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F Thoma

Publications and source records attributed to F Thoma.

16 recordsLinked to original sources
PubMed1992

Transcription, nucleosome stability, and DNA repair in a yeast minichromosome.

The template strand of the URA3 gene in the minichromosome YRpTRURAP is repaired of UV-induced cyclobutyl pyrimidine dimers (PD) much more efficiently than the nontemplate strand in growth-arrested Saccharomyces cerevisiae cells (Smerdon, M. J., and Thoma, F. (1990) Cell 61, 675). However, other regions of the plasmid are also efficiently repaired. We have examined the transcription and chromatin structure of these regions in growth-arrested cells to allow a more detailed comparison of transcription, nucleosome stability, and excision repair efficiency. Northern analysis, using strand-specific probes, indicates that four different transcripts are made from YRpTRURAP in addition to the URA3 mRNA in both growing and growth-arrested cells. The templates for these transcripts encompass all of the efficiently repaired regions outside of the URA3 gene. Nucleosome mapping indicates that the structure of the minichromosome in growth-arrested cells is indistinguishable from that of growing cells except for two nucleosomes in the region 500-800 base pairs 5' of the URA3 gene which become much less stable in growth-arrested cells. Comparison of the distribution of YRpTRURAP topoisomers in the two states, however, indicates that these nucleosomes are not lost from the majority of plasmid molecules. One of the four transcripts initiates in this region and increases by more than 5-fold in growth-arrested cells. Another transcript extends into a "slowly repaired" region which contains a very stable nucleosome. By determining the stability and relative amounts (at equilibrium) of the RNAs made from YRpTRURAP, transcription rates were determined and compared with the average PD repair rate for the different template regions. The results indicate that: 1) the rate of excision repair increases once a low, basal rate of transcription is achieved; 2) beyond this rate of transcription there is no simple correlation between the rates of transcription and PD repair; 3) nucleosome stability may "override" the coupling between transcription and repair if the transcription rate is low; and 4) at higher transcription rates, repair may be insensitive to nucleosome stability.

Base Sequence↗
PubMed1992

Species specific protein--DNA interactions may determine the chromatin units of genes in S.cerevisiae and in S.pombe.

Yeast genes, such as URA3, are chromatin units characterized by positioned nucleosomes and flanking nuclease sensitive regions (NSRs). To investigate the structural determinants at the chromatin level in vivo, the URA3 gene was dissected into three parts (U5', Umid and U3'), and the chromatin structures of the individual parts were analysed after insertion into minichromosomes and after chromatin assembly in vivo in Saccharomyces cerevisiae. While nucleosome positions were altered on Umid, the 5'-end and the 3'-end of URA3 maintained their native structures (a positioned nucleosome and a NSR each) independent of the site or orientation of insertion. This suggests that the chromatin unit of the native URA3 gene is dominated by strong protein boundaries at the 5'- and 3'-ends. In an alternative approach, we investigated whether nucleosome positions or NSRs were maintained when the whole URA3 gene was placed on a shuttle vector and assembled into chromatin by Schizosaccharomyces pombe providing different proteins, but the same nucleosomal spacing. In a complementary exchange experiment, the ade6 gene of S.pombe was shuttled to S.cerevisiae. In spite of a general conservation of histone proteins and nucleosome core structures, neither nucleosome positions nor NSRs were maintained in the heterologous background. The results demonstrate that chromatin structures are species specific and that the structural boundaries of yeast genes may be dominated by strong species specific protein-DNA interactions.

Chromatin↗
PubMed1992

Artificial nucleosome positioning sequences tested in yeast minichromosomes: a strong rotational setting is not sufficient to position nucleosomes in vivo.

DNA sequences that support bending around the histone octamer ('rotational setting') are considered to be a major determinant of nucleosome positions. TG5 is an artificial positioning sequence containing 100 bp of an (A/T)3NN(G/C)3NN motif repeated with a 10 bp period. It provides a strong rotational setting and is superior to natural sequences in nucleosome formation in vitro [Shrader, T.E. and Crothers, D.M. (1989) Proc. Natl. Acad. Sci. USA, 86, 7418-7422]. To investigate the contribution of the rotational setting to nucleosome positioning in vivo, TG sequences were inserted in a nucleosome, at the edge of a nucleosome and in a nuclease sensitive region of yeast minichromosomes and the chromatin structures were analysed. In none of the constructs were TG sequences folded in a positioned nucleosome, demonstrating that the rotational setting played a subordinate role in the rough positioning in vivo. The rotational setting might fine tune the positions. Positioned nucleosomes were found overlapping the ends of TG, indicating that a discontinuity of the 10 bp periodicity of (A/T)3 and (G/C)3 near the centre of a nucleosome might be favourable for positioning and serve as a translational signal.

Base Sequence↗
PubMed1990

Poly(dA).poly(dT) rich sequences are not sufficient to exclude nucleosome formation in a constitutive yeast promoter.

It was suggested that poly(dA).poly(dT) rich sequences in yeast Saccharomyces cerevisiae act as elements of constitutive promoters by exclusion of nucleosomes (Struhl, K. (1985). Proc. Natl. Acad. Sci. USA 82, 8419-8423). We have mapped the chromatin structure of the pet56-his3-ded1 region in minichromosomes and show that the poly(dA).poly(dT) sequences are located in nuclease sensitive regions. DNA fragments from the nuclease sensitive promoter region of DED1 were used for nucleosome reconstitution in vitro. We show that all sequences can form nucleosome cores and that the poly(dA).poly(dT) sequence can be incorporated in nucleosome cores. The results suggest that the nuclease sensitivity found in vivo is not established by poly(dA).poly(dT) mediated exclusion of nucleosomes.

