Full text of DOI:10.1080/15216540050212187

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IUBMB Life, 50: 145 149, 2000
c
°
Copyright 2000 IUBMB
1521-6543/01 $12.00 + .00
Original Research Article
Chain Length Analysis of ADP-Ribose Polymers
Generated by Poly(ADP-Ribose) Polymerase (PARP)
as a Function of ¯-NAD+ and Enzyme Concentrations
Hilda Mendoza-Alvarez, Susana Chavez-Bueno, and Rafael Alvarez-Gonzalez
Department of Molecular Biology and Immunology and Institute for Cancer Research, University of North
Texas Health Science Center at Fort Worth, Fort Worth, Texas
(
lational covalent modiŽ cation catalyzed by PARP itself EC
) ( )
2.4.2.30 1 . Other polypeptides with ADP-ribose polymeriz-
Summary
Bireactant autopoly ADP-ribosyl ation of poly ADP-ribose
) (
ing activity have recently been reported, for example, PARP-2
2, 3 , tankyrase 4 , and VAULT-PARP 5 , but little is known
about their biochemical properties.
(
)
(
)
(
)
polymerase PARP EC 2.4.2.30 was carried out by using either
(
)
( )
( )
increasing concentrations of ¯-NAD+ donor substrate at a Ž xed
(
)
(
protein concentration or increasing concentrations of PARP ac-
(
)
In the process of poly ADP-ribose synthesis, PARP cat-
+
32
)
[
]
ceptor substrate at a Ž xed ¯-NAD concentration. The P ADP-
ribose polymers synthesized were chemically detached from PARP
by alkaline hydrolysis of the monoester bond between the carboxy-
late moiety of Glu and the polymer. Nucleic acid–like polymers
were then analyzed by high-resolution polyacrylamide gel elec-
trophoresis and autoradiography. The ADP-ribose chain lengths
observed displayed substrate concentration–dependent elongation
from 0.2 µM to 2 mM ¯-NAD+ . Similar results were observed at
Ž xed concentrations of 4.5, 9, 18, 27, and 36 nM PARP. Therefore,
we conclude that the concentration of the ADP-ribose donor sub-
strate determines the average chain length of the polymer synthe-
sized. In contrast, the polymer sizewas unaltered when the concen-
tration of PARP was varied from 4.5 to 18 nM at a Ž xed ¯-NAD+
concentration. However, when PARP concentrations >18 nM were
used, the total amount of monomeric ADP-ribose produced was
noticeably less. Therefore, we conclude that high concentrations
of PARP lead to acceptor substrate inhibition at the level of the
ADP-ribose chain initiation reaction.
(
)
alyzes three chemically distinct reactions 6, 7 . The Ž rst step,
or “initiation” reaction, involves the speciŽ c attachment of one
ADP-ribose unit to a free carboxylate moietyon an acceptorpro-
tein 8 . For example, the intermolecular modiŽ cation of PARP,
the main protein target for poly ADP-ribosyl ation in DNA-
damaged cells, occurs stoichiometrically and quantitatively at
( )
four separate Glu residues 9 , the result of formation of a cat-
alytic homodimer of PARP 10 on nicked or broken DNA 11 .
The second and most efŽ cient reaction catalyzed by PARP is
( )
(
)
(
)
(
)
(
)
the protein-distal ADP-ribose polymerization 12, 13 reaction
or “chain elongation”. In this reaction, the 20-hydroxyl group of
the adenine proximal ribose of the acceptor unit, for example,
(
)
(
)
mono ADP-ribosyl ated-PARP 12, 13 , is used as the target for
ADP-ribose chain elongation, and a 20-100 O-glycosidic linkage
(
)
is formed 12 . The elongation step of ADP-ribose polymer syn-
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IUBMB
Life, 50: 145 149, 2000
(
)
thesis is highly processive 14 and occurs with an enzymatic
(
)
4 ( )
efŽ ciency kcat/kNAD > 2 £ 10 9 . Therefore, PARP may cat-
+
(
)
Keywords Mechanism; poly ADP-ribose polymerase, polymer
(
)
alyze >200 rounds of elongation for every initiation step 15 .
length; regulation; substrate availability.
