RESUMO
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.
Assuntos
DNA/metabolismo , Desoxirribonuclease BamHI/química , Desoxirribonuclease BamHI/metabolismo , Dobramento de Proteína , Composição de Bases , Sequência de Bases , Sítios de Ligação , Catálise , Gráficos por Computador , Cristalografia por Raios X , DNA/química , Desoxirribonuclease EcoRI/química , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
BACKGROUND: Type II restriction endonucleases recognize DNA sequences that vary between four to eight base pairs, and require only Mg2+ as a cofactor to catalyze the hydrolysis of DNA. Their protein sequences display a surprising lack of similarity, and no recurring structural motif analogous to the helix-turn-helix or the zinc finger of transcription factors, has yet been discovered. RESULTS: We have determined the crystal structure of restriction endonuclease BamHI at 1.95 A resolution. The structure was solved by combining phase information derived from multi-wavelength X-ray data by algebraic and maximum likelihood methods. The BamHI subunit consists of a central beta-sheet with alpha-helices on both sides. The dimer configuration reveals a large cleft which could accommodate B-form DNA. Mutants of the enzyme that are deficient in cleavage are located at or near the putative DNA-binding cleft. BamHI and endonuclease EcoRI share a common core motif (CCM) consisting of five beta-strands and two helices. It remains to be determined if other restriction enzymes also contain the CCM. CONCLUSIONS: The structure of BamHI provides the first clear evidence that there may be substantial structural homology amongst restriction enzymes, even though it is undetectable at the sequence level.
Assuntos
Desoxirribonuclease BamHI/química , Sequência de Aminoácidos , Cristalografia por Raios X/métodos , DNA/metabolismo , Análise Mutacional de DNA , Desoxirribonuclease BamHI/genética , Desoxirribonuclease EcoRI/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Homologia de Sequência de AminoácidosRESUMO
Amino acid residues Asn116 and Ser118 of the restriction endonuclease BamHI make several sequence-specific and water-bridged contacts to the DNA bases. An in vivo selection was used to isolate BamHI variants at position 116, 118 and 122 which maintained sequence specificity to GGATCC sites. Here, the variants N116H, N116H/S118G and S118G were purified and characterized. The variants N116H and N116H/S118G were found to have lost their ability to cleave unmethylated GGATCC sequences by more than two orders of magnitude, while maintaining nearly wild-type levels of activity on the N6-methyladenine-containing sequence, GGmATCC. In contrast, wild-type BamHI and variant S118G have only a three- to fourfold lower activity on unmethylated GGATCC sequences compared with GGmATCC sequences. The N116 to H116 mutation has effectively altered the specificity of BamHI from an endonuclease which recognizes and cleaves GGATCC and GGmATC, to an endonuclease which only cleaves GGmATCC. The N116H change of specificity is due to the lowered binding affinity for the unmethylated sequence because of the loss of two asparagine-DNA hydrogen bonds and the introduction of a favorable van der Waals contact between the imidazole group of histidine and the N6-methyl group of adenine.
Assuntos
Adenina/análogos & derivados , Desoxirribonuclease BamHI/genética , Desoxirribonuclease BamHI/metabolismo , Adenina/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Desoxirribonuclease BamHI/química , Escherichia coli/genética , Escherichia coli/metabolismo , Variação Genética , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , Engenharia de ProteínasRESUMO
Here, we investigate the highly specific interaction of the BamHI endonuclease with its cognate recognition sequence GGATCC by determining which amino acid residues can be substituted at the DNA interface while maintaining specificity. Mutational studies, together with the structural determination of the restriction endonuclease BamHI have revealed the amino acid residues which are involved in DNA catalysis and those which play a role in the specific binding of the enzyme to its cognate DNA recognition sequence. Amino acid residues N116, S118, R122, D154 and R155 are involved in DNA sequence recognition and are located in the major groove in close proximity to the nucleotide bases comprising the recognition sequence. Cassette mutagenesis of these amino acids, together with in vivo transcriptional interference selection, was used to identify an array of substitutions which maintain site-specific binding to the cognate GGATCC sequence. This approach has demonstrated the extent of acceptable variation among amino acid residues which are directly involved in site-specific binding. One variant, double mutant N116H, S118G was found to cleave DNA only when the adenine base in the recognition site is methylated.
