Novel features on H2AX role in Toxoplasma gondii

BOGADO, SS; DALMASSO, MC; ANGEL, SO

PUERTO MADRIN

Congreso; XLVI Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2010

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The histone variant H2AX prossesses a C-terminal conserved SQ(E/D) Φ motif (Φ being an hydrophobic amino acid), which makes it different from the rest of the H2A histones. Phospho-H2AX at this SQ motif (gammaH2AX) has become a DNA damage hallmark. In Toxoplasma gondii, H2AX is phosphorylated (S131) in the presence of oxidative damage. Interestingly, parasites grown under standard conditions show high levels of gammaH2AX. In addition, H2AX in enriched at repressed promoters and silenced regions, and its expression increases during bradyzoite development, suggesting an extra role for H2AX mutated at S131 for alanine, and two others expressing the wild type version of H2AX; both fused to c-myc tag at the N-terminus (c-mycH2AS131A and c-mycH2AX respectively). The presence of c-mycH2AX and c-mycH2AXS131A were determined by Western blot assays. The recombinant proteins were detected with anti-c-mycH2AX and c-mycH2AXS131A were detected with anti-c-myc in the four clones. Besides, c-mycH2AX was recognized by anti-gammaH2AX while c-mycH2AXS131A was not. Their localization was analyzed by indirect immunofluorescence using the same antibodies. In both cases the fusion proteins were localized in the nucleus. With all these clones the replication and growth rates as well as conversion to bradyzoites will be analyzed.