(Received for publication, September 6, 1994; and in revised form, October 26, 1994)
From the
Poly(ADP-ribose) polymerase (PADPRP) is biologically significant
in the rejoining of DNA strand breaks. Post confluent cultures of
3T3-L1 preadipocytes showed marked increases in PADPRP protein and
activity when the cells were induced to differentiate into adipocytes.
When this increase in PADPRP expression was prevented in stably
transfected 3T3-L1 cells by induction of PADPRP antisense RNA
synthesis, the cells did not differentiate nor undergo the two or three
rounds of DNA replication that are required for initiation of the
differentiation process. 3T3-Ll cells expressing PADPRP antisense RNA
under differentiation conditions were easily detached from plates and
in some cases eventually died. When newly expressed PADPRP protein and
DNA synthesis was assessed in cells at zero time or at 24 h after
induction of differentiation by incorporation of bromodeoxyuridine or
[H]thymidine into DNA, significant incorporation
was shown to occur in control cells after 24 h, but not in antisense
cells. Furthermore, during the first 24 h, the co-immunoprecipitation
of PADPRP and DNA polymerase
was observed in control cells,
whereas no such complex formation was noted in the induced antisense
cells, nor in uninduced control cells.
Poly(ADP-ribose) polymerase (PADPRP), ()a
chromatinassociated enzyme, has an absolute requirement for DNA for
activity and is proportionally activated by the presence of DNA strand
breaks (Smulson et al., 1977; Juarez-Salinas et al.,
1979; Benjamin and Gill, 1980; Berger et al., 1980). With
nuclear NAD as a substrate, PADPRP catalyzes the poly(ADP-ribosyl)ation
of a subclass of nuclear proteins, including histones, high mobility
group chromosomal proteins, topoisomerases (Kasid et al.,
1989), protein ICP4 of herpesvirus (Blaho et al., 1992), and
simian virus 40 (SV40) large T antigen (Baksi et al., 1987).
PADPRP and the nuclear protein modification it catalyzes are thought to
participate, together with other enzymes, in DNA repair and replication
as well as in other cellular processes in which cleavage and rejoining
of DNA segments may be required. These processes include cellular
differentiation and transformation, sister chromatid exchange, and gene
rearrangements and transpositions (Farzaneh et al., 1982;
Ferro and Olivera, 1984; Ueda and Hayaishi, 1985). Support for a role
of poly(ADP-ribosyl)ation in the differentiation process has been
provided by a number of studies, some of which examined the effects of
chemical inhibitors of PADPRP. Accordingly, DNA strand breakage, which
activates PADPRP as well as the enzyme NADase, has been implicated in
the terminal differentiation process of HL-60 cells (Farzaneh et
al., 1987). In this regard, in HL-60 cells induced to
differentiate with retinoic acid, the concentration of PADPRP mRNA
increased from an initially low value and remained elevated for
12-24 h, after which the concentration declined steadily (Bhatia et al., 1990b). A similar pattern was observed during dimethyl
sulfoxide-induced differentiation of HL-60 cells, with the exception
that the PADPRP mRNA concentration increased later in the
differentiation process (Bhatia et al., 1990b). Suzuki et
al.(1989) observed a reduction in PADPRP mRNA concentration after
exposure of HL-60 cells to retinoic acid for 3 days, after which time
45% of the cells showed the differentiated granulocytic phenotype.
Earlier studies suggested that an increase in PADPRP activity precedes
the differentiation process (Caplan and Rosenberg, 1975). In contrast,
decreased PADPRP activity has been correlated with the appearance of
specific gene products (Rastle and Swetly, 1978).
