Distinct Genes Encode Type II Topoisomerases for the Nucleus and Mitochondrion in the Protozoan Parasite Trypanosoma brucei

Abstract

Topoisomerases are essential for orderly nucleic acid metabolism and cell survival and are proven targets for clinically useful antimicrobial and anticancer drugs. Interest in the topologically intricate mitochondrial DNA (kinetoplast or kDNA) of Trypanosoma brucei brucei and related kinetoplastid protozoan parasites has led to many reports of type II topoisomerases that participate in kDNA metabolism (we term the T. brucei brucei gene TbTOP2mt). We have now identified and characterized two new genes for type II topoisomerases in T. brucei brucei, termed TbTOP2alpha and TbTOP2beta. Phylogenetically, they share a common node with other nuclear topoisomerases, clearly distinct from a clade that includes the previously reported kinetoplastid genes, all of which are homologs of TbTOP2mt. Southern blot analysis reveals the new genes are single copy and positioned approximately 1.7 kb apart. Cognate mRNAs are expressed in African trypanosomes, but only a single message is detected in Leishmania or Crithidia. TbTOP2alpha encodes an ATP-dependent topoisomerase that appears as a single approximately 170-kDa band on immunoblots and localizes to the nucleus; RNA interference leads to pleomorphic nuclear (but not kDNA) abnormalities and early growth arrest. The role of TbTOP2beta is unclear. Although transcribed in trypanosomes, TbTOP2beta is not detected by beta-specific antiserum, and RNAi silencing results in no obvious phenotype. These studies indicate that African trypanosomes and related kinetoplastid human pathogens are unusual in having independent topoisomerase II genes to service their nuclear and mitochondrial genomes, and they highlight TbTOP2alpha as a promising target for the development of much-needed new therapies.