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Year: 2004
Poly (ADP-ribose) reactivates stalled DNA topoisomerase I andinduces DNA strand break resealing
Malanga, M; Althaus, F R
Malanga, M; Althaus, F R (2004). Poly (ADP-ribose) reactivates stalled DNA topoisomerase I and induces DNAstrand break resealing. Journal of Biological Chemistry, 279(7):5244-5248.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:Journal of Biological Chemistry 2004, 279(7):5244-5248.
Malanga, M; Althaus, F R (2004). Poly (ADP-ribose) reactivates stalled DNA topoisomerase I and induces DNAstrand break resealing. Journal of Biological Chemistry, 279(7):5244-5248.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:Journal of Biological Chemistry 2004, 279(7):5244-5248.
Poly (ADP-ribose) reactivates stalled DNA topoisomerase I andinduces DNA strand break resealing
Abstract
Regulating the topological state of DNA is a vital function of the enzyme DNA topoisomerase I.However, when acting on damaged DNA, topoisomerase I may get trapped in a covalent complex withnicked DNA (stalled topoisomerase I), that, if unrepaired, may lead to genomic instability or cell death.Here we show that ADP-ribose polymers target specific domains of topoisomerase I and reprogram theenzyme to remove itself from cleaved DNA and close the resulting gap. Two members of thepoly(ADP-ribose) polymerase family, PARP-1 and 2, act as poly(ADP-ribose) carriers to stalledtopoisomerase I sites and induce efficient repair of enzyme-associated DNA strand breaks. Thus, bycounteracting topoisomerase I-induced DNA damage, PARP-1 and PARP-2 act as positive regulators ofgenomic stability in eukaryotic cells.
1
Poly(ADP-ribose) reactivates stalled DNA topoisomerase I and induces DNA strand break
resealing
Maria Malanga and Felix R. Althaus
Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 260, CH-
8057 Zurich, Switzerland
Corresponding author: Felix R. Althaus
Institute of Pharmacology and Toxicology, University of Zurich
Winterthurerstrasse 260, CH-8057 Zurich, Switzerland
Tel: ++41 01 6358762
Fax: ++41 01 6358910
Running title: Poly(ADP-ribose) reactivates stalled DNA topoisomerase I
JBC Papers in Press. Published on December 29, 2003 as Manuscript C300437200
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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SUMMARY
Regulating the topological state of DNA is a vital function of the enzyme DNA
topoisomerase I. However, when acting on damaged DNA, topoisomerase I may get trapped in a
covalent complex with nicked DNA (stalled topoisomerase I), that, if unrepaired, may lead to
genomic instability or cell death. Here we show that ADP-ribose polymers target specific
domains of topoisomerase I and reprogram the enzyme to remove itself from cleaved DNA and
close the resulting gap. Two members of the poly(ADP-ribose) polymerase family, PARP-1 and
2, act as poly (ADP-ribose) carriers to stalled topoisomerase I sites and induce efficient repair of
enzyme-associated DNA strand breaks. Thus, by counteracting topoisomerase I-induced DNA
damage, PARP-1 and PARP-2 act as positive regulators of genomic stability in eukaryotic cells.
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INTRODUCTION
DNA topoisomerase I (topo I) plays an essential role in controlling the level of DNA
supercoiling and relieving the torsional stress that is generated during replication, transcription,
recombination and chromatin remodeling (1,2). However, when scanning the topological state of
DNA, topo I may get trapped in the vicinity of DNA lesions and cause secondary DNA damage
by an abortive mechanism (3). This involves the formation of a DNA single strand break,
covalent attachment of the enzyme to the 3’-phosphate terminus (cleavage complex), and
inhibition of the DNA ligase activity of topo I (“stalled topo I”). Stalled topo I is a threat to
genomic stability and cell life since enzyme-bound nicked DNA can be converted into
irreversible single- or double-strand breaks. In fact, the efficacy of camptothecins, a prominent
class of anticancer drugs (also known as topo I poisons) is due to the stabilization of topo I
cleavage complexes (4). In eukaryotes, a specific repair enzyme, tyrosyl-DNA phosphodiesterase
1 (TDP1) has evolved to counteract the secondary DNA damage of stalled topo I by removing
the enzyme from the 3’-terminus. Nevertheless, a DNA single strand break is generated that
needs to be repaired by the base excision repair machinery (5,6).
