Centromere-associated topoisomerase activity in bloodstream form Trypanosoma brucei.
Topoisomerase-II accumulates at centromeres during prometaphase, where it resolves the DNA catenations that represent the last link between sister chromatids. Previously, using approaches including etoposide-mediated topoisomerase-II cleavage, we mapped centromeric domains in trypanosomes, early branching eukaryotes in which chromosome segregation is poorly understood. Here, we show that in bloodstream form Trypanosoma brucei, RNAi-mediated depletion of topoisomerase-IIα, but not topoisomerase-IIβ, results in the abolition of centromere-localized activity and is lethal. Both phenotypes can be rescued by expression of the corresponding enzyme from T. cruzi. Therefore, processes which govern centromere-specific topoisomerase-II accumulation/activation have been functionally conserved within trypanosomes, despite the long evolutionary separation of these species and differences in centromeric DNA organization. The variable carboxyl terminal region of topoisomerase-II has a major role in regulating biological function. We therefore generated T. brucei lines expressing T. cruzi topoisomerase-II truncated at the carboxyl terminus and examined activity at centromeres after the RNAi-mediated depletion of the endogenous enzyme. A region necessary for nuclear localization was delineated to six residues. In other organisms, sumoylation of topoisomerase-II has been shown to be necessary for regulated chromosome segregation. Evidence that we present here suggests that sumoylation of the T. brucei enzyme is not required for centromere-specific cleavage activity.
Obado
Samson O
SO
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK.
Bot
Christopher
C
Echeverry
Maria C
MC
Bayona
Julio C
JC
Alvarez
Vanina E
VE
Taylor
Martin C
MC
Kelly
John M
JM
D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D002503:Centromere / Q000201:enzymology*; D053837:DNA Cleavage; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D034622:RNA Interference; D058207:Sumoylation; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014349:Trypanosoma cruzi / Q000201:enzymology
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D004250:DNA Topoisomerases, Type II