DNA damage and the Trypanosoma brucei cell cycle

Trypanosoma brucei demonstrates unique features in its cell cycle and response to DNA damage, two aspects of biology which are intricately linked. Here, a technique for producing populations of bloodstream form parasites synchronised in S phase is developed and attempts made to use this technique to search for cell cycle linked changes in the levels of DNA damage response proteins. Additionally, two proteins central to the control of the DNA damage response in other organisms, ATM and ATR, are analysed in more detail by RNAi. Hydroxyurea (HU) was recently used to synchronise procyclic form parasites. Here HU synchronisation is demonstrated in the human infective bloodstream stage, with 10 μg.ml-1 shown to synchronise a population in S phase after 6 hours of exposure to the drug. Synchronised populations of T. brucei are then used to examine protein expression using western blots for the Rad51 paralogues Rad51-3 and Rad51-4, and to search for cell cycle regulated proteins more generally using Difference Gel Electrophoresis (DiGE). No cell cycle linked changes in protein levels were found using either method. RNAi-inducible cell lines were produced in both bloodstream and procyclic stages targeting the proteins ATM and ATR. Minor growth phenotypes were found for both proteins, post RNAi induction, in the procyclic stage, with 2 of 4 bloodstream stage ATM RNAi clones demonstrating a lethal phenotype and no growth phenotype being found in bloodstream stage ATR RNAi clones. Knockdown was demonstrated at the protein level in the ATM RNAi clones showing a lethal growth phenotype, but has not yet been shown in the remaining cell lines. The lethal phenotype appears to be linked to a failure to prevent DNA re-replication and segregate nuclei and kinetoplasts correctly