RESUMO
We describe the results of a systematic study, using electron microscopy, of the effects of ionic strength on the morphology of chromatin and of H1-depleted chromatin. With increasing ionic strength, chromatin folds up progressively from a filament of nucleosomes at approximately 1 mM monovalent salt through some intermediate higher-order helical structures (Thoma, F., and T. Koller, 1977, Cell 12:101-107) with a fairly constant pitch but increasing numbers of nucleosomes per turn, until finally at 60 mM (or else in approximately 0.3 mM Mg++) a thick fiber of 250 A diameter is formed, corresponding to a structurally well-organized but not perfectly regular superhelix or solenoid of pitch approximately 110 A as described by Finch and Klug (1976, Proc. Natl. Acad. Sci. U.S.A. 73:1897-1901). The numbers of nucleosomes per turn of the helical structures agree well with those which can be calculated from the light-scattering data of Campbell et al. (1978, Nucleic Acids Res. 5:1571-1580). H1-depleted chromatin also condenses with increasing ionic strength but not so densely as chromatin and not into a definite structure with a well-defined fiber direction. At very low ionic strengths, nucleosomes are present in chromatin but not in H1-depleted chromatin which has the form of an unravelled filament. At somewhat higher ionic strengths (greater than 5 mM triethanolamine chloride), nucleosomes are visible in both types of specimen but the fine details are different. In chromatin containing H1, the DNA enters and leaves the nucleosome on the same side but in chromatin depleted of H1 the entrance and exit points are much more random and more or less on opposite sides of the nucleosome. We conclude that H1 stabilizes the nucleosome and is located in the region of the exit and entry points of the DNA. This result is correlated with biochemical and x-ray crystallographic results on the internal structure of the nucleosome core to give a picture of a nucleosome in which H1 is bound to the unique region on a complete two-turn, 166 base pair particle (Fig. 15). In the formation of higher-order structures, these regions on neighboring nucleosomes come closer together so that an H1 polymer may be formed in the center of the superhelical structures.
Assuntos
Cromatina/ultraestrutura , Histonas/fisiologia , Nucleossomos/ultraestrutura , Técnicas Citológicas , Magnésio/farmacologia , Microscopia Eletrônica , Concentração Osmolar , Conformação Proteica , Cloreto de Sódio/farmacologiaRESUMO
Histone H1 is proposed to serve a structural role in nucleosomes and chromatin fibers, to affect the spacing of nucleosomes, and to act as a general repressor of transcription. To test these hypotheses, a gene coding for a sea urchin histone H1 was expressed from the inducible GAL1 promoter in Saccharomyces cerevisiae by use of a YEp vector for high expression levels (strain YCL7) and a centromere vector for low expression levels (strain YCL1). The H1 protein was identified by its inducibility in galactose, its apparent molecular weight, and its solubility in 5% perchloric acid. When YCL7 was shifted from glucose to galactose for more than 40 h to achieve maximal levels of H1, H1 could be copurified in approximately stoichiometric amounts with core histones of Nonidet P-40-washed nuclei and with soluble chromatin fractionated on sucrose gradients. While S. cerevisiae tolerated the expression of low levels of H1 in YCL1 without an obvious phenotype, the expression of high levels of H1 correlated with greatly reduced survival, inhibition of growth, and increased plasmid loss but no obvious change in the nucleosomal repeat length. After an initial induction, RNA levels for GAL1 and H1 were drastically reduced, suggesting that H1 acts by the repression of galactose-induced genes. Similar effects, but to a lower extent, were observed when the C-terminal tail of H1 was expressed.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Plasmídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Animais , Northern Blotting , Fracionamento Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Histonas/biossíntese , Cinética , Mitose , Nucleossomos/ultraestrutura , Ligação Proteica , RNA Fúngico/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/crescimento & desenvolvimento , Ouriços-do-Mar/genética , Fatores de TempoRESUMO
