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Figure 2. Structural and functional domains of PARP-1. PARP-1 has a highly conserved structural and functional organization including: (1) an N-terminal DNA binding domain with two Cys-Cys-His-Cys zinc finger motifs (FI and FII), (2) a nuclear localization signal (NLS), (3) a central automodification domain containing a BRCT ("BRCA1 C-terminus-like") protein-protein interaction motif, and (4) a C-terminal catalytic domain with a contiguous 50 amino acid sequence, the "PARP signature" motif, that forms the active site

Automodification of PARP-1 occurs in the AMD, which contains a number of glutamate residues that are the likely targets for PAR attachment, as well as a BRCT motif that is thought to function in protein-protein interactions (D'Amours et al., 1999; Rolli et al., 2000). AutoPARylated PARP-1 exhibits dramatically reduced DNA-binding activity and, hence, altered activities in a number of cellular endpoints. Together, the various biochemical activities of PARP-1 make it ideally suited as a regulator of a variety of signal-dependent nuclear processes.

1.3. Effects of PARylation on Target Proteins

Poly(ADP-ribose) (PAR) is a heterogenous branched polymer of repeating ADPR units linked via glycosidic ribose-ribose 1" ^ 2' bonds in the linear chain and glycosidic ribose-ribose 1''' ^ 2'' bonds at the branchpoints (Fig. 1c) (D'Amours et al., 1999; Kim et al., 2005). A single PAR molecule may contain as many as 200 ADPR units and has approximately one branch per 20 to 50 ADPR units. Based on its chemical similarity to DNA and RNA, PAR has been referred to as the "third type of nucleic acid" (D'Amours et al., 1999). Each ADPR residue in PAR contains an adenine moiety capable of base stacking and hydrogen bonding, as well as two negatively charged phosphate groups (Amé et al., 2000). Thus, PAR may form definitive structures through intramolecular interactions (Minaga and Kun, 1983a, b), and these structures have the potential for non-covalent attractive or repulsive interactions with other molecules (Mathis and Althaus, 1987; Wesierska-Gadek and Sauermann, 1988; Panzeter et al., 1992).

PAR may alter the activity of PARylated proteins by functioning as a site-specific covalent modification or a steric blocker. For example, inhibition of PARP-1's DNA binding activity by autoPARylation may be the result of charge repulsion between PAR and DNA, or steric effects of PAR that mask PARP-1's DBD (D'Amours et al., 1999). Furthermore, PAR may act as a protein binding matrix for a variety of nuclear proteins. In this regard, proteomic approaches have been used to identify a 20 amino acid PAR binding motif in a heterogeneous group of PAR-binding proteins, including core histones, p53, and XRCC-1 (Pleschke et al., 2000;

Gagne et al., 2003). Some non-sequence-specific DNA-binding proteins, such as the linker histone H1, may even have a greater affinity for PAR than DNA (Malanga et al., 1998). Thus, an understanding of PAR is an important aspect of understanding the biology of PARP-1 and other PARPs.

1.4. PAR Catabolism by PARG

The steady-state levels of PAR in vivo are regulated by the opposing actions of PARPs and poly(ADP-ribose) glycohydrolase (PARG). PARG is an enzyme with both exo- and endoglycosidase activities that hydrolyzes the glycosidic linkages between the ADP-ribose units of PAR producing free ADP-ribose (Amé et al., 2000; Davidovic et al., 2001) (Fig. 1c, see red arrow). Although PARP-1 may be present at a 5- to 20-fold molar excess over PARG in some cell types, PARG has a higher specific activity than PARP-1 and its enzymatic activity increases with increased PAR length (D'Amours et al., 1999). Furthermore, nucleo-cytoplasmic shuttling of the PARG protein may modulate the level of nuclear PARG activity (Bonicalzi et al., 2003; Ohashi et al., 2003). Ultimately, the activity of PARP-1 is intimately tied to the regulatory actions of PARG, yet this is an area of research that has not been explored in sufficient detail. Where appropriate below, we have described results that tie the actions of PARG to the biological function of PARP-1. For more details about the catabolism of PAR by PARG, as well as the interplay between PARP-1 and PARG, the reader is directed to the additional references listed in the bibliography (e.g., Davidovic et al., 2001).

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