As mentioned above, past approaches to determining the role of PADPRP in the differentiation process depended heavily on the use of chemical inhibitors (for example, nicotinamide, thymidine, benzamide, and coumadin) (Yamagoe et al., 1991) of poly(ADP-ribose) synthesis. However, the use of these inhibitors has limitations because of their potential effects on other biological processes (Milam and Cleaver, 1984). To alleviate these problems, we have previously explored the feasibility of establishing stably transfected mammalian cells that express PADPRP antisense RNA (Ding et al., 1992). Despite the fact that the half-life of PADPRP in cells is relatively long (48-72 h), the induction of PADPRP antisense RNA in HeLa cells decreases both PADPRP activity and protein in the nucleus (Ding et al., 1992). Additionally, chromatin of PADPRP-depleted cells has an altered structure, as assayed by deoxyribonuclease I susceptibility. Interestingly, given the proposed role for PADPRP in the repair of DNA strand breaks, cells depleted of PADPRP cannot initiate DNA rejoining after strand breaks, which, as mentioned above, is a process thought to have an important role in differentiation (Farzaneh et al., 1982).
The 3T3-L1 preadipocyte cell line represents a useful tool for studying mechanisms of cellular differentiation (Mackall et al., 1976; Reed and Lane, 1980; Pekala et al., 1981). When appropriately induced by a defined hormonal treatment (insulin, dexamethasone, and methylisobutylxanthine), 3T3-L1 preadipocytes differentiate in culture into cells that possess morphological and biochemical characteristics of adipocytes (Pekala et al., 1981). A marked increase in the concentrations of lipogenic and lipolytic enzymes as well as of other adipocyte-specific proteins accompanies acquisition of the adipocyte phenotype (Ntambi et al., 1988). Depending upon the assay used, both decreases as well as increases in PADPRP activity have been reported as one of the earliest events during differentiation in these cells (Pekala and Moss, 1983; Janssen and Hilz, 1989). Accordingly, with the antisense expression conditions for HeLa cells as a guide, we have analyzed how altering the concentration of PADPRP, by antisense RNA expression, may affect the onset and progression of cellular morphological changes in 3T3-L1 preadipocytes during differentiation. Additionally, a putative role for PADPRP in apoptosis in this system is discussed.
Figure 1:
Structure and restriction sites of the
pMAM-As plasmids containing murine PADPRP cDNA in antisense orientation
downstream of the MMTV LTR. The pMAM-As expression vector contained the
glucocorticoid-inducible MMTV promoter ligated to a 1.1-kb murine
PADPRP 5` cDNA fragment, in reverse orientation, that encoded the
DNA-binding domain and a portion of the automodification domain of the
enzyme. A transcription start site within the MMTV LTR was located 260
base pairs upstream of the cloning site, and the SV40 early splicing
and polyadenylation regions were located 1.0 kb downstream of the
PADPRP sequence. The expected transcript size was 2.4 kb. The
entire expression plasmid comprised 9.4 kb.
Southern
hybridization of genomic DNA and Northern analysis of PADPRP mRNA were
performed with P-labeled murine PADPRP cDNA that had been
prepared by random primer extension (>10
cpm/µg of
DNA). Antisense PADPRP RNA was detected by Northern analysis with a
single-stranded RNA probe that was generated from the plasmid, pGEM-3Z
(Promega). The plasmid was manipulated to contain the 1.1-kb EcoRI 5` fragment of murine PADPRP cDNA downstream of T7
promoter. The plasmid was linearized, and a
P-labeled RNA
probe complementary to PADPRP antisense RNA was synthesized with T7 RNA
polymerase.
Total
RNA from 1 10
cells from each antisense cell line
was isolated by guanidinium-phenol extraction (Chomczynski and Sacchi,
1987) and was analyzed by Northern analysis with the
P-labeled RNA probes complementary to PADPRP antisense RNA
or with
P-labeled murine PADPRP cDNA.
Figure 2:
Dexamethasone-induced synthesis of
PADPRP antisense RNA in 3T3-L1 cells stably transfected with plasmid
pMAM-As. A, control (nontransfected) 3T3-L1 cells and 3T3-L1
cells stably transfected with pMAM-As (lines As 5 and As
6) were incubated with dexamethasone (1 µM) for the
indicated time periods. Total RNA was isolated, and 10 µg were
subjected to Northern analysis as described under ``Materials and
Methods.'' B, Northern blots were hybridized with a P-labeled single-stranded RNA probe that was synthesized
from a pGEM-3Z vector containing the 1.1-kb mouse PADPRP cDNA fragment
used to construct pMAM-As. The riboprobe was synthesized in the sense
orientation with T7 RNA polymerase.