Transcription of yeast class III genes involves the formation of a transcription initiation complex that comprises RNA polymerase III (Pol III) and the general transcription factors TFIIIB and TFIIIC. Using a genetic screen for positive regulators able to compensate for a deficiency in a promoter element of the SNR6 gene, we isolated the NHP6A and NHP6B genes. Here we show that the high-mobility-group proteins NHP6A and NHP6B are required for the efficient transcription of the SNR6 gene both in vivo and in vitro. The transcripts of wild-type and promoter-defective SNR6 genes decreased or became undetectable in an nhp6ADelta nhp6BDelta double-mutant strain, and the protection over the TATA box of the wild-type SNR6 gene was lost in nhp6ADelta nhp6BDelta cells at 37 degrees C. In vitro, NHP6B specifically stimulated the transcription of SNR6 templates up to fivefold in transcription assays using either cell nuclear extracts from nhp6ADelta nhp6BDelta cells or reconstituted transcription systems. Finally, NHP6B activated SNR6 transcription in a TFIIIC-independent assay. These results indicate that besides the general transcription factors TFIIIB and TFIIIC, additional auxillary factors are required for the optimal transcription of at least some specific Pol III genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase III/genética , RNA Fúngico , RNA Nuclear Pequeno , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas HMGN , Proteínas de Grupo de Alta Mobilidade/genética , Mutagênese , Proteínas Nucleares/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , TATA Box , Transativadores/genética , Transcrição Gênica , Ativação TranscricionalRESUMO
Poly(dA.dT) sequences (T-tracts) are abundant genomic DNA elements with unusual properties in vitro and an established role in transcriptional regulation of yeast genes. In vitro T-tracts are rigid, contribute to DNA bending, affect assembly in nucleosomes and generate a characteristic pattern of CPDs (cyclobutane pyrimidine dimers) upon irradiation with UV light (UV photofootprint). In eukaryotic cells, where DNA is packaged in chromatin, the DNA structure of T-tracts is unknown. Here we have used in vivo UV photofootprinting and DNA repair by photolyase to investigate the structure and accessibility of T-tracts in yeast promoters (HIS3, URA3 and ILV1). The same characteristic photofootprints were obtained in yeast and in naked DNA, demonstrating that the unusual T-tract structure exists in living cells. Rapid repair of CPDs in the T-tracts demonstrates that these T-tracts were not folded in nucleosomes. Moreover, neither datin, a T-tract binding protein, nor Gcn5p, a histone acetyltransferase involved in nucleosome remodelling, showed an influence on the structure and accessibility of T-tracts. The data support a contribution of this unusual DNA structure to transcriptional regulation.
Assuntos
DNA Fúngico/química , DNA Fúngico/genética , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Conformação de Ácido Nucleico , Nucleossomos/fisiologia , Poli A/genética , Poli T/genética , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae , Leveduras/genética , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Bases , Cromossomos Fúngicos/química , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Cromossomos Fúngicos/efeitos da radiação , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Pegada de DNA , Reparo do DNA/genética , DNA Fúngico/metabolismo , DNA Fúngico/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribodipirimidina Fotoliase/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Genoma Fúngico , Histona Acetiltransferases , Hidroliases/genética , Maleabilidade , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Raios Ultravioleta , Leveduras/enzimologia , Leveduras/efeitos da radiaçãoRESUMO
DNA damage formation and repair are tightly linked to protein-DNA interactions in chromatin. We have used minichromosomes in yeast as chromatin substrates in vivo to investigate how nucleotide excision repair (NER) and repair by DNA-photolyase (photoreactivation) remove pyrimidine dimers from an origin of replication ( ARS1 ). The ARS1 region is nuclease sensitive and flanked by nucleosomes on both sides. Photoreactivation was generally faster than NER at all sites. Site-specific heterogeneity of repair was observed for both pathways. This heterogeneity was different for NER and photoreactivation and it was altered in a minichromosome where ARS1 was transcribed. The results indicate distinct inter-actions of the repair systems with protein complexes bound in the ARS region (ORC, Abf1) and a predominant role of photolyase in CPD repair of an origin of replication.