Base Sequence↗
PubMed1990

Site-specific DNA repair at the nucleosome level in a yeast minichromosome.

The rate of excision repair of UV-induced pyrimidine dimers (PDs) was measured at specific sites in each strand of a yeast minichromosome containing an active gene (URA3), a replication origin (ARS1), and positioned nucleosomes. All six PD sites analyzed in the transcribed URA3 strand were repaired more rapidly (greater than 5-fold on average) than any of the nine PD sites analyzed in the nontranscribed strand. Efficient repair also occurred in both strands of a disrupted TRP1 gene (ten PD sites), containing four unstable nucleosomes, and in a nucleosome gap at the 5' end of URA3 (two PD sites). Conversely, slow repair occurred in both strands immediately downstream of the URA3 gene (12 of 14 PD sites). This region contains the ARS1 consensus sequence, a nucleosome gap, and two stable nucleosomes. Thus, modulation of DNA repair occurs in a simple yeast minichromosome and correlates with gene expression, nucleosome stability, and (possibly) control of replication.

Base Sequence↗
PubMed1990

DNA repair in a small yeast plasmid folded into chromatin.

The question of whether excision repair of yeast plasmids accurately reflects the repair of yeast genomic chromatin has yielded conflicting answers. These conflicts could have arisen from differences in the conformation of plasmid molecules used during these studies. We have examined excision repair of UV photoproducts in a small (2619 bp) autonomously replicating plasmid (YRp-TRURAP), known to be folded into chromatin with positioned nucleosomes in vivo, in the yeast Saccharomyces cerevisiae. A quantitative assay was used to measure the yield of cyclobutane pyrimidine dimers (PD) in plasmid DNA by measuring the fraction of Form I molecules resistant to T4 endonuclease V. After a UV dose of 100 J/m2, which yields 1.2 PD/plasmid in irradiated cells, radiation insensitive (wt) cells repair approximately 70% of the PD in TRURAP chromatin in 2 hr (a rate comparable to that of genomic chromatin). On the other hand, no measurable repair occurs in TRURAP chromatin in radiation sensitive cells (rad1) during the same time period. Thus, this small plasmid contains sufficient chromatin structure in vivo to reflect the incompetent repair of genomic chromatin seen in a rad mutant, while maintaining the competent repair level in wt cells.

Base Sequence↗
PubMed1988

Chromatin folding modulates nucleosome positioning in yeast minichromosomes.

Based on the chromatin structures of the yeast URA3 gene and the TRP1ARS1 circle, we have designed circular minichromosomes of different sizes that should each form a tight tetranucleosome. This structure was assumed to be stiff and bulky and therefore likely to be sensitive to packaging into a three-dimensional structure. The structures of the minichromosomes were determined using micrococcal nuclease. Only one of the minichromosomes showed a protected region of about 570 bp, compatible with the predicted tight tetranucleosome, while all other constructs showed alternative structures. A comparison of the structures revealed that neither histone-DNA interactions nor influences from flanking boundaries are sufficient determinants of nucleosome positions. The data strongly suggest that chromatin folding modulates the nucleosome arrangement along the DNA.

Chromatin↗
PubMed1988

Topoisomerase I cleavage sites identified and mapped in the chromatin of Dictyostelium ribosomal RNA genes.

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.

Base Sequence↗
PubMed1987

Structure of the DNA gyrase-DNA complex as revealed by transient electric dichroism.

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.

Autoradiography↗
PubMed1985

Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure.

We describe a model system for study of chromatin structure at levels above that of the nucleosome. A series of fragments with lengths ranging from 172 to 207 bp tandemly repeated three to greater than 50 times was prepared; each repeat contains the region important in forming a positioned core particle on a sea urchin 5S rRNA gene upon in vitro association with histones. The tandemly repeated sequences can be studied as linear DNA fragments or as relaxed or supercoiled circular molecules. A number of criteria indicate that nucleosomes position correctly on all the tandemly repeated elements. Measurement of the change in linking number per core particle led to a value of -1.0. Both length and repeat number dependent changes in conformation of the nucleoproteins are observed. We discuss the possibility that some ordered higher level chromatin structure can form with DNA and core histones alone.

Animals↗
PubMed1979

Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin.

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.

Chromatin↗
PubMed1977

Influence of histone H1 on chromatin structure.

Removal of histone H1 produces a transition in the structure of chromatin fibers as observed by electron microscopy. Chromatin containing all histone proteins appears as fibers with a diameter of about 250 A. The nucleosomes within these fibers are closely packed. If histone H1 is selectively removed with 50-100 mM NaCl in 50 mM sodium phosphate buffer (pH 7.0) in the presence of the ion-exchange resin AG 50 W - X2, chromatin appears as "beads-on-a-string" with the nucleosomes separated from each other by distances of about 150-200 A. If chromatin is treated in the presence of the resin with NaCl at concentrations of 650 mM or more, the structural organization of the chromatin is decreased, yielding fibers of irregular appearance.

Animals↗
PubMedDate not supplied

The ade6 gene of the fission yeast Schizosaccharomyces pombe has the same chromatin structure in the chromosome and in plasmids.

We have analysed the chromatin structure of the ade6 gene of Schizosaccharomyces pombe and its flanking regions both in the chromosome and in plasmids. The chromatin structure is independent of the chromosomal or extrachromosomal location. The ade6 gene contains eight precisely positioned nucleosomes on the 5' half, 'not positioned' nucleosomes around the 3' end and a nuclease-sensitive promoter region. Precisely positioned nucleosomes, but no nuclease-sensitive region were also detected on the ura4 gene in the chromosome and on a plasmid. The results show that S. pombe chromosomal and extrachromosomal genes have chromatin structures similar to those of S. cerevisiae and higher eukaryotes.

Chromatin↗