(Received for publication, January 9, 1997, and in revised form, February 24, 1997)
From the The synthesis and rapid turnover of ADP-ribose
polymers is an immediate cellular response to DNA damage. We report
here the isolation and characterization of cDNA encoding
poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for
polymer turnover. PARG was isolated from bovine thymus, yielding a
protein of approximately 59 kDa. Based on the sequence of oligopeptides
derived from the enzyme, polymerase chain reaction products and partial
cDNA clones were isolated and used to construct a putative
full-length cDNA. The cDNA of approximately 4.1 kilobase pairs
predicted expression of a protein of approximately 111 kDa, nearly
twice the size of the isolated protein. A single transcript of
approximately 4.3 kilobase pairs was detected in bovine kidney
poly(A)+ RNA, consistent with expression of a protein
of 111 kDa. Expression of the cDNA in Escherichia coli
resulted in an enzymatically active protein of 111 kDa and an active
fragment of 59 kDa. Analysis of restriction endonuclease fragments from
bovine DNA by Southern hybridization indicated that PARG is encoded by
a single copy gene. Taken together, the results indicate that previous
reports of multiple PARGs can be explained by proteolysis of an 111-kDa enzyme. The deduced amino acid sequence of the bovine PARG shares little or no homology with other known proteins. However, it contains a
putative bipartite nuclear location signal as would be predicted for a
nuclear protein. The availability of cDNA clones for PARG should
facilitate structure-function studies of the enzyme and its involvement
in cellular responses to genomic damage.
The biological consequences of genomic damage include recovery of
normal cell function, cellular survival leading to malignant transformation, or cell death by necrosis or apoptosis (1). Among the
many variables that can affect the ultimate biological consequence of
DNA damage to a particular cell are (i) the amount, type, and location
of the DNA damage and (ii) the cellular response elicited by the
damage. An immediate cellular response to DNA damage is the activation
of poly(ADP-ribose) polymerase (PARP)1 by
DNA strand breaks (2). PARP catalyzes the conversion of NAD to
multibranched polymers containing up to 200 ADP-ribose residues (3).
Increases in polymer levels of more than 100-fold may occur within
minutes (4). PARP is a major acceptor for ADP-ribose polymers in an
automodification reaction (5-7), while histones and other DNA binding
proteins also are modified to a lesser extent (8, 9). Once synthesized,
polymers are rapidly turned over (10, 11), being converted to free
ADP-ribose by the action of poly(ADP-ribose) glycohydrolase (PARG) (12,
13). An ADP-ribosyl protein lyase has been proposed to catalyze removal of protein-proximal ADP-ribose monomers (14).
While the changes in chromatin structure that accompany the rapid
synthesis and turnover of this large polyanion are still poorly
understood, ADP-ribose polymer metabolism has been linked to the
enhancement of DNA repair (15-18), limitation of malignant transformation (19-21), enhancement of necrotic cell death (22), and
involvement in programmed cell death (23, 24). To date, studies of the
structure and function of the enzymes of ADP-ribose polymer metabolism
have been mainly limited to PARP (25). PARG has been isolated to
apparent homogeneity from bovine thymus (26, 27), guinea pig liver
(28), and human placenta (29), and basic enzymatic features have been
established (26). However, the structure of the protein has not been
elucidated. We report here the isolation and characterization of the
cDNA encoding bovine PARG. The availability of cDNA clones for
PARG should facilitate detailed studies of the enzyme and the
involvement of ADP-ribose polymer metabolism in cellular responses to
DNA damage.
