Fritz Thoma

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.

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.

Nuclease digestion of circular TRP1ARS1 chromatin reveals positioned nucleosomes separated by nuclease-sensitive regions

TRP1ARS1 is a circular yeast DNA of 1453 base-pairs that contains the N-5phosphoribosyl 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.

Protein-DNA interactions and nuclease-sensitive regions determine nucleosome positions on yeast plasmid chromatin.

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.

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.

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.

Modulations in Chromatin Structure During DNA Damage Formation and DNA Repair

It has long been recognized that the “target” of DNA-damaging agents and the “substrate” of DNA repair enzymes in eukaryotes is the highly compact and dynamic structure of chromatin. Understanding the modulation of DNA damage and repair in chromatin, as well as the modulation of chromatin structure by DNA damage and its repair processing, is necessary for understanding the fate of potential mutagenic and carcinogenic lesions in DNA. The central idea to be discussed in this chapter is that DNA damage, DNA repair (as well as other DNA-processing mechanisms), and chromatin structure are intimately associated in the cell (Fig. 1).