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Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosy1)ated nucleosomal proteins
A. GAUDREAU' AND L. M~NARD' Centre de recherche sur les rntcanisrnes de skcrttion, Facultk des sciences, Universite' de Sherbrooke,
Sherbrooke (Quk.), Canada JlK 2Rl G. DE MURCIA*
Institut de biologie rnolkculaire et cellulaire, Centre national de la recherche scientifique, 15, rue Descartes, 65 000 Strasbourg, France
AND
G. G. POIRIER"~
Centre de recherche sur les rnkcanisrnes de skcrttion, Facultt des sciences, Universite' de Sherbrooke, Sherbrooke (Qut . ) , Canada JlK 2Rl
Received July 19, 1985
Gaudreau, A., Mknard, L., de Murcia, G. & Poirier, G. G. (1986). Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase . . activity on poly(ADP-ribosy1)ated nucleosomal proteins. Biochern. Cell Biol. 64, 146-153
Hydrolysis of protein-bound 32~-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosy1)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosy1)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosy1)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations ( 2 1 0 pM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosy1)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.
Gaudreau, A., Mknard, L., de Murcia, G. & Poirier, G. G. (1986). Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosy1)ated nucleosomal proteins. Biochern. Cell Biol. 64, 146-153
L'hydrolyse par la glycohydrolase du poly(ADP-ribose) marquk au 32P et.lik aux protkines nuclkosomales a kvklk une accessibilitk diffkrentielle du polymkre attachk aux protkines poly(ADP-ribosy1)kes. L'hydrolyse rapide des formes hyper(ADP-ribosy1)Ces de I'histone H1 indique I'absence d'un complexe dimkrique entre deux molkcules d'histone HI . Lorsque le patron d'hydrolyse des histones poly(ADP-ribosy1)kes a btC analysk on a obsewk que le polymkre attachk a I'histone H2B est plus rksistant k I'hydrolyse que le polymkre attachk aux histones H1 et H2A et k la protkine A24. L'hydrolyse des protkines modifikes, qui sont marques ?I hautes concentrations de NAD ( 2 1 0 pM), indique que la seule protCine nucltosomale de haut poids rnol&ulaire marquee de f a ~ o n importante est la poly(ADP-ribose) polymkrase. Finalement une Ctude de rnicroscopie klectronique dkmontre que la poly(ADP-ribose) polymbrase hyper(ADP-ribosyl)ke, alors dissociCe de son complexe ADN-enzyme, retoume a 1'ADN lorsqu'elle est traitbe avec la poly(ADP-ribose) glycohydrolase.
Introduction The evidence implicating the involvement of poly-
(ADP-ribose) in DNA repair, replication, and transcrip- tion has been growing (1, 2). Poly(ADP-ribose) is
ABBREVIATIONS: poly(ADP-ribose), polymer of adenosine 5'-diphospho-5-P-D-ribose; LDS, lithium dodecyl sulfate; PCA, perchloric acid; DTT, threo-l,4-dimercapto-2,3-butane- diol (Cleland's reagent); TCA, trichloroacetic acid; CTAB, cetyltrimethylarnmonium bromide, TEA, triethanolamine; PAGE, polyacrylarnide gel electrophoresis.
'New address: Centre de recherche sur le cancer, LYH6tel- Dieu de QuCbec, 11 c6te du Palais, QuCbec (Quk.), Canada, G1R 2J6, and Dkpartement de biochimie, UniversitC de Laval, Laval (QuC.), Canada GIK 7P4.
'Dr. de Murcia was on leave on a North Atlantic Treaty Organization and National Research Council Fellowship.
3 ~ u t h o r to whom all correspondence should be addressed.
synthesized by a chromatin-bound enzyme which in response to DNA strand breaks (3), caused by different agents either chemical or physical, synthesizes the polymer on itself as well as on other protein constituents of the chromatin structure (4-7).
Poly(ADP-ribose) polymerase has been shown to be associated with the nucleosome structure (8-10) and to modulate chromatin structure by relaxation which does not exclude the aggregation of some adjacent nucleo- soma1 cores. Poly(ADP-ribose) polymerase enzymatic activity is regulated (1 1, 12) by a mechanism of auto(ADP-ribosylation) in which the enzyme synthe- sizes the polymer on itself, which in turn inhibits its ADP-ribosylating activity (13-15). This inhibition is due mainly to the fact that when the polymerase reaches a sufficient level of ADP-ribosylation it dissociates from DNA (13-15).