The influence of dexamethasone-induced PADPRP antisense RNA on the concentration of endogenous PADPRP mRNA in cell lines As 5 and As 6 was determined by Northern analysis with a murine PADPRP cDNA probe (data not shown). In control 3T3-L1 cells, treatment with dexamethasone for up to 72 h had no effect on the steady-state concentration of PADPRP mRNA. In contrast, treatment of As 5 and As 6 with dexamethasone at various time points reduced the concentration of PADPRP mRNA by 80-95%.
Figure 3: Effect of PADPRP antisense RNA synthesis on cellular PADPRP concentration. Control (nontransfected) 3T3-L1 cells (A) and As 5 and As 6 cells (B) were incubated with dexamethasone (1 µM). At the indicated times, cells were washed with PBS, and their protein concentration was determined. Equal amounts of total cellular protein (50 µg) were subjected to electrophoresis on an SDS-polyacrylamide gel, and the separated proteins were then transferred to a nitrocellulose filter. The filter was incubated with rabbit antiserum to porcine thymus PADPRP at a dilution of 1:2000. No Dex, cells incubated in the absence of dexamethasone. The positions of molecular mass standards (STD) are indicated.
In control cells, incubation with
dexamethasone for 72 h did not affect PADPRP concentration (Fig. 3A). Furthermore, the amounts of PADPRP in
uninduced As 5 and As 6 cells were approximately the same as that in
control cells. In contrast, the concentration of PADPRP was markedly
reduced in As 5 and As 6 cells after induction of PADPRP antisense RNA
synthesis. The PADPRP concentration in these cells decreased by
40, 60, and 80% after 48, 72, and 96 h of exposure to
dexamethasone, respectively (Fig. 3B). The 113-kDa
full-length PADPRP protein and the truncated 89-kDa protein decreased
proportionally with induction of antisense RNA synthesis. To verify
that the decrease in PADPRP concentration was not attributable to
decreased growth rate, we showed that the amounts of PADPRP in cells
incubated for 0 and 72 (As 6) or 96 (As 5) h in the absence of
dexamethasone were virtually identical (Fig. 3B).
Figure 4: PADPRP activity and concentration in control 3T3-L1 preadipocytes during differentiation. Two days after achieving confluency, 3T3-L1 cells were induced to differentiate with dexamethasone, methylisobutylxanthine, and insulin as described under ``Materials and Methods.'' At the indicated times, cells were assayed for PADPRP activity by the sonication method, as described under ``Materials and Methods,'' with 10-20 µg of protein for each determination. Values are the means of duplicate determinations. Inset, immunoblot analysis of PADPRP concentration. Equal amounts (50 µg) of total cellular protein were subjected to immunoblot analysis as described in Fig. 3. The arrow indicates the 113-kDa band corresponding to the full-length murine PADPRP.
The catalytic activity of PADPRP is influenced by the accessibility of acceptor molecules, and is also linearly dependent on the number of DNA strand breaks and effected by several other properties of chromatin (Butt et al., 1978, 1979; Benjamin and Gill, 1980; Berger et al., 1987; Jacobson and Jacobson, 1989). Because of these potential complications, we also examined the effect of differentiation on PADPRP by immunoblot analysis, which had not previously been examined. A marked increase in immunoreactive PADPRP was observed during the first 2 days of differentiation (Fig. 4, inset). Variability in the number of immunoreactive bands observed may be due to the fact that PADPRP has been reported to degrade in cells into peptides of discrete sizes (Hotlund et al., 1983; Surowy and Berger, 1985), which are also detectable with the porcine anti-PADPRP antibody. PADPRP activity decreased to below control values by 4 days, as the cells terminally differentiated into adipocytes. At the fully differentiated state (day 8), immunoreactive PADPRP was virtually undetectable. These results are consistent with previous studies showing low PADPRP activity in terminally differentiated cells (Cherney et al., 1985).