Assuntos
Desoxirribonuclease BamHI/genética , Desoxirribonuclease BamHI/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Desoxirribonuclease BamHI/química , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Conformação ProteicaRESUMO
The interaction of BamHI endonuclease with DNA has been studied crystallographically, but has not been characterized rigorously in solution. The enzyme binds in solution as a homodimer to its recognition site GGATCC. Only six base-pairs are directly recognized, but binding affinity (in the absence of the catalytic cofactor Mg(2+)) increases 5400-fold as oligonucleotide length increases from 10 to 14 bp. Binding is modulated by sequence context outside the recognition site, varying about 30-fold from the bes t (GTG or TAT) to the worst (CGG) flanking triplets. BamHI, EcoRI and EcoRV endonucleases all have different context preferences, suggesting that context affects binding by influencing the free energy levels of the complexes rather than that of the free DNA. Ethylation interference footprinting in the absence of divalent metal shows a localized and symmetrical pattern of phosphate contacts, with strong contacts at NpNpNpGGApTCC. In the presence of Mg(2+), first-order cleavage rate constants are identical in the two GGA half-sites, are the same for the two nicked intermediates and are unaffected by substrate length in the range 10-24 bp. DNA binding is strongly enhanced by mutations D94N, E111A or E113K, by binding of Ca(2+) at the active site, or by deletion of the scissile phosphate GpGATCC, indicating that a cluster of negative charges at the catalytic site contributes at least 3-4 kcal/mol of unfavorable binding free energy. This electrostatic repulsion destabilizes the enzyme-DNA complex and favors metal ion binding and progression to the transition state for cleavage.
Assuntos
DNA/metabolismo , Desoxirribonuclease BamHI/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Alquilação , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Cátions Bivalentes/farmacologia , DNA/química , Pegada de DNA , Desoxirribonuclease BamHI/química , Metabolismo Energético , Cinética , Sondas Moleculares , Peso Molecular , Oligodesoxirribonucleotídeos/química , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Soluções , Eletricidade Estática , TermodinâmicaRESUMO
Restriction endonuclease BamHI from Bacillus amyloliquefaciens has been co-crystallized with a 12 bp DNA fragment that encompasses its recognition site. The co-crystals diffract to at least 1.95 A resolution and belong to space group P2(1)2(1)2(1). The unit cell parameters are a = 108.8 A, b = 81.9 A, c = 68.8 A, consistent with one complex in the crystallographic asymmetric unit. The direction of the DNA appears to be along the b axis. In order to achieve end to end stacking of DNA, the complex must lie on the screw axis along b. A self-rotation function has determined the directions of the non-crystallographic 2-fold axes.
Assuntos
DNA/química , Desoxirribonuclease BamHI/química , Sequência de Bases , Cristalização , Cristalografia por Raios X , DNA/metabolismo , Desoxirribonuclease BamHI/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Molecular , Ligação ProteicaRESUMO
Crystal structures have now been determined for the R.PvuII restriction endonuclease as a protein-DNA complex [Cheng et al., EMBO J. 13 (1994) 3927-3935; this report] and in apo-form [Athanasiadis et al., Nature Struc. Biol. 1 (1994) 469-475; our unpublished result]. The structures indicate how the interaction with DNA might proceed [Riddihough, Nature 370 (1994) 78].
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/química , Estrutura Secundária de Proteína , Sítios de Ligação , Cristalografia por Raios X , Substâncias Macromoleculares , Modelos MolecularesRESUMO
The crystal structures of the DNA-N6-adenine-methyltransferase M.TaqI, in complexes with the cofactor S-adenosyl-L-methionine (AdoMet) and the competitive inhibitor sinefungin (Sf) show identical folding of the polypeptide chains into two domains. The N-terminal domain carries the cofactor-binding site, the C-terminal domain is thought to be implicated in sequence-specific DNA binding. Model building of the M.TaqI-DNA complex suggests that the adenine to be methylated swings out of the double helix as found previously in the cytosine-C5-MTase HhaI DNA co-crystal structure. A torsion of the methionine moiety of the cofactor is required to bring the methyl group within reach of the swung-out base and allow methyl group transfer.
Assuntos
DNA/metabolismo , Estrutura Secundária de Proteína , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Glicina , Metionina , Modelos Moleculares , Dados de Sequência Molecular , S-Adenosilmetionina/metabolismoRESUMO
The cleavage site for the restriction endonuclease EcoRV has been found to be 5'-GAT/ATC-3', rather than 5'- GATAT /C-3' as reported earlier by Kholmina et al. [ Dokl . Akad . Nauk . 253 (1980) 495-497].