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GAUDREAU ET AL.
Poly(ADP-ribose) glycohydrolase (1 6), on the other hand, being an exoglycosidase, degrades the poly(ADP- ribose) into ADP-ribose residues. T h e loss of ADP- ribose residues o n the polymerase reactivates the poly- merase and also restores the DNA-enzyme complex (12). Upon glycohydrolase action there is at least one ADP-ribose residue which remains bound to the accep- tor protein which is suggested to be hydrolysed by ADP-ribosyl lyase (17).
In this work, w e have studied the hydrolysis of poly(ADP-ribosy1)ated proteins in polynucleosomes by poly(ADP-ribose) glycohydrolase, to gain further know- ledge on the metabolism of poly(ADP-ribosy1)ated
' proteins in vivo where poly(ADP-ribose) turnover has been shown to be very fast (4, 5).
Materials and methods LSD and P-NAD were purchased from Boehringer Mann-
heim Co., MontrCal (QuC.). [ 3 2 ~ ] ~ ~ ~ was obtained from New England Nuclear, Montreal (QuC.), and acrylamide and
' bis-acrylamide were from Bio-Rad, Toronto, Ont. All other chemicals are from Sigma Co., St. Louis, MO.
Polynucleosome preparation Polynucleosomes of rat pancreatic tissue were isolated
essentially as previously elaborated (8,9). Purified nuclei were digested with 1 unit of micrococcal nuclease/mg DNA at 30°C to yield between 4 and 12% PCA-soluble nocleotides. Polynu- cleosomes were then resolved on a 5-29% sucrose gradient and spun for 12 hat 25 000 X gin a Beckman SW 27 rotor. For poly(ADP-ribosy1)ation reactions a fraction of three to six nucleosomes in size was selected from the gradient.
Poly(ADP-ribose) glycohydrolase purification Poly(ADP-ribose) glycohydrolase was purified essentially
as described by Burzio et al. (16). Testes from adult male Sprague-Dawley rats of 250g body weight were dissected, freed of fatty tissue, and decapsulated. Two homogenates were made with two groups of 16 testes and they were pooled after the acetic acid precipitation.
The pooled fractions were loaded on a phosphocellulose column preequilibrated in 50 rnM potassium phosphate buffer (pH 6.5) - 5 rnM 2-mercaptoethanol (buffer A). The glycohy- drolase was eluted from the phosphocellulose column with a linear gradient of buffer A and buffer A + 0.5 M KCI, 180 mL of each solution. The active fraction containing glycohy- drolases A and B were pooled and dialyzed overnight against buffer A containing 20% glycerol. This fraction was found to be free of DNase, RNase, phosphodiesterase, and protease activities.
P 0 ~ ( ~ - r i b a 3 y / ) a f l a f l O ~ of polynucleosomal fractio~c The pIy(ADP-ribosy1)ation reaction was typically carried
out in a final volume of 2mL with lOOpg nucleosomal DNA/mL pancreatic nucleosomes fmrn the sucrose gradient (see Pol ynucleosome preparation). The reaction mixture con- sisted of 50mM Tris-HC1 (pH 7.8) at 30°C with D'IT and MgClz at 8 mM each final concentration, with indicated concentrations of P - [ 3 2 ~ ] ~ ~ ~ (20 pCi; 1 Ci = GBq). The reaction was stopped after a 30-min incubation at 30°C by the addition of nicotinamide to a final concentration of 10 mM.
Hydrolysis of poly(ADP-ribosy1)ated nucleosomes Hydrolysis of free or nucleosomal-bound 32~-labelled
poly(ADP-ribose) was carried out by adding 1 enzyme unit of poly(ADP-ribose) glycohydrolase as described by Burzio et al. (16) per millilitre of the poly(ADP-ribosy1)ation reaction mixtures (previously stopped as above). A sample was taken before glycohydrolase addition and the other samples were analyzed every 15 min. The samples taken were immediately precipitated in TCA (20% final concentration) for a minimum of 1 h on ice and then centrifuged at 12 000 x g for 10 min. The pellet was washed with ether and nucleosomal proteins were solubilized by LDS solubilization buffer or histones were extracted as described by Aubin et al. (8, 9).
