(Received for publication, October 7, 1994; and in revised form, December 1, 1994)
From the
Cellular proteins extracted from normal and cancer cells bind
polymerizing ADP-ribose transferase (pADPRT) on nitrocellulose membrane
transblots. Histones at 1 mg/ml concentration completely prevent the
binding of pADPRT to cellular proteins, indicating that the binding of
histones to pADPRT sites competitively blocks the association of pADPRT
to proteins other than histones. The direct binding of pADPRT to
histones is shown by cross-linking with glutaraldehyde. The
COOH-terminal basic histone H1 tail binds to the basic polypeptide
domain of pADPRT. The basic domain present in the
NH-terminal part of core histones is the probable common
structural feature of all core histones that accounts for their binding
to pADPRT. Two polypeptide domains of pADPRT were identified, by way of
CNBr fragments, to bind histones. These two domains are located within
the 64-kDa fragment of pADPRT and are contiguous with the polypeptide
domains that were shown to participate in self-association of pADPRT,
ending at the 606th amino acid residue. The polypeptide domains of
pADPRT which participate in DNA binding are thus shown to associate
also with other proteins. Intact pADPRT binds to both the zinc-free or
zinc-reconstituted basic polypeptide fragments of pADPRT. Histones
activate auto-poly(ADP)-ribosylation of pADPRT by increasing the number
of short oligomers on pADPRT. This reaction is also dependent in a
biphasic manner on the concentration of pADPRT. Histones in solution
are only marginally poly(ADP)-ribosylated but are good polymer
acceptors when incorporated into artificial nucleosome structures.
Poly(ADP-ribose) polymerase (ADP-ribosyltransferase,
polymerizing, pADPRT, ()EC 2.4.2.30) is a highly abundant
nuclear protein of higher eukaryotes (1, 2) that
exhibits at least two catalytic functions: ADPR polymerizing and NAD
glycohydrolase activities, which are also regulated by the state of
differentiation of particular cell types(3) . Assuming the same
molecular activity in vitro and in vivo, pADPRT
activity in maximally stimulated cells appears to account for only one
percent of the molecular activity of this
enzyme(1, 2) , approximating the magnitude detectable
in the isolated homogeneous protein in the absence of coenzymic
DNA(4) , an activity present even in the 56-kDa polypeptide
fragment of pADPRT (5) that contains no DNA recognition
sites(6) . These observations suggest that the enzymatic
activity of pADPRT is highly regulated in physiologically operating
cells. Immunochemical estimation of native and automodified pADPRT in
intact cells in culture (AA-2 and MT-2 cells) indicated that only 4%
(for AA-2) or 20% (for MT-2) of pADPRT was auto-ADP-ribosylated, thus a
significant portion of this protein is available for macromolecular
associations(7) . Indeed recent results (8) show that in vitro pADPRT significantly stimulates DNA polymerase
but not
. Microinjection of the 46-kDa DNA-binding polypeptide
fragment of pADPRT into human fibroblasts inhibits MNNG-induced
unscheduled DNA synthesis(56) , further supporting the
contention that protein-protein or protein-DNA interactions involving
pADPRT may have biological consequences. Binding of pADPRT to the DNA
primer of human immunodeficiency virus reverse transcriptase was also
shown to inhibit this enzyme(9) . A potentially important
cellular function of pADPRT in NAD
-dependent repair of
damaged DNA in vitro has been also traced to the release of
the binding of pADPRT from DNA termini by auto-poly(ADP)-ribosylation
of the enzyme protein(10, 11) . Self-association
(dimerization) of pADPRT has been shown to be essential for
auto-poly(ADP)-ribosylation of the enzyme
protein(12, 13) . Furthermore, binding to and
trans-ADP-ribosylation (14) to an assortment of highly
significant enzymes that act on DNA, such as the
calcium/magnesium-dependent endonuclease(15) , DNA polymerase
and
, terminal deoxynucleotidyl transferase, DNA ligase
II(16, 17) , topoisomerase
I(18, 19) , and topoisomerase II (20) are also
of critical cell biochemical significance, because
trans-ADP-ribosylation to these enzymes produces a down-regulation of
their catalytic activities. As we show here, the binding of pADPRT to a
large number of proteins, extracted from 3T3, CHO, and PC12 cells,
occurs readily in protein transblots on nitrocellulose membranes (see Fig. 1), emphasizing the significant protein-protein associative
capacity of pADPRT. We also show that histones by apparent competition
can completely inhibit the binding of pADPRT to cellular proteins,
suggesting that pADPRT-histone association and the association of
pADPRT with other cellular proteins may occur at the same pADPRT site.
