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1.
Proc Natl Acad Sci U S A ; 98(17): 9766-71, 2001 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-11493711

RESUMO

Type I DNA topoisomerases are ubiquitous enzymes involved in many aspects of DNA metabolism. Escherichia coli possesses two type I topoisomerase activities, DNA topoisomerase I (Topo I) and III (Topo III). The gene encoding Topo III (topB) can be deleted without affecting cell viability. Cells possessing a deletion of the gene encoding Topo I (topA) are only viable in the presence of an additional compensatory mutation. In the presence of compensatory mutations, Topo I deletion strains grow normally; however, if Topo III activity is repressed in these cells, they filament extensively and possess an abnormal nucleoid structure. These defects can be suppressed by the deletion of the recA gene, suggesting that these enzymes may be involved in RecA-mediated recombination and may specifically resolve recombination intermediates before partitioning.


Assuntos
Anáfase , Proteínas de Bactérias/fisiologia , Cromossomos Bacterianos/fisiologia , DNA Topoisomerases Tipo I/fisiologia , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Bactérias/genética , DNA Topoisomerases Tipo I/deficiência , DNA Topoisomerases Tipo I/genética , Escherichia coli/enzimologia , Deleção de Genes , Microscopia de Fluorescência , Fenótipo , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Recombinação Genética/genética , Serina Endopeptidases/genética , Supressão Genética
2.
Nature ; 411(6841): 1077-81, 2001 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-11429611

RESUMO

A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes-DNA topoisomerases-alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5' phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 A resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements.


Assuntos
DNA Topoisomerases Tipo I/química , DNA de Cadeia Simples/química , Sítios de Ligação , Cristalografia por Raios X , DNA Topoisomerases Tipo I/metabolismo , DNA de Cadeia Simples/metabolismo , Escherichia coli/enzimologia , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
3.
Mol Cell ; 7(2): 301-7, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11239459

RESUMO

Type IA DNA topoisomerases possess several domains forming a toroidal molecule with a central hole large enough to accommodate single- or double-stranded DNA. The sign inversion model predicts several protein-DNA intermediates, including those in which DNA is trapped within the hole. Opposing cysteine residues were incorporated into two independent domains surrounding the putative DNA binding cavity of E. coli topoisomerase III, creating a molecule that can be covalently closed or opened by oxidizing or reducing the disulfide bond. The formation of the disulfide bond allowed the trapping of single- and double-stranded DNA within the cavity of the enzyme and the identification of other intermediates proposed by the sign inversion model.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA/química , DNA/metabolismo , Escherichia coli/enzimologia , Conformação de Ácido Nucleico , Sítios de Ligação , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/classificação , DNA Topoisomerases Tipo I/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/classificação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Ditiotreitol/farmacologia , Escherichia coli/genética , Dissulfeto de Glutationa/farmacologia , Modelos Moleculares , Mutagênese , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Especificidade por Substrato
4.
Mol Microbiol ; 35(4): 888-95, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10692165

RESUMO

A 17-amino-acid residue domain has been identified in Escherichia coli DNA topoisomerase III (Topo III) that is essential for Topo III-mediated resolution of DNA replication intermediates in vitro. Deletion of this domain reduced Topo III-catalysed resolution of DNA replication intermediates and decatenation of multiply linked plasmid DNA dimers by four orders of magnitude, whereas reducing Topo III-catalysed relaxation of negatively supercoiled DNA substrates only 20-fold. The presence of this domain has been detected in multiple plasmid-encoded topoisomerases, raising the possibility that these enzymes may also be decatenases.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Escherichia coli/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Replicação do DNA , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , Dimerização , Escherichia coli/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
5.
Mol Cell ; 3(5): 611-20, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10360177

RESUMO

E. coli RecQ protein is a multifunctional helicase with homologs that include the S. cerevisiae Sgs1 helicase and the H. sapiens Wrn and Blm helicases. Here we show that RecQ helicase unwinds a covalently closed double-stranded DNA (dsDNA) substrate and that this activity specifically stimulates E. coli topoisomerase III (Topo III) to fully catenate dsDNA molecules. We propose that these proteins functionally interact and that their shared activity is responsible for control of DNA recombination. RecQ helicase has a comparable effect on the Topo III homolog of S. cerevisiae, consistent with other RecQ and Topo III homologs acting together in a similar capacity. These findings highlight a novel, conserved activity that offers insight into the function of the other RecQ-like helicases.


Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Recombinação Genética , DNA/genética , DNA/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Escherichia coli/enzimologia , Regulação Fúngica da Expressão Gênica , RecQ Helicases , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
6.
Biopolymers ; 49(1): 55-70, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10070262

RESUMO

Structural and dynamic properties of opioid peptide E have been examined in an sodium dodecyl sulfate (SDS) micelle. Structural and dynamic studies both indicate that this peptide exhibits greater segmental mobility than typical structured proteins. An nmr structural analysis of adrenal peptide E in SDS micelles indicated the presence of two well-defined beta-turns, one at the N-terminus encompassing residues 3 to 6, and the second in the region between residues 15 and 18. Certain side chain dihedral angles were also remarkably well defined, such as the chi 1 angle of F4, which exhibited a trans configuration. These calculated structures were based on a set of 9.5 restraints per residue. The backbone dynamics of peptide E in SDS micelles were examined through an analysis of 15N-relaxation parameters. An extended model-free analysis was used to interpret the relaxation data. The overall rotational correlation time is 19.7 ns. the average order parameter S2 is 0.66 +/- 0.15. The N-terminal loop region residues including G3 to R6 have an average order parameter of 0.70 +/- 0.23. The average order parameter lies somewhere between that observed for a random coil (e.g., S2 = 0.3) and that of a well-defined tertiary fold (e.g., S2 = 0.86). This suggests that peptide E in SDS micelles adopts a restricted range of conformations rather than a random coil. Based on the helical structure recently obtained for the highly homologous kappa-agonist dynorphin-A(1-17) and the beta-turn in the same region of peptide E, it is reasonable to assume that these two elements of secondary structure reflect different receptor subtype binding geometries. The intermediate order parameters observed for peptide E in an SDS micelle suggest a degree of dynamic mobility that may enable facile interconversion between helical and beta-turn geometries in the N-terminal agonist domain.


Assuntos
Encefalinas/química , Encefalinas/metabolismo , Receptores Opioides/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Receptores Opioides/classificação , Termodinâmica
8.
Protein Expr Purif ; 11(1): 86-94, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9325143

RESUMO

Structural studies on soluble proteins using nuclear magnetic resonance (NMR) spectroscopy and other structural methods in general require large quantities of isotopically enriched proteins. Human interleukin-5 is a disulfide-linked homodimeric cytokine implicated in asthmatic response. The development of a high yield overexpression system for human interleukin-5 is an important prerequisite to using modern multidimensional NMR in the characterization of the solution structure of the protein and to characterize interactions with a soluble receptor domain. Significant amounts of the protein were expressed using an optimized synthetic gene in a high yield expression system. Gene synthesis was accomplished through the ligation of six oligonucleotides composed of optimized codons. The ligated fragments were further amplified by a polymerase chain reaction and then subcloned into the T7 RNA polymerase based overexpression vector pET11a. However, the induced protein accumulated in the form of inclusion bodies. Initially, the protein was solubilized under denaturing conditions and purified in these denaturing conditions by passage through a single S-200 HR sizing column. Finally, protein refolding was initiated in the presence of 2 M urea followed by dialysis. This protocol yielded 40 mg of biologically active, isotope-enriched protein from 4 liters of minimal medium thus facilitating structural studies by NMR. The strategy described may be of immense value in the production of significant quantities of recombinant, eukaryotic proteins for structural and other studies.


Assuntos
Clonagem Molecular , Genes Sintéticos , Interleucina-5/genética , Dobramento de Proteína , Sequência de Bases , Clonagem Molecular/métodos , DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Expressão Gênica , Humanos , Interleucina-5/biossíntese , Interleucina-5/química , Isopropiltiogalactosídeo/farmacologia , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Plasmídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , Desnaturação Proteica , Proteínas Virais
9.
J Biol Chem ; 272(31): 19582-7, 1997 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-9235964

RESUMO

The polypeptide encoded by the plasmid RP4 traE gene shows extensive protein sequence similarity to Escherichia coli topB, the gene encoding DNA topoisomerase III (Topo III). The traE gene product has been cloned into a bacteriophage T7-based transient expression system, and the polypeptide has been expressed and purified. The TraE protein exhibits topoisomerase activity similar to that of Topo III. Relaxation is stimulated by high temperature and low concentrations of Mg2+. In addition, similar to E. coli Topo III, the TraE protein is a potent decatenase and can substitute for Topo III activity in vivo. The biochemical properties of the TraE protein in vitro suggest that the protein may be involved in the resolution of plasmid DNA replication intermediates either during vegetative replication or in conjugative DNA transfer. Putative homologues of Topo III have been found to be encoded by other broad host range, conjugative plasmids isolated from both Gram-negative and Gram-positive organisms, suggesting that Topo III-like polypeptides may have an essential role in the propagation of many promiscuous plasmids.