–
Interestingly, Glu residue 988 of the carboxy terminal catalytic
domain of PARP has recently been proposed as the key residue
INTRODUCTION
(
)
in ADP-ribose chain elongation 16, 17 . Finally, the last step
(
)
)
The poly ADP-ribosyl ation of DNA-binding proteins, in-
(
)
of poly ADP-ribose synthesis corresponds to the polymeric
(
(
)
cluding poly ADP-ribose polymerase PARP , is a posttrans-
–
(
)
“branching” reaction00017 19 . This step involves the enzymatic
00
–
–
(
) ( )
ribose ribose glycosidic bond 18, 19
formation of a 2
1
Received 28 June 2000; accepted 29 June 2000.
and is believed to occur with a frequency of one branching point
per 40 rounds of elongation.
Address correspondence to Dr. RafaelAlvarez-Gonzalez,Associate
Director for Basic Research of the Institute for Cancer Research, Uni-
versityof North Texas Health Science Center at Fort Worth, 3500 Camp
Bowie Boulevard, Fort Worth, TX 76107-2699. Fax: 817-735-2133.
E-mail: ralvarez@hsc.unt.edu
(
)
Clearly, one may anticipatethat the hyperpoly ADP-ribosyl -
ation of DNA binding proteins with these highly branched and
–
complex nucleic acid like molecules results in a dramatic
145

146
MENDOZA-ALVAREZ ET AL.
32
+
electrostatic change of the acceptor protein surface. Not sur-
prisingly, the covalent attachment of ADP-ribose two negative
charges per nucleotide polymers to an acceptor molecule leads
to the shuttling off, or release, of this protein from the heli-
cal phosphodiester backbone of DNA 20 one negative charge
per nucleotide . Further, as a result of this ionic repulsion be-
tween ADP-ribose polymers and DNA, chromatin structure and
function may become uncoupled 21 . This uncoupling of chro-
matin structure and function may directly alterthe physiological
course of DNA replication 22 , DNA recombination 23 , gene
expression 24 26 , DNA base excision repair 27, 28 , and cell
survival or apoptosis 29 31 .
In spite of the multiple roles that ADP-ribose polymers play
in chromatin function, a termination of the ADP-ribose chain
length s required to modulate or regulate a speciŽ c DNA func-
[
]
a- P b-NAD was purchased from ICN. All other chemicals
(
used were of the highest purity commercially available.
Enzyme PuriŽ cation. PARP was puriŽ ed to homogeneity
from calf thymus by a previously published procedure 32 .
Autopoly ADP-Ribosyl ation of PARP. Increasing concen-
trations of PARP from 4.5 to 36 nM were incubated with vari-
)
(
)
(
) (
(
)
)
+
32
[
]
ous concentrations of P b-NAD in a 100-l l assay mixture
containing 100 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1 mM
dithiothreitol, and 20 lg/ml calf thymus DNA. After incuba-
tion for 3 min at 37 ±C, the reaction was stopped with ice-cold
(
)
(
)
(
)
)
–
(
)
(
(
)
trichloroacetic acid 20%, w/v .
–
(
)
Size Distribution of Enzyme-Bound ADP-Ribose Chains.
Acid-precipitable material was processed for qualitative anal-
ysis of the size distribution of the ADP-ribose polymers by a
( )
(
)
procedure published elsewhere 15 .
tion has not been documented. A major obstacle to identifying
the metabolic signals that dictate the chain length and complex-
RESULTS
ity of ADP-ribose polymers is our limited understanding of the
+
The size distribution of ADP-ribose polymers synthesized in
(
role that the substrate concentrations of b-NAD , DNA, and
(
)
the autopoly ADP-ribosyl ation reaction catalyzed by PARP as
)
perhaps PARP itself play in this process. Here, we report that
a function of the concentration of either b-NAD+ ADP-ribose
the concentration of b-NAD+ available to PARP determines the
average chain length of the polymer synthesized and that an
oversupply of PARP leads to substrate inhibition at the level of
chain initiation.
(
)
(
)
donor or PARP ADP-ribose acceptor was analyzed by high-
(
)
resolution polyacrylamide gel electrophoresis 15 .