Here we show that two members of the poly(ADP-ribose) polymerase family, i.e. PARP-
1 and PARP-2 can remove stalled topo I from DNA. PARP-1 and PARP-2 belong to the DNA
damage surveillance network (7,8). They are activated by DNA strand breaks to catalyze the
conversion of the respiratory coenzyme nicotinamide adenine dinucleotide (NAD+) into protein-
bound ADP-ribose polymers, with concomitant release of nicotinamide (9). While PARP-1 has
been found to poly (ADP-ribosyl)ate a number of nuclear proteins (“heteromodification”) (9
and references therein), both PARP-1 and PARP-2 preferentially modify themselves
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(“automodification”) (10, 11). We found that, with the attached polymers, PARP-1 and PARP-2
bind to specific domains of topo 1 and reprogram their functions. Even camptothecin-stabilized
cleavage complexes, resembling those formed by topo I in the vicinity of DNA lesions (3), could
be disjoined by this mechanism.
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Materials and Methods
Enzymes and oligonucleotides
Human DNA topoisomerase I, certified to be purified to homogeneity and free of nuclease and
topoisomerase II contaminations, was purchased from TopoGen, together with camptothecin and
polyclonal anti-human topoisomerase I antibodies.
Recombinant PARP-1 and PARP-2 (Alexis), [γ-32P]-ATP (7000 Ci/mmol) (ICN), and the
oligonucleotides (Microsynth) 5’-CTAGTAAAAGACTTGGAAAAATTTTTAAAAAA-3’, 5’-
AATTTTTTTTAAAAATTTTTCCAAGTCTTTTA-3’; 5’-CTAGTAAAA GACTTGGA-3’; 5’-
TTTTTTAAAAATTTTTCCAAGTCTTTTACTAG-p were of the highest purity commercially
available. The sequences of the oligonucleotides are essentially as reported (12,13). 5’-end
labeling of the scissile strand oligonucleotide was performed using T4 polynucleotide kinase and
[γ-32P]-ATP, annealed to the unlabeled complementary strand, and purified by polyacrylamide
gel eletrophoresis. The site of topo I cleavage is indicated by an arrow in Figures 1 and 2.
Automodified PARPs and poly(ADP-ribose)
In the automodification reaction , PARP-1 or PARP-2 were incubated for 30 min with 0.1-0.4
mM [α-32P]-NAD (4000 dpm/nmol), as described by Beneke et al. (14). Mock automodification
was carried out in the presence of 2 mM 3-aminobenzamide (AB). The products were purified by
ultrafiltration on Microcon YM-30 membrane. Automodified or mock modified enzymes were
used freshly for DNA binding/cleavage assays or stored at –80 °C in 50 mM Tris-HCl buffer,
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pH 7.4, containing 14 mM β-Mercaptoethanol, 0.5 mM PMSF, 20% glycerol. Free poly(ADP-
ribose) was prepared as previously described (15) and their size distribution determined (16).
Topo I reactions
Purified human topoisomerase I (0.5 pmol) was incubated with [32P]-5'-end labeled
oligonucleotide substrates (20’000-40’000 dpm; 20 fmoles) in 50 mM Tris-HCl, pH 8.0
including 0.1 mg/ml BSA either at 37 °C for 10 min or at 30 °C for 15 min (suicide substrate).
Reactions were carried out in 20 µl (final volume) and stopped by adding 0.5 volumes of 3x
Laemmli loading buffer. Covalent complexes of topoisomerase I with cleaved oligonucleotides
were separated from the uncleaved substrates by 10% SDS-PAGE and visualized by
autoradiography. Camptothecin (CPT) was used at 20 µM concentration. Autoradiographic
bands were quantified by scanning densitometry (Molecular Dynamics); for a representative set
of samples, the densitometric quantification of enzyme-bound and free DNA was paralleled by
liquid scintillation counting of excised radioactive bands. The fraction of the oligonucleotide
substrate in the cleavage complex ranged between 2-5% and 10-25%, in the absence or presence
of CPT, respectively.
