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Protease cleavage of histones in nucleosomes after their labelling in cell lysates using the endogenous poly(ADP-ribose) polymerase. Soluble oligonucleosomes, containing ADP-ribosylated histones were digested with 0 (a), 0.35 (b), 1.00 (c), or 2.06 ( d ) mg/mL of a-chymotrypsin as described in Materials and methods. Digestions with the Staphylococcus aureus V8 protease were with 0.65 (e), 1.43 (f), or 2.50 (g) mg/mL of enzyme. Samples were analyzed by electrophoresis on 18% polyacrylamide gels in the presence of SDS. In panel A, the Coomassie blue staining of the gel is shown. The autoradiogram of the same gel is shown in B. In panel C, the stained gel and the autoradiogram were superimposed and photographed together. Histones and the putative poly(ADP-ribose) polymerase displaying an apparent molecular weight of 110 000 to 120 000 on these gels are indicated to the right of C. Autorad., autoradiogram.
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Calf thymus histones (individually isolated or mixtures) and high mobility group proteins were ADP-ribosylated in vitro using [32P]NAD+ and immobilized purified poly(ADP-ribose) polymerase. The modified histones were then subjected to V8 protease or alpha-chymotrypsin digestion and the resulting peptides were separated by electrophoresis on acetic...
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... Coomassie brilliant blue staining of the gel showed the histone peptides after a-chymotrypsin digestion (Fig. 3A, lanes b and d). In this case histone H1 disappeared very early from the gel (Fig. 3A, lane b) g f e d c b a g f e d c b a g f e d c b a .*l"I-P ==*Im-pm - poly (ADP-R) synthetase ...
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... Coomassie brilliant blue staining of the gel showed the histone peptides after a-chymotrypsin digestion (Fig. 3A, lanes b and d). In this case histone H1 disappeared very early from the gel (Fig. 3A, lane b) g f e d c b a g f e d c b a g f e d c b a .*l"I-P ==*Im-pm - poly (ADP-R) synthetase ...
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... + Autorad. lished in previous studies (Weintraub and van Lente 1974). The same treatment seems to remove only 20-30 amino acids from each of the four core histones. The data in Fig. 3A displaying some preference in size of core histone peptides migrating just above, near, and below H4 are in agreement with the chromatin trypsinization studies of Weintraub and van Lente (1974). A similar conclusion can be drawn from the V8 protease cleavage of oligonucleosomes (Fig. 3A, lanes e and g). Again histone H1 disappeared ...
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... amino acids from each of the four core histones. The data in Fig. 3A displaying some preference in size of core histone peptides migrating just above, near, and below H4 are in agreement with the chromatin trypsinization studies of Weintraub and van Lente (1974). A similar conclusion can be drawn from the V8 protease cleavage of oligonucleosomes (Fig. 3A, lanes e and g). Again histone H1 disappeared rapidly from the gel. The bands in lanes e-g in Fig. 3A migrating slightly below H1 were intact V8 protease bands. That these bands represented indeed V8 and not H1 could also be seen from the fact that they increased in concentration from lane e to f to g, where the V8 concentration ...
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... of core histone peptides migrating just above, near, and below H4 are in agreement with the chromatin trypsinization studies of Weintraub and van Lente (1974). A similar conclusion can be drawn from the V8 protease cleavage of oligonucleosomes (Fig. 3A, lanes e and g). Again histone H1 disappeared rapidly from the gel. The bands in lanes e-g in Fig. 3A migrating slightly below H1 were intact V8 protease bands. That these bands represented indeed V8 and not H1 could also be seen from the fact that they increased in concentration from lane e to f to g, where the V8 concentration ...
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... autoradiogram of the same gel (Fig. 3B) showed that the histone peptides arising by protease digestion were mono- or oligo-(ADP-ribosy1)ated about to the same extent as the intact histones. In the absence of protease digestion, histones H3 and H2B, as well as the putative poly(ADP-ribose) polymerase (indicated to the right of C), were preferentially labelled followed by H2A, ...
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... showed that the histone peptides arising by protease digestion were mono- or oligo-(ADP-ribosy1)ated about to the same extent as the intact histones. In the absence of protease digestion, histones H3 and H2B, as well as the putative poly(ADP-ribose) polymerase (indicated to the right of C), were preferentially labelled followed by H2A, H4, and H1 (Fig. 3B, lane a). The putative poly(ADP-ribose) polymerase radioactively labelled band disappeared even faster than histone H1 during both a-chymotrypsin and V8 protease digestion of oligonucleosomes (Fig. 3B). These data showed that this enzyme was highly accessible to proteases in chromatin. This conclusion was not modified even in the case that not ...