The electrophoretic distribution of the ADP-ribose polymers
synthesized with 4.5 nM PARP a suboptimal amount of en-
(
+
)
zyme at increasing concentrations of b-NAD from 200 nM to
EXPERIMENTAL PROCEDURES
2 mM is shown in Fig. 1A. The short ADP-ribose oligomers
of no more than 8 residues when synthesized with 200 nM
Chemicals and Materials. Dithiothreitol, lithium dodecyl
b-NAD+ Fig. 1A, lane 1 increased to 18 ADP-ribose units
(
)
(
)
sulfate LDS , and calf thymus DNA were obtained from Sigma;
Figure 1. Effect of the b-NAD+ concentration on the ADP-ribose polymer size distribution of the products generated by PARP at
( )
( )
a Ž xed, suboptimal enzyme concentration of either 4.5 nM A or 9 nM B , as shown by autoradiographic analysis of the protein-
free polymers synthesized in the automodiŽ cation reaction of PARP after high-resolution polyacrylamide gel electrophoresis. The
(
)
(
)
relative electrophoretic migration of xylene cyanol 20-mer , bromophenol blue 8-mer , and AMP are indicated to the left of the
autoradiograph. The concentration of b-NAD+ is indicated at the bottom of each lane.

147
We alsodetermined the sizedistribution of ADP-ribose chains
PARP CONCENTRATION AFFECTS ADP-RIBOSE CHAIN LENGTH
when b-NAD+ was increased to 400 nM Fig. 1A, lane 2 . No
(
)
(
)
synthesized with 9 nM PARP Fig. 1B , that is, at half of the op-
(
)
sign of branched polymers at the origin of the gel 15 was de-
(
)
timal enzyme concentration see below . Results of these exper-
iments were similar to those using 4.5 nM PARP. Under closer
analysis, however, an obvious difference between the patterns
tected in this population of polymers. In contrast, the presence
(
)
of a radioactive signal at the origin of the gel Fig. 1A, lane 3
indicated that branched polymers were synthesized with 1 l M
b-NAD+ . As expected, greater micromolar concentrations of
(
)
of ADP-ribose polymers synthesized at 4.5 Fig. 1A and 9 nM
(
)
PARP Fig. 1B became evident when comparing lanes 9 and 10
(
)
the ADP-ribose donor 1.0 lM to 2 mM increased the length
32
[
]
of both panels. In Fig. 1B the intensity of the P AMP bands
synthesized with 1 and 2 mM b-NAD+ slightly decreased when
the concentration of enzyme was increased to 9 nM PARP.
–
and complexity of ADP-ribose polymers generated, as shown
by the band at the origin of the gel Fig. 1A, lanes 3 10 . Inter-
estingly, the overall amount of P AMP observed in Fig. 1A,
–
(
)
32
[
]
(
)
–
Fig. 2 lanes 1 10 shows the results for polymer synthe-
sis with increasing concentrations of b-NAD+ at a Ž xed PARP
concentration of 18 nM, the optimal enzyme concentration for
lanes 3 8, a hallmark of ADP-ribose chain initiation, gradu-
ally disappeared when b-NAD+ concentrations were between
1 and 500 lM. That is, under those conditions, the elongation
(
)
(
)
PARP autopoly ADP-ribosyl -ation in vitro 10 . These results
and branching reactions were favored. Surprisingly, however,
32
(
)
conŽ rm our preliminary observation 10 that the Ž nal chain
[
]
the P AMP band reappeared at millimolar concentrations of
length of the polymers synthesized is a bNAD+ concentration
b-NAD+ Fig. 1A, lanes 9 and 10 . Thus, the efŽ ciency of elon-
gation and branching decreased as a result of substrate inhibition
by millimolar concentrations of b-NAD+ .
–
(
)
–
(
)
dependent phenomenon lanes 1 10 . Note that the intensity
+
32
[
]
P AMP band observed with 1 and 2 mM b-NAD
of the
Figure 2. Effect of the b-NAD+ concentration on the ADP-ribose polymer size distribution of the products generated by PARP
(
)
at a Ž xed optimal enzyme concentration of 18 nM. The relative electrophoretic migration of xylene cyanol 20-mer , bromophenol
+
(
)
blue 8-mer , and AMP are indicated to the left of the autoradiograph. The concentration of b-NAD is indicated at the bottom of
each lane.

148
MENDOZA-ALVAREZ ET AL.