Assuntos
Reparo do DNA , DNA Fúngico/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Origem de Replicação , Saccharomyces cerevisiae/genética , Dano ao DNA , DNA Fúngico/efeitos da radiação , Cinética , Dímeros de Pirimidina/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Raios UltravioletaRESUMO
Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in a yeast minichromosome, having a galactose-inducible GAL1:URA3 fusion gene, a constitutively expressed HIS3 gene and varied regions of chromatin structure. Transcription of GAL1:URA3 increased >150-fold, while HIS3 expression decreased <2-fold when cells were switched from glucose to galactose medium. Following galactose induction, four nucleosomes were displaced or rearranged in the GAL3-GAL10 region. However, no change in nucleosome arrangement was observed in other regions of the minichromosome following induction, indicating that only a few plasmid molecules actively transcribe at any one time. Repair at 269 cis-syn CPD sites revealed moderate preferential repair of the transcribed strand of GAL1:URA3 in galactose, consistent with transcription-coupled repair in a fraction of these genes. Many sites upstream of the transcription start site in the transcribed strand were also repaired faster upon induction. There is remarkable repair heterogeneity in the HIS3 gene and preferential repair is seen only in a short sequence immediately downstream of the transcription start site. Finally, a mild correlation of repair heterogeneity with nucleosome positions was observed in the transcribed strand of the inactive GAL1:URA3 gene and this correlation was abolished upon galactose induction.
Assuntos
Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Reparo do DNA , Dímeros de Pirimidina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Cromossomos Fúngicos/efeitos da radiação , DNA Fúngico/análise , DNA Fúngico/efeitos da radiação , Genes Reporter/genética , Modelos Genéticos , Plasmídeos , RNA Fúngico/análise , RNA Fúngico/efeitos da radiação , Fatores de Tempo , Transcrição Gênica , Raios UltravioletaRESUMO
To study mechanisms of nucleosome positioning, small circular plasmids were constructed, assembled into chromatin in vivo in Saccharomyces cerevisiae, and their chromatin structures were analysed with respect to positions of nucleosomes and nuclease-sensitive regions. Plasmids used include insertions of the URA3 gene into the TRP1 gene of the TRP1ARS1 circular plasmid in the same (TRURAP) or opposite (TRARUP) orientation. The URA3 gene has six precisely positioned, stable nucleosomes flanked by nuclease-sensitive regions at the 5' and 3' ends of the gene. Three of these nucleosome positions do not depend on the flanking nuclease-sensitive regions, since they are formed at similar positions in a derivative plasmid (TUmidL) that contains the middle of the URA3 sequence but not the 5' and 3' ends. These positions are probably due to protein-DNA interactions. In both TRURAP and TRARUP, the positions of the nucleosomes on the TRP1 gene were, however, shifted compared with the positions on the parental TRP1ARS1 circle and TUmidL. These changes are interpreted to be due to changes in the positions of flanking nuclease-sensitive regions that might act as boundaries to position nucleosomes. Thus, two independent mechanisms for nucleosome positioning have been demonstrated in vivo. The ARS1 region contains the 3' end of the TRP1 gene and the putative origin of replication. Since in TRURAP and TRARUP the TRP1 gene is interrupted, but the ARS1 region remains nuclease sensitive, this non-nucleosomal conformation of the ARS1 region probably reflects a chromatin structure important for replication.