Assuntos
Enzimas de Restrição do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II , Sequência de Bases , Escherichia coli/enzimologia , Especificidade por SubstratoRESUMO
The nucleotide sequence of the genes encoding methyltransferase TaqI (M.TaqI) and restriction endonuclease TaqI (R.TaqI) with the recognition sequence, TCGA, were analyzed in clones isolated from independent libraries. The genes, originally reported as 363 and 236 codons long [Slatko et al., Nucleic Acids Res. 15 (1987) 9781-9796] were redetermined as 421 and 263 codons long, respectively. The C terminus of the taqIM gene overlaps the N terminus of the taqIR gene by 13 codons, as observed with the isoschizomeric TthHB8I restriction-modification system [Barany et al., Gene 112 (1992) 13-20]. Removal of the overlapping codons did not interfere with in vivo M.TaqI activity. We postulate the overlap plays a role in regulating taqIR expression.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Homologia de Genes/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Thermus/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Códon/genética , Desoxirribonucleases de Sítio Específico do Tipo II/química , Regulação Bacteriana da Expressão Gênica/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Thermus/genéticaRESUMO
M.FokI, a type-IIS modification enzyme from Flavobacterium okeanokoites, was purified, and its activity was characterized in vitro. The enzyme was found to be a DNA-adenine methyltransferase and to methylate both strands of the asymmetric FokI recognition sequence: (formula; see text) M.FokI does not methylate single-stranded DNA, nor does it methylate double-stranded DNA at sequences other than FokI sites.
Assuntos
Adenina/metabolismo , Flavobacterium/enzimologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Sequência de Aminoácidos , Cromatografia em Camada Fina , DNA/metabolismo , Flavobacterium/genética , Metilação , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/isolamento & purificação , Especificidade por SubstratoRESUMO
FokI is a type IIs restriction endonuclease which recognizes an asymmetric DNA sequence and cleaves DNA a short distance away from the sequence. The enzyme is bipartite in nature with its DNA recognition and cleavage functions located on distinct domains. We report here cocrystals of the complete FokI enzyme (579 amino acids) bound to a 20-bp DNA fragment containing its recognition sequence. The complex is amongst the largest protein-DNA complexes to be crystallized, and required macroseeding techniques for optimal crystal growth. The cocrystals diffract to at least 2.8 A in resolution and belong to space group P2(1) with unit cell dimensions of a=67.9 A, b=119.8 A, c=69.1 A, beta = 96.6 degrees. Using specific amino acid analysis we show that asymmetric unit contains a single FokI molecule bound to the 20-bp DNA fragment. This paper reports the first cocrystals of a type IIs restriction endonuclease.
Assuntos
DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/química , Cristalografia por Raios X , Fragmentação do DNA , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Especificidade por SubstratoRESUMO
This report describes the use of a biochemical tool that has been developed to aid in the manipulation of DNA. A DNA binding-proficient and cleavage-deficient BamHI mutant protein, E113K, was used in vitro to protect its recognition sequence (5'-GGATCC-3') against the catalytic action of site-specific endonuclease, exonuclease and methylase. In vitro conditions are reported here in which the E113K protein protects BamHI sites (5'-GGATCC-3') from cleavage by BamHI endonuclease or Sau3AI endonuclease (5'-GATC-3'); protects a neighboring restriction site 5'-CCCGGG-3' from SmaI endonuclease digestion; blocks methylation of 5'-GGATCC-3' by Dam methylase (5'-GATC-3'); and blocks Bal31 exonuclease progression at a BamHI site. The Bal31 procedure could be used to generate unidirectional deletions of a DNA fragment. The use of mutant endonucleases that are binding-proficient and cleavage-deficient to shield DNA from nuclease digestion or methylase modification expands the repertoire of methods to manipulate DNA in vitro.
Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA/química , Desoxirribonuclease BamHI/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica) , Sequência de Bases , Desoxirribonuclease BamHI/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Endodesoxirribonucleases/metabolismo , Metiltransferases/metabolismo , Dados de Sequência Molecular , MutaçãoRESUMO
A chemiluminescent DNA detection method is described and its application shown for both single-vector and multiplex DNA sequencing using the standard dideoxy chain-termination process. This recently developed detection method, which utilizes the light emitted by an enzyme-catalyzed dioxetane reaction, is highly sensitive and affords significant advantages in safety and speed over the traditional radioactive labeling method. When adapted to a multiplex strategy, this chemiluminescent detection method constitutes a safe, simple and rapid method for increasing the throughput of DNA sequencing procedures.
Assuntos
Sequência de Bases , DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Técnicas Genéticas , Vetores Genéticos/genética , Medições LuminescentesAssuntos
Bromodesoxicitidina/farmacologia , Desoxicitidina/análogos & derivados , Simplexvirus/metabolismo , Animais , Bromodesoxicitidina/metabolismo , Bromodesoxicitidina/uso terapêutico , Desoxicitidina/metabolismo , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Herpes Simples/tratamento farmacológico , Camundongos , Simplexvirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
An enzyme activity of Bacillus subtilis has been found that catalyzes the dephosphorylation and deamination of phosphohomoserine to alpha-ketobutyrate, resulting in a bypass of threonine in isoleucine biosynthesis. In crude extracts of a strain deficient in the biosynthetic isoleucine-inhibitable threonine dehydratase, phosphohomoserine was converted to alpha-ketobutyrate. Phosphohomoserine conversion to alpha-ketobutyrate was shown not to involve a threonine intermediate. Single mutational events affecting threonine synthetase also affected the phosphohomoserine-deaminating activity, suggesting that the deamination of phosphohomoserine was catalyzed by the threonine synthetase enzyme. It was demonstrated in vivo, in a strain deficient in the biosynthetic threonine dehydratase, that isoleucine was synthesized from homoserine without intermediate formation of threonine.
Assuntos
Bacillus subtilis/metabolismo , Butiratos/biossíntese , Homosserina/metabolismo , Transferases/metabolismo , Aminoidrolases/metabolismo , Bacillus subtilis/enzimologia , Isótopos de Carbono , Sistema Livre de Células , Desaminação , Hidroliases/metabolismo , Isoleucina/biossíntese , Cetonas/biossíntese , Mutação , Fosfatos/metabolismo , Treonina/metabolismo , TrítioRESUMO
A mutant BamHI endonuclease, E77K, belongs to a class of catalytic mutants that bind DNA efficiently but cleave DNA at a rate more than 10(3)-fold lower than that of the wild-type enzyme (S. Y. Xu and I. Schildkraut, J. Biol. Chem. 266:4425-4429, 1991). The preferred cofactor for the wild-type BamHI is Mg2+. BamHI is 10-fold less active with Mn2+ as the cofactor. In contrast, the E77K variant displays an increased activity when Mn2+ is substituted for Mg2+ in the reaction buffer. Mutations that partially suppress the E77K mutation were isolated by using an Escherichia coli indicator strain containing the dinD::lacZ fusion. These pseudorevertant endonucleases induce E. coli SOS response (as evidenced by blue colony formation) and thus presumably nick or cleave chromosomal DNA in vivo. Consistent with the in vivo result, the pseudorevertant endonucleases in the crude cell extract display site-specific partial DNA cleavage activity. DNA sequencing revealed two unique suppressing mutations that were located within two amino acid residues of the original mutation. Both pseudorevertant proteins were purified and shown to increase specific activity at least 50-fold. Like the wild-type enzyme, both pseudorevertant endonucleases prefer Mg2+ as the cofactor. Thus, the second-site mutation not only restores partial cleavage activity but also suppresses the metal preference as well. These results suggest that the Glu-77 residue may play a role in metal ion binding or in enzyme activation (allosteric transition) following sequence-specific recognition.
Assuntos
Desoxirribonuclease BamHI/metabolismo , Genes Supressores , Magnésio/metabolismo , Manganês/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Deleção Cromossômica , Códon/genética , DNA/metabolismo , Desoxirribonuclease BamHI/genética , Desoxirribonuclease BamHI/isolamento & purificação , Escherichia coli/genética , Dados de Sequência Molecular , Mutagênese , Transformação Genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
A new site-specific endonuclease, Sfi I, has been isolated from Streptomyces fimbriatus . This is the first report of a type II restriction endonuclease whose recognition specificity requires eight nucleotides. Sfi I cleaves the sequence, GGCCNNNN / NGGCC , symmetrically to produce a three base, 3' extension.