PARG was purified from bovine thymus
tissue (Pel-Freez) by modifications of previously published procedures
(26, 27). The enzyme was isolated up to the polyethylene glycol
(PEG)-6,000 fractionation step as described previously (27). However,
DNA-agarose and heparin-Sepharose chromatographic steps used previously
were omitted, and the PEG-6,000 fraction was applied directly to an affinity matrix of poly(ADP-ribose)-dihydroxyboronyl-Sepharose (PADPR
DHB-Sepharose). The active fractions eluted from PADPR DHB-Sepharose
(25 ml) were pooled, placed in dialysis tubing, concentrated against
dry PEG-20,000 to approximately 12 ml, and dialyzed against 2 liters of
20 mM potassium phosphate buffer, pH 8.0, 0.1% Triton
X-100, 5 mM The purification procedure for the bovine thymus PARG summarized in
Table I is typical for results obtained from six
separate preparations of the enzyme. Purification from 500 g of
bovine thymus achieved approximately 50,000-fold purification and
yielded approximately 20 µg of purified protein. Analysis of the
final preparation by SDS-PAGE revealed that more than 95% of the
protein migrated at an apparent molecular mass of approximately 59 kDa (Fig. 1).
Table I.
Purification of poly(ADP-ribose) glycohydrolase from bovine thymus
Prior to proteolytic fragmentation, the
purified PARG (40 µg in 100 µl of 0.4 M ammonium
bicarbonate buffer, pH 8.0, 8 M urea) was incubated in a
final concentration of 2.2 mM dithiothreitol at 56 °C
for 15 min. Iodoacetamide was added to a final concentration of 2.0 mM, and the sample was incubated at 25 °C for 15 min.
After dilution with an equal volume of water, 1.5 units of immobilized L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (Pierce) was added, and the sample was incubated at 37 °C
for 18 h with gentle rotary shaking. Finally, the mixture was
subjected to centrifugation at 16,000 × g for 5 min to
separate the tryptic fragments from the immobilized trypsin. The
tryptic fragments were adjusted to 0.05% in trifluoroacetic acid and
separated on a 4.6 mm × 25 cm, Microsorb MV, C4
reversed-phase HPLC column (Rainin) eluted with an 80-min linear
gradient from 4 to 44% acetonitrile in 0.05% trifluoroacetic acid.
Four oligopeptide fractions, with approximate elution times of 61, 63, 68, and 75 min, were selected for peptide sequence analysis by the
Edman degradation method (34). Amino acid sequence data of four
oligopeptides, designated by their approximate HPLC elution times from
the reversed-phase column, are shown in Table II.
Table II.
Amino acid sequence of oligopeptides derived from poly(ADP-ribose)
glycohydrolase
Division of Medicinal Chemistry and
Pharmaceutics,
Lucille P. Markey Cancer
Center, University of Kentucky, Lexington, Kentucky 40536
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Purification of PARG
-mercaptoethanol, 0.1 mM
thioglycolic acid, 0.4 M KCl (buffer A). The sample was
loaded onto a 1.0 × 11-cm Toyopearl AF-Red (Supelco) column, and
PARG was eluted with an 80-ml linear gradient of 0.4-2 M
KCl in buffer A. The active fractions, eluting at approximately 1.25 M KCl, were pooled, placed in dialysis tubing, concentrated
against solid sucrose to approximately 9 ml, and dialyzed against 20 mM potassium phosphate buffer, pH 7.2, 0.75 M
KCl, 0.1% Triton X-100, 10% glycerol, 5 mM
-mercaptoethanol, 0.1 mM thioglycolic acid. PARG
activity was determined as described by Ménard and Poirier (30),
and protein content was determined by the method of Bradford (31). The
final preparation was quantified by SDS-PAGE (32) and Coomassie Blue
staining to compare the intensity of the protein band with a known
amount of bovine serum albumin (33).
Step
Protein
Total activity
Specific
activity
Yield
Purification
mg
units
units/mg
protein
%
-fold
Crude
extract
27,800
57,400
2.06
100
1.0
Protamine
sulfate
12,500
58,000
4.64
101
2.3
Ammonium
sulfate
4,480
30,000
6.70
52
3.3
CM-Sepharose
171
19,100
112
33
55
PEG
6000
23.0
7,530
327
13
160
PADPR-DHB-Sepharose
1.30
6,730
5,180
12
2,500
Toyopearl
AF-Red
0.023
2,260
98,300
4
48,000
Fig. 1.