Poly(ADP-ribose), either PARP-bound or as free polymer, was present in the reaction
mixtures at the amounts indicated in the Figures (pmol quantities refer to the ADP-ribose unit);
reactions were started by the addition of the radioalabeled oligonucleotide substrates. For
reversal experiments, topo I - DNA complexes were allowed to form over 10 min incubation in
the conditions described above, then either poly(ADP-ribose) or 0.35 M NaCl (12,17) were
added and incubation was extended for the time indicated in the figure legends. After SDS-
PAGE and autoradiography, remaining cleavage complexes were quantified by scanning
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densitometry. For reversal kinetics, the complex formed before any addition was set at 100% and
percentage data were plotted on a semilogarithmic scale against time. The religation constant (kr)
was calculated by fitting these data to the equation Ct = C0e-krt (where Ct = % remaining cleavage
complex at the end of the incubation time, t; C0 = % cleavage complex at t0 = 100). Kr values
were converted to half life (t1/2) values according to the equation t1/2 = ln 2/kr (18).
Binding of topo I to dsDNA was determined by electrophoretic mobility shift (19). Topo
I (45 ng) was incubated with 20 fmol of radiolabeled oligonucleotide, in the conditions described
above. At the end of the incubation time, half the reaction mixtures (10 µl) were withdrawn,
mixed with 0.25 volumes of loading buffer (10 mM Tris-HCl, pH 8.0, 15% Ficoll, 0.05%
Bromophenol Blue) and electrophoresed on native 6% polyacrylamide gel. The remaining half of
the reactions was analysed by SDS-PAGE to evaluate the potential contribution of covalent
complexes to the mobility shift. In the conditions used to visualize the noncovalent topo I-DNA
complex, the cleavage conjugate was undetectable.
Poly (ADP-ribose) binding assay
The noncovalent binding of poly(ADP-ribose) to topo I (and other proteins) was determined as
previously described (20). Purified proteins were either dot blotted or transferred onto
nitrocellulose membrane after electrophoretic separation on 10% SDS-polyacrylamide gel.
Bound poly(ADP-ribose) was visualized by autoradiography and quantified by scintillation
counting of excised radioactive dots. Western blotted proteins were gold stained using the
Protogold staining kit (British BioCell International), as recommended by the manufacturer.
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RESULTS AND DISCUSSION
Poly (ADP-ribose) regulates the formation of topo I cleavage complexes
The data in Fig. 1A show that PARP-1 and PARP-2 block the formation of topo I covalent
complex with DNA only in the poly ADP-ribosylated form, while the unmodified enzymes,
incubated under identical conditions with the inhibitor 3-aminobenzamide (AB), were
completely ineffective. Moreover, PARP-1 and 2 were still effective, when topo I complex
formation was strongly enhanced in the presence of camptothecin (CPT, Fig. 1A, bottom), but
again only after the enzymes had been automodified with ADP-ribose polymers. These data
suggested that the ADP-ribose polymers attached to PARP-1 or PARP-2 could be responsible for
the inhibitory effects on topo I complex formation. Direct evidence for this is shown in Fig. 1B.
Free ADP-ribose polymers detached from PARP-1 or PARP-2 expressed the full inhibitory
activity in a dose-dependent manner (Fig. 1B), while a 4-fold excess (w:w) of a 39-nucleotide
single stranded DNA did not influence the reaction (Fig. 1C, right panel). Degradation of
polymers to monomeric ADP-ribose, using poly(ADP-ribose)glycohydrolase (PARG),
completely abolished the inhibitory principle (Fig. 1C, left panel).
Poly(ADP-ribose) could prevent the initial DNA binding of topo I which then would
preclude subsequent formation of covalent complexes. Fig. 2A shows that DNA binding, as
measured by electrophoretic mobility shift analysis, was not affected by ADP-ribose polymers at
quantities up to 20 pmol that completely blocked covalent complex formation (Fig. 1). Also,
(ADP-ribose) polymers did not accelerate dissociation of topo I already bound to DNA after a 10
min preincubation (Fig. 2A, lane 5). On the other hand, 0.35 M NaCl was sufficient to prevent
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formation of topo I - DNA noncovalent complexes as well as to dissociate them (Fig. 2A, lanes 4
& 6). It should be mentioned that in these types of analyses (see also Fig. 2D) the shifted topo I –
DNA complexes always migrated as two discrete bands. The high purity of the topo I
preparations (see “Materials and Methods”) and the fact that both bands were reduced when
incubation was carried on in the presence of anti-topo I antibodies, but not in the presence of
non-specific immunoglobulins (data not shown), argue against DNA binding by protein
contaminants. It seems also unlikely that the two complexes result from DNA binding of
differently sized topo I fragments. In fact, in western blot analyses, a single component of the
expected molecular weight was detected by polyclonal anti-topo I antibodies (Fig. 2A, right
panel); in addition, topo I, either labeled by covalent binding to radiolabeled oligonucleotides
(Fig. 1-3) or by noncovalent interaction with [32P]-poly (ADP-ribose) (Fig. 4), migrated as a
single band in SDS-polyacrylamide gels. Thus, generation of different conformers of topo I-
DNA complexes as well as binding of one or two topo I molecules/oliglonucleotide molecule
might underlie the peculiar electrophoretic behaviour under native conditions. On the other hand,
topo I dimers have been shown to form both in vitro (21) and in vivo (22).
