(Received for publication, September 13, 1994; and in revised form, October 28, 1994)
From the
The utilization of dCTP derived from de novo synthesis
through ribonucleotide reductase in exponentially growing CCRF-CEM
cells was compared with the metabolic fate of dCTP produced by the
salvage pathway. Exogenous dCyd was not effectively incorporated into
replicating DNA; instead, dCTP derived from ribonucleotide reductase
(labeled by [5-H]Cyd) was the main precursor for
that purpose, apparently because of functional compartmentation of the
dCTP pool in these cells. Studies of the metabolic route of
incorporation of exogenous [5-
H]dCyd into DNA of
growing CCRF-CEM cells demonstrated that it was mainly incorporated
through the DNA repair pathway. Incorporation of
[5-
H]dCyd into DNA of synchronized cell
populations was maximal in G
cells, whereas
[
H]dThd incorporation occurred predominantly in S phase cells. When cellular DNA was density labeled by
incubation with BrdUrd, repaired DNA, which was less dense than
replicated DNA, was preferentially labeled by
[5-
H]dCyd. In contrast, replicated DNA was
labeled by both [
H]dThd and
[5-
H]Cyd. The DNA-damaging agents
methylmethanesulfonate, ultraviolet irradiation, and
-irradiation
inhibited [
H]dThd incorporation, whereas they
stimulated the accumulation of [5-
H]dCyd in DNA.
Based on these results, we propose that the dCTP pool is functionally
compartmentalized in growing CCRF-CEM cells. dCTP derived from the
salvage pathway is utilized predominantly for DNA repair, whereas the de novo pathway supplies dCTP for DNA replication.
Replicative DNA synthesis consumes the major portion of cellular deoxynucleoside triphosphates (dNTPs), whereas other processes such as repair of damaged DNA and deoxynucleotidyl intermediates of lipid metabolism probably place lesser demands on the cellular pools. Evidence from metabolic studies indicates that ribonucleotide reductase-mediated de novo synthesis of deoxynucleotides is tightly coupled to replicative DNA synthesis (Nicander and Reichard, 1983; Mathews and Ji, 1992; Reddy and Fager, 1993). Indeed, multiprotein complexes containing enzymes, including ribonucleotide reductase, that participate in both dNTP synthesis and DNA replication have been isolated and characterized (Noguchi et al., 1983; Harvey and Pearson, 1988; Hammond et al., 1989; Wu et al., 1994). This concept that the de novo pathway for dNTP synthesis is linked with DNA replication is further supported by indications that DNA is replicated at structurally distinct sites in the nucleus (Nakamura, et al., 1986; Mills et al., 1989; Cox and Laskey, 1991; Horzak et al., 1993; Coverley and Laskey, 1994). Kinetic studies of ribonucleoside uptake into the DNA of whole cells indicate that dNTP pools derived from de novo pathways are likely to be rather small and turn over rapidly (Nicander and Reichard, 1983). If the dNTP pool utilized for DNA replication derived from de novo synthesis is localized to the region surrounding a replication center, it is reasonable to ask whether dNTPs derived from different sources, such as the salvage pathways, could also be used for DNA replication.
It is clear from
investigations in many cell types that [5-H]dCyd
is a poor substrate for labeling replicating DNA (Plagemann et
al., 1978; Cohen et al., 1983; Karle et al.,
1983; Nicander and Reichard, 1983; Balzarini et al., 1984;
Taljanidisz et al., 1986; Leeds and Mathews, 1987;
Sasvari-Szekely et al., 1989; Xu and Plunkett, 1992; 1993).
This stands in contrast to [5-
H]Cyd which
specifically labels dCMP in replicating DNA and to
[
H]dThd, which has been taken as the standard for
measuring this process (Reichard, 1988). Incorporation of
[5-
H]dCyd and [
H]dThd
become comparable only in cells that are not active in DNA replication
(Holmberg et al., 1988) or when de novo synthesis of
dCDP is inhibited (Nicander and Reichard, 1983).