Analysis of purified bovine thymus PARG by
SDS-PAGE. An aliquot of the purified enzyme was precipitated by
trichloroacetic acid, washed with acetone, resuspended in SDS-PAGE
sample buffer, separated on a 10% SDS-PAGE gel, and stained with
Coomassie Blue. The positions of molecular weight marker proteins are
shown.
[View Larger Version of this Image (22K GIF file)]
Oligopeptide
Amino acid sequence
10 20 30
68
LFTEVLDHNE CLIITGTEQY SEYTGYAETY R
63
AYCGFLRPGV SSENLSAVAT GNXGCGAFG
61
FLINPELIVS R
75
IALXLPNIXT QPIPLL
To obtain cDNA clones encoding bovine
PARG, PCR amplification experiments were followed by the screening of
two different bovine cDNA libraries. Fig. 2 depicts two PCR
products and eight cDNA clones that were isolated to provide a
putative full-length cDNA clone encoding bovine PARG. For each of
the cDNA inserts characterized, the sequence of both strands was
determined by the dideoxynucleotide chain termination method (35) using
Sequenase from U.S. Biochemical Corp.
The first step leading to the isolation of cDNA clones was to
synthesize two multidegenerate 17-mer primers, GAYCAYAAYGARTGYYT and
CKRTANGTYTCNGCRTA (where Y represents T/C, R is A/G, K is T/G, and N is
A/T/C/G), based on two regions of the oligopeptide 68 sequence (DHNECL
and YATEYR, Table II). Using the multidegenerate primers and an
oligo(dT)-primed bovine thymus cDNA gt11 library BL1019b
(CLONTECH), PCR amplification generated a 74-bp DNA
fragment with a deduced amino acid sequence identical to the
corresponding region of oligopeptide 68. Next, two specific 24-mer
oligonucleotide primers (ATCATCACAGGTACTGAGCAGTAC and
GCCTGTGTATTCACTGTACTGCTC) based on the sequence of this 74-bp DNA were
used in combination with
gt11 forward and reverse primers to amplify
PCR products 1 and 2 from the bovine thymus library. PCR product 1 contained 231 bp of sequence including the region encoding the
N-terminal region of oligopeptide 68 and the entire sequence of
oligopeptide 61. PCR product 2 contained 757 bp, which included
sequence encoding the C-terminal region of oligopeptide 68 and the
entire sequence of oligopeptide 63. The sequence information obtained
from PCR products 1 and 2 was used to isolate cDNA clones obtained
by the screening of bovine thymus and bovine kidney cDNA libraries.
A 518-bp EcoRI-HindIII fragment from PCR product
2 was used as a probe to screen approximately 1 × 106
independent clones from the bovine thymus library. Two positive cDNA clones (clones 1 and 2) were isolated, which overlapped PCR products 1 and 2. However, attempts to obtain clones from the bovine
thymus library that contained sequence 5
to clone 2 were unsuccessful.
Thus, a 231-bp EcoRI-KpnI fragment from clone 2 was used as a probe to screen approximately 5 × 105
independent clones of the bovine kidney 5
stretch plus cDNA
gt11 library BL3001b (CLONTECH). Three positive
cDNA clones (clones 3-5) were obtained, all of which contained
sequence 5
to clone 2. Each of these clones also contained sequence
encoding oligopeptide 75. Clones 1-5 provided multiple overlapping
sequences in the 3
-terminal portion of a consensus cDNA, but
additional clones were sought to obtain overlapping sequences for the
5
-terminal region. Thus, a 436-bp EcoRI-KpnI
fragment located at the 5
end of clone 3 was used as a probe to screen
approximately 6 × 105 independent clones of the
bovine kidney library. Clones 6-8 provided overlapping sequences for
the 5
-terminal region. The full-length cDNA was constructed by
ligating a 3.9-kb XbaI-NsiI fragment from pWL11
(clone 1 cDNA insert in pTZ18R (36)) and a 3.0-kb
NsiI-XbaI fragment from pWL13 (clone 4 cDNA
insert in pTZ18R). The resulting plasmid, termed pWL30, contained the
4,070-bp full-length cDNA.