It follows that a more detailed analysis of pADPRT-histone association
may shed light on the general pADPRT-protein binding sites of pADPRT.
Clarification of pADPRT-protein binding in terms of identification of
binding domains on pADPRT may have cell biological importance since
inhibitors of pADPRT which do not inhibit poly(ADP-ribose) catabolism
eventually convert cellular pADPRT to a protein- and DNA-binding
cellular component that can regulate complex cellular
processes(5, 7, 10, 11, 21) .
As a beginning to this highly involved series of pADPRT-protein
interactions we first determined histone and self-association sites of
pADPRT by nitrocellulose transblot binding and protein cross-linking
methods. The study of binding kinetics of proteins in solution is
subject to further experimental work.
Figure 1:
The binding of
pADPRT to cellular proteins, their inhibition by histone H1 and 0.5 M NaCl. Panel A, electrophoretically separated
cellular proteins on transblots were stained with Ponceau S (see
``Materials and Methods''). Lane 1, 3T3 cells; lane 2, CHO cells; lane 3, PC12 cells. Panel
B, autoradiography identifying the binding of I-labeled pADPRT (10 µg, 4
10
cpm) to the cellular proteins on transblots, as described in Panel A. Lane 1, 3T3 cells; lane 2, CHO cells; lane 3, PC12 cells. Panel C, the binding experiment,
as shown in Panel B, was performed in the presence of 1 mg/ml
solution of histone H1. Panel D, the binding assay shown in Panel B was performed in the presence of 0.5 M NaCl. Panel E, immunochemical assay for pADPRT antibody-reactive
proteins shown in Panel A. The same amounts of cellular
proteins were employed in experiments shown in Panels
A-E, hence the faint immunopositive pADPRT bands shown in Panel E, lanes 1 and 2, indicate apparently
smaller concentrations of pADPRT in 3T3 and CHO
cells.
In and of itself, pADPRT-histone binding is a significant biochemical problem. By far the most abundant nuclear proteins that are trans-ADP-ribosylated by pADPRT are histones (22) and we have demonstrated the in vivo occurrence of poly(ADP)-ribosylated histones by specific antibodies directed against the polymer(23) . Besides serving as ADPR acceptors (24) under certain conditions histones also activate rates of poly(ADP)-ribosylation(25) . In stimulated thymocytes histone H3 is selectively poly(ADP)-ribosylated (26) . Apart from the binding of the zinc fingers of pADPRT to DNA termini(27) , molecular details of protein-protein associations of pADPRT are unknown. We report here the identification of binding domains on pADPRT for histones which probably represent general protein binding sites including a self-association site of the pADPRT enzyme protein, and provide evidence for a regulatory effect of these sites on enzymatic activity.
Electrophoretically 98% homogeneous pADPRT and coenzymic DNA
were isolated from calf thymus as reported
elsewhere(28, 29) . Unavoidable traces of lower mass
bands were peptides of pADPRT produced by proteases as identified by
immunotransblots(28) . The isolated protein did not contain
covalently bound poly(A)DP-ribose as determined by the absence of
binding to phenylboronate affinity columns(7) . The commercial
sources of reagents were as follows: CNBr-activated Sepharose 4B and
the Mono S column from Pharmacia Biotech Inc., Affi-Gel 10 from
Bio-Rad, [P]NAD
and the
I-labeled Bolton-Hunter reagent from ICN (Irvine, CA),
ZnCl
from DuPont-NEN), and Centricon
concentrators from Amicon (Danvers, MA). Histones were obtained from
Sigma. All other chemicals used were of the highest purity available.