Poly (ADP-ribose) affects both the forward and reverse DNA transesterification reaction of
topo I, but with opposite effects
The reduced yield of cleavage complexes in the presence of poly (ADP-ribose) could be
explained by either inhibition of DNA cleavage (forward transesterification reaction) or by a
shift in the cleavage/religation equilibrium in favour of religation (reverse transesterification
reaction). To examine whether poly (ADP-ribose) could affect DNA cleavage, the
oligonucleotide shown in Fig. 2B was used as a suicide substrate for topo I: in fact the enzyme
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becomes trapped in the covalent complex (suicide complex), since the cleavage reaction
generates a free trinucleotide with the 5’-OH end that is lost by diffusion and hence religation
becomes impossible (12). The results of Fig. 2B demonstrate that poly(ADP-ribose) is a potent
inhibitor of DNA cleavage by topo I: as little as 1 pmol of polymeric ADP-ribose was sufficient
for complete inhibition (Fig. 2B). Conversely, poly (ADP-ribose) had no effect on preformed
suicide complexes (Fig. 2B, compare lanes 1 & 6). Again, inhibition of DNA cleavage was also
achieved with poly(ADP-ribosyl)ated PARP-1 and PARP-2 (Fig. 2C & D), but not with the
unmodified enzymes (Fig. 2C, lanes 4 & 5). Incidentally, noncovalent topo I binding to the
suicide substrate was equally unaffected by poly(ADP-ribosyl)ated PARP-1 and PARP-2 (Fig.
2D), as has been observed with the reversible cleavage substrate (Fig.2A).
Next we determined whether poly(ADP-ribose) could reverse preformed covalent topo I-
DNA conjugates by stimulating the religation activity of topo I. In the experiment shown in Fig.
3A, complex formation was allowed to proceed for 10 and 20 min in the absence or presence of
camptothecin. Under these conditions, a steady state level of cleavage complexes was reached
within 10 min. Following addition of ADP-ribose polymers, the topo I – DNA complexes
disappeared (Fig. 3A, lane 3). Strikingly, this was also observed with the complexes stabilized
in the presence of camptothecin (Fig. 3A, lane 6), indicating that poly (ADP-ribose) is able to
overcome the poisoning effect of the drug. Removal of cleavage complexes was achieved with
poly (ADP-ribosyl)ated PARP-1 (Fig. 3B) or PARP-2 (not shown), but not with the unmodified
enzymes (Fig. 3B, lane 6), emphasizing that polymers are essential for this effect. It should be
noted that due to the strong inhibition exerted by poly (ADP-ribose) on the DNA cleavage
reaction (Fig. 2B-D), the disappearance of cleavage complexes in these reversal experiments was
essentially dependent on the rate of religation. Uncoupling of cleavage and religation reactions
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can also be achieved by increasing salt concentration. In fact 0.35 M NaCl blocks cleavage,
probably by interfering with de novo binding of the enzyme to DNA (12,17, and Fig 2A), but has
no effect on the DNA ligation activity of topo I. The effect of CPT is also reversed under such
conditions (17). A comparison of the religation kinetics in the presence of either poly (ADP-
ribose) or 0.35 M NaCl indicated that DNA strand break resealing by topo I is indeed accelerated
by poly (ADP-ribose) (Fig. 3C & D): the half life of cleavage complexes was more than 2 fold
shorter in the presence of poly (ADP-ribose). Taken together, these results suggest that the low
yield of cleavage complexes (Fig. 1) and the efficient reversal of such complexes (Fig. 3) may
arise from two opposing actions of poly(ADP-ribose): inhibition of DNA cleavage (Fig. 2) and
stimulation of the topo I religation activity (Fig. 3).