PARG was expressed
using two different bacterial expression systems, the pTrcHis Xpress
System (Invitrogen), in which the expressed protein contains a leader
polyhistidine sequence, and the glutathione S-transferase
(GST) gene fusion system (Pharmacia Biotech Inc.). For expression in
the pTrcHis Xpress system, three different DNA fragments were amplified
and inserted into the pTrcHis expression plasmid. Construct A,
containing the cDNA sequence 3 to 2,946, was prepared by
subcloning a 2.9-kb XhoI-EcoRI DNA fragment
amplified from pWL30 with primers WIN34 (GCTGCGGGTCTCGAGCATGAGTGCGGGC) and WIN15 (GCGTCTAGAATTCACTTGGCTCCTCAGGC). Construct B,
containing the cDNA sequence
3 to 3,813, was prepared by
subcloning a 3.8-kb XhoI-EcoRI DNA fragment
amplified from pWL30 with primers WIN34 and WIN33
(CCGGAATTCGGGTTTTTTGTTAATGAAAATTTATTAAC). Construct C, containing
cDNA sequence 964-2,946, was prepared by subcloning a 2.0-kb DNA
fragment amplified from pWL13 with primers WIN14 (TCAGAGCAGATGAACTCGAGCAGTCCAGG) and WIN15. Constructs A, B, and C were
used to transform E. coli TOP10 cells for expression
experiments.
For expression of PARG as a GST fusion protein, an insert containing the cDNA sequence from position 1138 to 2946 was prepared by subcloning a 1.8-kb EcoRI-EcoRI fragment amplified from pWL30 with the oligonucleotide CCAATTTGAAGGAGGAATTCCCGCCGCCACCATGAATGATGTGAATGCCAAACGACCTGGA and WIN15 as primers. The resulting DNA fragment was inserted into the EcoRI site of the pGEX-2T expression vector, and the plasmid was used to transform E. coli NM522 cells.
For expression experiments, bacterial cultures were grown at 37 °C
in 1% Bacto-tryptone, 0.5% yeast extract, and 0.5% NaCl to
approximately 0.6 A600/ml and were induced with
1 mM isopropyl--D-thiogalactoside (IPTG).
Cells were collected by centrifugation, and crude extracts were
prepared by sonication (10 A600/ml) in 10 mM sodium phosphate buffer, pH 7.2, 150 mM
NaCl, 0.5 mg/ml lysozyme, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mM EDTA, 0.7 µg/ml pepstatin A, 0.5 µg/ml leupeptin, and 1 µg/ml aprotinin. Cell extracts were subjected to
centrifugation, and the supernatant fraction was used for assay. PARG
assay conditions were as described previously (30). Following
incubations, portions of reaction mixture were analyzed by thin layer
chromatography (30) or subjected to anion exchange HPLC. Anion exchange
HPLC utilized a Whatman Partisil SAX column equilibrated with 7 mM potassium phosphate buffer, pH 4.0, at a flow rate of 1 ml/min. The sample was diluted in the same buffer, applied to the
column, and eluted with a 30-min linear gradient from 7 mM
potassium phosphate buffer, pH 4.0, to 250 mM potassium
phosphate buffer, 0.5 M KCl, pH 4.0. Activity gel assays
for PARG were done by casting polyacrylamide gels with automodified
PARP containing [32P]ADP-ribose polymers as described
previously (37). Following electrophoresis, PARG was renatured by
incubating the gels at 25 °C in 5 volumes of 50 mM
sodium phosphate buffer, pH 7.5, 50 mM NaCl, 10% glycerol,
1% Triton X-100, 10 mM
-mercaptoethanol, changing the
buffer every 3 h for a total of five changes. After an additional
incubation at 37 °C for 3 h, gels were dried, and PARG activity
was detected following autoradiography as a clear band on a black
background. Cell extracts containing PARG fused to GST were examined
for binding to glutathione-Sepharose 4B (GSH-Sepharose) (Pharmacia
Biotech Inc.) according to the specifications of the manufacturer.
