Polynucleosomes electron microscopy

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. 

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). 

Sedimentation coefficient determinations [4,21] on polynucleosomes ADP-ribosylated in the presence of 200 fjM NAD for different time intervals, gave values of S20 w ing from 51.5 0.4 S for control nucleosomes to 44.9 0.2 S for fully relaxed nucleosomes (Fig. 3). The time course was in good agreement with the relaxation process visualized by electron microscopy. 

In order to investigate the modification of DNA accessibility in chromatin structures relaxed by ADP-ribosylation, the DNA polymerase a activity has been determined [22]. As shown in Table 1, this activity is more than twofold higher in polynucleosomes ADP-ribosylated for 25 min than in control polynucleosomes. Moreover, this increase in template capacity is correlated with the ADP-ribosylation induced relaxation time course as demonstrated by electron microscopy and sedimentation velocity analysis. 

Fig. 1. Electron microscopic visualization of reconstituted poly(ADP-ribosyl)ated Hl-depleted chromatin. Hl-depleted polynucleosomes were incubated at 30 C with I purified poly(ADP-ribose) in the presence of 200 NAD. The reaction was stopped by adding 1 mM 3-AB and nucleosomes were reconstituted by adding histone HI slowly at a ratio (Hl/nucleosome 1/1). Samples were diluted and treated for electron microscopy as described by Poirier et al. (5) (a and c) represent control and poly(ADP-ribosyl)-ated Hl-depleted chromatin reconstituted with histone HI.

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