The effect of PADPRP antisense RNA was subsequently examined during differentiation (Fig. 5). The marked increases in PADPRP activity and protein observed during the first 2 days of differentiation of control 3T3-L1 cells (Fig. 4) were not apparent in antisense cells; PADPRP activity and protein remained constant (Fig. 5). A slightly higher immunological reaction of the truncated PADPRP was noted at day 2, although this was not associated with increased PADPRP at this time point. Furthermore, in contrast to control 3T3-L1 cells (both nontransfected and transfected with pMAM-neo only), As 5 and As 6 cells did not accumulate cytoplasmic triglyceride as visualized by phase-contrast microscopy and oil red O staining (Fig. 6). In both control cell lines, triglycerides were observed from day 3 of differentiation and were visible in almost 90% of the cells by day 7. As 5 and As 6 cells also did not exhibit any of the morphological changes observed for control cells after induction of differentiation.
Figure 5: PADPRP activity and concentration in 3T3-L1 As 5 cells after incubation with inducers of differentiation. Two days after achieving confluency, 3T3-L1 cells were exposed to inducers of differentiation. At the indicated times, cells were assayed for PADPRP activity by the sonication method. Values are the means of duplicate determinations. Inset, equal amounts (20 µg) of total cellular protein were subjected to immunoblot analysis of PADPRP as described in Fig. 4. The arrow indicates the 113-kDa band corresponding to the full-length murine PADPRP.
Figure 6: Cytoplasmic triglyceride accumulation in control and pMAM-As-transfected 3T3-L1 cells. Seven days after exposure to inducers of differentiation, cells were stained with Oil Red O dye and examined by phase-contrast microscopy. A control (nontransfected) 3T3-L1 cells. B, mock-transfected 3T3-L1 cells (cells with transfected with pMAM-neo vector devoid of PADPRP cDNA. C, As 5 cells; D, As 6 cells.
These antisense cells did not undergo the normal two or three rounds of cell division during the initial 2 days after exposure to inducers of differentiation (Fig. 7). Furthermore, the cells became easily detached from plates after 5 days of exposure to differentiation inducers and eventually died, as revealed by their failure to exclude trypan blue. In some instances, the antisense cells progressed through one round of cell division (Fig. 8, inset); however, this process resulted in even more extensive cytotoxicity. These results are consistent with a potential role of PADPRP in apoptosis (Nosseri et al., 1994; Kaufmann et al., 1994) and antisense 3T3-L1 cells might prove useful to further study this important biological process. Although the role of PADPRP in cells that undergo apoptosis is still unclear, it has been observed that inhibitors of PADPRP may either enhance or reduce the extent of apoptosis (Rice et al., 1992, Nosseri et al., 1994). Confluent antisense cells, treated with dexamethasone alone, did not undergo cell death, even after 2 weeks of exposure to this agent. Thus, the cytotoxicity observed in the antisense cell lines, exposed to differentiation inducers, was probably not attributable to antisense expression per se. The expression of several other antisense transcripts in 3T3-L1 cells, such as those to actin or ferritin heavy chain, has been shown not to affect differentiation or to have adverse effects on growth (Wenz et al., 1992).
Figure 7: Proliferation of control and pMAM-As-transfected 3T3-L1 cells after incubation with inducers of differentiation. Two days after achieving confluency, control (nontransfected) 3T3-L1 cells and antisense cells were incubated with inducers of differentiation as described under ``Materials and Methods.'' Cells were collected at the indicated times and counted with a hemocytometer and trypan blue dye and with a Coulter counter.
Figure 8:
Time course of DNA replication in 3T3-L1
control and antisense cells during differentiation, as measured by the
rate of [H]-TdR incorporation into DNA.
