Identification of a Regulatory Segment of Poly(ADP-ribose) Glycohydrolase
Coordinate regulation of PARPs-1/2 and PARG is required for cellular responses to genotoxic stress. While PARPs-1/2 are regulated by DNA breaks and covalent modifications, mechanisms of PARG regulation are poorly understood. We report here discovery of a PARG regulatory segment far removed linearly from residues involved in catalysis. Expression and analysis of human PARG segments identified a minimal catalytically active C-terminal PARG (hPARG59) containing a 16residue N-terminal mitochondrial
... targeting sequence (MTS). Deletion analysis and site-directed mutagenesis revealed that the MTS, specifically hydrophobic residues L473 and L474 were required for PARG activity. This region of PARG was termed "Regulatory Segment/MTS" (REG/ MTS). The overall α-helical composition of hPARG59, determined by circular dichroism (CD), was unaffected by mutation of the REG/MTS leucine residues, suggesting that activity loss was not due to incorrect protein folding. REG/MTS was predicted to be in a loop conformation since the CD spectra of mutant Δ1-16 lacking the REG/MTS showed a higher α-helical content compared to hPARG59, indicating a secondary structure other than α-helix for this segment. Deletion of the REG/MTS from full-length hPARG111 also resulted in complete loss of activity, indicating that all PARG isoforms are subject to regulation at this site. Presence of the REG/MTS raises the possibility that PARG activity is regulated by interactions of PARPs-1/2 and other proteins at this site, raises interesting questions concerning mitochondrial PARG since MTS residues are often removed after transport, and offers a potentially novel site for drug targeting of PARG. The synthesis of polymers of ADP-ribose (PAR)1 is an immediate cellular response to DNA strand breaks caused by oxidative stress, ionizing radiation or alkylating agents (1-3). A primary enzyme involved in this post-translational modification is poly (ADP-ribose) polymerase-1 (PARP-1), which belongs to a growing family of PARPs that includes a second member (PARP-2) that also responds to DNA strand breaks (4,5). Upon binding to DNA strand breaks, PARPs-1/2 use nicotinamide adenine dinucleotide (NAD + ) as a substrate to synthesize PAR targeted to the PARPs themselves and other nuclear acceptor proteins, such as histones, p53 and enzymes involved in DNA repair (6). PARP-1 is inactivated and released from the DNA strand break by its automodification. Poly(ADP- † Supported by research grant CA 43894 from the National Institutes of Health to M.K.J.