Assuntos
DNA Topoisomerases Tipo I/genética , Escherichia coli/enzimologia , Plasmídeos , Sequência de Aminoácidos , Replicação do DNA , DNA Super-Helicoidal/metabolismo , Dados de Sequência Molecular
10.
Biochemistry ; 36(19): 5658-68, 1997 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-9153405

RESUMO

Mutational studies directed at evaluating the effect of the axial ligand plane orientation on electrochemical properties of cytochrome b5 have been performed. As described in the previous paper, structural consequences of one of these mutations, the A67V mutation, have been evaluated using NMR solution methods. The lack of large shifts relative to the wild-type protein in both the imidazole Ndelta nitrogen and proton resonances of the H63 imidazole ring indicates that the hydrogen bond between the carbonyl of F58 and the imidazole ring of H63 remains intact in this mutant. Effects of the imidazole plane reorientation on the Fe d-orbitals were evaluated on the basis of interpretation of EPR spectra, near-infrared bands associated with ligand-to-metal charge transfer transitions, reorientation of the anisotropy of the paramagnetic center determined by calculation of pseudocontact shifts, and the temperature dependence of the contact-shifted resonances. The dominant effect of the imidazole reorientation appears to have been a destabilization of the d(xz) orbital energy and a reorientation of the d(pi) orbitals. This is surprising in light of the -20 mV shift in the reduction potential of the mutant relative to the wild-type protein and indicates that a destabilization of d(yz)-orbital energy level of the reduced state dictates the observed change in reduction potential. Measured values for the reorganizational energy and heterogeneous electron transfer rates were indistinguishable for wild-type and mutant proteins. This is perhaps surprising, given significant differences in the pattern of electron delocalization into the porphyrin ring observed as significantly altered contact shift patterns. Mutational studies perturbing the H39 imidazole were also performed but with more limited success.


Assuntos
Citocromos b5/química , Citocromos b5/genética , Mutagênese Sítio-Dirigida , Animais , Citocromos b5/isolamento & purificação , Eletroquímica , Espectroscopia de Ressonância de Spin Eletrônica , Heme/química , Ligantes , Espectroscopia de Ressonância Magnética , Oxirredução , Ratos , Espectroscopia de Luz Próxima ao Infravermelho
11.
Biochemistry ; 36(19): 5645-57, 1997 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-9153404

RESUMO

Mutants of cytochrome b5 were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V mutation achieved this goal through the substitution of a bulkier, hydrophobic ligand for a residue, in the sterically hindered hydrophobic heme binding pocket. Solution structures of mutant and wild-type proteins in the region of the mutation were calculated using restraints obtained from 1H and 15N 2D homonuclear and heteronuclear NMR spectra and 1H-15N 3D heteronuclear NMR spectra. More than 10 restraints per residue were used in the refinement of both structures. Average local rmsd for 20 refined structures was 0.30 A for the wild-type structure and 0.38 A for the A67V mutant. The transfer of amide proton resonance assignments from wild-type to the mutant protein was achieved through overlays of 15N-1H heteronuclear correlation spectra of the reduced proteins. Side chain assignments and sequential assignments were established using conventional assignment strategies. Calculation of the orientation of the components of the anisotropic paramagnetic susceptibility tensor, using methods similar to procedures applied to the wild-type protein, shows that the orientation of the in-plane components are identical in the wild-type and mutant proteins. However, the orientation of the z-component of the susceptibility tensor calculated for the mutant protein differs by 17 degrees for the A-form and by 11 degrees for the B-form from the orientation calculated for the wild-type protein. The rotation of the z-component of the susceptibility tensor (toward the delta meso proton) is in the same direction and is of the same magnitude as the rotation of the H63 imidazole ring induced by mutation.


Assuntos
Citocromos b5/química , Citocromos b5/genética , Imidazóis/química , Mutagênese Sítio-Dirigida , Amidas/química , Animais , Heme/química , Heme/genética , Ligantes , Espectroscopia de Ressonância Magnética , Conformação Proteica , Ratos , Soluções , Estereoisomerismo
12.
J Biomol NMR ; 8(2): 171-83, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8914273

RESUMO

Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain 13C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b5. The assignment of backbone 13C resonances was used to confirm previous 1H and 15N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329-8340]. On the basis of short- and medium-range NOEs and backbone 13C chemical shifts, the solution secondary structure of rat cytochrome b5 has been determined. The striking similarity of backbone 13C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the 13C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.