Post-confluent control and antisense cells were induced to
differentiate, harvested at indicated time intervals, pulse labeled for
15 min with [
H]TdR (0.2 µCi/ml), and the acid
insoluble radioactivity was then measured by TCA precipitation and
liquid scintillation counting as described under ``Materials and
Methods.''
Figure 9: Determination of BrdU incorporation and expression of PADPRP during the first 24 h of differentiation in intact cells by immunocytochemical methods. Two days after achieving confluency, control (non-transfected) 3T3-L1 cells (A) and antisense cells (B) were incubated with inducers of differentiation for 24 h. Selected nuclei from A were enlarged (C). Cells were collected at zero time (prior to adding differentiation inducers) and 24 h after induction of differentiation as described under ``Materials and Methods.'' For BrdU incorporation, the cells were incubated with 10 µM BrdU for 1 h and immunodetected by a mouse monoclonal anti-BrdU, followed by rhodamine-labeled anti-mouse IgG. To detect PADPRP immunologically, fixed cells were incubated with rabbit anti-PADPRP, followed by fluoresceinlabeled anti-rabbit IgG, as described under ``Materials and Methods.''
Selected nuclei from Fig. 9A were enlarged (Fig. 9C). Both BrdU and PADPRP appear to be concentrated in distinct intranuclear granular foci, although it is not clear if they represent similar substructures of the nuclei. As evident in Fig. 9B, there was negligible BrdU and PADPRP immunocytochemical staining in nuclei of antisense 3T3-L1 cells after 24 h of induction of differentiation.
In the experiment in Fig. 10, both control and antisense
cells were examined for interaction of PADPRP and DNA pol at zero
time and 24 h after induction of differentiation. Crude extracts
derived from these cells were immunoprecipitated with a monoclonal
antibody to DNA pol
. The immunocomplex was then isolated by
addition of protein A-Sepharose beads and separated by SDS-gel
electrophoresis, followed by Western blotting using antibody to PADPRP.
Figure 10:
PADPRP and DNA polymerase associate
during early stages of differentiation, as determined by
co-immunoprecipitation. Both control and antisense cells were treated
essentially as described in the legend to Fig. 9. At zero time
(prior to differentiation) and 24 h after induction of differentiation,
cells were collected, washed, lysed, and aliquots of the cell extracts
(50 µg of protein) were immunoprecipitated with a monoclonal
anti-DNA pol
antibody, as described under ``Materials and
Methods.'' The immunocomplex was then separated by SDS-gel
electrophoresis, transferred to nitrocellulose by Western transfer,
probed with a rabbit anti-PADPRP antibody, and detected by
electrochemiluminescence. Lanes 1, 3, 4, 7, and 8 are samples from control cells, at zero time (lane 1), or 24 h after induction of differentiation (lanes 3, 4, 7, and 8); lanes 3 and 4 are duplicate determinations. In lane 7,
control cell extracts were incubated with only protein A-Sepharose,
prior to electrophoresis, and, in lane 8, preimmune serum was
substituted for the anti-DNA pol
antibody. Lanes 1, 5, and 6 represent samples from antisense cells,
either at zero time (lane 1) or at 24-h differentiation (lanes 5 and 6); lanes 5 and 6 are
duplicate determinations.
With control cells at zero time (Fig. 10, lane 1),
PADPRP was not observed to co-immunoprecipite with DNA pol ,
consistent with the low levels of PADPRP in cells prior to
differentiation (Fig. 4). However, after 24 h of induction of
differentiation (lanes 3 and 4) binding of DNA pol
and PADPRP was observed, as shown by the immunostained band for
murine PADPRP at
113 kDa. Co-immunoprecipitation of the two
enzymes did not occur if only protein A-Sepharose was present (lane
7), nor if preimmune serum replaced anti-DNA pol
(lane
8). As anticipated, we did not observe the co-immunoprecipitation
of PADPRP with DNA pol
in antisense cells, either at zero time or
after 24 h of differentiation (lanes 2, 5, and 6). The data in Fig. 8Fig. 9Fig. 10further support our earlier
results using purified enzymes (Simbulan et al., 1993) and
suggest that PADPRP and DNA pol
may associate at some stage
during the round of DNA replication required for differentiation.