Total cytoplasmic RNA and poly(A)+ RNA were isolated from bovine kidney MDBK cells (ATCC CCL22) using TRIzol reagent (Life Technologies, Inc.) following the manufacturer's recommendations. RNA was fractionated on denaturing agarose gels (38), transferred to nylon membranes, and hybridized with clone 4 (Fig. 2) radiolabeled by a random hexamer priming method (39). Total genomic DNA was prepared from bovine thymus tissue as described previously (38), and DNA (10 µg) was digested with EcoRI, BglII, XbaI or PstI, fractionated on a 1% agarose gel, transferred to a nylon membrane (Hybond N+, Amersham), and hybridized using an 828-bp HindIII fragment of clone 1 radiolabeled as described for clone 4 above (39). Prehybridizations and hybridizations were carried out at 42 °C in 50% formamide, 0.25 M sodium phosphate buffer, pH 7.2, 0.25 M NaCl, 7% SDS, 1 mM EDTA.
The
combined nucleotide sequence of clones 1-8 (Fig. 2)
predicted a full-length cDNA clone of 4,070 bp containing 257 bp of 5-noncoding sequence, a single open reading frame of 2,931 bp, and a
3
-noncoding region of 882 bp. Fig. 3 shows the complete nucleotide sequence (GenBankTM accession number U78975[GenBank]) and
the deduced amino acid sequence, which predicts a protein of 977 amino
acids and a molecular mass of 110.8 kDa.
Northern Blot Analysis of Bovine Kidney RNA
An unexpected
feature of the consensus full-length cDNA clone was that it
predicted the expression of a protein of approximately 111 kDa (Figs. 2
and 3), while the enzymatically active PARG from thymus had a molecular
mass of approximately 59 kDa (Fig. 1). To determine the size of the RNA
transcript for PARG, total RNA and poly(A)+ RNA were
isolated from MDBK cells and annealed using clone 4 as the
hybridization probe. A single transcript of approximately 4.3 kb was
detected in the poly(A)+ RNA (Fig.
4B, lane 2). Thus, the transcript
size was consistent with the expression of a 111-kDa PARG protein.
Expression of PARG in E. coli
To determine whether the
isolated cDNA encoded PARG, three different constructs containing
specific regions of the cDNA were inserted into the pTrcHisB
expression vector. Constructs A and B contained the entire open reading
frame of 110.8 kDa, which together with the fusion partner predicted a
protein of approximately 115 kDa. Construct B also contained the
3-untranslated region of the clone. Since the isolated PARG of
approximately 59 kDa contained enzymatic activity, construct C
contained only the 75-kDa carboxyl-terminal region of the PARG, which
predicted a fusion protein of approximately 79 kDa. Following
transformation of cells with the three expression constructs, cell
extracts were examined for PARG activity using three different assay
methods. Using a thin layer chromatography assay that measures release
of [32P]ADP-ribose from [32P]ADP-ribose
polymers (30), PARG activity was detected in extracts from cells
transformed by each of the constructs. Fig.
5A shows results obtained with constructs B
and C. No activity was detected in cells transformed with the empty
vector, but activity was detectable without induction by IPTG,
indicating a leaky lac promoter. The addition of IPTG
resulted in a time-dependent increase of up to approximately 4.5-fold in enzymatic activity. Fig. 5A also
shows that the enzymatic activity was strongly inhibited by the
presence of ADP-hydroxymethylpyrrolidine diol, a specific inhibitor of PARG (40, 41). The material released from ADP-ribose polymers was shown
to be exclusively ADP-ribose by strong anion exchange HPLC (Fig.
5B), demonstrating that the cell extracts did not contain any other ADP-ribose polymer-degrading enzymes such as
phosphodiesterase, which catalyzes the formation of AMP and
phosphoribosyl-AMP (42).
Expression of PARG in E. coli.
