Poly native chromatin

Fig. 1. Time course study of poly(ADP-ribosyl)ation of chicken erythrocyte core particles (O—O), calf thymus native chromatin (A—A), and HI-depleted chromatin (A—A) by purified calf thymus poly(ADP-ribose) polymerase at 30°C. Poly(ADP-ribose) polymerase (2 Mg/OD unit), which was purified free of its DNA according to Zahradka and Ebisuzaki [15], was incubated with various chromatin preparations at 200 nM NAD. At various times, the reaction was stopped by the addition of 10% TCA (Cl3AcOH)/2% PPi, and the activity was determined according to Aubin et al. [8, 9]. — represents the endogenous activity of the poly(ADP-ribose) polymerase found on calf thymus native chromatin...

In order to elucidate the nature of the poly(ADP-ribose) polymerase interaction with the various components of chromatin, biochemical and morphological studies on various levels of organization of histone-DNA complexes were carried out. The poly-(ADP-ribosyl)ation of histone Hl-DNA complexes, core particles, chromatin depleted of histone HI, as well as native chromatin, were systematically studied using purified DNA-free calf thymus poly(ADP-ribose) polymerase in an in vitro reaction system. 

Biochemical and Morphological Study of the Poly( ADP-Ribosyl)ation of Native Chromatin, Histone HI Depleted Chromatin, and Core Particles... 

First, we have analyzed the stimulatory effect of various levels of chromatin structure on the enzymatic activity of purified calf thymus poly(ADP-ribose) polymerase free of its DNA (Fig. 1). We found that native chromatin stimulates the purified polymerase maximally. Chromatin depleted of non-histone proteins with 0.3 M NaCl has almost the same capacity to stimulate the polymerase as native chromatin (data not shown). Core particles were also found to stimulate the activity of the purified polymerase, but to a lesser degree. Finally, chromatin depleted of histone HI was the least stimulatory, having one-third the effect that native chromatin has on the polymerase. No... 

Fig. 2A,B. Acid-urea gel electrophoresis of [ Pj poly(ADP-ribosyl)ated histones isolated from native chromatin (at), core particles b) and Hl-depleted chromatin (c). These different fractions were incubated as described in the legend of Fig. 1 and precipitated with 20% TCA (ClgAcOH). Histones were extracted as described previously [8, 9] and separated by acid-urea gel electrophoresis according to Panyim and Chalkley [17]. A Stained gel, B autoradiogram of the gel...

Furthermore, the acceptor proteins were analyzed by acid urea polyacrylamide gel electrophoresis (Fig. 2). Histones H2B, HI and protein A24 were found to be poly-(ADP-ribosyl)ated in native chromatin while in histone Hl-depleted chromatin and core particles it was found that histone H2B and protein A24 were poly(ADP-ribosyl)-ated. The presence of the hyper(ADP-ribosyl)ated forms of histone H2B on each of these various levels of chromatin structure have been confirmed by western blot analysis and by two-dimensional polyacrylamide gel electrophoresis (data not shown). The poly(ADP-ribosyl)ation of protein A24 have also been demonstrated by this last technique. 

Fig. 3. Electron microscopic visualization of control and poly(ADP-ribosyl)ated native chromatin, HI-depleted chromatin and core particles. Following poly(ADP-ribosyl)ation at 200 yM NAD as described in the legend of Fig. 1, the samples were diluted to 0.01 OD unit at 260 nm and fixed for 1 h at 20 C in buffer containing 40 mM NaCl, 10 vaM TEACL, 0.2 mM EDTA and 0.1% (v/v) glutaraldehyde and processed for electron microscopy according to Poirier et al. [6]. Control native chromatin, Hl-depleted chromatin and core particles (a-c). Poly(ADP-ribosyl)ated native chromatin, Hl-depleted chromatin and core particles (d-f). Notice the dissociation of the DNA from the nucleosome cores in the poly(ADP-ribosyl)ated core particles. Also notice the relaxation of chromatin structure in the native chromatin. Big arrows indicate the automodified enzyme and small arrows indicate the DNA (145 bp) free after poly(ADP-ribosyl)ation. Th bars indicate 1000 A...

Concomitantly, structures resulting from the poly(ADP-ribosyl)ation of native chromatin, chromatin-Hl and core particles were examined by electron microscopy. We found, as described earlier for pancreatic chromatin [6, 7], that calf thymus chromatin adopts a more relaxed conformation upon poly(ADP-ribosyl)ation by purified calf thymus poly(ADP-ribose) polymerase free of its DNA (Fig. 3a,d). It was also found that this chromatin exhibited a lower sedimentation velocity as compared to control chromatin [6]. And recently, it has been shown that DNA polymerase a activity is more than twofold higher in the presence of pancreatic polynucleosomes ADP-ribosylated as compared to control polynucleosomes [16]. In striking contrast, no ultrastructural effect was observed when chromatin depleted of histone HI was poly(ADP-ribosyl)ated (Fig. 3b, e). 