Cyanogen bromide (CNBr) fragments of pADPRT were prepared by the following technique. Homogeneous pADPRT (200 µg) was precipitated with 20% trichloroacetic acid, and pelleted by centrifugation, washed with 70% ethanol, and dried. The dried protein was dissolved in 100 µl of 88% formic acid, 300 µl of 0.1 M HCl and 20 µl of CNBr (100 mg/ml in ethanol) were added, and the mixture was allowed to stand for 48 h at room temperature. Then the solvent was evaporated by freeze-drying and the residue dissolved in 400 µl of ``renaturation buffer''(32) . Digestion of pADPRT with plasmin was carried out as described earlier(6) .
pADPRT-Sepharose columns (1-ml bed volume) were loaded with either 50 µg of histone H1 or its chymotryptic polypeptides. Chromatography was carried out exactly as described above.
The binding of pADPRT to cellular proteins extracted from
three cell types is shown in Fig. 1. Among the numerous protein
fractions separated by gel electrophoresis (Fig. 1, Panel
A) upon transblot to nitrocellulose membranes approximately
16-20 major bands adsorbed I-labeled pADPRT to
varying extents. (Fig. 1, Panel B). When the binding
experiment was repeated in the presence of 1 mg/ml of histone H1 added
to the binding mixture, pADPRT exhibited no association with the
transblotted cellular proteins (Fig. 1, Panel C).
Histone H1 could be replaced by any other histone (not shown). A
similar inhibition of binding occurred in the presence of 0.5 M NaCl, a salt concentration usually required to extract pADPRT from
nuclei(28) . It was also found that the assay for pADPRT
binding to other polypeptides or the binding of pADPRT-derived peptides
to histones was limited by the size of the polypeptide, which was
6-10 kDa, and no association by this technique could be detected
if the polypeptide was of a smaller size. This size limitation puts
some constraints on binding experiments employing the transblot
technique, as will be shown below. When the presence of pADPRT in
extracts of 2
10
cells was probed with
pADPRT-specific polyclonal antibody(28) , faint bands were seen
in 3TC and CHO cells (Fig. 1, Panel E, lanes 1 and 2) and stronger bands, including a proteolytic
degradation product of pADPRT, were detectable in the extract of the
more malignant PC12 cells (Fig. 1, Panel E, lane
3).
Since histones prohibited the association of pADPRT with
other cellular proteins, implying that histones effectively competed at
protein binding sites of pADPRT with other proteins, we reasoned that
determination of histone-binding polypeptide domains of pADPRT may
identify general protein association sites. The prerequisite for this
assumption was a direct demonstration of the association of histones
with pADPRT, which was carried out by the glutaraldehyde cross-linking
technique (12) as shown in Fig. 2. In Fig. 2, Panel A, Coomassie Blue-stained protein bands identify pADPRT
and histone 2B separately but not in the cross-linked complex (lanes 4 and 6) because of the limited resolving
power of SDS-PAGE. In the autoradiography shown in Panel B of Fig. 2, unlabeled pADPRT (lane 7) gave no signal,
whereas I-labeled histone 2B (lane 8) and
glutaraldehyde cross-linked histone 2B (lane 9) clearly
separated from the pADPRT-
I-labeled-histone 2B
cross-linked product, which is shown in lane 10. The pADPRT
component of the cross-linked pADPRT-histone 2B complex was identified
by immunoblot (Fig. 2, Panel C). Lane 11 is
pADPRT, lane 12 is glutaraldehyde cross-linked pADPRT, and lane 14 is the adduct of pADPRT with glutaraldehyde
cross-linked
I-labeled histone 2B, containing also some
glutaraldehyde cross-linked dimers of pADPRT. Lane 13 contains
histone H2B which does not react with anti-pADPRT serum.