Relatively little is known of the metabolic fate of dNTPs derived from the salvage of deoxynucleosides. Evidence exists that salvaged dCyd is utilized in the formation of thymidylate used in replicative DNA synthesis in cell lines (Jackson, 1978; Xu and Plunkett 1992). Insight into the utilization of the dCTP pool derived from the salvage pathway for purposes other than replicative DNA synthesis was provided by Spyrou and Reichard(1987, 1989) who demonstrated that dCTP precursors for deoxyliponucleotide synthesis were derived from salvaged dCyd. Although some studies have indicated that labeled dCyd may be used to measure DNA repair (Snyder, 1984; Elliott and Downes, 1986; McKenna and McKelvey, 1986; Holmberg et al., 1988), it was only recently suggested that dCTP derived from the salvage pathway might be used as a specific substrate for DNA repair (Spasokukotskaja et al., 1992).
Following this lead, we sought to identify the source of dCTP
utilized for DNA repair. To this end, we have compared the formation of
dCTP from Cyd and dCyd and have investigated its utilization in human T
lymphoblast CCRF-CEM cells. Our results support the hypothesis that
there are two functionally separate dCTP pools in these cells;
exogenous [5-H]dCyd labels one dCTP pool, and one
is preferentially used for DNA repair.
Figure 1:
Incorporation
of [5-H]dCyd and [
H]dThd in
exponentially growing CCRF-CEM cells. CCRF-CEM cells were labeled with
0.2 µCi/mL [5-
H]dCyd (
) or
[
H]dThd (
) to quantitate the rate of
incorporation of each into DNA. Aliquots of cells were withdrawn at
indicated times and were subjected to HClO
extraction. The
specific activity of [5-
H]dCTP and
[
H]dTTP pools of HClO
-soluble
extracts (A) and the radioactivity incorporated into
HClO
-insoluble material (B) were determined as
described in ``Experimental Procedures.'' The data represent
the mean ± S.D. of three
determinations.
Figure 2:
Effect of exogenous dCyd on
[5-H]Cyd incorporation into DNA. Exponentially
growing CCRF-CEM cells were preincubated with indicated concentrations
of dCyd for 2 h before labeling with [5-
H]Cyd
(0.2 µCi/ml) for 30 min. Cells were then subjected to HClO
extraction. The specific activity of
[5-
H]dCTP pool (
) and the radioactivity
incorporated into DNA (
) were quantitated as described under
``Experimental Procedures.''
Figure 3:
Incorporation of
[5-H]dCyd and [
H]dThd in
synchronized CCRF-CEM cells. Cells were synchronized by double
aphidicolin block as described under ``Experimental
Procdures.'' After cells were released from the second aphidicolin
treatment, aliquots of cells were withdrawn at indicated times and were
labeled with either [5-
H]dCyd (
) or
[
H]dThd (
) for 10 min. The radioactivity
incorporated into HClO
-insoluble material was quantitated
by liquid scintilation counting. Cell number was determined separately
at the time of assay. The data were expressed as dpm/10
cells. Flow cytometry analysis demonstrated that about 70% of the
cells entered mid-S phase between 4 and 6
h.
As shown in Fig. 4A, when exponentially growing CCRF-CEM cells were
labeled with [H]dThd for 39 h without BrdUrd and
an additional 13 h in the presence of 1 µM BrdUrd, a
bimodal distribution of [
H]dThd-labeled DNA was
observed. Radioactivity was associated with a UV-absorbing peak that
banded at a high density (fractions 32-45) and was also
incorporated into a peak of lesser density (fractions 5-15).
These peaks represent newly replicated and unreplicated DNA after
BrdUrd addition, respectively. The higher density peak shows less UV
absorbance due to the effects of the BrdUrd treatment on DNA
replication. When [
H]dThd labeling and BrdUrd
incorporation were carried out simultaneously for half of a cell cycle,
only the high density peak was labeled (Fig. 4B). On
the other hand, when cells were incubated with
[5-
H]dCyd, radioactivity was incorporated into a
peak of intermediate density (Fig. 4C, fractions
16-25) in addition to incorporation into the high density peak.
As was the case for dThd, when cells were incubated with
[5-
H]Cyd, only the high density peak was labeled (Fig. 4D), indicating that both
[
H]dThd and [5-
H]Cyd served
mainly as precursors for DNA replication.
