In vivo-regulation of DNA-topoisomerase II isoenzymes by interacting proteins
Final Report Abstract
All cellular DNA metabolic processes require the catalytic activity of DNA- topoisomerases. The cleavage of double-stranded DNA, which is required e.g. for the segregation of sister chromatids during mitosis, is provided by topoisomerase II. Vertebrates have two genetically distinct isoforms with similar enzymatic activities in vitro but different properties in vivo. Topoisomerase IIα has essential functions during cell proliferation that cannot be complemented by IIβ. Topoisomerase IIβ is the only topoisomerase II expressed in non-proliferating tissues, and is essential for neuronal development. Consistent with isoform specific functions in proliferating cells, where IIα (IIβ not) is vital for proper chromosome organization and sister chromatid segregation, the IIα isoform is bound to chromosomes throughout mitosis, a distribution not found for the IIβ isoform. This isoform specific functionality is conferred by the last 210 amino acids of topoisomerase IIα. To further investigate differences between the two isoform, we developed a human expression and purification system. Until now, human topoisomerase II isoforms have been purified from yeast, a system enabling purification of large enzyme quantities. However, topoisomerase II thus purified cannot be used to study secondary modifications specific to human cells. We therefore believe that the human system developed within the frames of this project is an appropriate tool to purify topoisomerase II isoforms with their native secondary modifications. This would be an important contribution to the field, as it is clear that secondary modifications of the enzymes are important in the regulation of their biological function. So far we purified topoisomerase IIα and IIβ from cycling cells and used these enzyme preparation to demonstrate that the genotoxic properties of the food contaminant alternariol, is mainly due to the toxin acting as a topoisomerase II poison preferentially affecting the IIα isoform and to show that the different genotoxic potential of several dietary, environmental and therapeutic topoisomerase II poisons is explained by differences in persistence time of the topoisomerase II DNA cleavage complex. Finally, we could show that topoisomerase IIα regulate DNA entanglements, that in concert with protein-mediated compaction act to fold chromatin into mitotic chromosomes.
Publications
- 2009. Alternariol acts as a topoisomerase poison, preferentially affecting the IIalpha isoform. Mol Nutr Food Res. 53:441-51
Fehr, M., G. Pahlke, J. Fritz, M.O. Christensen, F. Boege, M. Altemoller, J. Podlech, and D. Marko
- 2010. Mitotic chromosomes are constrained by topoisomerase II-sensitive DNA entanglements. J Cell Biol. 188:653-63
Kawamura, R., L.H. Pope, M.O. Christensen, M. Sun, K. Terekhova, F. Boege, C. Mielke, A.H. Andersen, and J.F. Marko