Figure 2:
Cross-linking with glutaraldehyde of
histone 2B with pADPRT. Panel A, Coomassie Blue-stained
proteins. Lane 1, molecular weight standards; lane 2,
pADPRT and degradation products; lane 3, I-labeled histone 2B; lane 4,
glutaraldehyde-cross-linked pADPRT; lane 5,
glutaraldehyde-cross-linked
I-labeled histone H2B; lane 6, glutaraldehyde-cross-linked
I-labeled
histone H2B with pADPRT. Panel B, autoradiography of labeled
proteins given in Panel A. Lane 7, unlabeled pADPRT; lane
8,
I-labeled histone H2B; lane 9, same as lane 8 except cross-linked with glutaraldehyde; lane
10, glutaraldehyde cross-linked pADPRT with
I-labeled histone H2B. Panel C, identification
of pADPRT component in Panel B by immunoblot. Lane
11, pADPRT; lane 12, cross-linked pADPRT; lane
13, histone H2B; lane 14, cross-linked pADPRT with
I-labeled histone H2B.
Figure 3:
Binding of pADPRT to histone H1 and to its
chymotryptic fragments. A, the binding of
[P]ADP-ribosylated pADPRT to histone H1 and to
its chymotryptic polypeptide fragments. 10 µg of histone H1 (lanes 1 and 3) and 10 µg of
chymotrypsin-digested histone H1 (lanes 2 and 4) were
separated by SDS-PAGE, transblotted onto nitrocellulose membranes, and
probed with [
P]ADP-ribosylated pADPRT (3 µg,
10
cpm; obtained by incubating pADPRT with 25 nM [
P]NAD
for 10 min followed
by gel filteration on a Sephadex G75 column) and then processed (see
``Materials and Methods''). Lanes 1 and 2 are Ponceau Red-stained polypeptides and lanes 3 and 4 are autoradiograms. The doublets in lanes 2 and 4 (17 and 18 kDa) are the COOH-terminal basic fragments of
histone H1. B, the binding of histone H1 and its chymotryptic
fragments to pADPRT-Sepharose affinity columns. The pADPRT-Sepharose
columns were loaded either with chymotryptic fragments of 50 µg of
histone H1 (lanes 1-7) or with 50 µg of histone H1 (lanes 8-14). The columns were then eluted with NaCl
solutions with increasing ionic strength, and aliquots from each
fraction were loaded onto SDS-PAGE gels, electrophoresed, and Coomassie
Blue-stained. Lane 1 shows polypeptides of histone H1 obtained
by chymotryptic digestion; lanes 2 and 9 are
flow-through fractions with buffer only; lanes 3 and 10 are eluates with 50 mM NaCl; lanes 4 and 11 are eluates with 100 mM NaCl; lanes 5 and 12 are eluates with 200 mM NaCl. Lanes 6 and 13 are eluates with 400 mM NaCl and lanes 7 and 14 are eluates with 1000 mM NaCl, indicating that
there are no further absorbed polypeptides present in the adsorbed
form.
Figure 4:
Binding of labeled histones to
transblotted chymotryptic fragments of pADPRT. The digestion of pADPRT,
separation of polypeptides, electroblotting onto a nitrocellulose
membrane, and the procedure for binding I-labeled
histones are described under ``Materials and Methods.'' Lane 1, Coomassie Blue-stained peptides, lane 2,
autoradiography of bound
I-labeled histones to
transblotted peptides of pADPRT.
The correctness of these conclusions was tested also by
``opposite'' labeling, i.e. radioiodination of
polypeptides of pADPRT. The same results as shown in Fig. 4were
obtained, i.e. only the basic half of pADPRT bound to
histones, regardless which protein species was labeled with I. Fig. 5, lane 1, shows the Coomassie
Blue-stained histones (mixed histones; 8 µg/lane). Lane 2 illustrates that the 56-kDa polypeptide (labeled with
I) of pADPRT (the catalytic domain) did not bind to
transblotted histones at all. The binding of the labeled basic 64-kDa
polypeptide to transblotted histones is apparent from lane 3 together with lane 4, a positive control where the
binding of labeled intact pADPRT protein to histones is shown. The
intact protein bound to transblotted histones in the same manner as the
64-kDa basic polypeptide (compare lanes 3 and 4).
