Time course of polynucleosome relaxation and ADP‐ribosylation

Abstract

Isolated rat pancreatic polynucleosomes were poly(ADP-ribosylated) with purified calf thymus poly(ADPribose) polymerase. A time course study was performed using an NAD concentration of 200 pM and changes in nucleosomal structure were investigated by means of electron microscopy visualization and sedimentation velocity determinations. In parallel, analyses of histone H1 poly(ADP-ribosylation) and determinations of DNA polymerase CI activity on ADP-ribosylated polynucleosomes were done at different time intervals.A direct kinetic correlation between ADP-ribose incorporation, polynucleosome relaxation amd histone HI hyper-ADP-ribosylation was established. In addition, DNA polymerase CI activity was highly stimulated on ADPribosylated polynucleosomes as compared to control ones, suggesting increased accessibility of DNA to enzymatic action.Because of the strong evidence implicating histone HI in the maintenance of higher-ordered chromatin structures, the present study may provide a basis for the interpretation of the involvement of the histone HI ADP-ribosylation reaction in DNA rearrangements during DNA repair, replication or gene expression.Poly(ADP-ribose) polymerase, an enzyme ubiquitous to eucaryotes, catalyses the synthesis of a homopolymer of ADP-ribose units at the expense of the cellular NAD pool. The poly(ADP-ribose) formed is generally covalently bound to various nuclear proteins, mainly the enzyme itself and histones, and it has been suggested to be involved in DNA replication, DNA repair and cell differentiation (for reviews, see [I -41).Post-translational modifications of histones, i. e. phosphorylation, acetylation and poly(ADP-ribosylation), may be potential modulators of the nucleosomal structure during DNA transcription and replication (for reviews, see [5 -71). Amongst the histones it was suggested that given the localization of histone HI at the region of entry and exit of the nucleosomal DNA, this histone would stabilize the nucleosomal structure such as to modulate, and subsequently maintain, the organization and higher-order coiling transitions of a polynucleosomal chain into a helix or solenoid of pitch 11 nm [8, 91 (see also the Discussion).We focused on histone poly(ADP-ribosylation) since poly(ADP-ribose) polymerase is believed to be located preferentially in the internucleosomal regions of chromatin [ 10, 111 and, amongst the histones, HI is the best acceptor of poly(ADP-ribose) in vitro in isolated nuclei [12-141 and in vivo in untreated tissue [I 51. Moreover, ADP-ribosylation was recently shown to cause the relaxation of the polynucleosome structure in vitvo, presumably through its effect of modificaAbbreviations. ADP-ribose, adenosine(5')diphospho(5)-P-~-ribose; poly(ADP-ribose), polymer of ADP-ribose.Enzymes. Poly(ADP-ribose) polymerase (EC 2.4.99.-); micrococcal nuclease (EC 3.1.31.1); DNA polymerase c( (EC 2.7.7.7); pyruvate kinase (EC 2.7.1.40).tion of histone H1, using either exogenously added purified calf thymus enzyme [ 161 or the intrinsic poly(ADP-ribose) polymerase [17]. I...

Inhibition of calf thymus type II DNA topoisomerase by poly(ADP-ribosylation).

“…Thus if poly(ADPribosylation) were to be of significance in vivo, one would expect these proteins to be major acceptors. Although any proposal for the significance of the inhibition is clearly speculative, inhibition of topoisomerase II at times of chromatin relaxation resultant from histone HI poly(ADP-ribosylation) (Aubin et al, 1983;Niedergang et al, 1985) might be necessary to restrict the introduction of topological changes in the chromatin. wt.…”

Poly(ADP-ribosyl)ation of Histone H1 Correlates with Internucleosomal DNA Fragmentation during Apoptosis

“…Evidence has been accumulated showing the correlation between the change of chromatin structure and poly-(ADP-ribosyl)ation of histone H1. Electron microscopic studies have revealed that the solenoid chromatin structure was relaxed by the poly(ADP-ribosyl)ation of histone H1 protein exposing internucleosomal DNA regions from the interior of the structure (54,55). De Murcia et al (56) further demonstrated that the degradation of ADP-ribose units on poly(ADP-ribosyl)ated histone H1 by poly(ADP-ribose) glycohydrolase restored the solenoid chromatin structure.…”

Mechanisms of poly(ADP‐ribose) polymerase catalysis; mono‐ADP‐ribosylation of poly(ADP‐ribose) polymerase at nanomolar concentrations of NAD

“…[3]) and the histone adducts thus formed can be further elongated by poly(ADPribose) polymerase [7]. Mechanistic interpretation of the enzymology of poly-ADP-ribosylation and of important supramolecular events in chromatin that appear to coincide with shifts in protein patterns of ADP-ribosylation [8,9] depend on a more detailed understanding of the initial steps of ADPribosylation at the poly(ADP-ribose) polymerase level [lo]. Pursuing this question, the existence of an early product [l l] was indicated.…”