Panel A, analysis of bacterial extracts using a thin layer
chromatography assay (30). Reaction mixtures contained approximately
15,000 cpm of [32P]ADP-ribose polymers, and the cpm shown
represent ADP-ribose released from the ADP-ribose polymers. Bar
1, a strain transformed by pTrcHis without an insert but induced
with 1 mM IPTG for 5 h at 37 °C. A strain
containing construct B is shown without the addition of IPTG (bar
2) or after the addition of 1 mM IPTG for 1.5 h
(bar 3) or 5 h (bar 4). A strain containing
construct C 5 h after induction by IPTG is shown in the absence
(bar 5) and presence (bar 6) of 167 µM ADP-hydroxymethylpyrrolidine diol (40, 41). Panel B, analysis of material
released from ADP-ribose polymers by anion exchange HPLC. Extracts from
a strain containing construct B were incubated with
[32P]ADP-ribose polymers (30), and a portion was analyzed
by anion exchange HPLC as described under "Experimental
Procedures." The elution times for AMP, ADPR, and PR-AMP are
indicated by arrows. Panel C, analysis of PARG
activity following SDS-PAGE. Crude extracts (1 µl each) of cells
transformed by pTrcHis without an insert (lane A) or
containing construct A (lane B) were analyzed by an activity
gel assay in which the gel was incubated following electrophoresis in
50 mM sodium phosphate buffer, pH 7.5, 50 mM
NaCl, 10% glycerol, 1% Triton X-100, 10 mM
-mercaptoethanol to allow renaturation of PARG activity (37). The
positions of molecular weight marker proteins are shown. Panel
D, expression of a carboxyl-terminal region of PARG cDNA as a
fusion protein with GST. A construct corresponding to a 69-kDa
carboxyl-terminal region of the PARG cDNA was prepared and used to
transform E. coli NM522 cells as described under
"Experimental Procedures." Cell extracts were subjected to
SDS-PAGE, and proteins were stained by Coomassie Blue. Lane
1, extract from uninduced cells; lane 2, extract from cells induced with 1 mM IPTG for 2 h; lane
3, proteins in extracts from cells shown in lane 2 that
bound to GSH-Sepharose; lane 4, material released from
GSH-Sepharose by treatment with thrombin.
To determine the size of the expressed enzymatic activity, an activity gel assay (37) was used. With construct A, activity was observed at approximately 115 and 59 kDa (Fig. 5C). No bands were produced from extracts from the IPTG-induced pTrcHisB vector that did not contain an insert. Extracts from cells transformed with construct B showed bands at approximately 115 and 59 kDa, and extracts from cells transformed with construct C showed bands at approximately 79 and 59 kDa (data not shown). During storage at 4 °C, cell extracts lost activity migrating at the higher molecular weight, while the activity at approximately 59 kDa increased (data not shown). Expression of PARG in the pTrcHisB expression vector did not result in detectable amounts of protein by staining with Coomassie Blue. Thus, another construction was designed to overexpress a 69-kDa carboxyl-terminal region of the PARG as a fusion with GST, which allows convenient protein purification by affinity chromatography on a GSH-Sepharose column. Two hours after induction with IPTG, strong expression of a protein migrating at approximately 90 kDa was observed (Fig. 5D). This protein bound to GSH-Sepharose and was eluted by GSH. The construct contained a thrombin cleavage site between the GST and the 69-kDa region of PARG, and treatment of the material bound to GSH-Sepharose with thrombin resulted in the release of a protein that migrated at approximately 59 kDa. This result suggests that the protein purified from the bovine thymus may be larger than suggested by its migration on SDS-PAGE. Attempts to obtain high level expression of the full-length protein were unsuccessful.