Figure 3. Effect of the b-NAD+ concentration on the ADP-ribose polymer size distribution of the products generated by PARP at
( )
( )
a Ž xed above-optimal enzyme concentration of either27 nM A or 36 nM B , as shown by autoradiographic analysis of the protein-
free polymers synthesized in the automodiŽ cation reaction of PARP after high-resolution polyacrylamide gel electrophoresis. The
(
)
(
)
relative electrophoretic migration of xylene cyanol 20-mer , bromophenol blue 8-mer , and AMP are indicated to the left of the
autoradiograph. The concentration of b-NAD+ is indicated at the bottom of each lane.
(
)
(
)
Fig. 2, lanes 9 and 10 is unchanged from that in Figs. 1A that modulate the activity ies of PARP—particularly, those that
(
and 1B.
shift this versatile DNA-dependent enzyme from mono ADP-
( )
)
Finally, to fully evaluate the effects of the enzyme concen- ribosyl ating activity 9 to ADP-ribose elongation and branch-
(
)
tration on the structural complexity of the products formed by ing functions 12, 13, 17 . A clear understanding of the signals
(
)
PARP, we also carried out enzymatic autopoly ADP-ribosyl - that regulate PARP enzymatic behavior is crucial, given that
ation at 27 and 36 nM PARP. Indeed, we had previously re- the biological function of ADP-ribose polymers in transcription
–
(
)
(
)
ported a decline in overall enzymatic activity under these high
23 26 and DNA base excision repair 27, 28 may be chain-
(
)
nanomolar concentrations of PARP 10 apparently because of length dependent.
acceptor substrate inhibition. The results observed under higher Here, we conclusively demonstrate that the size and com-
(
)
nanomolar concentrations of PARP are shown in Figs. 3A and plexity of poly ADP-ribose is speciŽ cally determined by the
3B. Again, even at high nanomolar PARP concentrations, our b-NAD+ concentration and not by the concentration of PARP
–
(
)
results indicate that the concentration of the ADP-ribose donor,
Figs. 1 3 . Indeed, the size distribution pattern of ADP-ribose
b-NAD+, determines the Ž nal size and complexity of the poly- polymers as a function of b-NAD+ concentration from 200 nM
(
mers synthesized compare the overall polymer size distribu- to 2 mM, at Ž ve distinct Ž xed nM concentrations of PARP,
)
tion with that in Figs. 1 and 2 . In addition, Figs. 3A and 3B remains unchanged. Therefore, the length and complexity of
(
)
show that, although the size distribution of poly ADP-ribose
ADP-ribose polymers synthesized by PARP are determined by
+
32
(
)
[
]
did not change substantially, the P AMP band observed at the ADP-ribose donor b-NAD concentration. Our results
1 and 2 mM b-NAD+ decreased until undetectable. Thus, our are also consistent with the notion that PARP is a highly pro-
(
(
)
results also indicate that the inhibition of the autopoly ADP- cessive enzyme 14 at high micromolar concentrations of
ribosyl ation reaction at high nanomolar concentrations of b-NAD+ . Furthermore, our data suggest that when the ADP-
)
(
)
PARP acceptor substrate and millimolar concentrations of ribose donor substrate concentration is limiting, PARP does not
b-NAD+ donor substrate is the result of inhibition by acceptor display much processivity. This is particularly relevant to the
(
)
substrate of the initiation step of polymer synthesis.
putative roles of PARP in DNA base excision repair and apop-
tosis after DNA damage, conditions in which the intracellular
+
(
pools of b-NAD are usually depleted. Finally, our data Figs. 1
DISCUSSION
)
and 2, lanes 9 and 10 are also consistent with the conclusion
To elucidate the biological role of ADP-ribose polymers in that high nanomolar concentrations of PARP result in acceptor
eukaryotic cells, we must uncover the regulatory mechanisms substrate inhibition at the level of ADP-ribose chain initiation.

149
PARP CONCENTRATION AFFECTS ADP-RIBOSE CHAIN LENGTH
ACKNOWLEDGMENT
This project was supported fully by grant GM45451 from the
National Institutes of Health to R.A.-G.
Evidence for active site similarities to the ADP-ribosylating toxins. J. Biol.
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17. Rolli, V., O’Farrell, M., Menissier-de Murcia, J., and de Murcia, G. 1997
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)
Random mutagenesis of the poly ADP-ribose polymerase catalytic do-
main reveals amino acids involved in polymer branching. Biochemistry 36,
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