Low pH LDS-polyacrylamide gel electrophoresis The electrophoretic separation of 32~-labelled nucleosomal
proteins by LDS-polyacrylamide gel electrophoresis was performed essentially as described by Jones et al. (18) as modified by Huletsky et al. (19). The nucleosomes labelled with 32~-radioactive poly(ADP-ribose) were resuspended in sample buffer of 0.25 M citric acid- 0.032 M phosphoric acid, adjusted to pH 4.0 with solid Tris base - 5% LDS (w/v) - 4% (v/v) 2-mercaptoethanol - 8 M urea.
Polyacrylamide gels using a 4% stacking gel and a separat- ing gel of 8% were used to resolve 32~-labelled poly(ADP- ribose) polymerase. Gels were run at 50 V overnight in electrode buffer of 0.05 M glutamic acid - 1% LDS. A maximum amount of protein from 10 pg nucleosomal DNA was deposited in each well to achieve optimal separation of acceptor proteins.
Separation of histone by acid-urea-polyacrylarnide gel elec- trophoresis
The histones were extracted from [32P]ADP-ribosylated nucleosomal pellets and then separated as described by Aubin et al. (8,9) and West and Bonner (20). For best resolution on the gels protein extracted from 20 p g of nucleosomal DNA was deposited in each well.
Two-dimensional gel electrophoresis of [32~]AD~-ribosylated histones
[ 3 2 ~ ] ~ ~ ~ - r i b o s y l a t e d histones were extracted as described by Aubin et al. (8,9) and electrophoresed on 1 M acetic acid - 6 M urea - 6 mM Triton X-100 vertical slab gels in the first dimension and in the second dimension on 1 M acetic acid - 6 M urea - 0.15% CTAB vertical slab gels according to West and Bonner (20) and as modified by Huletsky et al. (19).
Autoradiography Following electrophoresis and staining of [ 3 2 ~ ] ~ ~ ~ - r i b o -
sylated histones, gels were dryed on a Bio-Rad gel dryer and autoradiographed using Fuji RX films.
Auto-poly(ADP-ribosy1)ation and electron microscopy For auto-poly(ADP-ribosy1)ation experiments the poly-
(ADP-ribose) polymerase was purified as described by de Murcia et al. (15). The DNA-bound enzyme was poly(ADP- ribosy1)ated for 30 min at 30°C in incubation buffer containing 25 mM Tris-HC1 (pH 8.0), 4 mM MgC12, 4 mM DTT, and 200 pM NAD. The reaction was stopped as described above and a sample was taken before glycohydrolase addition. One unit of poly(ADP-ribose) glycohydrolase (16) was then added per 100 pg DNA/mL (100 ~g of polymerase/ml) and incu-
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148 BIOCHEM. CELL BIOL. VOL. 64. 1986
T I M E (min)
FIG. 1. Comparative studies of glycohydrolase activity on purified poly(ADP-ribose) (A) and on nucleosomal bound polymer (0).
bated for 5 and 20 min. Following the various treatments the samples were diluted to 0.5 pg/mL at 20°C in buffer contain- ing20mMNaCI,5 mMTEA.Cl (pH7.4), and0.2 mMEDTA and fixed for 1 h in 0.14% (v/v) glutaraldehyde in the same buffer and processed for electron microscopy. For bright field observations, 400 mesh grids covered with a very thin carbon film were activated by a glow discharge in pentylamine vapor as described by Dubochet et al. (21). The specimens were adsorbed as described (22) on the activated grids. The grids were shadowed with tungsten at angle of 7" in an Edwards evaporatory equipped with electron beam guns (E085 -1 1, Edwards). The thickness of the metal deposition was mon- itored on a quartz thin crystal monitor (lTM4, Edwards). The grids were examined in a Philips 201 electron microscope.
Isolation of 32P-labelled poly(ADP-ribose) Pancreatic polynucleosomes were purified as described
above and were incubated with 1 mM NAD ( 3 2 ~ labelled) in 50 mM Tris-HC1 (pH 7.8), 8 mM MgC12, and 8 mM D l T for 30min at 30°C. the 32P-labelled poly(ADP-ribose) was then purified as described by de Murcia et al. (15).
