Arndt Richter

Expression of the topoisomerase i gene in serum stimulated human fibroblasts

We have determined the levels of mRNA coding for human type I DNA topoisomerase (EC 5.99.1.2.) in resting and proliferating human cells in culture. After addition of serum to growth arrested cells, we observed an continuous increase in the amount of topoisomerase I mRNA, starting after serum addition and reaching a maximum at 25 h after stimulation. At the end of the S-phase, a 6-fold higher amount of topoisomerase I mRNA was present in these cells. Nuclear run on transcription experiments showed, that the increase of topoisomerase I mRNA was preceded by a 3- to 4-fold increase in de novo mRNA synthesis. In contrast, during the same time period the amount of topoisomerase I increased only by a factor of 2, and the specific activity (enzymatic activity/mg protein) remained constant.

Simultaneous Purification of DNA Topoisomerase I and II from Eukaryotic Cells

We have developed a procedure for the simultaneous purification of DNA topoisomerase I and II from calf thymus. Both enzymes were first extracted from isolated nucleoprotein complexes. After batchwise chromatography on hydroxylapatite the two enzyme activities were separated on a FPLC phenylsuperose column. The enzymes were further purified by a second chromatography on phenylsepharose (topo I) or FPLC Mono Q (topo II). The purification can be finished within three days, yielding 0.5-1.0 mg quantities of homogeneous, enzymatic active preparations of the two proteins from 200 g of starting material.

Conserved regulatory elements in the type I DNA topoisomerase gene promoters of mouse and man

The gene for mammalian type I DNA topoisomerase is constitutively expressed, but also regulated by a number of external stimuli. We compared the nucleotide sequences of the human and the mouse topoisomerase I gene promoters because promoter elements, essential for basic as well as regulated gene expression, should be conserved during evolution. We found that proximal upstream sequences are highly conserved and include potential binding sites for ubiquitous transcription factors, a regulatory CRE site as well as two novel promoter elements that have been shown to be important for the expression of the human gene. The more distal parts of the upstream sequences are less well conserved but include two regions that are almost identical in the human and the mouse gene. One of these regions contains a binding site for a basic-helix-loop-helix/leucine-zipper protein, and the other contains an AT-rich element with the potential for DNA bending.

Comparison of replicative and non-replicative chromatin assembly pathways in HeLa cell extracts

It has been reported that chromatin assembly in mammalian cell extracts depends exclusively or preferentially on ongoing DNA replication (Stillman, B. (1986) Cell 45, 555-565). More recently, this view has been challenged demonstrating that, in the same extracts, chromatin can also be formed efficiently in the absence of DNA replication (Gruss et al. (1990) EMBO J. 9, 2911-2922). The experiments, described in this communication, were performed to resolve this apparent contradiction. We found that there are at least two distinct in vitro pathways for chromatin assembly in HeLa cell extracts. The replicative pathway requires a nuclear protein, most likely identical with the chromatin assembly factor, described by Stillman (1986, Cell 45, 555-565), and the free soluble histones present in the cytosol of S phase cells. In contrast, a non-replicative pathway was identified that depends on isolated nuclear histones. As one component of the non-replicative assembly pathway we identified a cytosolic factor that was purified to apparent homogeneity and shown to be an acidic 50 kDa polypeptide. The isolated cytosolic 50 kDa protein efficiently promoted nucleosome assembly as demonstrated by one- and two-dimensional gel electrophoresis of in vitro packaged plasmid DNA.

Histone H1 inhibits eukaryotic DNA topoisomerase I

Histone H1 inhibits the catalytic activity of topoisomerase I in vitro. The relaxation activity of the enzyme is partially inhibited at a molar ratio of one histone H1 molecule per 40 base pairs (bp) of DNA and completely inhibited at a molar ratio of one histone H1 molecule per 10 base pairs of DNA. Increasing the amount of enzyme at a constant histone H1 to DNA ratio antagonizes the inhibition. This indicates that topoisomerase I and histone H1 compete for binding sites on the substrate DNA molecules. Consistent with this we show on the sequence level that histone H1 inhibits the cleavage reaction of topoisomerase I on linear DNA fragments.