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Biochemistry and Biophysics |
Department of Radiation Oncology [S. J. D., Z-C. Z., W. J. C., G. I.], Kimmel Cancer Center, and Biostatistics Section of Clinical Pharmacology, Department of Medicine [R. C., T. H.], Jefferson Medical College, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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INTRODUCTION |
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A separate line of investigation suggests the involvement of the rad50/mre11/p95 complex in NHEJ, and biochemical studies ascribe an exonuclease activity to the mre11/rad50 complex (reviewed in Refs. 11 and 32 )). It is interesting that the mre11/rad50 complex, together with ligase I, mediates rejoining of nonhomologous ends in vitro (33 , 34) . The contribution of mre11/rad50 complex to DNA DSB rejoining in vivo is not clear, but cells deficient in p95, a component of the mre11/rad50 complex, show normal kinetics of DNA DSB rejoining (35) . Thus, a number of factors potentially involved in NHEJ have been recently identified in mammalian cells, but their functions and interactions during rejoining of DNA DSB remain to be elucidated.
Results of experiments evaluating DNA DSB rejoining in
DNA-PKcs-deficient cells frequently indicate a cessation of rejoining
after an initial short period of repair (36, 37, 38, 39, 40, 41)
.
According to such data, a significant fraction of DNA DSBs (
50%,
depending on cell line, repair conditions, dose of radiation, and other
factors) remain unrejoined in DNA-PKcs-deficient cells. Such a high
proportion of unrepaired DNA DSBs is hard to reconcile with the general
properties of DNA-PK-deficient cells (42
, 43)
. This is
because DNA DSBs are also induced during DNA replication, after
exposure to background levels of radiation, as well as by the reactive
oxygen intermediates generated as by-products of cellular metabolism.
If nearly half of these DNA DSBs remained unrejoined, cell growth and
viability would be affected. These arguments are further reinforced by
the fact that DNA-PKcs-deficient mice (scid or DNA-PK
knockout mice) develop normally, and several of their phenotypic
alterations are directly related only to immune defects resulting from
defective V(D)J recombination rather than genomic instability or
unrejoined breaks.
Similar difficulties become also apparent when the levels of unrepaired
DNA DSBs are compared with cell radiosensitivity to killing in
DNA-PKcs-deficient cells. Comparison of the number of unrepaired DNA
DSBs, calculated assuming 50% probability of rejoining, with the slope
of the survival curve suggests that surviving cells tolerate a large
number of unrejoined DNA DSBs (10). This result is in contrast to
observations in yeast, where one unrejoined DNA DSB/cell, on the
average, is a lethal event (1)
, and raises the question as
to why higher eukaryotes should tolerate high levels of such a lethal
and mutagenic lesion.
The above complications could be resolved if DNA-PKcs-deficient cells used DNA-PK-independent mechanisms to rejoin the large majority of DNA DSBs, but the experimental design and technical difficulties generated by DNA degradation during incubation for repair prevented its quantitative evaluation. This hypothesis is directly supported by results obtained with scid cells showing complete, albeit slow, rejoining of radiation-induced DNA DSBs when long incubations for repair are allowed and methods of pulsed-field gel electrophoresis are used that are capable of separating degraded from nondegraded DNA (44) . Because the leaky phenotype of the scid mutation (45) raises the possibility that the complete rejoining observed is attributable to residual DNA-PK activity, we wished to study systematically rejoining of DNA DSBs in human cells devoid of detectable DNA-PK activity and DNA-PKcs.
Here, we report on the overall capacity for and the kinetics of DNA DSB rejoining in a human tumor cell line known to be deficient in DNA-PK activity and its normal counterpart in the presence or absence of a DNA-PK inhibitor, wortmannin. The results indicated that, like mouse cells (44) , human cells are capable of rejoining the majority of DNA DSBs induced in their genome, regardless of DNA-PK status. The kinetics of rejoining are biphasic under all conditions examined, with a fast and a slow component. The slow component removes the majority of DNA DSBs in cells lacking DNA-PK activity, as well as in DNA-PK-proficient cells exposed to wortmannin. In the presence of DNA-PK, fast rejoining of DNA DSBs is activated and increases the proportion of DNA DSB rejoining with fast kinetics. The implications of these observations for our understanding of the mechanism of DNA DSB rejoining in mammalian cells are discussed.
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MATERIALS AND METHODS |
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106/dish and were
irradiated to measure cell survival or the kinetics of DNA DSB
rejoining.