Results and discussion We have compared the hydrolysis by poly(ADP-
ribose) glycohydrolase of 32P-labelled poly(ADP- ribose) in free form with that covalently bound to nucleosomal proteins (Fig. 1). This figure shows that equivalent amounts of poly(ADP-ribose) covalently bound to nucleosomes are hydrolysed much more slowly than in the free form. The activity of the enzyme on purified poly(ADP-ribose) is much greater since more than 70% of the polymer is digested after 10 min of incubation, whereas only 15% of the protein-bound polymer was hydrolysed during the same time. After 30min all of the free polymer was digested, whereas more than 60% of the protein-bound polymer remained.
These results suggest that poly(ADP-ribose) glyco-
FIG. 2. LDS-PAGE of poly(ADP-ribosy1)ated proteins at low NAD concentrations and treated by poly(ADP-ribose) glycohydrolase. Nuclwsomes were labelled with P- [~~P]NAD at 500 nM NAD. After the labelling period the reaction was stopped by the addition of nicotinamide to a final concentration of 10mM; treatment with glycohydrolase was started and aliquots samples were taken at various times; proteins were extracted as described in Materials and methods. S, stained gel. Tracks 1-5 represent autoradiogram at 0, 15,30,45, and 60 min of glycohydrolase hydrolysis. E, enzyme.
hydrolase is probably inhibited by DNA strand breaks and by the free staggered end termini of DNA in the nucleosomal structure. Indeed it has been shown that single-stranded DNA inhibits the enzymatic activity of glycohydrolase (23). Another possible explanation for the faster digestion of purified polymer, as compared with the protein-bound polymer is that the enzyme is able to hydrolyse from both ends, as compared with only one end available for the protein-bound polymer (24, 25). Also a differential accessibility of protein-bound polymer as compared with the free polymer might explain the results described in Fig. 1.
Figure 2 shows a time course study of the hydrolysis, analyzed by acid LDS-PAGE, of the total poly(ADP- ribosy1)ated nucleosomal proteins which were labelled at low NAD concentrations. Identified by the comigra- tion of this labelled band with purified poly(ADP- ribose) polymerase (19), the polymerase itself shows extensive modification, and hydrolysis of the polymer bound to the polymerase shows that it is the only modified protein of molecular weight higher than 50 000. Furthermore, most of the poly(ADP-ribose) is found attached to the enzyme and to other nonhistone proteins (26, 27). While the stained gel does not reveal any protein band at the polymerase level, the autoradio- graphy shows intense labelling, demonstrating the fact that the polymerase is the best poly(ADP-ribose) accep-
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GAUDREAU ET AL. 149
FIG. 3. Acid-urea-PAGE of poly(ADP-ribosy1)ated protein at low NAD concentrations and treated by poly(ADP-ribose) glycohydrolase. Nucleosomes were labelled with [ 3 2 ~ ] ~ ~ ~ and incubated with poly(ADP-ribose) glycohydrolase as described in the legend of Fig. 2. Histones were extracted as described in Materials and methods. S, stained gel. Tracks 1-5 of the autoradiogram represent 0, 15, 30, 45, and 60 min of glycohydrolase hydrolysis.
FIG. 4. Acid-urea-PAGE of poly(ADP-ribosy1)ated nucleosomal proteins at 100 pM NAD and treated with poly(ADP- ribose) glycohydrolase. Nucleosomes were labelled with [ 3 2 ~ ] N ~ D at 100 yM NAD and incubated with poly(ADP-ribose) glycohydrolase as described in the legend of Fig. 2. Histones were extracted and separated as described in the legend of Fig. 3. S, stained gel. Tracks 1-5 of the autoradiogram represent 0, 15, 30,45, and 60 min of glycohydrolase hydrolysis.
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150 BIOCHEM. CELL BIOL. VOL. 64. 1986
FIG. 5 . Two-dimensional electrophoresis of ADP-ribosylated histones. Nucleosomcs were labelled with ["PINAD at 10 FM NAD and incubated with poly(ADP-ribose) glycohydrolase as described in the legend of Fig. 2. Histones were extracted and electrophoresed in a acid-urea-Triton (AUT) gel in the first dimension and in acid-urca-CTAB (AUC) in the second dimension as described in Materials and methods. S, stained two-dimensional gcl. A l , autorad~ogram of gel at 10 pM NAD. A2 and A3, autoradiograms of gels aftcr 15 and 45 min of exposure to poly(ADP-ribose) glycohydrolase.
tor on a molar ratio basis in chromatin. This feature has also been observed by other investigators (14, 26, 28).