Assuntos
Citocromos b5/química , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Carbono/química , Isótopos de Carbono , Compostos Férricos/química , Hidrogênio/química , Espectroscopia de Ressonância Magnética/métodos , Magnetismo , Microssomos Hepáticos/enzimologia , Dados de Sequência Molecular , Nitrogênio/química , Isótopos de Nitrogênio , Ratos
13.
J Biol Chem ; 271(15): 9039-45, 1996 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-8621552

RESUMO

The role that the carboxyl-terminal amino acids of Escherichia coli DNA topoisomerase I (Topo I) and III (Topo III) play in catalysis was examined by comparing the properties of Topo III with those of a truncated enzyme lacking the generalized DNA binding domain of Topo III, Topo I, and a hybrid topoisomerase polypeptide containing the amino-terminal 605 amino acids of Topo III and the putative generalized DNA binding domain of Topo I. The deletion of the carboxyl-terminal 49 amino acids of Topo III decreases the affinity of the enzyme for its substrate, single-stranded DNA, by approximately 2 orders of magnitude and reduces Topo III-catalyzed relaxation of supercoiled DNA and Topo III-catalyzed resolution of DNA replication intermediates to a similar extent. Fusion of the carboxyl-terminal 312 amino acid residues of Topo I onto the truncated molecule stimulates topoisomerase-catalyzed relaxation 15-20-fold, to a level comparable with that of full-length Topo III. However, topoisomerase-catalyzed resolution of DNA replication intermediates was only stimulated 2-3-fold. Therefore, the carboxyl-terminal amino acids of these topoisomerases constitute a distinct and separable domain, and this domain is intimately involved in determining the catalytic properties of these polypeptides.


Assuntos
DNA Topoisomerases Tipo I/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Catálise , Replicação do DNA , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/química , Escherichia coli/enzimologia , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Proteínas Recombinantes de Fusão , Relação Estrutura-Atividade , Especificidade por Substrato
14.
Nat Struct Biol ; 3(4): 333-9, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8599759

RESUMO

The geometry of the ferricytochrome b5-ferricytochrome c complex has been analysed using long-range interprotein paramagnetic dipolar shifts. Heteronuclear filtered NMR spectra of samples containing 15N-labelled cytochrome b5 in complex with unlabelled cytochrome c allowed unambiguous assessment of pseudocontact shifts relative to diamagnetic reference states. Because pseudocontact shifts can be observed for protons as much as 20 A from the paramagnetic centre, this approach allows study of electron transfer proteins in fast exchange. Our findings provide the first physical evidence confirming hypotheses presented in previous theoretical studies. This absence of certain predicted shifts that are expected based on the best fit to a static model of the complex suggests that cytochrome b5 is more dynamic in solution than in the crystal, in agreement with molecular dynamics simulations.


Assuntos
Grupo dos Citocromos c/química , Citocromos b5/química , Espectroscopia de Ressonância Magnética/métodos , Animais , Simulação por Computador , Grupo dos Citocromos c/metabolismo , Citocromos b5/metabolismo , Elétrons , Microssomos Hepáticos/química , Radioisótopos de Nitrogênio , Ratos
15.
J Biol Chem ; 270(40): 23700-5, 1995 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-7559540

RESUMO

The binding of DNA topoisomerase III (Topo III) to a single-stranded DNA substrate containing a strong cleavage site has been examined. The minimal substrate requirement for Topo III-catalyzed cleavage has been determined to consist of 7 bases; 6 bases 5' to the cleavage site and only 1 base 3' to the site. Nuclease P1 protection experiments indicate that the enzyme also binds to its substrate asymmetrically, protecting approximately 12 bases 5' to the cleavage site and only 2 bases 3' to the cleavage site. A catalytically inactive mutant of Topo III shows the same protection pattern as the active polypeptide, indicating that Topo III is a site-specific binding protein as well as a topoisomerase. Consistent with this view, an oligonucleotide containing a cleavage site is a more effective inhibitor and is bound more efficiently by Topo III than an oligonucleotide without a cleavage site.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/enzimologia , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Escherichia coli/genética , Modelos Biológicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Especificidade por Substrato
17.
J Biol Chem ; 269(12): 9052-9, 1994 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-7510701

RESUMO

The nucleic acid-binding domain of Escherichia coli DNA topoisomerase III (Topo III) has been identified using a selection procedure designed to isolate inactive Topo III polypeptides. Deletion of this binding domain, contained in the carboxyl terminus of Topo III, results in a drastic reduction in the ability of the enzyme to bind to single-stranded DNA and RNA substrates. Successive truncation of the enzyme within this region results in the gradual loss of nucleic acid binding activity and in a gradual change in the mechanism of Topo III-catalyzed relaxation of negatively supercoiled DNA. The reduction of nucleic acid binding activity of the truncated polypeptides does not result in a loss of cleavage site specificity for the enzyme, suggesting that other amino acids are involved in the positioning of the nucleic acid within the nicking/closing site of the topoisomerase.


