INTECH   27907
INSTITUTO TECNOLOGICO DE CHASCOMUS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Proteins involved in DNA homologous recombination repair in Toxoplasma gondii: BRCA2 and RAD51 characterization
Autor/es:
ANA SALDARRIAGA; LAURA VANAGAS; CONSTANZA CRISTALDI; SERGIO O. ANGEL; AGUSTINA GANUZA
Lugar:
Mar del Plata
Reunión:
Congreso; ReuniĆ³n anual de sociedades de Biociencia SAIC.SAFE.SAB.SAP. AACYTAL.NANOMED-ar.HCS; 2019
Resumen:
Toxoplasmagondii is an obligateintracellular parasite, belonging to the phylum Apicomplexa and is responsibleof toxoplasmosis infection. Although there are treatments againsttoxoplasmosis, due to the toxicity of the drugs used there is an intensive searchfor new treatments against the parasite and innocuous to the host cell. Thereare conserved components of the homologous recombination DNA repair (HRR) pathwayin T.gondii that could present unique characteristics which make themattractive therapeutic targets. Among them, a putative T. gondii BRCA2 was identified insilico because of the presence of conserved domains with the humanhomologous. In higher eukaryotes BRCA2interacts with the recombinase RAD51, which is also present in T. gondii, generating an essential complex for the HRR. T.gondii BRCA2 and RAD51 genes were cloned and expressed inbacteria to obtain recombinant proteins used to produce specific mousepolyclonal antibodies. RAD51 was expressed as an entire recombinant protein,but for BRCA2 only the OB1 domain was expressed due to its high mass, near 480 kDa. The antibodies were titrated by ELISA, and used to detect theirpresence in T.gondii by Western Blot(WB) and their subcellular localization by Indirect Immunoflourescence (IFA) either in normal conditions or using DNA damaging agents such as phleomycin and metylmethanesulfonate (MMS). The resultsshowed no differences in the protein expressionby WB in a DNA damage context, compared to non-treated parasites, for bothproteins. When parasites were analyzed by IFA TgBRCA2 showed a spotteddistribution along the whole parasite (nucleus included) in normal and DNA damageconditions. The antibodiesobtained against these two important proteins will allow us to make progress inthe understanding of the complex BRCA2-RAD51, which is fundamental tostudy the DNA repair by HRR observed in other eukaryotes.