Although chemical inhibitors have proven useful in
investigating potential functions of PADPRP (Caplan et al.,
1979; Morioka et al., 1979; Brac and Ebisuzaki, 1985) they are
not without potential complications (Milam and Cleaver, 1984). Thus, we
have applied a more specific approach, that of antisense RNA synthesis,
to determine the effect of inhibition of poly(ADP-ribosyl)ation on the
differentiation of 3T3-L1 preadipocytes. Both PADPRP protein and
activity show a transient, marked increase on exposure of cells to
inducers of differentiation (Fig. 5). The increase in PADPRP
expression was prevented in this study by antisense RNA synthesis.
Under these conditions, both cellular proliferation and differentiation
did not occur ( Fig. 6and Fig. 7). The synthesis of
antisense RNA corresponding to -actin or to ferritin heavy chain
has previously been shown to have no influence on the differentiation
of these cells (Wenz et al., 1992). Alternatively, Fang-Tysr
and Lane(1992) have shown that antisense expression of an RNA
associated with adipocyte differentiation (C/EBP) prevented expression
of a number of adipocyte specific mRNAs and also prevented
differentiation. The observation that the increases in both PADPRP
protein and activity occur at the same time as cellular proliferation
at the onset of differentiation does not directly prove a correlation
between these two events. However, the increased stability of PADPRP
mRNA during DNA replication (Bhatia et al., 1990a), as well as
the high concentrations of poly(ADP-ribosyl)ated intermediates at the
middle and end of S phase (Kidwell and Mage, 1976; Wong et
al., 1983), supports the concept that the increase in PADPRP
expression during the early stages of differentiation may be critical
for the differentiation of preadipocytes because of a role of the
enzyme in a stage in the cell cycle prior or during DNA replication.
Various models, all of which involve chromatin restructuring, have been proposed to explain the necessity for DNA replication prior to differentiation (Villarreal, 1991). Implicit in many of these models is the repositioning or alteration of nucleosomes, which may result in the activation or inhibition of specific genes and may involve various nuclear protein modifications, including poly(ADP-ribosyl)ation. In this regard, poly(ADP-ribosyl)ation may aid in either relaxation (de Murcia et al., 1986) or condensation of chromosomal proteins around the replicating regions of chromatin (Butt et al., 1980).
A role for poly(ADP-ribosyl)ation in the regulation of chromatin structure is supported by various previous observations. In HeLa cells synthesizing PADPRP antisense RNA, we observed a hypersensitivity of chromatin to deoxyribonuclease 1 (Ding et al., 1992). Chromatin regions corresponding to replicating regions of DNA shows transient hypersensitivity to nuclease digestion (Smerdon, 1989), which may be related to localized changes in nuclear organization of the 300-Å fiber of chromatin. Additional experimental systems have indicated that poly(ADP-ribosyl)ation of nucleosomal proteins may influence the structure of chromatin and thereby facilitate DNA replication and other nuclear processes (Jump et al., 1980; Althaus et al., 1985; Chatterjee et al., 1989; Jacobson and Jacobson, 1989). Recently, Mathis and Althaus(1986) showed a significant influence of poly(ADP-ribosyl)ation and chromatin structure on DNA repair: in the presence of chemical inhibitors of PADPRP, DNA adducts were inaccessible for repair and tended to accumulate in nonnucleosomal regions of chromatin.
The initial stages in the rejoining of DNA strand breaks were shown to be inhibited in HeLa cells depleted of PADPRP as a result of PADPRP antisense RNA synthesis (Ding et al., 1992). Thus, the low viability of 3T3-L1 cells induced both to differentiate and to synthesize PADPRP antisense RNA may result from a toxic accumulation of Okazaki fragments generated during the early stages of differentiation. In this regard, Lonn and Lonn(1988) showed that the accumulation of unligated Okazaki fragments is increased significantly in human cells incubated with the PADPRP inhibitor, 3-aminobenzamide.
