DNA TOPOISOMERASE II AND CHROMOSOME STRUCTURE

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The long-term goal of this research is to determine the underlying biochemical/genetic properties that differentiate the DNA on eukaryotic chromosomes into its distinct classes - euchromatin and heterochromatin. Euchromatin and heterochromatin differ in very fundamental characteristics with euchromatin containing genes that have distinct phenotypic effects, and the heterochromatin, originally defined cytologically as highly condensed DNA, being a repository of repeated, non-coding DNA sequences. Although heterochromatic DNA has largely unknown function, it is essential for the normal division of the genetic material during meiosis and mitosis. It is therefore likely that some or most heterochromatic DNA is not "junk" but rather a collection of sequences that function via non-transcriptional mechanisms. This research will elucidate one aspect of this repeated DNA that has been proposed as important in causing heterochromatin to behave as it does - interactions with topoisomerase II (topo II). One hypothesis proposes that heterochromatic behavior is a function of its interactions with the DNA modifying protein, topo II. Topo II, an important component of the nuclear matrix in Drosophila, has been shown to be important in many of the features usually associated with heterochromatin from DNA condensation to affecting the frequency of recombination. Topo II poisons are also potent antitumor drugs. Preliminary evidence indicates that heterochromatin is highly enriched for sites essential for topo II function, leading to the hypothesis that chromatin structure can be explained by the differing density of topo II sites. This hypothesis is primarily based on conceptualization of DNA sequences extracted from the GenBank database. Therefore, to fully test this idea it is necessary to determine the generality of the observation. This will be done using two experimental approaches. First, clones of the sequences subjected to computer analysis will be used for in vitro analysis to test for topo II binding and cutting. Clones from heterochromatic regions should have a higher density of legitimate topo II sites. Second, the distribution of functional topo II sites in euchromatin and heterochromatin will be compared in vivo using cell lines. A topo II inhibitor, VP-16, that causes easily discernible topo II cleavage will be applied to Drosophila cell cultures and DNA will then be fractionated as a function of topo II cleavage. Southern blots of the DNA from inhibitor treated cells from both standard and pulse field gels will be probed with large PI clones from euchromatic and heterochromatic locations. The hypothesis predicts that heterochromatic regions will contain many more active topo II sites as determined by the number of cleavable sites per kilobase.