02 May 2003
Defining the minimal catalytic domain of poly(ADP-ribose) glycohydrolase
T. Sizemore, R. Meyer, M. Meyer-Ficca, E. Jacobson, M. JacobsonMed Sci Monit 2003; 9(1): 62-0 :: ID: 15201
Abstract
Poly(ADP-ribosyl)ation alters the function of numerous regulatory proteins within the cell. The posttranslational modifications are mediated by the poly(ADP-ribose) polymerase (PARP) family of enzymes. Only one enzyme, poly(ADP-ribose) glycohydrolase (PARG), is known to catalyze the hydrolysis of the polymer to free ADP-ribose by exo/endo-glycosidic cleavage. Previous research has identified amino acid residues essential for enzymatic activity in a sub region in the catalytic C-terminal region of PARG. Secondary structure predictions indicate that this sub region of PARG may contain a minimal catalytic domain with an ADP-ribosyltransferase fold similar to that observed in PARP-1 and bacterial toxin ADP-ribosyltransferases. In order to identify, purify and characterize the smallest catalytically active fragment of human PARG (hPARG), we designed a PCR strategy that was based on the exon/intron organization of the hPARG gene. Amplified regions of various portions of the PARG catalytic domain were ligated into a bacterial expression vector pBAD102/D-TOPO and the presence of the desired inserts was confirmed by sequence analysis. PARG fragments carrying engineered His-tags were expressed and purified and the presence of catalytic activity was detected by glycohydrolase assays using 32P labeled ADP-ribose polymers. This approach allowed the identification of a minimal catalytic domain of PARG, which should facilitate our understanding of PARG structure and function relationships and potentially allow structural analysis of the PARG catalytic center by crystallography in the future.
Keywords: PARG, PARP, gene cloning, ADP-ribose, ADP-ribose polymers, glycohydrolase assay
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