the two domains, perhaps indicating the bound ATP. Observed crystal contacts between actin molecules and a model for the F-actin filament are discussed. Two high-affinity Ca2+-binding sites which also bind Ba2+ have been located at the surface of the DNase I molecule.

• Rhodes D, Klug A
• Helical periodicity of DNA determined by enzyme digestion.
• Nature. 1980; 286: 573-8
• Display abstract

• The periodicity of DNA has been determined by binding short, stiff pieces of DNA to a flat surface and using DNase I to probe the accessibility of the phosphodiester bonds. We have found that the distance between successive DNase I cutting sites is 10.6 +/- 0.1 bases for the DNA immobilized on three different surfaces. We identify this value with the number of base pairs per turn of the DNA double helix in solution.

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