Assuntos
Cromatina/análise , DNA Fúngico/metabolismo , Proteínas Fúngicas/metabolismo , Nucleossomos/análise , Plasmídeos , Saccharomyces cerevisiae/genética , Sequência de Bases , Mapeamento Cromossômico , Desoxirribonuclease I , Desoxirribonucleases , Genes Fúngicos , Nuclease do MicrococoRESUMO
We have attacked H1-containing soluble chromatin by alpha-chymotrypsin under conditions where chromatin adopts different structures. Soluble rat liver chromatin fragments depleted of non-histone components were digested with alpha-chymotrypsin in NaCl concentrations between 0 mM and 500 mM, at pH 7, or at pH 10, or at pH 7 in the presence of 4 M-urea. alpha-Chymotrypsin cleaves purified rat liver histone H1 at a specific initial site (CT) located in the globular domain and produces an N-terminal half (CT-N) which contains most of the globular domain and the N-terminal tail, and a C-terminal half (CT-C) which contains the C-terminal tail and a small part of the globular domain. Since in sodium dodecyl sulfate/polyacrylamide-gel electrophoresis CT-C migrates between the core histones and H1, cleavage of chromatin-bound H1 by alpha-chymotrypsin can be easily monitored. The CT-C fragment was detected under conditions where chromatin fibers were unfolded or distorted: under conditions of H1 dissociation at 400 mM and 500 mM-NaCl (pH 7 and 10); at very low ionic strength where chromatin is unfolded into a filament with well-separated nucleosomes; at pH 10 independent of the ionic strength where chromatin never assumes higher order structures; in the presence of 4 M-urea (pH 7), again independent of the ionic strength. However, hardly any CT-C fragment was detected under conditions where fibers are observed in the electron microscope at pH 7 between 20 mM and 300 mM-NaCl. Under these conditions H1 is degraded by alpha-chymotrypsin into unstable fragments with a molecular weight higher than that of CT-C. Thus, the data show that there are at least two different modes of interaction of H1 in chromatin which correlate with the physical state of the chromatin. Since the condensation of chromatin into structurally organized fibers upon raising the ionic strength starts by internucleosomal contacts in the fiber axis (zig-zag-shaped fiber), where H1 appears to be localized, it is likely that in chromatin fibers the preferential cleavage site for alpha-chymotrypsin is protected because of H1-H1 contacts. The data suggest that the globular part of H1 is involved in these contacts close to the fiber axis. They appear to be hydrophobic and to be essential for the structural organization of the chromatin fibers.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Cromatina , Histonas , Animais , Cromatina/ultraestrutura , Quimotripsina , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Modelos Moleculares , Concentração Osmolar , RatosRESUMO
We have studied in reconstitution experiments the conditions under which peptides derived from histones H1 and H5 are bound in chromatin and to what extent they are involved in the organization of chromatin fibers. The fragments of rat liver histone H1 (rH1) and chicken erythrocytes H1 (cH1) and H5 (cH5) used were the globular domains (rG-H1, cG-H1, cG-H5), the globular domain and the N-terminal tail (rCT-N), about half of the globular domain and the C-terminal tail (rNBS-C) and the C-terminal tail (rCT-C). Fragments containing the C-terminal tail (rNBS-C and rCT-C) dissociate from H1-depleted rat liver chromatin at 300 mM-NaCl and above (similar to uncleaved H1) and fragments lacking the C-terminal tail (rG-H1 and rCT-N) dissociate between 100 and 200 mM-NaCl. This suggests that at putative physiological ionic strengths the binding of rH1 is dominated by its C-terminal tail, whereas the globular region and the N-terminal tail might only be loosely bound or not bound at all and by this modulate chromatin structure. The globular domain of cH5 binds more tightly than that of the chicken and rat H1 and is only partially released at 200 mM. Since in the transcriptionally silent erythrocytes of birds H5 replaces H1 to a large extent, we suggest that the globular domain of H1 serves as a temporary seal and that of H5 as a permanent seal of the nucleosome. All the H1 and H5 peptides tested condensed and precipitated chromatin and H1-depleted chromatin: rNBS-C and rCT-C at lower peptide per nucleosome ratios than rG-H1, cG-H1 and rCT-N. At about one peptide per nucleosome none of the H1 fragments induced condensation similar to that of native chromatin. At a peptide per nucleosome ratio close to the point of precipitation, all H1 fragments, but not poly-L-lysine, induced similar compact forms which were fiberlike, although more irregular than the compact fibers of native chromatin. These reconstitution experiments suggest that both halves of H1 as well as the globular domain by itself are involved and capable in forming higher-order chromatin structures. Details of these structures are not known.