Poly (ADP-ribose) binds noncovalently to topo I
With their long polymeric arms, members of the PARP family can reach out to bind specific
proteins (23-25) and alter their domain functions (20,26). Several DNA damage checkpoint
proteins are targeted in this manner and the mechanism involves one or several poly(ADP-
ribose)-binding sequence motifs in the target protein (27). The motif, a 20 to 25 amino acid
sequence with regularly spaced hydrophobic residues, interspersed with basic residues and
flanking arginines and lysines, was found to occur at three conserved sites in the topo I sequence
(Fig. 4A). These sites are located in structurally and functionally distinct protein domains (28):
two sequences, comprising amino acids 261-280 and 532-550, are in the core DNA binding
domain, but at opposite sites of the clamp formed by the enzyme around DNA; a third polymer-
binding site (sequence 669-688) is in the linker domain. The latter connects the DNA binding
and C-terminal domains and has a regulatory role on the overall reaction as it slows down the
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religation step and thereby facilitates DNA relaxation (18,29). Biochemical analysis using a
nitrocellulose blot assay (20) directly confirmed that topo I belongs to the family of poly(ADP-
ribose)-binding proteins (Figs. 4B & C). In the dot blot assay (Fig. 4B), about 1 pmol polymeric
ADP-ribose/pmol protein bound to topo I and histone H1 (positive control) immobilized on
nitrocellulose. Poly (ADP-ribose) binding also occurred after electrophoretic separation and
western blotting of these proteins (Fig. 4C).
Mapping the poly (ADP-ribose)-binding sites into the published crystal structure of
human topo I (28) provided a plausible explanation for the differential effects of ADP-ribose
polymers on specific domain functions. First, the polymer-binding regions have no primary role
in the DNA-binding of topo I, which may explain why poly (ADP-ribose, while interacting with
the free enzyme (Fig. 4B &C) does not affect topo I - DNA noncovalent complexes (Fig. 2A &
D). On the other hand, the sequence comprising amino acids 532-550 is located into core sub-
domain III that largely contributes to the formation of the catalytic site: binding of poly (ADP-
ribose) at this site may underlie the strong inhibition of the DNA cleavage reaction. The cap
region of topo I (residues 175-433), which includes the poly (ADP-ribose)-binding sequence
261-280, is also essential for DNA cleavage (30).
How poly (ADP-ribose) counteracts CPT action and accelerates DNA religation is less obvious
and requires further considerations. The x-ray crystal structures of human topo I in covalent
complex with DNA in the absence and presence of a CPT analogue, topotecan, have revealed the
mode of binding of the drug and clarified important mechanistic aspects of topo I poisoning (31).
Topotecan binds to the DNA-enzyme complex by intercalating into DNA at the site of cleavage
and displacing the nucleophilic 5’-OH end of the cleaved strand: thus religation is prevented.
The comparison of the structure of the ternary (enzyme - DNA - drug) and binary (enzyme –
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DNA) complexes also showed that the most dramatic changes involved the linker domain, which
could be visualized in the electron density map only after drug binding. Interestingly, deletion of
the linker domain, either total (18) or partial (32), or even a single amino acid substitution (33)
make the enzyme insensitive to CPT. Alltogether, these observations point to an involvement of
this domain in the stabilization of the ternary complex. It should be noted that the linker-
associated poly (ADP-ribose)-binding site (amino acids 669-688) is likely to be the most (if not
the only) accessible one to the polymer when topo I is engaged in the cleavage complex. Thus,
we speculate that targeting of the linker domain by poly (ADP-ribose) may have a dual effect: i)
it may induce conformational changes that destabilize the CPT binding pocket and reposition the
broken DNA strand so that ligation can occur (neutralization of CPT poisoning; Fig. 3); ii) it
may alleviate the restrains imposed by the linker domain on DNA religation (18,29), which
would explain the reduced half life of cleavage complexes (Fig. 3C & D).