Figure 5:
Binding of I-labeled pADPRT
and its N- and COOH-terminal fragments to transblotted histones. Four
µg of mixed histones were electrophoresed (36) then
transblotted onto nitrocellulose membranes and probed with
I-labeled pADPRT. Lane 1 shows Coomassie
Blue-stained histones, all the other lanes are autoradiograms. The
absence of binding of radiolabeled 56-kDa polypeptide of pADPRT to
histones is shown in lane 2. Lanes 3 and 4 illustrate
the histone binding of the labeled 64-kDa polypeptide and of the entire
pADPRT protein as a control.
Figure 6: Electrophoretic separation of CNBr fragments of pADPRT eluted from a histone-Sepharose affinity column. Lane 1 shows CNBr-generated peptides prior to passing through the affinity matrix; lane 2, the flow-through CNBr fragments; lanes 3-7, the eluates with 50, 100, 200, 400, and 1000 mM NaCl, respectively. For technical details, see ``Materials and Methods.''
The 14-kDa polypeptide was identified by sequencing and corresponds
to residues 186-290 on the sequence of pADPRT (105 residues, M = 11,826), its NH
-terminal
being GFSVL. . . . This polypeptide is located between the 29- and
36-kDa domains of pADPRT obtained by digestion with
plasmin(6) . It is only 22 residues downstream of the second
zinc finger as apparent from the domain diagram (Fig. 7). We
also sequenced the fragments which elute at 0.4 M NaCl
(apparent molecular mass of 10 and 18 kDa, shown in Fig. 6, lane 6). The NH
terminus of the 18-kDa peptide was
XXLTLGXLSQ. . . . This sequence places the peptide on the bovine enzyme
at 396-525 (130 residues, M
=
14,002). Sequencing of the 10-kDa fragment yielded an
NH
-terminal sequence of XEVKEANIRV. . . . The position of
this peptide is at 446-525 (80 residues, M
= 8,603). These two polypeptides thus partly overlap, the
larger includes the smaller one which is adjacent to the 56-kDa
catalytic domain. These two polypeptides are part of the automodifiable
region (45) of pADPRT and only the shorter one is shown as
histone binder in Fig. 7.
Figure 7:
Self- and histone-binding sites of pADPRT.
In the upper diagram shaded rectangles indicate
histone-binding CNBr peptides and open rectangles are peptides
participating in self-binding. The lower diagram shows cutting
sites by proteases. Small ellipses in the upper diagram mark the position of zinc fingers. N, the
NH-terminal; C, the COOH-terminal; pl,
plasmin cutting sites; chy, chymotryptic cutting sites. The
sign ∨ identifies the NH
-terminals of polypeptides
obtained by direct sequencing. Established polypeptide domains of
pADPRT are also indicated with double-ended
arrows.
Our results are consistent with the existence of two histone-binding regions, one between residues 186 and 290, which is close to the second zinc finger, and a second histone-binding sequence that is between residues 446 and 525 that is part of the automodifiable portion of pADPRT (Fig. 7).