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Animais , Sítios de Ligação , Precipitação Química , Galinhas , Cromatina/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Eritrócitos/análise , Fígado/análise , Microscopia Eletrônica , Concentração Osmolar , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Ratos , TripsinaRESUMO
Sites of an endogenous activity that has the properties of a DNA topoisomerase I have been identified on the palindromic ribosomal RNA genes of the slime mould Dictyostelium discoideum. This was done in vitro, by treating isolated nuclei with sodium dodecyl sulphate, which denatures topoisomerase during its cycle of nicking, strand passing and resealing, and hence reveals the DNA cleavages. It was also done in vivo using the drug camptothecin, which is believed to stabilize the cleavable complex of topoisomerase I plus DNA, hence increasing the chances of cleavage when sodium dodecyl sulphate is subsequently added. The cleavages in vitro and in vivo were mapped by indirect end-labelling. Both treatments cause what appear to be strong double-stranded cleavages at 200 and 2200 base-pairs and at 17 X 10(3) base-pairs upstream from the rRNA transcription start. The cleavage at 200 base-pairs was analysed in greater detail using RNA hybridization probes specific for single DNA strands. The cleavage is in fact composed of three closely spaced nicks on each DNA strand. The DNA sequence at each of the nicks is strongly homologous across 15 base-pairs. Sodium dodecyl sulphate-induced cleavage by eukaryotic topoisomerase I is known to yield enzyme covalently attached to the 3' cut end of the DNA. We show that protein-linked DNA restriction fragments with their 3' ends at the cleavage sites are selectively retarded on denaturing gels, which provides strong evidence that the unusual cluster of cleavages is caused by a topoisomerase I. Additionally, the camptothecin results revealed cleavages not only at the specific upstream sites, but also across the transcribed region. Interestingly, the zone of camptothecin-assisted cleavage does not extend as far at the 3' end of the gene as the zone of endogenous nuclease sensitivity.
Assuntos
Cromatina/metabolismo , Mapeamento Cromossômico , DNA Topoisomerases Tipo I/metabolismo , Genes , RNA Ribossômico/genética , Sequência de Bases , Sítios de Ligação , DNA Ribossômico/metabolismo , Dictyostelium , Transcrição GênicaRESUMO
To characterize nucleosome structure and positioning in the chromosomal context, the chromatin structure of the whole URA3 gene was studied in the genome and in a minichromosome by testing the accessibility of DNA to micrococcal nuclease and DNase I. The cutting patterns and hence the chromatin structures were almost indistinguishable in the genome and in the minichromosomes. The only notable exception was enhanced cutting between nucleosomes U3/U4 and U4/U5 in the minichromosomes. The results demonstrate that there is no severe constraint acting from outside the URA3 gene in chromosomes and minichromosomes. While low-resolution mapping showed six regions with a positioned nucleosome (U1 to U6), each region resolved in a complex pattern consistent with multiple overlapping positions. Some regions (U1, U4, U5 and U6) showed multiple positions with a dominant rotational setting (DNase I pattern), while U2 showed positioning within 10 bp but with no defined rotational setting, demonstrating that nucleosome positions were not in phase and not coordinately regulated. Reduced DNase I cutting from about 50 bp form the 5' end towards 3' end was common to all nucleosome regions. This polarity has been observed on isolated core particles. The results demonstrate that the DNase I pattern observed in vitro indeed reflects a structural property of nucleosomes in the chromosomal context. It is emphasized that despite the local heterogeneity revealed by high-resolution mapping, the low-resolution map is a reasonably accurate representation of the chromatin structure.