In conclusion, both PARP-1 and PARP-2 are able to disjoin covalent DNA complexes
and induce the reversal of DNA breaks that are formed when topo I gets stalled in the vicinity of
DNA lesions. DNA breaks are also the activating signal for PARP-1 and PARP-2 (7-11) to
produce (ADP-ribose)n-polymers which we show here to be the molecules responsible for
removing topo I from DNA. Thus, an important aspect of PARP-1 and PARP-2 function in
preserving genomic stability of cells is to deploy polymers to sites of stalled topo I. By contrast
to the topo I removal mechanism catalyzed by tyrosyl-DNA phosphodiesterase 1 (5,6), this
PARP-based rescue mechanism has an immediate impact on the genomic stability of cells, as it
is activated within seconds and does not leave an unfilled DNA gap.
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Our data also bear on the design of new therapeutic strategies for the treatment of
cancers. As shown in Figs. 1 & 3, poly(ADP-ribose) may counteract the effects of the cancer
therapeutic drug camptothecin and contribute to drug resistance. Thus, the efficacy of
camptothecin-mimetic drugs can be enhanced by combination with PARP inhibitors. The in vivo
relevance supporting this concept has already been demonstrated: cancer cells with compromised
PARP-1 function are hypersensitive to camptothecin (34) and the cytotoxicity of topo I-targeted
drugs is enhanced by PARP inhibitors (34,35). Finally, the differential effects of poly(ADP-
ribose) on particular domain functions of topo I, i.e. the DNA cleavage and ligation reactions can
be exploited by site-specific targeting and this opens new perspectives for the design of novel
topo I – based anticancer drugs.
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Acknowledgments
This work was supported by the Swiss National Science Foundation.
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Figure legends
Figure 1 – Poly(ADP-ribose), either PARP-1 or PARP-2 bound, or as a free polymer, regulates
the level of cleavage complexes formed on a topo I substrate (top). 1A, complex formation in the
absence or presence of either automodified (lanes 4 and 7) or mock poly (ADP-ribosyl)ated
PARPs (PARP/AB, lanes 5 and 6). PARP-1*, PARP-2* (20 pmol enzyme-bound poly(ADP-
ribose). Lanes 2 and 3, native PARP-2 (250 and 500 fmol, respectively). 1B, cleavage complexes
(mean +/- S.D., n ≥ 4) in the presence of increasing amounts (pmol) of poly(ADP-ribose) and
SDS-PAGE analysis (insert). 1C, left, effect of either intact (lanes 3 and 5) or PARG-digested
(lanes 2 and 4) poly(ADP-ribose); right, effect of a 39mer ss-oligonucleotide.
Figure 2– Poly(ADP-ribose) does not interfere with topo I binding to, or dissociation from
DNA, but it inhibits DNA cleavage. 2A, left: electrophoretic mobility shift analysis of topo I
DNA-binding in the absence (lane 2) or presence of either poly (ADP-ribose) (20 pmol; lane 3)
or 0.35 M NaCl (lane 4). Alternatively, buffer (lane 7), poly (ADP-ribose) (20 pmol; lane 5) or
NaCl (0.35 M; lane 6) were added after 10 min preincubation of topo I with the radiolabeled
oligonucleotide and reactions were continued for 10 min further. About 4% of input DNA
(sequence shown in Fig. 1A) was shifted by topo I. Lane 1: topo I omitted. 2A, right: western
blot analysis. Topo I binding by specific polyclonal antibodies was detected by enhanced
chemiluminescence (ECL). 2B, lanes 1 – 4, 15 min incubation of suicide substrate (sequence
shown on top) with topo I +/- the indicated amounts of poly(ADP-ribose); lanes 5 and 6:
incubation was for 30 min: buffer (lane 5) or 5 pmol poly(ADP-ribose) (lane 6) were added after
the first 15 min of incubation. 2C, reaction was carried on in the absence (lane 1) or presence of
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either native (lanes 4,5) or poly(ADP-ribosylated) PARPs (*PARP: 1 pmol PARP-bound
polymeric ADP-ribose). 2D, native PAGE analysis of topo I noncovalent and covalent (suicide)
complexes (*PARP: 20 pmol PARP-bound polymeric ADP-ribose). The fraction of input DNA
in the suicide complex increased with incubation time and was 7-10% after 15 min.
