Poly(ADP-ribose)polymerase metabolism in Trypanosoma cruzi.

BALTANÁS R; ALONSO GD; TORRES HN; FLAWIÁ MM; FERNÁNDEZ VILLAMIL SH

Mendoza, Argentina.

Congreso; VII Congreso Argentino de Protozoologia y Enfermedades Parasitarias.; 2005

Sociedad Argentina de Protozoologia.

Poly(ADP-ribose)polymerase is a nuclear enzyme which has been mainly involved in DNA repair. Recent evidences have implicated this enzyme in normal cell physiological functions, including chromatin remodeling and gene regulation. Screening T.cruzi genome databases (TIGR) a PARP homologous sequence was identified. The PCR amplification product encodes a 592 amino- acids protein, of about 65 kDa and isoelectric point of 9.5. Primary structure showed high identity with PARP family members, mainly PARP-2 type. The carboxyl-terminal region contains the catalytic domain with PARP signature motif (TGYMFGKG). Putative automodification domain rich in E (13%) and WGR were also present. The amino-terminal domain does not reveal any sequence similarity with the classical PARP Zn finger N-terminal. Southern blot showed a single-copy gene and a single transcript of 1.8 kb was observed in epimastigotes by Northern blot. Nuclear and kinetoplastic localization in epimastigotes was ascertained by IFI. Full length PARP was expressed in E.coli as 6xHis fusion protein using pET22b+. Activity assay was performed on partial purified recombinant enzyme and was dependent on protein content and time course. PARP automodification was suggested by SDS/PAGE and the increase of activity after releasing ADP-ribose polymers by alkaline treatment. We also found the catabolic counterpart, poly(ADP-ribose)glycohydrolase (PARG), in T. cruzi genome databases suggesting an analogy with the known poly(ADP-ribose) metabolism in higher eukaryotes. Our findings provide an initial biochemical characterization of this covalent post-traslational modification which is an interesting contribution to the DNA metabolism study in trypanosomatids.