Theodor Koller

Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks.

The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.

Disruption of the nucleosomes at the replication fork.

The fate of parental nucleosomes during chromatin replication was studied in vitro using in vitro assembled chromatin containing the whole SV40 genome as well as salt‐treated and native SV40 minichromosomes. In vitro assembled minichromosomes were able to replicate efficiently in vitro, when the DNA was preincubated with T‐antigen, a cytosolic S100 extract and three deoxynucleoside triphosphates prior to chromatin assembly, indicating that the origin has to be free of nucleosomes for replication initiation. The chromatin structure of the newly synthesized daughter strands in replicating molecules was analysed by psoralen cross‐linking of the DNA and by micrococcal nuclease digestion. A 5‐ and 10‐fold excess of protein‐free competitor DNA present during minichromosome replication traps the segregating histones. In opposition to published data this suggests that the parental histones remain only loosely or not attached to the DNA in the region of the replication fork. Replication in the putative absence of free histones shows that a subnucleosomal particle is randomly assembled on the daughter strands. The data are compatible with the formation of a H3/H4 tetramer complex under these conditions, supporting the notion that under physiological conditions nucleosome core assembly on the newly synthesized daughter strands occurs by the binding of H2A/H2B dimers to a H3/H4 tetramer complex.

Characterization of the DNA binding activity of stable RecA-DNA complexes: interaction between the two DNA binding sites within RecA helical filaments

The DNA-binding, annealing and recombinational activities of purified RecA-DNA complexes stabilized by ATP gamma S (a slowly hydrolysable analog of ATP) are described. Electrophoretic analysis, DNase protection experiments and observations by electron microscopy suggest that saturated RecA complexes formed with single- or double-stranded DNA are able to accommodate an additional single strand of DNA with a stoichiometry of about one nucleotide of added single-stranded DNA per nucleotide or base-pair, respectively, of DNA resident in the complex. This strand uptake is independent of complementarity or homology between the added and resident DNA molecules. In the complex, the incoming and resident single-stranded DNA molecules are in close proximity as the two strands can anneal in case of their complementarity. Stable RecA complexes formed with single-stranded DNA bind double-stranded DNA efficiently when the added DNA is homologous to the complexed strand and then initiate a strand exchange reaction between the partner DNA molecules. Electron microscopy of the RecA-single-stranded DNA complexes associated with homologous double-stranded DNA suggests that a portion of duplex DNA is taken into the complex and placed in register with the resident single strand. Our experiments indicate that both DNA binding sites within RecA helical filaments can be occupied by either single- or double-stranded DNA. Presumably, the same first DNA binding site is used by RecA during its polymerization on single- or double-stranded DNA and the second DNA binding site becomes available for subsequent interaction of the protein-saturated complexes with naked DNA. The way by which additional DNA is taken into RecA-DNA complexes shows co-operative character and this helps to explain how topological problems are avoided during RecA-mediated homologous recombination.

High resolution physical mapping of specific binding sites of Escherichia coli RNA polymerase on the DNA of bacteriophage T7

Abstract The number and positions of the specific binding sites of RNA polymerase holoenzyme on bacteriophage T7 DNA were re-evaluated at a molar enzyme to DNA ratio of 14. From the genetically left end of the T7 genome specific binding sites were found to be located at 0·581(±0·056), 1·221(±0·083), 1·539(±0·088), 1·834(±0·10), 3·728(±0·095), 7·821(±0·137) and at 92·123(±0·218) map units (one unit is equal to 1% the length of T7 DNA). The data on the enzyme binding sites and the initiation sites for transcription reported in the literature were fitted into a model in which the T7 genome has four RNA polymerase binding sites in the so-called early promoter region at the extreme left, two sites at the beginnings of gene 0.7 and 1 , respectively, and one site close to the right end of the DNA. In conjunction with the biochemical data reported by Hsieh & Wang (1976), these results suggest that binding of RNA polymerase and initiation of RNA synthesis take place at the same or at nearby sites.

Chromatin structure of a hyperactive secretory protein gene (in Balbiani ring 2) of Chironomus.

We examined the chromatin structure of a Balbiani ring (secretory protein gene) in the salivary glands of Chironomus larvae in its hyperactive state after stimulation with pilocarpine. For the inactive state of the gene an established tissue culture cell line, not expressing the gene, was used. Electron microscopy showed an RNA polymerase density of approximately 38/microns. Micrococcal nuclease digestion of purified nuclei followed by DNA transfer and hybridization revealed a smear with no recognizable discrete DNA fragments. Without pilocarpine stimulation a faint nucleosomal repeat was superimposed upon the smear, and in tissue culture cells a clear nucleosomal repeat was revealed. The restriction enzyme XbaI, which has a 6‐bp recognition sequence, cut the gene in the hyperactive chromatin state, but not in its inactive conformation. The combined results are best explained by the absence of most of the nucleosomes in this hyperactive RNA polymerase II transcribed gene.