Cells were irradiated using a Pantak X-ray machine operated at 320 kV,
10 mA with a 2-mm Al filter (effective photon energy, 90 kV), at a
dose rate of 2.7 Gy/min. Dosimetry was performed with a Victoreen
dosimeter that was used to calibrate an in-field ionization monitor.
Cell radiosensitivity to killing was determined by the clonogenic assay. Cells were trypsinized at 37°C immediately after irradiation at room temperature and seeded into 100-mm tissue culture dishes at various densities aiming at 30–150 colonies/dish. To evaluate the effect of wortmannin, cells were pretreated at 20 µM for 45 min and plated 6 h after irradiation. After an incubation period of up to 3 weeks, cells were stained with crystal violet, and colonies of >50 cells were counted.
Cells for DNA DSB repair experiments were labeled with 0.1 µCi/ml [14C]thymidine plus 2.5 µM cold thymidine for the entire period of growth. When indicated by the experimental protocol, cells were treated with wortmannin (Sigma) for 1 h before irradiation. Cells were cooled to 4°C prior to irradiation and were irradiated on ice. After irradiation, the medium was replaced with fresh growth medium prewarmed at 42°C (to rapidly restore 37°C to the cultures), and cells were returned to the incubator at 37°C to allow for repair. Cells were prepared for DNA DSB analysis at various times thereafter.
It proved essential to allow cells to repair DNA DSBs under conditions
optimal for growth. Cells maintained in suspension during repair or
cells allowed to repair after embedding in agarose displayed suboptimal
kinetics of rejoining, where the slow component was difficult to
discern. After completion of the repair time interval, cells were
trypsinized (30 min on ice for the first 4 h, and 5 min at 37°C
at later times), centrifuged, and resuspended in serum-free medium at a
concentration of 6 x 106
cells/ml. This cell suspension was mixed with an equal volume of 1%
agarose (InCert agarose; FMC), pipetted into 3-mm diameter glass tubes,
and placed on ice to allow for solidification. The solidified
cell-agarose suspension was extruded from the glass tubes and cut into
3 x 5-mm cylindrical blocks containing 1 x 105 cells/block (48)
. Blocks
were then placed in lysis buffer containing 10 mM Tris (pH
8.0), 50 mM NaCl, 0.5 M EDTA, 2%
N-lauryl sarcosyl, 0.1 mg/ml proteinase E and O, and
incubated first at 4°C for 45 min and then at 50°C for 16–18 h.
After lysis, agarose blocks were washed for 1 h at 37°C in a
buffer containing 10 mM Tris (pH 8.0) and 0.1
M EDTA and were then treated for 1 h at
37°C in the same buffer, at pH 7.5, with 0.1 mg/ml RNase A. Cells
from identically treated nonirradiated cultures were also processed at
predefined times to determine the signal generated by nonirradiated
cells (background). A similar protocol was also used to determine
induction of DNA DSBs except that in this case, cells were embedded in
agarose prior to irradiation on ice, and were lysed immediately
thereafter.
AFIGE (48) was carried out in 0.5% Seakem agarose (FMC), cast in the presence of 0.5 µg/ml ethidium bromide, in 0.5x TBE [45 mM Tris (pH 8.2), 45 mM boric acid, and 1 mM EDTA] at 10°C for 40 h. During this time, cycles of 1.25 V/cm for 900 s in the direction of DNA migration alternated with cycles of 5.0 V/cm for 75 s in the reverse direction. The agarose gels were quantified to estimate DNA damage by means of a PhosphorImager (Molecular Dynamics). Gels were dried and exposed to radiation-sensitive screens for 48–96 h. DNA DSBs were quantitated by calculating the FAR (from the well into the lane) in irradiated and nonirradiated samples. The FAR measured in nonirradiated cells (background) was subtracted from the results shown with irradiated cells. Gel images were obtained either by photographing ethidium bromide-stained gels under UV light or from the PhosphorImager.
Repair kinetics were fitted assuming two exponential components of rejoining according to the equation FAR = Ae-bt + Ce-dt (49) . The first term in the equation was fitted to the slow component of rejoining, and the second term was fitted to the fast component of rejoining. Fitting was achieved using the nonlinear regression analysis routines of a commercially available software package (SAS). Parameters A and C describe the amplitudes, and parameters b and d are the rate constants of the slow and the fast components of rejoining, respectively. From these parameters, the half-times for the rejoining of the slow and the fast components were calculated as t50,fast = ln2/b, and t50,slow = ln2/d, respectively. The fraction of DSBs rejoined by fast kinetics was calculated as Ffast = A/A + C and Fslow = C/A + C.
