Substrate specificity plays an important role in uncoupling the catalytic and scaffolding activities of rat testis DNA topoisomerase II alpha

Topoisomerase II (topo II) is a dyadic enzyme found in ail eukaryotic cells . Topo II is involved in a number of cellular processes related to DNA meta bolism, including DNA replication, recombination and the maintenance of gen omic stability. We discovered a correlation between the development of post natal testis and increased binding of topo II alpha to the chromatin fracti on. We used this observation to characterize DNA-binding specificity and ca talytic properties of purified testis topo II alpha. The results indicate t hat topo II alpha binds a substrate containing the preferred site with grea ter affinity and, consequently, catalyzes the conversion of form I to form IV DNA more efficiently in contrast to substrates lacking such a site. Inte restingly, topo II alpha displayed high-affinity and cooperativity in bindi ng to the scaffold associated region. In contrast to the preferred site, ho wever, high-affinity binding of topo II alpha to the scaffold-associated re gion failed to result in enhanced catalytic activity. Intriguingly, competi tion assays involving scaffold-associated region revealed an additional DNA -binding site within the dyadic topo II alpha. These results implicate a du al role for topo II alpha in vivo consistent with the notion that its seque stration to the chromatin might play a role in chromosome condensation and decondensation during spermatogenesis.