ANALYSIS OF FUNCTIONAL DOMAIN ORGANIZATION IN DNA TOPOISOMERASE-II FROM HUMANS

The functional domain structure of human DNA topoisomerase II alpha an d Saccharomyces cerevisiae DNA topoisomerase II was studied by investi gating the abilities of insertion and deletion mutant enzymes to suppo rt mitotic growth and catalyze transitions in DNA topology in vitro. A lignment of the human topoisomerase II alpha and S. cerevisiae topoiso merase II sequences defined 13 conserved regions separated by less con served or differently spaced sequences. The spatial tolerance of the s pacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual dom ains. We found that the exact spacing between most of the conserved do mains is noncritical, as insertions in the spacer regions were tolerat ed with no influence on complementation ability. All conserved domains , however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II car boxy-terminal truncations were investigated with respect to the abilit y to support viability, cellular localization, and enzymatic propertie s, The analysis showed that the divergent carboxy-terminal region of h uman topoisomerase II alpha is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.