The application of synthetically modified oligonucleotides to the study of DNA topoisomerase I

The DNA topoisomerases are ubiquitous cellular enzymes that are responsible for the overall topological state of chromosomal DNA. Topoisomerase I functions by the introduction of transient single-strand breaks at the level of the phosphodiester backbone and requires neither metal cofactors nor ATP in order to facilitate supercoil relaxation. As a consequence of its central role in replication, transcription and chromatin recombination, topoisomerase I has long been viewed as an attractive target for therapeutic strategies.; In a continuing effort to further characterize topoisomerase I, numerous oligonucleotide probes have been constructed using automated oligonucleotide synthesis protocols. For instance, alteration of the phosphodiester connectivity at positions surrounding the single cleavage sequence of vaccinia topoisomerase I contrast analogous experiments performed upon the human enzyme. The synthesis of isomerically pure methylphosphonate dinucleotide cassettes was also accomplished followed by their successful incorporation into positions in and about the high affinity cleavage sites of both vaccinia and human topoisomerases I. The contrasting effects of single-site charge neutralization upon the extent and rates of each topoisomerase I-mediated transesterification are described. The observation of a latent endonuclease activity resident in vaccinia topoisomerase I is also discussed.; Camptothecin (CPT) is a known eukaryotic topoisomerase I poison that is thought to function by the stabilization of the transient enzyme-DNA covalent binary complex through non-covalent interactions. While numerous models exist that attempt to reconcile the accumulated biochemical and spectroscopic data, debate continues regarding the putative ternary complex. Detailed within are the synthetic efforts aimed at the construction of a CPT-DNA conjugate oligonucleotide, covalently linked on the scissile strand of the human topoisomerase I high affinity cleavage substrate.