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Functional characterisation of cohesin subunit SMC3 and separase and their roles in the segregation of large and minichromosomes in Trypanosoma brucei

Bessat, Mohamed Said Nooh


Mohamed Said Nooh Bessat


Klaus Ersfeld


The genome of the African trypanosome, Trypanosoma brucei, presents an unusual karyotype in which two main classes of chromosomes, large and small minichromosomes, need to be faithfully replicated and segregated during the cell cycle. Although the large and minichromosomes are colocalised and segregated by association with the mitotic spindle, minichromosomes exhibit segregation patterns that differ from those observed for large chromosomes. To address whether this difference is reflected at a molecular level, two different proteins that have highly conserved functions in eukaryotic chromosomes segregation were characterised in this study. The first protein, SMC3, is a component of the chromosome cohesion apparatus that holds sister chromatids together after their replication until segregation at anaphase. The second protein, separase, is a cysteine protease that resolves sister chromatid cohesion at the onset of anaphase and has, in other organisms, additional functions during mitosis. The T. brucei cohesin subunit, TbSMC3, localised to the nucleus as a chromatin-bound protein from G1 phase until metaphase and dissociated from chromatin during anaphase until the completion of cell division. On the other hand, cytoplasmic localisation of separase with nuclear exclusion was prevalent until the onset of metaphase when the protein re-localised to the nucleus, thus providing a potential control mechanism to prevent premature cohesin cleavage. Interference with the normal expression of SMC3 and separase by RNA interference resulted in defects in growth rate, cell cycle progression and chromosomes segregation. TbSMC3 depletion produced a lethal phenotype and inhibition of cell cycle progression. Similarly, lethality with severe inhibition of cell cycle progression was the main feature of separase depletion. Using fluorescence in situ hybridisation (FISH), it was shown that SMC3 depletion had no visible effect on the symmetric segregation of the minichromosome population, but interferes with the faithful mitotic segregation of large chromosomes. In contrast, separase depletion blocks the segregation of both large and minichromosomes. In separase-depleted mitotic cells, cohesins remained bound to chromatin, which is in contrast to rapid dissociation of cohesins from chromatin in wild-type mitotic cells. The severity of segregation phenotypes after separase depletion was additionally explained by defects in the mitotic spindle assembly. In both SMC3 and separase depleted cells, cytokinesis in the absence of mitosis/karyokinesis was not inhibited in procyclic cells, resulting in the generation of anucleate 'zoid' cells. The lethality imposed on trypanosome cells after depletion of both SMC3 and separase proteins indicate that they can serve as potential drug targets for anti-parasite chemotherapy.


Bessat, M. S. N. (2009). Functional characterisation of cohesin subunit SMC3 and separase and their roles in the segregation of large and minichromosomes in Trypanosoma brucei. (Thesis). University of Hull. Retrieved from

Thesis Type Thesis
Deposit Date Aug 15, 2011
Publicly Available Date Feb 22, 2023
Keywords Biological sciences
Public URL
Additional Information Department of Biological Sciences, The University of Hull
Award Date May 1, 2009


Thesis (11.1 Mb)

Copyright Statement
© 2009 Bessat, Mohamed Said Nooh. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

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