Histone shuttling by poly(ADP-ribosylation).

Abstract

We have found that two nuclear enzymes, i.e. poly(ADP-ribose) polymerase (EC 2.4.2.30) and poly(ADP-ribose) glycohydrolase, may cooperate to function as a histone shuttle mechanism on DNA.The mechanism involves four distinct reaction intermediates that were analyzed in a reconstituted in vitro system.In the first step, the enzyme poly(ADP-ribose) polymerase is activated in the presence of histone-DNA complexes and converts itself into a protein carrying multiple ADP-ribose polymers.These polymers attract histones that dissociate from the DNA as a histonepolymer-polymerase complex.The DNA assumes the electrophoretic mobility of free DNA and becomes susceptible to nuclease digestion (second step).In the third step, poly(ADP-ribose) glycohydrolase degrades ADPribose polymers and thereby eliminates the binding sites for histones.In the fourth step, histones reassociate with DNA, and the histone-DNA complexes exhibit the electrophoretic mobilities and nuclease susceptibilities of the original complexes prior to dissociation.Our results are compatible with the view that the poly(ADP-ribosylation) system acts as a catalyst of nucleosomal unfolding of chromatin in DNA excision repair.In DNA excision repair of higher eukaryotes, the processing of NAD+-derived ADP-ribosyl residues by the poly(ADPribosylation) system may be stimulated several thousand-fold.Most of this turnover is associated with the automodification of the nuclear enzyme poly(ADP-ribose) polymerase with multiple ADP-ribose polymers and their rapid degradation by poly(ADP-ribose) glycohydrolase.The biological significance of this automodification cycle is not understood (for reviews, see Ueda and Hayaishi (1985), Althaus andRichter (1987), andDe Murcia et al. (1988)).We have tested the hypothesis that this automodification cycle catalyzes the reversible dissociation of histones from DNA, thereby exposing local DNA domains to other proteins.For this purpose, an in vitro poly(ADP-ribosylation) system was reconstituted in the presence of histone-DNA complexes, and the consequences of poly(ADP-ribosylation) on histone sequestration were analyzed by mobility shift gel electrophoresis and enzyme protection assays.The results show that the two major enzymes responsible for poly(ADP-ribose) turnover in DNA excision repair may catalyze a reaction cycle in which