Assuntos
DNA Topoisomerases Tipo I/química , DNA Super-Helicoidal/metabolismo , Escherichia coli/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA Topoisomerases Tipo I/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Ligação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Relação Estrutura-Atividade
18.
J Biol Chem ; 269(3): 2093-9, 1994 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-8294462

RESUMO

oriC and pBR322 DNA replication, reconstituted with purified replication proteins, has been used to study the functional activities of Escherichia coli topoisomerase I, DNA gyrase, and topoisomerase III during the final stages of DNA replication. In the oriC system, DNA gyrase-catalyzed decatenation of daughter DNA molecules was very inefficient, whereas topoisomerase III could catalyze complete decatenation. In the pBR322 DNA replication system, almost all the daughter DNA molecules could be decatenated by DNA gyrase alone in the absence of salt. Decatenation by DNA gyrase in the pBR322 system was completely inhibited, without a concomitant inhibition of DNA synthesis, by the addition of physiological concentrations of salt. Topoisomerase III, however, could decatenate all of the daughter DNA molecules in the pBR322 system, even in the presence of high concentrations of salt. A similar effect could not be observed in the oriC system, because the addition of salt inhibited DNA synthesis. Topoisomerase I was incapable of catalyzing decatenation under any conditions examined in either the oriC or pBR322 replication system. The addition of topoisomerase I to the replication systems resulted only in an inhibition of DNA synthesis.


Assuntos
Replicação do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Bacteriano/biossíntese , Escherichia coli/enzimologia , Escherichia coli/genética , Plasmídeos , Trifosfato de Adenosina/metabolismo , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo II/isolamento & purificação , DNA Circular/biossíntese , Eletroforese em Gel de Poliacrilamida , Cinética
19.
J Biol Chem ; 267(29): 20532-5, 1992 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-1383203

RESUMO

Relaxation of superhelical DNA by Escherichia coli topoisomerase III (Topo III) was inhibited by the inclusion of tRNA in the reaction mixture. Investigation of the basis of this inhibition revealed that Topo III could bind RNA and establish a cleavage-religation equilibrium. The addition of SDS to these reaction mixtures induced cleavage of the RNA by Topo III. The nucleotide sequences of RNA and DNA cleavage sites were identical, although cleavage site preference differed. Thus, the possibility that Topo III can pass strands of RNA as well as strands of DNA must be considered in accounting for the role of this enzyme in nucleic acid metabolism.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Escherichia coli/enzimologia , RNA/metabolismo , Sequência de Bases , Cinética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/metabolismo , Oligorribonucleotídeos/metabolismo , RNA/biossíntese , Cloreto de Sódio/farmacologia , Especificidade por Substrato
20.
J Biol Chem ; 266(21): 13988-95, 1991 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-1856227

RESUMO

The Escherichia coli DNA replication proteins n and n" function in vitro in the assembly of the primosome, a mobile multiprotein replication priming complex thought to operate on the lagging-strand template at the E. coli DNA replication fork. Both proteins have been purified from E. coli HMS83 cells based on their requirement for the reconstitution of bacteriophage phi X174 complementary strand DNA synthesis in vitro with purified proteins. As a step toward understanding the role of these proteins in vivo, the genes for primosomal proteins n and n", designated priB and priC, respectively, have been cloned molecularly. priB encodes a 104-amino acid 11.4-kDa polypeptide and corresponds to an previously identified open reading frame between rpsF and rps R within a ribosomal protein operon at 95.5 min on the E. coli chromosome. priC encodes a 175-amino acid 20.3-kDa polypeptide. These two gene products were overexpressed at least 1000-fold in E. coli using a bacteriophage T7 transient expression system. Both proteins have been purified to apparent homogeneity from extracts prepared from these overproducing strains.


Assuntos
Proteínas de Bactérias/genética , Replicação do DNA , Proteínas de Escherichia coli , Escherichia coli/genética , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Códon , DNA Bacteriano/genética , Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Proteínas Recombinantes/isolamento & purificação
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