Southern Blot Analysis of PARG Genomic ComplexityPrevious
studies have reported that PARG isolated from nuclear fractions had a
molecular mass of approximately 75 kDa (28, 29), while PARG isolated
from whole cell homogenates or postnuclear supernatant fractions had a
molecular mass of approximately 59 kDa (26, 27, 43). These results
suggest either that two or more genes may code for PARG or that
proteolysis generates lower molecular weight forms from higher
molecular weight forms. The cDNA isolated encoded a protein
considerably larger than any PARG proteins previously described,
consistent with the possibility that the different forms of PARG are
derived from a single form by proteolytic cleavage. To test the
hypothesis that PARG is encoded by a single copy gene, the genomic
complexity of the PARG gene was analyzed by a Southern hybridization
experiment. Genomic DNA was digested with four different restriction
enzymes, which did not contain restriction sites within the
carboxyl-terminal region of the PARG cDNA. Following
electrophoresis, the restriction digests were subjected to
hybridization with a probe that corresponded to the carboxyl-terminal
region of the PARG cDNA. The analysis displayed in Fig.
6 shows that, in each restriction digest, the probe
hybridized primarily with a single restriction fragment. The fainter
signals probably reflect the presence of introns in the PARG gene. This
result indicates that PARG is encoded by a single copy gene in the
bovine genome.
The rapid synthesis of ADP-ribose polymers that occurs in response to DNA strand breaks is accompanied by very rapid polymer turnover (4, 10, 11), indicating that PARP and PARG activities are closely coordinated as cells respond to DNA damage. While PARP has been widely studied, information concerning structure and function relationships of PARG is much more limited.
The isolation of partial PARG cDNA clones (Fig. 2) has allowed the
construction of a cDNA clone that codes for the sequence of all
four oligopeptide sequences present in the isolated protein (Table II,
Fig. 3) and contains PARG activity when expressed in E. coli
(Fig. 5). The cDNA clone (Fig. 3) has features typical of cDNAs
that code for mammalian proteins. These include (i) an oligo A
(putative poly(A)+) sequence at the 3 end, (ii) a
polyadenylation signal (AATAAA) (44) 12 bp upstream from the oligo A
sequence, (iii) a sequence of ATTTA in the 3
-untranslated region
thought to play a role in selective mRNA degradation in mammalian
cells (45), (iv) a single open reading frame, and (v) a nucleotide
sequence around the first start codon commonly found at known sites of
initiation of translation (46). The likelihood that the cDNA clone
constructed represents a full-length or nearly full-length clone for
PARG is supported by the observation that hybridization of
poly(A)+ RNA from bovine kidney cells with the cDNA
showed a single band of hybridization of approximately the same size as
the cDNA (Fig. 4).
The nucleotide sequence encoding bovine PARG indicates that PARG shares
little or no homology with other known sequences. A search of sequence
data banks has failed to reveal significant homology with any sequences
coding for known proteins. A strong sequence homology has been observed
with human and rat cDNA clones that likely represent partial clones
for PARG from these species. Examination of protein sequence data bases
also has shown that the deduced amino acid sequence of PARG lacks any
sequence homology with known proteins. However, the amino acid sequence
shares a significant homology with a protein sequence from
Caenorhabditis elegans that may represent the PARG protein
from this organism (47). The deduced amino acid sequence of PARG has
been examined for a number of structural motifs that can be predicted
from the primary amino acid sequence. We have noted that the expressed PARG protein can form dimers stable to SDS-PAGE conditions. In that
regard, residues 871-907 show significant homologies to known leucine
zipper dimerization sequences (48). Another motif identified is a
putative bipartite nuclear location signal (NLS) (49). It is
interesting that PARP also contains a bipartite NLS (50). Fig.
7 compares deduced amino acid sequences in the NLS
region of the bovine PARG and homologous regions of putative PARG
sequences from human, mouse, and C. elegans with the NLS
region of PARP from seven different organisms. The putative NLS of PARG
fulfills the criteria for bipartite NLS in that it contains conserved
acidic and basic amino acid residues at two different locations each within the region of homology to the NLS of PARP (50).