Nucleosome assembly in mammalian cell extracts before and after DNA replication.

Protein‐free DNA in a cytosolic extract supplemented with SV40 large T‐antigen (T‐Ag), is assembled into chromatin structure when nuclear extract is added. This assembly was monitored by topoisomer formation, micrococcal nuclease digestion and psoralen crosslinking of the DNA. Plasmids containing SV40 sequences (ori‐ and ori+) were assembled into chromatin with similar efficiencies whether T‐Ag was present or not. Approximately 50‐80% of the number of nucleosomes in vivo could be assembled in vitro; however, the kinetics of assembly differed on replicated and unreplicated molecules. In replicative intermediates, nucleosomes were observed on both the pre‐replicated and post‐replicated portions. We conclude that the extent of nucleosome assembly in mammalian cell extracts is not dependent upon DNA replication, in contrast to previous suggestions. However, the highly sensitive psoralen assay revealed that DNA replication appears to facilitate precise folding of DNA in the nucleosome.

Asymmetry of Dam remethylation on the leading and lagging arms of plasmid replicative intermediates

In Escherichia coli, adenine methylation at the sequence GATC allows coupling of cellular processes to chromosome replication and the cell cycle. The transient presence of hemimethylated DNA after replication facilitates post‐replicative mismatch repair, induces transcription of some genes and allows transposition of mobile elements. We were interested in estimating the half‐life of hemimethylated DNA behind the replication fork in plasmid molecules and in determining whether Dam methyltransferase restores N6 adenine methylation simultaneously on both replicative arms. We show that remethylation takes place asynchronously on the leading and lagging daughter strands shortly after replication. On the leading arm the fully methylated adenine is restored ∼2000 bp (corresponding to 2 s) behind the replication fork, while remethylation takes twice as long (at 3500–4000 bp or ∼3.5–4 s) on the lagging replicative arm. This observation suggests that Dam remethylation of the lagging arm requires ligated Okazaki fragments.

Torsional stress induces left-handed helical stretches in DNA of natural base sequence: circular dichroism and antibody binding.

Above a threshold of torsional stress, the c.d. spectrum of covalently closed circular DNA of natural base sequence acquires a Z‐like contribution and antibodies raised against Z‐DNA are bound. Mapping of the antibody binding sites by electron microscopy reveals sites which correlate with stretches enriched in alternating purine‐pyrimidine sequences and GC base pairs.

Formation and characterization of soluble complexes of histone H1 with supercoiled DNA

We have analyzed the interaction of rat liver histone H1 with superhelical DNA. Depending on the ratio of H1 to DNA and the concentration of salt, two different types of complexes were found. Above a critical ratio of H1 to DNA, called the aggregation point, large aggregates are formed, which have a cable-like appearance in the electron microscope. Below the aggregation point, individual soluble complexes are formed, which are the subject of this study. With increasing ionic strength, the aggregation point is shifted towards lower ratios of H1 to DNA. In the soluble complexes, H1 appears to bind along superhelically intertwined DNA strands, forming a polymer. Partial digestion of the complexes with protease suggests protection of the N-terminal tail and the globular domain of H1. Similar soluble complexes were observed with various H1 fragments but not with the core histones. In the soluble complexes, similar regions of the H1 molecule are considered to be protected from cleavage by protease, as in chromatin. Therefore, these complexes appear to be a valuable model for the interaction of H1 in chromatin fibers.

The integrity of the histone-DNA complex in chromatin fibres is not necessary for the maintenance of the shape of mitotic chromosomes

In this study we addressed the question of whether scaffold structures produced from purified mitotic chromosomes are an artefact of dehistonization, and whether the integrity of the chromatin fibres is necessary for the maintenance of the well-known shape of mitotic chromosomes. Purified mitotic chromosomes from Friend erythroleukemia cells were treated either with increasing NaCl concentrations up to 500 mM, or with 6 M urea in the presence or absence of 10 mM 2-mercaptoethanol. The main criterion for the intactness of the overall chromosome shape as seen by electron microscopy was the characteristic X-or U-like appearance with clearly discernable chromatid axes. Histone H1 is known to be essential for the integrity of chromatin fibres. Its removal in sucrose gradients containing 500 mM NaCl did not lead to loss of the overall chromosome shape. However, treatment of chromosomes in sucrose gradients containing 10 mM 2-mercaptoethanol and 6 M urea led to loss of the structure probably due to dissociation (or denaturation) of shape-determining (scaffolding) components. Under these conditions most of the histones remained bound to the chromosomes, and the fibres in this chromatin material, after removal of excess urea and 2-mercaptoethanol, still showed condensation of the nucleosome filaments into the characteristic fibre structures upon increasing ionic strength. Our observations are compatible with the model that specific non-histone components, independently of histone-DNA interactions, organize or stabilize the structure of metaphase chromosomes.