Figure 3 – Poly(ADP-ribose) reverses preformed topo I DNA cleavage complexes. 3A, cleavage
complexes were allowed to form in 10 min incubation (lanes 1 and 4), then either buffer (lanes 2
and 5) or 20 pmol poly(ADP-ribose) (lanes 3 and 6) were added and incubation was continued
for additional 10 min. 3B, time course of the disappearance of CPT-stabilized cleavage
complexes upon addition of 20 pmol PARP-bound poly(ADP-ribose) (PARP-1*); lane 1:
preformed complexes; lanes 2 – 4: cleavage complexes 1, 2 and 5 min after PARP-1* addition.
Buffer (lane 5) or native PARP-1 (lanes 6) served as controls. 3C, comparison of the kinetics of
cleavage complex reversal in the presence of either poly (ADP-ribose) (20 pmol) or NaCl.
Following additions, incubations were continued for 1 (lanes 2 and 5), 2 (lanes 3 and 6) or 5 min
(lanes 4 and 7). 3D, semilogarithmic plot of data from 3C. Remaining cleavage complexes are
expressed as percentage of the complexes formed during 10 min incubation, before any addition
(lane 1 in 3C, control). Reactions were carried on in the presence of CPT.
Figure 4 – Poly(ADP-ribose) binds noncovalently to topo I. 4A, top: sequence alignments with
a poly (ADP-ribose)-binding motif. For each peptide, the positions of the first and last amino
acids in the human topo I sequence are indicated. 4A, bottom: domain structure of topo I (18);
white boxes with vertical dotted lines locate the poly (ADP-ribose)-binding sites. 4B, [32P]-poly
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(ADP-ribose) probing of proteins dot-blotted on nitrocellulose membrane; 4C, proteins were
electrophoretically separated before transfer on nitrocelluose and poly (ADP-ribose) binding
assay. Left panel: autoradiogram; right panel: gold staining of proteins on nitrocellulose. H: 1 µg
calf thymus histone H1; M: molecular weight markers, Precision Plus, BioRad; T: 0.2 µg
purified topo I.
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A
1 2 3 4 5 6 7
No additi
on
PARP-2
PARP-2
PARP-2*
PARP-1*
PARP-2/A
B
PARP-1/A
B
- CPT
+ CPT
AT T T T C T G AACC T T T T T AAAAAT T T T T T T T AA-5’3’-C T AG T AAAAG AC T T GG AAAAAT T T T T AAAAAA-3’5’-[ P]-32
C
+ CPT
PARG + + - + -
- CPT+ CPT1 2 3 4 5 6 7
no additi
on
ss-3
9mer
B
0 5 10 20 0 5 10 20
- CPT + CPT
Poly(ADP-ribose)(ADP-ribose pmol)
20
40
60
80
100
0 5 10 15 20clea
vag
e co
mp
lex
(% o
f co
ntr
ol)
Fig. 1
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B
C
-3’C T A G T A A A A G A C T T G G A5’-[ P]-32
-5’-P3’-G A T C A T T T T C T G A A C C T T T T T A A A A A T T T T T T
1 2 3 4 5 6
0 1 2.5 5 0 5pmol
1 2 3 4 5
No additi
on
PARP-1
PARP-2
PARP-2*
PARP-1*
D
Fig. 2
A
noncovalentcomplexes
No additi
on
PARP-2*
PARP-1*
free probe
suicidecomplex
1 2 3 4 5 6 7
(ADP-ribose)n - - + - + - -- - - + - + -0.35 M NaCl
ECL
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Fig. 3
A
(ADP-ribose)n - - + - - +
1 2 3 4 5 6- CPT + CPT
B
PARP-1* - + + + - -
1 2 3 4 5 6
(ADPR)n0.35 M NaCl
- + + + - - -
- - - - + + +
1 2 3 4 5 6 7
C
D
clea
vag
e co
mp
lex
(% o
f co
ntr
ol)
time (min)
100
10
10 1 2 3 4 5 6
+ (ADPR)n
+ NaCl
time (min)
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Fig. 4
A
hx b bb bx h hh h
K A D A K V M K D A K T K K - V V E S K K
A K M L D H E Y T T K E I F - R K N F F KK D S I R Y Y N K V P V E K R - V F K N
261 280532 550
669 688
B
H1 Prot K DNase I H110 ng 1 µg 1 µg 50 ng
11 22 45 90 topo I (ng)
H M T H M T250150
10075
50
37
C
Core domainN-terminal domain Linker
C-terminal domain
1 215 636 713 765N- -C
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