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... H2B, as well as the putative poly(ADP-ribose) polymerase (indicated to the right of C), were preferentially labelled followed by H2A, H4, and H1 (Fig. 3B, lane a). The putative poly(ADP-ribose) polymerase radioactively labelled band disappeared even faster than histone H1 during both a-chymotrypsin and V8 protease digestion of oligonucleosomes (Fig. 3B). These data showed that this enzyme was highly accessible to proteases in chromatin. This conclusion was not modified even in the case that not all of the polymerase seen in lane a of Fig. 3B was bound to nucleosomes, since the protease digestion of the polymerase was rapid and quantitative. This was concluded from the complete absence ...
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... polymerase radioactively labelled band disappeared even faster than histone H1 during both a-chymotrypsin and V8 protease digestion of oligonucleosomes (Fig. 3B). These data showed that this enzyme was highly accessible to proteases in chromatin. This conclusion was not modified even in the case that not all of the polymerase seen in lane a of Fig. 3B was bound to nucleosomes, since the protease digestion of the polymerase was rapid and quantitative. This was concluded from the complete absence of the putative poly(ADP-ribose) polymerase band in lanes b-g in Fig. ...
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... to proteases in chromatin. This conclusion was not modified even in the case that not all of the polymerase seen in lane a of Fig. 3B was bound to nucleosomes, since the protease digestion of the polymerase was rapid and quantitative. This was concluded from the complete absence of the putative poly(ADP-ribose) polymerase band in lanes b-g in Fig. ...
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... have then performed protease digestion on histones in intact oligonucleosomes isolated from cultured mouse cells after their labelling with I~'P]NAD+ using the endogenous poly(ADP-ribose) polymerase (Fig. 3). In this case, the labelled mono(ADP-ribosy1)ated histones were digested to the same extent as bulk nonlabelled histones by the a-chymotrypsin or Staphylococcus aureus V8 protease. This was deduced from the Coomassie blue stained histone peptides, showing that the bulk nonlabelled histones coin- cided with the peptide labelling ...
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... between in vitro and cell lysates ADP-ribosylated histones may be due to the following two reasons. First, during in vitro ADP-ribosylation using isolated histones or HMGs and poly(ADP-ribose) polymerase, the ADP-ribosylation species of histones were different from those obtained during ADP-ribosylation in cell lysates (cf. Figs. 1 and 2 with Fig. 3). Poly(ADP-ribose) polymerase seems to be specifi- cally located in polynucleosomes ( Leduc et al. 1986), and thus histone modification must be specific and depend on histone topology in the intact nucleosome. Furthermore, we have found when purified components were used such as polymerase and glycohydrolase that different acceptor ...
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... mono-or oligo-(ADP-ribosy1)ated histones were obtained during labelling of oligonucleosomes in cell lysates (Fig. 3). In addition they displayed the same sensitivity to proteases as nonmodified molecules (Fig. 3). This finding is in contrast to the results in Fig. 1, for example, where the mono(ADP-ribosy1)ated species of histone H1 failed to be efficiently digested by a-chymotrypsin; there was no major H1 peptide in Fig. lA, lane g, that was ...
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... mono-or oligo-(ADP-ribosy1)ated histones were obtained during labelling of oligonucleosomes in cell lysates (Fig. 3). In addition they displayed the same sensitivity to proteases as nonmodified molecules (Fig. 3). This finding is in contrast to the results in Fig. 1, for example, where the mono(ADP-ribosy1)ated species of histone H1 failed to be efficiently digested by a-chymotrypsin; there was no major H1 peptide in Fig. lA, lane g, that was sufficiently mono(ADP-ribosy1)ated in Fig. lB, lane g. This observation leads to the conclusion that ...
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... the differences in the accessibility to protease digestion between in vitro or in cell lysates arises from differences in the method of protease digestion. In the first case, isolated histones were subjected to protease digestion ( Figs. 1 and 2). In the second case, histones in intact native oligonucleosomes were digested with proteases (Fig. 3). However, when mono(ADP-ribosy1)ated histones were extracted with 0.4 M HC1 from this oligonucleosome fraction and were subjected to protease digestion, similar data to those shown in Fig. 3 were obtained (not shown). Thus, the differences in protease sensitivity between ADP-ribosylated histones obtained with exogenous or endogenous ...
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... histones were subjected to protease digestion ( Figs. 1 and 2). In the second case, histones in intact native oligonucleosomes were digested with proteases (Fig. 3). However, when mono(ADP-ribosy1)ated histones were extracted with 0.4 M HC1 from this oligonucleosome fraction and were subjected to protease digestion, similar data to those shown in Fig. 3 were obtained (not shown). Thus, the differences in protease sensitivity between ADP-ribosylated histones obtained with exogenous or endogenous poly(ADP-ribose) polymerase have to be explained solely on the basis of differences in the modifica- tion sites within the primary structures of the histone molecules and in the number of ...