After hydrolysis times of 45 and 60 min, poly(ADP- ribose) polymerase returns to its normal molecular weight as observed on the autoradiogram (Fig. 2, lanes 4 and 5). This suggests that most of the aggregation of labelled material originating from the top of the gel came from the polymerase itself. A minor amount of labelling was present below the poly(ADP-ribose) polymerase; this could be DNA topoisomerase I because this enzyme has been shown to be poly(ADP-ribosy1)ated and to migrate below the polymerase (26). That poly(ADP-
ribose) polymerase returns to its original molecular weight as a single band indicates that the polymerase, at least in pancreatic nucleosomes, is not processed by specific proteases as observed for lymphocytes by Surowy and Berger (29).
Figure 3 shows the separation of poly(ADP-ribo- sy1)ated histones by acid-urea gel electrophoresis from the same experiment shown in Fig. 2. It reveals the presence of ADP-ribosylated species of histones H2B at 500 nM NAD (19) and that some of the species are more resistant to hydrolysis after a 60-min exposure to glycohydrolase as compared with the polymer attached
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GAUDREAU ET AL. 151
FIG. 6. LDS-PAGE of poly(ADP-ribosy1)ated proteins at 10 and 100 pM NAD and treated by poly(ADP-ribose) glycohydrolase. Nucleosomes were labelled with [ 3 2 P ] ~ ~ ~ and incubated with poly(ADP-ribose) glycohydrolase as described in the legend of Fig. 2. Proteins were extracted as described in Materials and methods. S, stained gel. Tracks 1-5 of the autoradiogram represent digestions of 0, 15, 30, 45, and 60min of nucleosomes labelled at 10 pM NAD and tracks 6-10 represent the same digestion times of nucleosomes labelled at 100 p M NAD. E, enzyme.
to the polymerase. This observed resistance of poly- (ADP-ribosy1)ated histone H2B to glycohydrolase is probably due to the inaccessibility of some polymer residues on the protein, since histone H2B is a nucleo- some core protein. Furthermore, the fact that poly(ADP- ribosy1)ated nucleosomes, purified by affinity chroma- tography, showed more single-stranded breaks (30) suggests that the presence of single-stranded breaks in these nucleosomes would prevent glycohydrolase hy- drolysis of the polymer attached to histone H2B.
When polymer hydrolysis was analyzed on histones which were labelled at 100 pM NAD by acid-urea gel electrophoresis to generate hyper(ADP-ribosy1)ated forms of histone H1 (9, 19), it was observed that the hyper(ADP-ribosy1)ated forms were digested quite ex- tensively and much faster than other less modified species (Fig. 4). This suggests the absence of histone H 1 dimer complexes postulated to explain the electro- phoretic mobility of hyper(ADP-ribosy1)ated species of histone H1 (2, 24, 25). However, the labelling being present in the region of histone H2B seems to be much more resistant to hydrolysis than that of histone HI.