Figure 4:
Separation of DNA fragments labeled by
[5-H]dCyd and [
H]dThd by
alkaline CsCl gradient centrifugation. Each experiment was carried out
with 2
10
exponentially growing CCRF-CEM cells
suspended in 60 ml of media. A, cells were labeled with 1
µM [
H]dThd (0.2 µCi/ml) for 39
h. BrdUrd (1 µM) and fresh [
H]dThd
were added for the last 13 h of the incubation. B, cells were
incubated with 1 µM [
H]dThd (0.2
µCi/ml) for 13 h in the presence of 1 µM BrdUrd. C, cells were labeled with tracer amounts of
[5-
H]dCyd (0.2 µCi/ml) for 13 h in the
presence of 1 µM BrdUrd. D, cells were labeled
with tracer amounts of [5-
H]Cyd (0.2 µCi/ml)
for 13 h in the presence of 1 µM BrdUrd. Isolation of DNA
and CsCl centrifugation were performed as described under
``Experimental Procdures.'' Fractions of 0.2 ml were
collected and diluted for the measurement of radioactivity (
) and
UV absorption at 260 nm (
).
We hypothesize that the
DNA labeled by dCyd that banded at the intermediate density was newly
repaired DNA. Again, this is a relatively low level of incorporation
and probably represents a background level of repair and possibly that
stimulated by the actions of BrdUrd (Ashman et al., 1981;
Shewach et al. 1992). Incorporation of dThd or Cyd into this
peak was too minor to be detected. The overall incorporation of dCyd
was still low compared with either Cyd or dThd in the high density
peak. When both the intermediate density peak and the high density peak
fractions were combined, the DNA specific activity (dpm/UV absorbance
unit) from [H]dThd and
[5-
H]Cyd labeling was 23- and 13-fold higher,
respectively, than that labeled by [5-
H]dCyd.
These results suggest the existence of functionally compartmentalized
dCTP pools for DNA replication and DNA repair in CCRF-CEM cells. The
portion of [5-
H]dCyd that was incorporated into
the high density peak may represent the upper limit of mixing of
salvage pathway products with the dCTP pool used in replication. It is
also possible that some DNA repair was taking place in the newly
replicated DNA, probably because of the BrdUrd, which itself evokes DNA
repair processes (Hopkins and Goodman, 1980; Shewach et al.,
1992).
Figure 5:
Effect of BrdUrd on
[5-H]dCyd incorporation into DNA. CCRF-CEM cells
were incubated with BrdUrd at the indicated concentrations for 16 h.
Cells were then incubated without (
) or with (
) 1 µM non-radioactive dCyd for additional 2 h before they were labeled
with [5-
H]dCyd for 60 min. The radioactivity
incorporated into DNA was quantitated as described under
``Experimental Procedures.''
Figure 6:
Effects of MMS and UV on
[5-H]dCyd and [
H]dThd
incorporation into DNA fragments analyzed by CsCl gradient
centrifugation. Exponentially growing CCRF-CEM cells were treated
without (
), or with 6 J/m
UV (
), or with 0.3
mM MMS (
). Cells were then labeled with
[
H]dThd (A) or with
[5-
H]dCyd (B) in the presence of 1
µM BrdUrd for 13 h as described in Fig. 4. For
MMS-treated samples, labeling of [
H]dThd or
[5-
H]dCyd was carried out in the presence of MMS.
Isolation of DNA and CsCl centrifugation were performed as described
under ``Experimental
Procedures.''
The functional compartmentation of dCTP derived from the de novo and salvage pathways has important implications for
cellular metabolism. Comparison of the rate of
[5-H]Cyd incorporation into DNA with that of
[5-
H]dCyd indicates that Cyd is efficiently used
for DNA replication in exponentially growing cells, whereas dCyd is a
relatively poor precursor for this purpose. On the other hand, it is
clear that cells utilize salvaged dCyd for the synthesis of dTTP
through the dCMP deaminase pathway (Jackson, 1978; Xu and Plunkett,
1992) and for the synthesis of deoxyliponucleotides (Spyrou and
Reichard, 1987, 1989). The origin of deoxynucleotides used for
mitochondrial DNA synthesis may be derived from a distinct
ribonucleotide reductase associated with this organelle (Young et
al., 1994). The present study provides evidence that
[5-
H] dCyd was used selectively as a precursor of
DNA repair in exponentially growing CCRF-CEM cells. Induction of DNA
repair in growing cells with DNA-damaging agents enhanced the
incorporation of [5-
H]dCyd into DNA, whereas such
treatments disrupted DNA replication (Fig. 6). Furthermore,
density labeling experiments demonstrated that DNA undergoing repair
was specifically labeled with [5-
H]dCyd ( Fig. 4and Fig. 6).
