Covalent linkage of ADP-ribose polymers to proteins is generally considered essential for the posttranslational modification of protein function by poly(ADP-ribosyl)ation. Here we demonstrate an alternative way by which ADP-ribose polymers may modify protein function. Using a highly stringent binding assay in combination with DNA sequencing gels, we found that ADP-ribose polymers bind noncovalently to a specific group of chromatin proteins, i.e., histones H1, H2A, H2B, H3, and H4 and protamine. This binding resisted strong acids, chaotropes, detergents, and high salt concentrations but was readily reversible by DNA. When the interactions of variously sized linear and branched polymer molecules with individual histone species were tested, the hierarchies of binding were branched polymers greater than long, linear polymers greater than short, linear polymers and H1 greater than H2A greater than H2B = H3 greater than H4. For histone H1, the target of polymer binding was the carboxy-terminal domain, which is also the domain most effective in inducing higher order structure of chromatin. Thus, noncovalent interactions may be involved in the modification of histone functions in chromatin.

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...

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The enzymes poly(ADP-ribose)polymerase and poly(ADP-ribose) glycohydrolase may cooperate to drive a histone shuttle mechanism in chromatin. The mechanism is triggered by binding of the N-terminal zinc-finger domain of the polymerase to DNA strand breaks, which activates the catalytic activities residing in the C-terminal domain. The polymerase converts into a protein carrying multiple ADP-ribose polymers which displace histones from DNA by specifically targeting the histone tails responsible for DNA condensation. As a result, the domains surrounding DNA strand breaks become accessible to other proteins. Poly(ADP-ribose)glycohydrolase attacks ADP-ribose polymers in a specific order and thereby releases histones for reassociation with DNA. Increasing evidence from different model systems suggests that histone shuttling participates in DNA repair in vivo as a catalyst for nucleosomal unfolding.

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