Proteins involved in DNA Homologous Recombination Repair in Toxoplasma gondii: BRCA2 and RAD51 characterization

CRISTALDI CONSTANZA; SALDARRIAGA CARTAGENA, ANA M.; GANUZA AGUSTINA; ANGEL, SERGIO O.; VANAGAS, LAURA

Mar del Plata

Congreso; XXXI Reunión Científica Anual, Sociedad Argentina de Protozoología (SAP) ? Reunión Anual de Sociedades de Biociencia: SAIC. SAFE. SAB. SAP. AACYTAL. NANOMED- ar. HCS; 2019

Toxoplasma gondii is an obligate intracellular parasite, belonging to the phylum Apicomplexa and is responsible of toxoplasmosis infection. Although there are treatments against toxoplasmosis, due to the toxicity of the drugs used there is an intensive search for new treatments against the parasite and innocuous to the host cell. There are conserved components of the homologous recombination DNA repair (HRR) pathway in T.gondii that could present unique characteristics which make them attractive therapeutic targets. Among them, a putative T. gondii BRCA2 was identified in silico because of the presence of conserved domains with the human homologous. In higher eukaryotes BRCA2 interacts 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 in bacteria to obtain recombinant proteins used to produce specific mouse polyclonal 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 their presence 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 results showed no differences in the protein expression by WB in a DNA damage context, compared to non-treated parasites, for both proteins. When parasites were analyzed by IFA TgBRCA2 showed a spotted distribution along the whole parasite (nucleus included) in normal and DNA damage conditions. The antibodies obtained against these two important proteins will allow us to make progress in the understanding of the complex BRCA2-RAD51, which is fundamental to study the DNA repair by HRR observed in other eukaryotes.