Our approach involved investigating
incorporation of dCyd and of Cyd into the dCMP of DNA and determining
whether it occurred through DNA replication or DNA repair. The two
pathways were distinguished by differential density labeling;
BrdUrd-labeled replicating DNA banded in CsCl gradients at a high
density, whereas the density of DNA in which BrdUrd was incorporated
during repair was intermediate to replicating DNA and that of parental
DNA without BrdUrd (Fig. 4). Greater than 60% of incorporated
dCyd was found in the intermediate density peak, and so it appeared to
be incorporated through DNA repair. Because some of dCyd identified in
the high density peak was likely due to repair induced by BrdUrd, the
actual proportion of dCyd used for DNA repair was probably even
greater. In contrast, the majority of Cyd was incorporated into the
high density peak of replicating DNA. Due to the much greater amount of
replicating DNA relative to repaired DNA in these growing cells,
estimation of Cyd incorporation into the intermediate density peak was
uncertain, but appeared to be less than 5%. In control cells, the rate
of [5-H]Cyd incorporation (59 pmol/10
cells/min, Fig. 2) was significantly greater than the rate
of dThd incorporation (7.8 pmol/10
cells/min, Fig. 1). Assuming that dThd incorporation represents the true
rate of DNA replication (Nicander and Reichard, 1983; Reichard, 1988),
the apparent greater rate of [5-
H]Cyd
incorporation may be attributed to functional compartmentation of dCTP.
These calculations were based on the average cellular pool-specific
activities; because ribonucleotide reductase activity varies with the
cell cycle (Eriksson et al., 1984) whereas the activity of
dCyd kinase is relatively stable (Liliemark and Plunkett, 1986; Arner et al., 1988), it is possible that the specific activity of
[
H]dCTP depends on cell cycle stage and also on
cell type. For example, Chinese hamster ovary cells exhibit a 10-fold
difference in [
H]dCTP-specific activity between
G
and S phase populations (Leeds and Mathews, 1987); in
contrast, the [
H]dCTP-specific activity in
exponentially growing CEM cells was only 60% greater than in S phase
cells (Xu and Plunkett, 1993). Additionally, a compartmentalized dCTP
pool used for DNA replication could have had a much higher specific
activity. If so, the calculated rate of [5-
H]Cyd
incorporation into DNA would be decreased to a value comparable to that
of [
H]dThd incorporation. Although the average
cellular [
H]dCTP-specific activity decreased in
cells that were preincubated with dCyd (Fig. 2), the rate of
[5-
H]Cyd incorporation into DNA was apparently
unchanged. This suggests that the de novo metabolic route via
ribonucleotide reductase is largely restricted from mixing with the
dCTP pool generated by the salvage pathway. Furthermore, using
synchronized cells, we found that the peak of
[5-
H]dCyd incorporation did not coincide with S
phase DNA replication (Fig. 3), another indication that the dCTP
pool labeled by [5-
H]dCyd was excluded from DNA
replication.
A compelling body of evidence supports the role of ribonucleotide reductase as the key enzyme in the functional compartmentation of dCTP (Moyer and Henderson, 1985; Nguyen and Sadee, 1986; Spyrou and Reichard, 1989; Mathews and Ji, 1992; Reddy and Fager, 1993). It has been calculated that CDP reduction is the most rapid among the four ribonucleoside diphosphate substrates (Jackson, 1992). Consistent with its central role in supplying dNTPs for DNA replication, the activity of this enzyme is known to be elevated during S phase. Because dCTP pools also increase in S phase cells (Liliemark and Plunkett, 1986; Arner et al., 1988) it is reasonable to conclude that ribonucleotide reductase is capable of producing an excess of dCTP beyond that consumed by DNA replication. In contrast, the activity of deoxycytidine kinase, the rate-limiting step in the salvage pathway, is fairly constant throughout the cell cycle (Liliemark and Plunkett, 1986; Arner et al., 1988). This is consistent with the notion that dCTP generated by the salvage pathway is not specifically required in S phase. Thus it is unlikely that dCTP derived from the salvage pathway would compete effectively with dCTP from the de novo pathway for incorporation into replicating DNA. If ribonucleotide reductase were localized near the DNA replication apparatus or were functionally part of that process, the preferential utilization of dCTP generated by the de novo pathway could be enhanced.