To obtain further insight on the relative distribution of the poly(ADP-ribose) residues after hydrolysis of the polymer attached to the various histones, we have separated the poly(ADP-ribosy1)ated proteins by two- dimensional gel electrophoresis (Fig. 5) (19). Exarnina-
tion of the autoradiograms reveals that the polymers attached to histones H1 and H2A and protein A24 are much more accessible to hydrolysis than the polymer attached to histone H2B. We can easily see in frame A3 of Fig. 5, the autoradiography after 45 min of glycohyd- rolase digestion, that on H2B the labelling is still very intense as compared with the other labelled histones. To further investigate the kinetics of poly(ADP-ribose) hydrolysis on nucleosomal histone, the two-dimen- sional gels for each time course were cut for each labelled histone species, being A24, H2B, H2A, and H1. Frame A1 shows no glycohydrolase digestion or a 100% polymer level for each labelled species of histone. Frame A3 indicates that on histone H2A and A24 only 19% of the label remains after 45 min of glycohydrolase digestion. For H1 there is still 31 %, while H2B has still 52% of its original labelling. Indeed the reports by Bonner and Stedman (3 1) and Boulikas et al. (32) that histone H2A, protein A24, and histone H1 are proximal in the nucleosome structure explains their parallel sensitivity to glycohydrolase action, as opposed to histone H2B which is likely to be more buried within the nucleosome core. Figure 6 shows an electrophoretic separation of total nucleosomal poly(ADP-ribosy1)ated proteins which were labelled at 10 and 100 pM NAD and thereafter incubated with poly(ADP-ribose) glyco- hydrolase. The hydrolysis of the polymer appears to be
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152 BIOCHEM. CELL BIOL. VOL. 64, 1986
FIG. 7. Electron microscopic visualization of poly(ADP-ribose) polymerase under control and hypermodified forms. The enzyme at a concentration of 1.6 mg/mL was poly(ADP-ribosy1)ated as described in Materials and methods and hydrolysed by poly(ADP-ribose) glycohydrolase. Following the various treatments, the samples were diluted, fixed, and processed for electron microscopy (see Materials and methods). (a) Control DNA-polymerase complex; (b and c) poly(ADP-ribosylated) polymerase; (d and e) 5 and 20min after poly(ADP-ribose) glycohydrolase addition, respectively. Arrowheads indicate automodified poly(ADP-ribose) polymerase. The bar indicates 1000 A ( I A = 0.1 nm).
very rapid as a whole, with the labelling at the core histones seeming to be more sensitive to glycohydrolase activity. As H2B is a minor component of the core his- tone, the resistance of its polymer to glycohydrolase activity is masked.
By analyzing the relative distribution of the polymer on total nucleosomal proteins in three regions of the polyacrylamide gel (Fig. 6) we did not observe any major difference in the hydrolysis of the polymer in the regions of the polymerase, HI, or the core histones. In Fig. 7, upon incubation at high substrate concentration we have generated hyper(ADP-ribosy1)ated poly(ADP- ribose) polymerase and we have shown that it dissoci- ates itself from DNA as described by de Murcia et al. (15). When treated with poly(ADP-ribose) glycohydro- lase it reassociated with the DNA as observed by Zahradka and Ebisuzaki (13), thus showing that when a good part of the polymer is hydrolyzed the polymerase returns to its DNA and recovers its activity (13).
In this study we have demonstrated that the kinetics of
poly(ADP-ribose) formation and digestion. Further- more, we have recently found preferential modification of only histone H2B on carcinogen-damaged SV40 minichromosomes (33) isolated from permeabilized cells, whereas we found on the cellular chromatin poly(ADP-ribosy1)ation of protein A24 and histones H2B, H2A, and H1; thus this suggests a differential turnover of the polymer on the SV40 minichromosomes, such as described in the present work.
Acknowledgements We would like to thank S. Bilodeau, C. Gouin, and E.
Lemay for expert secretarial assistance. We also thank Dr. Teni Boulikas and Dr. Remy Aubin for excellent discussion. This research was supported by the Medical Research Council of Canada, the Natural Sciences and Engineering Research Council of Canada, and the Fonds pour la formation de chercheurs et l'aide a la recherche.
p o l y ( ~ ~ ~ - r i b o s e ) glycohydrolase activity is dependent mainly on the accessibility of the polymer. *lso 1. Shall, S. (1982) in ADP-ribosylation Reactions (Hays-
ishi, 0. & Ueda, K. , eds.), pp. 478-520, Academic demonstrated is the return of the poly(ADP-ribose) Press, New York and London ~0 l~merase '0 its DNA after glycoh~drolase action, thus 2. ~ ~ d ~ l , p, , 0kazaki, H, & ~ i ~ d ~ ~ ~ ~ ~ , C. (1982) Prog,
reactivating the polymerase for future activity (13). This Nucleic Acids Res. Mol. ~ i ~ l . 27, 1-51 implies that these two enzymes, poly(ADP-ribose) 3. Benjamin, R. C . & Gill, D. M. (1980) J. Biol. Chem. polymerase and the glycohydrolase, are the two main 255, 10 502 - 10508 enzymes which modulate the nucleosomal structure and 4. Jacobson, E. L., Antol, K. M., Juarez-Salinas, H. & maybe even chromatin function by the kinetics of Jacobson, M. K. (1983) J . Biol. Chem. 258, 103-107
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