CLONING AND CHARACTERIZATION OF A POLY(ADPRIBOSE) GLYCOHYDROLASE IN Trypanosoma brucei

SCHLESINGER M; VILCHEZ LARREA S C; ALONSO G D; TORRES H N; FLAWIA M M; FERNANDEZ VILLAMIL SH

Puerto Madryn

Congreso; 46 Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2010

SAIB

Poly-ADP-ribose (PAR), generated by poly(ADPribose) polymerase (PARP) in the presence of DNA strand breaks (DSB), is common to nuclear processes related to DNA metabolism, such as structural chromatin remodeling during DNA repair, transcription, DNA replication and cell death pathways. Poly(ADP-ribose)glycohydrolase (PARG) is the main PAR hydrolyzing activity in the cell. T. brucei evades the immune system by antigenic variation of surface glycoproteins (VSG), which requires recombination sites within DNA inducing transient DSB. To further investigate this process, here we present the cloning and characterization of TbPARG. Southern-blot analysis confirmed a single copy of this gene in T. brucei genome. Sequence alignments of the catalytic domain showed the PARG signature containing the three essential acidic residues (D-E-E) conserved within trypanosomatids. Expression of the 60 kDa protein in procyclic stage was demonstrated by Western-blot. Nuclear localization of the enzyme in the basal state of the parasite, as well as after hydrogen peroxide treatment, was observed by IFI. PAR generation after a genotoxic insult was also detected in the nucleus by using antiPAR antibodies. Currently, we are working on TbPARG assay in procyclic extracts and of the recombinant protein rTbPARG-His, expressed in bacteria.