These modifications were also associated with a high level of poly(ADP-ribosyl)ation of the enzyme. Furthermore we have found that upon poly(ADP-ribose) glycohydrolase action on poly(ADP-ribosyl)ated chromatin, modification of histone H2B was more resistant than modification of other nucleosomal proteins (8). Menard et aL (9) have also shown, in a reconstituted in vitro poly(ADP-ribose) turnover system, that the half life on various acceptor proteins is quite different. These results suggest that the preferential distribution of poly(ADP-ribose) on nuclear proteins is related on the one hand to the localization of the enzyme on active (Hl-depleted) and inactive (native) chromatin and on the other hand to the turnover rate of poly(ADP-ribose) on the enzyme which then determines the pattern of poly(ADP-ribosyl)ation of nuclear proteins. We will discuss in the last section the preferential distribution of die enzyme in the nucleus. 

Modulation of chromatin structure by poly(ADP-ribosyl)ation effect of poly(ADP-ribosyl)ation on chromatin superstructure. We have shown that poly(ADP-ribosyl)ation of native chromatin in vitro from endogenous and exogenous activity of the enzyme led to decondensation of the 30 nm chromatin fiber (3, 5, 10). This decondensation was associated with h5 rmodified forms of histone HI at high NAD concentrations. We have thus studied the stmctural effect of poly(ADP-ribosyl)ation at a high NAD level on HI-depleted chromatin where modification of core histones was observed (7). This specific poly(ADP-ribosyl)ation prevented the recondensation of polynucleosomes reconstituted with native histone HI (Fig. 1). 

Since the major poly(ADP-ribose) acceptor protein in chromatin and in intact cells is poly(ADP-ribose) polymerase (5, 11, 12), we have studied the interaction of the highly modified polymerase with native and Hl-depleted chromatin in order to define its role as a potential modulator of chromatin structure. When poly(ADP-ribosyl)ated native chromatin was resolved on sucrose gradients, it was found that most of the modified enzyme was... 

Fig. 3. Binding of polyclonal and monoclonal antibodies against histone to poly(ADP-ribosyl)ated native chromatin. ELISA were carried out on chromatin with rabbit antisera as described by Muller et al. (24) and with monoclonal antibodies by Muller et al. (25). Native chromatin direcdy diluted in PBS-T (H), native chromatin which was incubated in the presence of Mg+ and poly(ADP-ribose) polymerase ( ), native chromatin incubated in the presence of Mg+2, poly(ADP-ribose) polymerase and 200 iM NAD (El), in the presence of Mg+2, poly(ADP-ribose) polymerase, 200 iM NAD and inhibitors, NAM 10 mM ( ), 3-AB 5 niM (0). Controls were also done with no enzyme in the presence of Mg+, 200 lM NAD and inhibitors, NAM (DQ), 3-AB (S). Antibodies used were A monoclonal antibody A21 supernatant (recognizes the N-terminal part of H2B in residues 1-25), B monoclonal antibody C13 supernatant (recognizes region situated in residues 10-35 of H2B), C antiserum to peptide 130-135 H3, D antiserum against H2A.

When rat pancreatic polynucleosomes were poly(ADP-ribosylated) with purified calf thymus poly(ADPR) polymerase and examined by electron microscopy a relaxation of their native zigzag structure was observed, even at high ionic strengths they showed a close resemblance to chromatin depleted of histones HI. The relaxed state of poly(ADP-ribosylated) polynucleosomes was also confirmed by sedimentation velocity analysis [19, 20]. Locally relaxed regions can also be generated within poly-nucleosome chains by the activity of their intrinsic poly(ADPR) polymerase and appeared to be correlated with the formation of hyper(ADP-ribosylated) forms of histone HI and an increase of DNA polymerase activity [21]. The posttranslational transitory modifications of histones are potential modulators of chromatin stmcture. This may be involved in DNA transcription, replication, and repair. 

The poly(ADP-ribosyl)ation of histones HI and H2B and poly(ADP-ribose) polymerase in vivo during DNA repair (12, 18) destabilizes chromatin superstructure and core nucleosome structure. On the one hand, poly(ADP-ribosyl)ation of these histones causes local disruption of the 30 nm fiber of Hl-containing chromatin (3) and prevents the recondensation of Hl-depleted chromatin (Fig. 1 and 4). On the other hand, as shown in Fig. 3, core histones become accessible to core histone antibodies upon poly(ADP-ribosyl)ation of native and Hl-depleted chromatin, thus suggesting a destabilization of core nucleosome structure. This effect would cause an increased accessibility of core DNA to various multienzymatic repair enzyme complexes in poly(ADP-ribosyl)ated chromatin. Indeed Althaus et al. (23), have shown that poly(ADP-ribosyl)ation is associated with the removal of bulky adducts from chromatin by repair enzymes. Upon resealing of DNA strand breaks, poly(ADP-ribose) polymerase redistributes to the three types of nuclear compartments (Fig. 4). It would re-equilibrate with open (Hl-depleted chromatin) or condensed (Hl-containing chromatin)... 

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