Endoglycosidic cleavage of branched polymers by poly(ADP‐ribose) glycohydrolase

Abstract

Post-translational modification of nuclear proteins with poly(ADP-ribose) modulates chromatin structure and may be required for DNA processing events such as replication, repair and transcription. The polymer-catabolizing enzyme, poly(ADP-ribose) glycohydrolase, is crucial for the regulation of polymer metabolism and the reversibility of the protein modification. Previous reports have shown that glycohydrolase digests poly(ADP-ribose) via an exoglycosidic mechanism progressing from the protein-distal end of the polymer. Using two independent approaches, we investigated the possibility that poly(ADP-ribose) glycohydrolase also engages in endoglycosidic cleavage of polymers. First, partial glycohydrolase digestion of protein-bound poly(ADP-ribose) led to the production of protein-free oligomers of ADP-ribose. Second, partial glycohydrolase digestion of a fixed number of protein-free poly(ADP-ribose) polymers resulted in a transient increase in the absolute number of polymers while polymer size continuously decreased. Furthermore, endoglycosidic activity produced linear polymers from branched polymers although branch points themselves were not a preferential target of cleavage. From these data, we propose a mechanism whereby poly(ADPribose) glycohydrolase degrades polymers in three distinct phases ; (a) endoglycosidic cleavage, (b) endoglycosidic cleavage plus exoglycosidic, processive degradation, (c) exoglycosidic, distributive degradation.Poly(ADP-ribose) glycohydrolase is the only nuclear enzyme known to hydrolyze a-( 1"-2') glycosidic linkages. The natural substrate for this enzyme is poly(ADP-ribose), which is formed by the nuclear enzyme poly(ADP-ribose) polymerase and can contain branches. The polymers are covalently bound to various acceptor proteins, all of which bind to nucleic acids. Synthesis and degradation of these polymers is thought to regulate protein-DNA interactions during DNA processes involving single-strand and double-strand breaks (reviewed in [l -31). Polymer catabolism by poly(ADP-ribose) glycohydrolase is stimulated in mammalian cells exposed to increasing doses of DNA-damaging agents [4] and is reduced in a murine lymphoma cell line deficient in a specific repair function [5].The molecular mechanism by which poly(ADP-ribose) glycohydrolase degrades polymers has been investigated by several groups in the past. Most of these studies indicated that the glycohydrolase hydrolyzes ribose-ribose linkages from the protein-distal end of the polymer, i.e. exoglycosidically [6-lo]. The results from Ikejima and Gill, however, suggested the possibility of endoglycosidic attack [ 111. We present an in-depth study in which purified poly(ADP-ribose) glycohydrolase was incubated with protein-bound or protein-free ADP-ribose polymers, and the reaction products were directly analyzed on high-resolution DNA sequencing gels and by high-performance liquid chromatography. The results show that poly(ADP-ribose) glycohydrolase produces endoglycosidic reaction products in that (a) glycohydrolase activity releases...

Mutational Analysis of the Poly(ADP-Ribosyl)ation Sites of the Transcription Factor CTCF Provides an Insight into the Mechanism of Its Regulation by Poly(ADP-Ribosyl)ation

“…A further form of CTCF that migrated at 180 kDa (CTCF-180) and carried a PARylation mark was later reported (84,92). We verified this modification in a PARG enzyme assay using MCF7 cell nucleoplasmic extracts enriched with CTCF-180 following treatment with sodium butyrate (15). In this experiment, a shift from CTCF-180 to CTCF-130 was observed with increasing doses of PARG, suggestive of the removal of the PARylation mark from CTCF-180 by PARG (Fig.…”

“…The nucleoplasmic extracts from MCF7 cells (10 6 ), enriched with the CTCF-180 isoform (84), were obtained as described previously (92). The extracts were incubated with 1, 2, 3, or 5 mU of PARG enzyme (Axxora) as reported earlier (15). The incubated extracts were analyzed by Western blotting using an anti-CTCF antibody (Abcam).…”

Nuclear ADP-Ribosylation Reactions in Mammalian Cells: Where Are We Today and Where Are We Going?

“…These products may be important signaling molecules involved in distinct cellular processes, such as cell death or cell growth. In addition, branched and short polymers are degraded more slowly by PARGs than long and linear poly-ADP-ribose polymers (18,52,53). This mode of action of PARGs may explain the very short half-life of poly-ADP-ribose synthesized in the presence of DNA damage in vivo (Ͻ40 s) compared with the far longer half-life (Յ7.7 h) of constitutively synthesized poly-ADP-ribose in unstimulated cells (15,435,437).…”

“…Of these two enzymatic activities, only the Parg genes and their gene products have been identified and characterized to date (18,230,297). The major mammalian poly-ADP-ribose-ribose-glycohydrolase, PARG, has both endoglycosidase and exoglycosidase activities (18,52,53), which are responsible for the hydrolysis of glycosidic riboseribose bonds internal to and at the ends of ADP-ribose polymers, respectively. The endoglycosidase activity releases free poly-ADP-ribose from PARPs and is of particular physiological importance because it may provide a mechanism for the generation of various types of free poly-ADP-ribose.…”

A Quantitative Assay Reveals Ligand Specificity of the DNA Scaffold Repair Protein XRCC1 and Efficient Disassembly of Complexes of XRCC1 and the Poly(ADP-ribose) Polymerase 1 by Poly(ADP-ribose) Glycohydrolase

“…PAR Turnover by PARG Efficiently Disassembles PARP1-XRCC1-PARG degrades the PAR PTM by a combination of exo-and endo-glycohydrolase activity (19,30,31), leaving a single ADP-ribose moiety attached to PARP1 that is a substrate for the recently identified mono(ADP-ribose) (MAR) hydrolases (32). Because XRCC1 binds weakly to ADP-ribose and iso-ADPr (Fig.…”