Regions within the N-terminal domain of human topoisomerase I exert important functions during strand rotation and DNA binding.

The human topoisomerase I N-terminal domain is the only part of the enzyme still not crystallized and the function of this domain remains enigmatical. In the present study, we have addressed the specific functions of individual N-terminal regions of topoisomerase I by characterizing mutants lacking amino acid residues 1-202 or 191-206 or having tryptophane-205 substituted by glycine in a broad variety of in vitro activity assays. As a result of these investigations we find that mutants altered in the region 191-206 distinguished themselves from the wild-type enzyme by a faster strand rotation step, insensitivity towards the anti-cancer drug camptothecin in relaxation and the inability to ligate blunt end DNA fragments.

Recombinogenic flap ligation mediated by human topoisomerase I.

Aberration of eukaryotic topoisomerase I catalysis leads to potentially recombinogenic pathways by allowing the joining of heterologous DNA strands. Recently, a new ligation pathway (flap ligation) was presented for vaccinia virus topoisomerase I, in which blunt end cleavage complexes ligate the recessed end of duplex acceptors having a single-stranded 3'-tail. This reaction was suggested to play an important role in the repair of topoisomerase I-induced DNA double-strand breaks.

The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation.

Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis.

Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells.

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (alpha and beta) with similar properties in vitro.

Multiple collagen I gene regulatory elements have sites of stress-induced DNA duplex destabilization and nuclear scaffold/matrix association potential.

The availability of the complete nucleotide sequences of numerous prokaryotic and eukaryotic organisms should stimulate the development and application of computer-based approaches for studying genome organization and function. Earlier work has shown that distinct regulatory DNA elements can be identified by computational analysis as sites of stress-induced DNA duplex destabilization (SIDD). Here we report the results of computational and experimental analyses of previously identified regulatory elements in the murine alpha1(I) collagen (Col1a1) gene domain.