Assuntos
Cromatina/química , Cromossomos Fúngicos/genética , Proteínas Fúngicas/genética , Nucleossomos/química , Saccharomyces cerevisiae/genética , Sequência de Bases , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Pegada de DNA , Primers do DNA , Desoxirribonuclease I/metabolismo , Eletroforese em Gel de Ágar , Genoma Fúngico , Histonas/química , Histonas/metabolismo , Nuclease do Micrococo/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Nucleossomos/genética , Nucleossomos/ultraestrutura , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestruturaRESUMO
TRP1ARS1 is a circular yeast DNA of 1453 base-pairs that contains the N-5'phosphoribosyl anthranilate isomerase (TRP1) gene and a sequence important for autonomous replication (ARS1). It exists extrachromosomally in 100 to 200 copies/cell and is presumably packed in nucleosomes. TRP1ARS1 has been partially purified as chromatin from lysed spheroplasts of yeast using gel filtration. A structural analysis of mapping micrococcal nuclease and DNAase I cutting sites with an accuracy of +/- 20 base-pairs is presented. Comparison of nuclease cleavage sites in chromatin and in purified DNA reveals that regions which are protected against nuclease attack are not distributed randomly. These regions are big enough to accommodate nucleosome cores. Three nucleosomes are positioned in the so-called ARS sequences, and are stable at low and high levels of digestion. The TRP1 gene region is covered by four nucleosomes, but they are neither randomly arranged nor precisely positioned. They are not stable and rearrange or disintegrate during digestion. The nucleosomal regions are separated by two segments of DNA (A, B), each about 180 base-pairs long, which are very sensitive to DNAase I and micrococcal nuclease and therefore presumably not packed in nucleosomes. Region B is found 5' to the TRP1 gene and might be related to transcription, whereas region A is centered around the termination codon of the TRP1 gene and the putative origin of replication.
Assuntos
Aldose-Cetose Isomerases , Cromatina , DNA Circular , DNA Fúngico , Saccharomyces cerevisiae/genética , Composição de Bases , Carboidratos Epimerases/genética , Cromatina/isolamento & purificação , Mapeamento Cromossômico , Replicação do DNA , DNA Circular/isolamento & purificação , DNA Fúngico/isolamento & purificação , Desoxirribonuclease I , Eletroforese em Gel de Ágar , Genes , Nuclease do Micrococo , Nucleossomos , Transcrição GênicaRESUMO
We have analyzed the structure of complexes between DNA gyrase and four defined DNA fragments by electric dichroism. Both the extrapolated dichroism and relaxation time of these complexes suggest that a single turn of DNA is wrapped around the enzyme with the entry and exit points located close together. The average angle between the DNA tails emerging from the particle is about 120 degrees. This structure is consistent with that seen by electron microscopy. Addition of ATP or the non-hydrolyzable ATP analog 5'-adenylyl-beta, gamma-imidodiphosphate results in a structural change of the complex, consistent with the DNA tails now being wrapped around the protein. The significance of these observations with respect to the mechanism of DNA supercoiling by DNA gyrase is discussed.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Autorradiografia , Dicroísmo Circular , DNA/efeitos dos fármacos , DNA/ultraestrutura , Microscopia Eletrônica , Norfloxacino/farmacologiaRESUMO
Soluble rat liver chromatin was studied at pH 7 and at pH 9. In order to remove selectively non-histone components or non-histone components and histone H1, fractionation of chromatin was performed at pH 7 and pH 9 at different ionic strengths. The salt-dependent condensation of the fractionated chromatin was analysed in the electron microscope. There is no difference between the appearance of H1-depleted chromatin at poH 7 and pH 9. In H1-containing chromatin the shift from pH 7 to pH 9 leads to the following morphological changes: a) at very low ionic strength the nucleosomes unravel partially or totally and the zigzag-shaped fibres disappear in favour of beads-on-a-string; b) with increasing ionic strength the filaments condense into fibres, however, these fibres appear distorted and clearly less ordered than at pH 7. There is no indication of a release or displacement of histone H1. The pH-effect is completely reversible. The data suggest a pH-induced change in the mode of action of histone H1 in the formation of nucleosome beads and higher order chromatin structures.