Since
both direct chemical (12) and kinetic (13) evidence
supports the significance of self-association of pADPRT molecules, we
attempted to identify polypeptide domains of pADPRT that are
participatory in the self-binding reaction. Chemical cross-linking
experiments (12) earlier identified the 36- and 29-kDa
polypeptides as binding to each other, implying self-association. When
the pADPRT protein was digested with plasmin (6) a degradation
pattern of polypeptides occurs as visualized after electrophoresis on
10% SDS-PAGE and transblot to nitrocellulose membranes followed by
Western blot with anti-pADPRT antiserum(28) , as shown in Fig. 8(lanes 1 and 2). The native enzyme
protein (Fig. 8, lane 1) contains traces of polypeptide
degradation products of pADPRT having masses of 98, 80, 67, and 56
kDa(6) . As shown in Fig. 8, lane 2, digestion
of pADPRT with plasmin (see legend to Fig. 8) yielded 64-, 56-,
36-, and 29-kDa polypeptides (6) and some minor peptides. When
these transblotted polypeptides were probed with I-labeled pADPRT for self-binding, the intact pADPRT, and
the 64-, 36-, and the 29-kDa polypeptides exhibited strongest binding (Fig. 8, lanes 3 and 4). The same polypeptides
also bound DNA (Fig. 8, lanes 5 and 6), and
the 29- and 64-kDa peptides incorporated
Zn (Fig. 8, lane 7). Since it is known that zinc finger
polypeptides of the 29-kDa fragment are required for the binding of DNA
termini(27) , the role of Zn
in self-binding
of pADPRT had to be clarified. As illustrated in Fig. 8, lanes 8 and 9, zinc-depleted (lane 8) or
zinc-replenished (lane 9) polypeptides of 64-, 36-, and 29-kDa
mass equally bound radioiodinated pADPRT, suggesting that the basic (64
kDa) polypeptide fragment of pADPRT participates in self-binding and
Zn
may not be critical in this self-association
reaction. Densitometric tracings of lanes 8 and 9 confirm the conclusions deduced from visual inspection of the
bands and it is apparent that self-association of pADPRT at the 64- and
29-kDa peptide domains is Zn
-independent. As shown in Fig. 7, the two discrete polypeptide domains specific for
histone binding are also part of the 64-kDa proteolytic enzyme
fragment.
Figure 8:
Identification of the basic polypeptide
domain (64 kDa) as the self-binding portion of pADPRT. Lanes 1 and 2 are immunostained blots of pADPRT (lane 1)
and its plasmin-digested (lane 2) polypeptides. Lanes 3 and 4 are autoradiograms of transblotted pADPRT and its
polypeptide fragments, both probed with I-labeled pADPRT. Lanes 5 and 6 are autoradiograms of transblotted
pADPRT and its polypeptide fragments, both probed with
[
P]DNA. Lane 7 shows two polypeptide
fragments (64 and 29 kDa) of pADPRT labeled with
Zn
. Lanes 8 and 9 are
polypeptide fragments of pADPRT obtained by plasmin digestion of pADPRT
which on nitrocellulose membrane transblots bind
I-labeled pADPRT (64-, 39-, and 29-kDa peptides). The
polypeptides shown in lane 8 contain no Zn
,
which was lost during electrophoresis and renatured in the presence of
EDTA (see ``Materials and Methods''). In lane 9,
Zn
had been reincorporated into polypeptides on
nitrocellulose transblots. Both Zn
-free and
Zn
-replenished polypeptides bound pADPRT equally, as
shown in the densitometric tracing made from lanes 8 and 9.
In further experiments, CNBr-generated peptides were
separated by electrophoresis followed by transblot to PVDF membranes
and incubation with radioiodinated pADPRT to detect self-association.
This technique identified two discrete pADPRT-binding polypeptide
fragments (separation not shown) and their NH-terminal
amino acid sequence was VKTQT. . . for the 13-kDa fragment
(326-445, 120 residues, M
13056) and KKTLK.
. . for the 9-kDa fragment (526-606, 81 residues, M
9070). The same 13-kDa polypeptide coeluted with
the pADPRT molecule on a Sephadex G75 column as identified by SDS-PAGE
(not shown), indicating self-binding in solution. Positioning of the
13- and 9-kDa CNBr fragments together with the histone-binding
polypeptides (Fig. 7) shows the existence of contiguous and
intermittent histone- and self-binding regions in the pADPRT molecule.
The 29-kDa NH
-terminal polypeptide, which was not further
fragmented, comprises the third domain involved in self-association. As
stated above, CNBr fragmentation of the 29-kDa polypeptides yielded
fragments too small for binding assays in our test system.
Figure 9:
The effect of histone H3 on the catalytic
activity of pADPRT. A, effect of pADPRT concentration on the
specific activity of the enzyme in the absence and presence of histone
H3, determined in the presence of 200 µM [P]NAD
and 200 µg/ml
coenzymic DNA. Enzymatic activity of pADPRT at varying enzyme
concentrations was assayed (6, 54) either in the
absence (open circles) or in the presence (closed
circles) of 200 µg/ml of histone H3 for 1 min at 23 °C.
