Topoisomerase I action on the heterochromatic DNA from the brine shrimp Artemia franciscana: studies in vivo and in vitro.

The genomes of higher eukaryotes contain various amounts of tandem repeated DNA sequences (satellite DNA) typically located in the constitutive heterochromatin, the most highly condensed region of interphase chromosomes. We have previously demonstrated that an AluI DNA family of repeats is the major component of constitutive heterochromatin in the brine shrimp Artemia franciscana. The analysis of cloned heterochromatic fragments revealed that this repetitive DNA shows a stable curvature conferring a solenoidal geometry to the double helix. In this paper we provide evidence, using the antitumour drug camptothecin, that, in vivo, topoisomerase I cleaves heterochromatin with a frequency comparable with that observed in the whole genome. The analysis of the break sites shows that the enzyme cleaves heterochromatic DNA at specific sites characterized by a degenerate consensus sequence. Moreover the enzyme-mediated breaks have, in vitro, a degenerate consensus sequence similar to, but not identical with, the in vivo one. Some of these sites are influenced by the DNA flanking the heterochromatic insert, suggesting that structural variations could modify the enzyme specificity.

Nucleotide variation and molecular structure of the heterochromatic repetitive AluI DNA in the brine shrimp Artemia franciscana.

It has been suggested that DNA bending could play a role in the regulation of gene expression, chromosome segregation, specific recombination and/or DNA packaging. We have previously demonstrated that an AluI DNA family of repeats is the major component of constitutive heterochromatin in the brine shrimp A. franciscana. By the analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments we verified that the repetitive AluI DNA shows a stable curvature that determines a solenoidal geometry to the double helix. This particular structure could be of relevant importance in conferring the characteristic heterochromatic condensation. In this paper we evaluate how the point mutations that occurred during the evolution of the AluI sequence of A. franciscana could influence the sequence-dependent tridimensional conformation. The obtained data underline that, in spite of the high sequence mutation frequency (10%) of the repetitive DNA, the general structure of the heterochromatic DNA is not greatly influenced, but rather there is a substantial variation of the copy number of the repetitive AluI fragment. This variation could be responsible for the hypothetical function of the constitutive heterochromatin.