Uncoupling of DNA excision repair and nucleosomal unfolding in poly (ADP-ribose)-depleted mammalian cells

Abstract

The repair of DNA damage in eukaryotic cells is closely coupled with local changes of chromatin structure such that newly synthesized repair patches transiently appear in 'free' DNA domains with increased accessibility to enzymatic and chemical probes. We have isolated these domains from mammalian cells repairing bulky DNA adducts. During the first 3 h of repair, excision of adducts occurred exclusively in free DNA and was closely linked with the appearance of newly synthesized repair patches. Following depletion of chromatin-bound poly(ADP-ribose), the repositioning of repair patches into these domains was completely blocked, although overall repair patch synthesis was unaltered. Concomitantly, DNA adducts were no longer excised and tended to accumulate in free DNA domains. Our results suggest a tight coupling of the excision step with the formation of free DNA domains by a mechanism involving poly ADPrnbosylation of chromatin proteins.In chromatin of mammalian cells, newly synthesized DNA repair patches exhibit a transient micrococcal nuclease hypersensitivity. This hypersensitivity is thought to reflect local disruptions in the tightly packed nucleosomal organization of chromatin, causing exposure of 'free' DNA domains (for review see 1,2). The function of these domains as well as the mechanisms involved in their formation are unknown. We have speculated that the post-translational poly ADP-ribosylation of chromatin proteins might be involved in the formation of free DNA domains in DNA excision repair. Poly ADP-ribosylation is catalyzed by the enzyme poly(ADP-ribose)polymerase (1,3,4; EC 2.4.2.30). Following activation by DNA nicks, this enzyme operates in a strictly processive manner (5). Continuous treatment of living cells with benzamide, a competitive inhibitor of this enzyme (3,4,6), results in the degradation of chromatin-bound ADP-ribose polymers (7) by the enzyme poly(ADP-ribose)glycohydrolase (3,4). Using non-replicating adult rat hepatocytes in primary monolayer culture, we have established conditions for the complete depletion of chromatin-associated poly(ADP-ribose) (7,8). Hepatocytes survive up to 9 days under these conditions and maintain expression of liver-specific functions (8). Thus, poly(ADPribose)-depleted hepatocytes represent a convenient model system to study the role of poly ADP-ribosylation in specific steps of DNA repair.We have previously shown that unfolded free DNA domains can be isolated from the chromatin of intact mammalian cells by taking advantage of their preferential accessibility to 8-methoxypsoralen (9). Upon intercalation into free DNA domains of living cells, 8-methoxypsoralen can be photoactivated to form bifunctional DNA adducts crosslinking the two DNA strands. Crosslinked (free) DNA domains can then be isolated quantitatively following a denaturation/renaturation treatment and subsequent nuclease SI digestion of non-crosslinked DNA strands (for details see 9).Here we have isolated free DNA domains from non-replicating hepatocytes (9

Deletion Mutants of Poly(ADP-Ribose) Polymerase A Support a Model of Cyclic Association and Dissociation of Enzyme from DNA Ends During DNA Repair

“…These observations, together with the data fromour current study (Figures 2 and 3), may begin to clarify earlier observations showing a significant increase in the accessibility of 3'-OH primer sites to DNA polymerase in nuclei preincubated with NAD (Mathis & Althaus, 1990;Roberts et al, 1974). The ability of NAD to release this template restriction and expose DNA primer sites in chromatin has been observed to vary during the cell cycle (Roberts et al, 1974).…”

“…Immunochromatography with antibodies to poly(ADP-ribose) has revealed that chromatin regions adjacent to DNA strand breaks in HeLa cells as well as in polyoma-and SV40-infected cells possess a high concentration of poly(ADP-ribosy1)ated proteins (Baksi et al, 1987;Thraves et al, 1985). In this regard, Mathis and Althaus (1990) demonstrated an influence of chromatin structure on DNA repair while studying the inhibition of repair with chemical inhibitors of PADPRP. An open chromatin structure became apparent during the repair of bulky alkylation adducts.…”

The role of poly(ADP-ribose) in the DNA damage signaling network

“…This mechanism might act in vivo as a catalyst of nucleosome unfolding, by transiently displacing histones from DNA and hence facilitating DNA transactions by increasing template accessibility. In poly(ADP-ribose)-depleted cells, nucleosome unfolding accompanying the repair of bulky DNA adducts as well as removal of such lesions are severely impaired (Mathis and Althaus 1990).…”

Chain Length Analysis of ADP‐Ribose Polymers Generated by Poly(ADP‐Ribose) Polymerase (PARP) as a Function of ß‐NAD and Enzyme Concentrations

“…Not surprisingly, the covalent attachment of ADP-ribose (two negative charges per nucleotide) polymers to an acceptor molecule leads to the shuttling off, or release, of this protein from the helical phosphodiester backbone of DNA (20) (one negative charge per nucleotide). Further, as a result of this ionic repulsion between ADP-ribose polymers and DNA, chromatin structure and function may become uncoupled (21). This uncoupling of chromatin structure and function may directly alter the physiological course of DNA replication (22), DNA recombination (23), gene expression (24)(25)(26), DNA base excision repair (27,28), and cell survival or apoptosis (29)(30)(31).…”