Poly(ADP-ribose) may signal changing metabolic conditions to the chromatin of mammalian cells.

Abstract

In mammalian cells, NADI serves a dual role as a respiratory coenzyme and as a substrate for the posttranslational poly(ADP-ribose) modification of chromatin proteins, catalyzed by the nuclear enzyme poly(ADP-ribose) polymerase EC 2.4.2.30]. Biological evidence strongly suggests that poly(ADP-ribosyl)ation modulates chromatin functions, although the precise molecular mechanisms involved have not yet been elucidated. Here we describe conditions for the rapid uptake of exogenously supplied NADI by living hepatocytes in primary monolayer culture. Raising the intracellular NADI concentration by 70% caused a 5-fold increase of chromatin-bound poly(ADP-ribose). We conclude that the constitutive level of posttranslational poly(ADP-ribose) modifications of chromatin proteins in mammalian cells is related to the availability of NAD+, which varies in different physiological and pathological states. We propose that poly-(ADP-ribose) may serve a hitherto unrecognized function by signaling altered metabolic conditions to the chromatin and thus modulate its functions in tune with changing metabolic states.Many lines of biological evidence support the concept that the posttranslational modification of chromatin -roteins by the nuclear enzyme, poly(ADP-ribose) polymei.Xe [NAD+ ADP-ribosyltransferase, EC 2.4.2.30], modulates the phenotypic expression of various chromatin function, in higher eukaryotes (1, 2). The respiratory coenzyme NAD-serves as the specific substrate of this chromatin enzyme. Thi nrompts the question, Why is there such a direct linkage b, w-veen a posttranslational regulatory mechanism peculiar to chromatin and cellular redox reactions? A partial explanation of this is provided by the concept of "suicidal NAD+ depletion" as proposed by Berger (3). This concept emphasizes that carcinogen-inflicted DNA damage in mammalian cells dtamatically stimulates the utilization of NAD+ for poly(ADPribose) biosynthesis and concomitantly depletes the cellular NAD+ pool(s). Thus, the chromatin-associated enzyme poly-(ADP-ribose) polymerase may serve as part of a metabolic "shut-off' mechanism under conditions of excessive DNA damage (3). Here we examined the hypothesis that metabolic conditions affecting the availability of the respiratory coenzyme NAD+ may be signaled to chromatin by poly(ADPribose). Therefore, experimental conditions were set up to determine whether an altered level of posttranslational poly(ADP-ribose) modification could be forced upon the chromatin of mammalian cells by directly manipulating the intracellular NAD+ pool with exogenously supplied NAD+.MATERIALS AND METHODS NAD+ Loading of Hepatocytes. Hepatocytes were isolated from adult rats (male SIVZ rats, 180-240 g, fed ad lib) and cultured in 100-mm Coming tissue culture dishes (seeding density, 1.2 x i07 cells per dish, in 10 ml of medium L-15 per dish) as described (4). Following a 4-hr adaptation to culture conditions, the monolayers were washed and maintained under serum-free conditions with another medium change after 24 hr. The hepatocy...

PARP-1 enhances the mismatch-dependence of 5′-directed excision in human mismatch repair in vitro

“…2D, PARP-1 effects on the 5′- or 3′-directed reactions in the presence of 10 μM NAD + were similar to those observed in the absence of the nucleotide except that higher PARP-1 concentrations are required to achieve maximal effects. We also evaluated PARP-1 effects in the presence of 500 μM NAD + , which approximates the intracellular concentration [29, 30]. Except for a shift to somewhat higher PARP-1 concentrations, results were similar to those obtained in the presence of 10 μM NAD + (Fig.…”

Regulation of Poly(ADP-ribose) Polymerase-1 by DNA Structure-specific Binding

“…The above described ability of PARP-1 to form macromolecular complexes with DNA in a structure-specific fashion prompted us to investigate whether such physical interactions result in the catalytic activation of PARP-1. To address this question, we evaluated the auto-poly(ADP-ribosyl)ation rates upon PARP-1 binding to non-B DNA structures under conditions optimal for catalytic activity of mammalian PARP-1 proteins and with NAD ϩ at an upper limit of physiological concentrations (30). The in vitro auto-poly(ADP-ribosyl)ation of PARP-1 was stimulated by the addition of different non-B DNA constructs (Fig.…”

Poly-ADP-ribosylation in health and disease

“…The synthesis of pADPr is directly proportional to the number of single stand breaks (SSBs) and double strand breaks (DSBs) present in genomic DNA [10]. Also, both constitutive and stimulated levels of ADP-ribose are directly related to the concentration of NAD + in cells [58,59]. pADPr resulting from DNA damage has a very short half-life compared to constitutive polymers (< 1 min compared to 7.7 h) [13].…”