The reactions were started by adding 1 µl of pADPRT to reaction
mixtures of varying volumes. The specific activity of the enzyme (ordinate) is plotted against the log of enzyme concentration (abscissa). Fig. 9B shows the effect of
varying histone concentration on the specific activity of pADPRT. The
pADPRT concentration in Fig. 9B was 5.5 nM and
the histone concentration varied between 0.03 and 200
µg/ml.
Figure 10:
The influence of variation in pADPRT
concentration, in the absence and presence of a constant concentration
of mixed histones, on the chain length distribution of
poly(ADP-ribose). The concentrations of pADPRT in the reaction solution
assayed were 2 nM (Lanes 1 and 2), 20 nM (lanes 3 and 4) and 200 nM (lanes 5 and 6). Odd numbered solutions contained no histones, even numbered ones contained mixed histone (200 µg/ml).
The volumes of the reaction solutions were each 100 µl when the
pADPRT concentrations were 20 and 200 nM, and the volume was
1000 µl when the pADPRT concentration was 2 nM. The assays
were carried out under conditions described in legend to Fig. 9.
At the end of incubation for 5 min at 23 °C the reaction was
stopped with 20% trichloroacetic acid (4 °C), the precipitates were
sedimented by centrifugation, washed four times with 10%
trichloroacetic acid (4 °C), twice with absolute ethanol (200
µl/wash), and then air-dried and dissolved in 2% SDS containing TE
buffer (50 µl) and 100 µg of proteinase K. Aliquots of the P-labeled poly(ADP-ribose) representing 30,000 cpm were
withdrawn and adjusted to 20 µl with TE buffer, then loaded onto
20% PAGE with TBE as the running buffer. Xylene cyanol and bromphenol
blue were used as polymer size markers.
Figure 11: Poly(ADP)-ribosylation of pADPRT and histones in reconstructed nucleosome. Nuceosome reconstruction and assay of pADPRT activity was done as described under ``Materials and Methods.'' Proteins were separated by acidic SDS-PAGE(55) . Lane 1, pADPRT (1.2 µg) + coDNA (10 µg); lane 2, pADPRT (1.2 µg) + coDNA (10 µg) + histone H3 (10 µg); lane 3, pADPRT (1.2 µg) + reconstructed nucleosome (equal to 10 µg of DNA).
The apparent contradiction between these enzymological results and the known poly(ADP)-ribosylation of histones in nuclei was resolved by demonstrating that artificially reconstructed nucleosomes (40) indeed provided favorable conditions for extensive poly(ADP)-ribosylation of histones, as illustrated in Fig. 11. In Fig. 11, lane 1, auto-ADP-ribosylation of pADPRT is shown in the presence of DNA. Addition of mixed histones (lane 2) increased auto-ADPR-ribosylation of pADPRT without detectable histone ADP-ribosylation. However, in reconstructed nucleosomes extensive poly(ADP)-ribosylation of histones was apparent (Fig. 11, lane 3).
The previously outlined domain structure of pADPRT, which consists of DNA binding, self ADP-ribosylation and catalytic domains(31) , has now been expanded to include protein- and self-association participitating polypeptide regions. Because histones completely block the association of pADPRT to all proteins (Fig. 1), that in the absence of histones bind pADPRT, it seems reasonable to postulate that a more detailed analysis of histone binding sites of pADPRT may be a first approximation to the identification of general protein binding domain of pADPRT. Our technique of protein-protein binding assays has a size limitation (approximately 6-10 kDa) thus its accuracy is relatively limited. Furthermore binding strength is estimated only by salt gradients sufficient to disrupt association. Despite these shortcomings, our results clearly show that protein-protein binding regions coincide with DNA binding and automodification domains (Fig. 7). Since the DNA-binding zinc-finger region regulates, by as yet unknown mechanisms, the enzymatic as well as DNA binding activities of pADPRT, the proximity of protein binding sites implies that the two types of macromolecular associations, i.e. protein/DNA and protein/protein interactions, may have mutual regulatory functions, which are clearly of highly complex nature. Since auto-poly(ADP)-ribosylation by electrostatic repulsion inhibits pADPRT-DNA binding (9, 29) and an oscillating poly(ADP)-ribosylation) of histones has been recognized(46) , the inhibition of poly(ADP)-ribose synthesis, without altering the catabolism of polymers, would be expected to have profound effects on the above outlined macromolecular interactions. Consistent with this prediction we have observed large cellular phenotypic changes upon treatment of intact cells with specific pADPRT ligands, reflecting pleiotropic responses(57) . A more specific understanding of macromolecular association of pADPRT requires spectroscopic and fluorometric techniques, implementation of which is now made more feasible by our outline of protein binding domains of this complex protein.