Due to technical limitations, the cellular location of ribonucleotide reductase has remained uncertain. Although some cellular fractionation studies (Leeds, et al., 1985) and investigations using immunocytochemistry (Engstrom and Rozell, 1988) have suggested that ribonucleotide reductase is a cytosolic enzyme, there is evidence that it may be associated with the nuclear membrane (Sikorska et al., 1990). Furthermore, several laboratories have characterized multienzyme complexes that contain ribonucleotide reductase and other enzymes involved in dNTP synthesis and DNA replication (Noguchi et al. 1983; Harvey and Pearson, 1988; Hammond et al., 1989, Reddy and Fager, 1993). A possible resolution to these apparently contradictory findings could be that ribonucleotide reductase exists both in a multiprotein DNA replication complex in the nucleus and as a free enzyme in the cytosol. It is possible that immunocytochemical methodologies may detect the soluble enzyme in the cytosol, but perhaps not a complexed form in the nucleus due to blockage of the epitope by other protein components.
Evidence from recent studies demonstrating that DNA replication forks are arranged in defined spatial patterns within the nucleus provides a structural context for DNA replication in which functionally compartmentalized dNTP pools may be a central component. It is now known that replication forks are tightly clustered in foci within the nucleus (Nakamura, et al., 1986; Mills et al., 1989; Cox and Laskey, 1991; Hozak et al., 1993; Coverley and Laskey, 1994). This arrangement is likely to facilitate rapid consumption of large amounts of dNTPs in each replication focus. It is doubtful that this rate of DNA synthesis can be supported by the relatively low concentration of dNTPs estimated assuming that the dNTPs are uniformly distributed in total cell water.
In contrast to the focal nature of DNA replication, it is likely that DNA repair in response to alkylating agents or radiation is dispersed throughout the genome, possibly with foci at transcriptionally active sites (Jackson et al., 1994). In this situation, DNA synthesis associated with nucleotide excision repair probably utilizes dCTP from a more general pool. We envision that this pool is composed of a mixture of dCTP from the salvage pathway and dCTP which has escaped from its source at ribonucleotide reductase in the replicating foci. In the process of diffusion, its concentration has been diluted relative to that within the replicating focus. When considering the utilization of exogenous dCyd, we propose that the salvage pathway contributes to this generalized pool of dCTP, which our experiments have demonstrated comprises the bulk of the nucleotide used for DNA repair, but makes only a small contribution to replicative DNA synthesis.
The relatively minor utilization of
exogenous dCyd in replicating DNA should not be viewed as contradictory
to the fact that a number of dCyd analogs, such as arabinosylcytosine,
2`,2`-difluoro-2`-deoxycytidine, 5-aza-2`-deoxycytidine, and
2`,3`-dideoxycytidine require the same salvage pathway for activation
prior to incorporation into DNA (Major et al., 1981;
Momparler, 1985; Huang et al., 1991, Starnes and Cheng, 1987).
The extent to which these analogs are incorporated as a result of
repair synthesis relative to DNA replication is unknown, although it
has been assumed that the latter pathway is utilized in the absence of
DNA damaging stimuli. Although the rate of exogenous dCyd incorporation
into replicating DNA (0.36 pmol/10 cells/min) is about 5%
of that of dThd incorporation (Fig. 1), the rate of
2`,2`-difluoro-2`-deoxycytidine incorporation was even less than that
of dCyd (Huang et al., 1991). On the other hand, in comparison
with dCyd, there are factors that favor the incorporation of analogs
via the replication pathway. For example, cytidine nucleotide analogs
accumulate to greater cellular concentrations than does dCTP (Plunkett
and Gandhi, 1993), and the presence of the nucleotides of these drugs
in the cell is prolonged because they are less likely to be eliminated
by dCMP deaminase (Momparler, 1985; Heinemann et al., 1992).
Thus, the incorporation of cytidine analogs may be taken as an
indication of the upper limit of the mixing that occurs between the
general dCTP pool generated by the salvage pathway with the dCTP pool
produced by the de novo pathway for DNA replication.
In summary, it appears most of the dCTP generated by ribonucleotide reductase goes to a high throughput, low volume pool; some of this dCTP appears to become available to a more general pool in the nucleus. The salvage pathway also contributes dCTP and cytidine nucleotide analogs to this general pool. One of the functions of this pool in CCRF-CEM cells is to supply dCTP for repair of DNA. It is possible that this functional compartmentation could be used to advantage in chemotherapy by combining cytidine nucleotide analogs with agents and modalities that evoke a DNA repair response in tumor cells. Recent reports suggest the utility of this approach (Gregoire et al., 1994; Shewach et al., 1994).