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Induction of DNA damage repair defect by the exogenous expression of BRCA2 BRC repeat in Trypanosoma brucei
Author(s) -
Hall Mack,
Misra Smita,
Chaudhuri Minu,
Chaudhuri Gautam
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.876.3
Subject(s) - rad51 , biology , trypanosoma brucei , homologous recombination , microbiology and biotechnology , dna repair , dna , dna damage , genetics , gene
The DNA recombination repair protein BRCA2 is conserved and is present in many eukaryotes including the protozoan parasite Trypanosoma brucei . One of the major functions of BRCA2 is to haul the DNA recombinase RAD51 from the cytosol to the nucleus. Binding of BRCA2 to RAD51 is mediated through the consensus BRC repeats of BRCA2 and this binding is critical for homologous recombination. Yeast 2‐hybrid analysis studies revealed that a single BRC repeat from TbBRCA is enough to bind to different isoforms of TbRAD51. We tested whether over‐expression of a single BRC repeat unit of TbBRCA2 will increase the parasites sensitivity to DNA damage. In order to evaluate the importance of BRCA2/RAD51 interactions on the DNA recombination/repair processes in T. brucei , we have amplified a single BRC repeat from its ORF. We cloned this amplicon into pLew79MHTAP vector, and stably transfected the procyclic form of the parasite. Transfected cells were selected with phleomycin. Expression of the BRC repeat was evaluated by RT‐PCR and Western blot analyses. To evaluate the effects of the over‐expression of a single BRC repeat on DNA damage sensitivity, procyclic forms of T. brucei over‐expressing a single BRC repeat were exposed to increasing concentrations of methyl‐methane sulfonate. Over‐expression of a single BRC repeat significantly increased the sensitivity of the parasite cells to this DNA damaging reagent.