PADPRP contains two zinc fingers, which are required for the binding of the enzyme to both single-strand and double-strand DNA breaks in a sequence-independent manner (de Murcia et al., 1983; Cherney et al., 1987). Furthermore, the catalytic activity of PADPRP is dependent on DNA strand breaks. The replication forks associated with differentiation-related DNA synthesis contain numerous free ends that could activate the poly(ADP-ribosyl)ation of nucleosomal proteins, which, in turn, might alter the structure of these regions. In this regard, Satoh and Lindahl(1992) have proposed a model, on the basis of in vitro data, in which non-poly(ADP-ribosyl)ated PADPRP binds to ends of DNA during DNA replication or repair. Subsequently, the large negative charge resulting from extensive automodification of bound PADPRP would promote removal of the enzyme from the DNA ends to allow DNA ligation. This proposed mechanism is in agreement with our previous data showing that the addition of NAD (the substrate of PADPRP) to chromatin in vitro increases the accessibility of either endogenous or exogenously added DNA polymerase to DNA primers (Roberts et al., 1974). In further support of the putative association of poly(ADP-ribosyl)ation with DNA synthesis is the observation that the replicating regions of chromatin are selectively retained relative to nonreplicating regions, when either polyoma or SV40 minichromosomes are subjected to anti-poly(ADP-ribose) antibody affinity chromatography (Baksi et al., 1987).
We recently tested the above model with bacterially expressed mutants of PADPRP with deletions in the three major functional domains of the enzyme (Smulson et al., 1994). Deletion mutants with an intact amino-terminal DNA binding domain inhibit DNA repair, whereas a mutant with a deletion in the DNA-binding domain does not inhibit the in vitro assay. However, whether the deletion is in the NAD-binding, active site domain, or the automodification domain, the inhibition of repair exerted by these mutant proteins is not alleviated by NAD.
In
the current work, we have provided data showing the
co-immunoprecipitation of PADPRP and DNA pol in crude extracts
derived from control cells during the initial stages of
differentiation, but this was not observed in the antisense cells (Fig. 10). In this regard, we previously showed that PADPRP
binds to purified calf thymus DNA pole
and stimulates its
activity by about 10-60-fold in a dose-dependent manner. It was
also observed that the stimulatory activity produced by this physical
association of PADPRP and DNA pol
is lost when PADPRP is
automodified. Apparently, in the presence of PADPRP, the saturation
curve for the DNA template primer becomes sigmoidal; at very low
concentrations of DNA, PADPRP inhibits the reaction in competition with
template DNA, while at higher DNA doses the reaction is significantly
stimulated by increasing the V
of the reaction,
which suggests an allosteric effect of PADPRP on the activity of DNA
pol
(Simbulan et al., 1993).
Reddy and Pardee(1980)
provided interesting data earlier and Reddy and Fager(1993), more
recently, which identify a nuclear complex of ``DNA
precursor-synthesizing enzymes juxtaposed with the replication
apparatus comprising DNA polymerase, other enzymes and structural
proteins.'' By immunofluorescent imaging analysis, Li et
al.(1993) further showed that DNA pol and DNA ligase, both
of which exists in a 21 S multienzyme complex for DNA synthesis, can be
localized to distinct ``granular-like foci,'' whereas DNA pol
, not associated with the complex, appears to be diffusely
distributed in the nucleus. We have also previously shown that, in
crude extracts of calf thymus, a portion of PADPRP exists in a 400-kDa
as well as a large 700-kDa complex, containing DNA pol
(Simbulan et al., 1993). We anticipate that the 3T3-L1 antisense cell
lines characterized in our current study may help facilitate a better
understanding of the significance of the association of PADPRP and DNA
pol
and also what other biological roles poly(ADP-ribosyl)ation
may participate in during differentiation.