An intriguing finding was that the PARG cDNA clone codes for a protein of approximately 111 kDa, which is nearly twice the size of the PARG protein isolated from bovine thymus (Fig. 1). It seems likely that PARG contains a protease sensitive site that, following proteolysis, yields a protein fragment of approximately 59 kDa that still retains enzymatic activity. Several pieces of evidence favor this possibility. (i) Expression of the carboxyl-terminal portion of the cDNA resulted in enzymatic activity (Fig. 5). (ii) All of the oligopeptides sequenced were located in the carboxyl-terminal half of the protein (Figs. 2 and 3). (iii) The only protein, other than the 59-kDa protein detected in the thymus preparation was approximately 111 kDa (Fig. 1). (iv) The PARG activity expressed in bacteria was sensitive to proteolysis, yielding a protein of approximately 59 kDa (Fig. 5). (v) The cleavage site in PARG is in the region of the putative NLS, and the PARP NLS is located in a protease-sensitive site (24). Taken together with the data suggesting that bovine PARG appears to be coded for by a single copy gene (Fig. 6), proteolysis seems likely to explain the presence of PARG activity with a molecular mass of approximately 74 and 59 kDa in bovine thymus preparations (51). Likewise, a similar mechanism could explain previous reports of a PARG of 74 kDa isolated from nuclear fractions of guinea pig liver and human placenta (28, 29) and a PARG of 59 kDa isolated from postnuclear fractions of guinea pig liver (43).
While proteolysis of a larger protein to yield smaller proteins retaining PARG activity seems likely to explain the size heterogeneity of PARG previously reported, it remains to be determined if proteolysis normally occurs in vivo or whether it occurs during purification of the enzyme. While our results show that a full-length protein can be expressed containing PARG activity (Fig. 5C), the molecular size of PARG in vivo also remains to be determined. If PARG occurs as a larger protein, an interesting possibility is that the amino-terminal region may be involved in the regulation of enzymatic activity.
The availability of cDNA clones coding for PARG should facilitate studies of structural and functional aspects of this enzyme and the metabolic pathway in which it is involved. The isolation and characterization of cDNAs encoding PARG from other sources will allow information concerning changes in the structure of the protein during evolution. Studies in progress of cDNA clones from human and mouse indicate that PARG is highly conserved in mammals.2 The availability of PARP cDNA has allowed a number of molecular genetic approaches to study the function(s) of ADP-ribose polymer metabolism, and the availability of PARG cDNA should allow the design of additional molecular genetic approaches for studying this metabolism. Recently, a mouse strain containing a genetic inactivation of the gene encoding PARP has been reported (53). Although not extensively characterized, mice homozygous for the disrupted PARP gene are viable, indicating that the absence of PARP (and ADP-ribose polymers) has no major consequences for development. Disruption of the gene encoding PARG in mice containing a normal PARP gene will allow the determination of whether other cellular enzymes can replace PARG in the turnover of ADP-ribose polymers and/or whether development will occur in the absence of PARG. Alternatively, disruption of the PARG gene in mice containing a disrupted PARP gene may provide insights for the coordinated function of PARP and PARG.
Understanding the involvement of ADP-ribose polymer metabolism in determining the frequency of alternative cellular outcomes of DNA damage may also have a therapeutic benefit. For example, the efficacy of many antitumor agents appears to be closely related to the ability to increase the frequency of apoptosis (1, 23). The ability of inhibitors of PARP to chemosensitize specifically cycling cells to the cytotoxic effects of DNA-damaging agents has demonstrated the potential of targeting ADP-ribose polymer metabolism for cancer therapy (16). In that regard, PARG is a potentially attractive target due to its low abundance, its structurally unique substrate, and the high turnover of ADP-ribose polymers that occurs following DNA damage. Modulation of PARG activity by chemical inhibitors or by molecular genetic approaches should allow assessment of the value of PARG as a new target for therapeutic development.
We thank Donna Coyle for excellent technical assistance, Arnold Huang for growth of MDBK cells, and Dr. Guy Poirier for many helpful discussions. The PARG inhibitor ADP-hydroxymethylpyrrolidine diol was a gift from Dr. James Slama. Peptide sequence determinations were done by the University of Kentucky Molecular Structure and Analysis Facility directed by Dr. Thomas Vannaman.