Our results suggests that the presence of Zn in the basic polypeptide fragments of pADPRT appears to be
unnecessary for binding to intact pADPRT. However the presence of
Zn
in the pADPRT molecule which is used as a binding
reagent may weaken this argument since the Zn
in the
intact pADPRT may not be required for the binding to zinc-free
polypeptides. A direct test of this assumption is technically not
possible, because removal of Zn
from pADPRT by
specific reagents (47) results in large changes in the physical
properties of pADPRT that make it experimentally unsuited for binding
assays. (
)
Both histone- and self-association have
significant influence on the specific polymerase activity of this
enzyme (Fig. 9), and it appears meaningful that both
protein-binding domains reside within the 64-kDa basic part of the
pADPRT molecule, which also contains DNA binding domains(31) ,
the 29-kDa part with its zinc fingers binding to DNA
termini(27) , and the 36-kDa part (6) that binds to
internal regions of certain double-stranded
DNAs(48, 49) . The effect of histones on the enzymatic
activity of pADPRT is not confined to an action on apparent specific
activity, as illustrated at various protein concentrations (Fig. 9). Histones also induce the synthesis of many short chain
oligomers (Fig. 10), an observation that agrees with previously
obtained results with the carboxyl-terminal domain of human pADPRT (50) . The apparent nonselectivity of histones as
pADPRT-binding proteins is in contrast to the well known specific
biological function of histones in the nucleus(43) . It has
been shown (51) that in isolated polynucleosomes the
concentration of NAD determines the rate of
poly(ADP)-ribosylation of various histone species, indicating that the
concentration of NAD
and the nucleosomal structure, or
both, are both regulators of histone-specific poly(ADP)-ribosylation.
The absence of poly-ADP-ribosylation of histones in solution by
purified pADPRT has been previously reported (52) , but the
changes in the polymer-accepting function of histones, as a consequence
of supramolecular structural modifications, as we have shown here (Fig. 11), have not so far been considered. The apparent
nonspecificity of histones to bind pADPRT in our model system appears
to be explained by the commonality of lysine-rich polypeptide domains
present in all histones (42, 43) as experimentally
demonstrated here with proteolytic fragments of histone H1 (Fig. 3). It has been shown that the free poly(ADP-ribose)
polymer binds to lysine-rich histone tails(41) , the same
domain which associates with specific basic polypeptides of pADPRT, as
documented here for H1. The binding of the acidic poly(ADP-ribose) to
lysine-rich polypeptides appears to be explainable by electrostatic
forces, but the association of basic polypeptide domains of histones to
lysine-rich polypeptides is not consistent with ionic binding and may
require hydrophobic mechanisms.
Our results, identifying the 29-kDa and two smaller domains (9 and 13 kDa) of pADPRT to be participatory in self-association, may be correlated with predictions made from the cDNA structure of pADPRT isolated from Drosophila(53) . A leucine zipper motif has been identified in the automodification domain of the Drosophila enzyme, and it is noteworthy that the amphipathic helix at 395-417 residues of the bovine pADPRT includes a degenerate ``bZip'' domain identified in Drosophila(53) . The functional importance of this domain in the self-association of pADPRT appears to be supported by our results.