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RESULTS |
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indicate a steady increase in FAR, a measure of DNA DSBs present, with
increasing dose of radiation. The increase in FAR can be approximated
by a straight line up to 40 Gy and is similar in both cell lines.
Because induction of DNA DSBs as a function of radiation dose is
considered linear (1)
, the nearly linear relationship
between FAR and radiation dose in the range of interest also implies a
nearly linear relationship between FAR and DNA DSBs present. This
allows evaluation of DNA DSBs rejoining directly from FAR
versus time plots, obviating corrections otherwise required
when the relationship between FAR and dose deviates significantly from
linearity (50)
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shows the results obtained in experiments where rejoining was followed
for up to 6 h after irradiation. Rejoining of DNA DSBs in M059-K
cells is practically complete at 6 h and follows biphasic
kinetics. The solid line in Fig. 2
was obtained by fitting the results
to the sum of two exponential functions as outlined in "Material and
Methods" and discussed in greater detail later in this section. From
this fitting, half-times of 22 min (95% CI, 17–31) and 12 h
(95% CI, 11–15) were calculated for the fast and the slow components
of rejoining, respectively. Approximately 83% of the DNA DSBs are
rejoined with fast kinetics with only 17% rejoining with slow
kinetics. Thus, a fast component dominates DNA DSBs rejoining in M059-K
cells, but a slow component is also clearly identifiable, handling
approximately one of five DNA DSBs.
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The results in Fig. 2
show a significant number of unrepaired DNA DSBs
in M059-J cells after 6 h of repair but do not suggest a cessation
or even a slow-down in rejoining with progressing repair time. We
conducted, therefore, a second set of experiments in which DNA DSB
rejoining was followed for up to 30 h after irradiation. Fig. 3
shows the results of this family of experiments. As expected from the
results in Fig. 2
, rejoining of DNA DSBs is complete within 6 h
after irradiation in M059-K cells. At this time, FAR reaches levels
approaching detection limits and remains unchanged for up to 30 h
after irradiation. In M059-J cells, on the other hand, DNA DSB
rejoining proceeds steadily in the time interval between 6 and 30 h following slow kinetics. At 30 h, FAR reaches values only
slightly higher than those measured in M059-K cells, suggesting nearly
complete rejoining of DNA DSBs. The solid line drawn through the data
points of M059-K cells in Fig. 3
corresponds to half-times of 22 min
(95% CI, 17–31) and 12 h (95% CI, 11–15) for the fast and the
slow component of rejoining, respectively, and a proportion of 83% of
DNA DSB rejoining with fast kinetics. These repair half-times are
identical to those estimated in Fig. 2
. The solid line drawn through
the data points of M059-J cells corresponds to the same half-times for
the fast and the slow components of DNA DSBs rejoining and a proportion
of 28% of DNA DSBs rejoining with fast kinetics. Thus, M059-K and
M059-J cells have a similar overall capacity for DNA DSB rejoining, but
M059-K cells complete rejoining faster. The lack of detectable DNA-PK
activity in M059-J cells (36)
suggests that DNA DSB
rejoining must be predominantly through a DNA-PK-independent process.
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First, we studied the effect of wortmannin on M059-J cells. An
additional goal of these experiments was to evaluate the contribution
of DNA-PK to wortmannin-induced inhibition of DNA DSB rejoining. This
is important because, as mentioned above, wortmannin inhibits the
entire family of PI 3-Ks and probably also other cellular kinases. We
reasoned that if wortmannin inhibited DNA DSB rejoining mainly by
inhibiting DNA-PK activity, it should have no effect on M059-J cells
because they lack this activity. The results in Fig. 4
indicate that wortmannin only has a relatively small effect on DNA DSB
rejoining in M059-J cells (compare with the broken line showing results
of untreated cells), compatible with the notion that DNA-PK is the
principal target of the drug for DNA DSB rejoining. Fitting of the
results indicates that wortmannin treatment does not affect the
half-times of DNA DSB rejoining (see above) but reduces the proportion
of DNA DSBs rejoined with fast kinetics from 28 to 7%. The
ramifications of these observations are discussed in the following
section.
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, diamonds; compare with the dotted line showing results of untreated
cells). The qualitative characteristics of the inhibition are similar
to those observed in M059-J cells, but significantly more inhibition is
observed in M059-K cells. As a result, the kinetics of DNA DSB
rejoining after treatment with wortmannin are practically identical in
the two cell lines. Fitting of the results can be achieved with the
same half-times as for M059-J cells, and it is estimated that only 11%
of the DNA DSBs are rejoined with fast kinetics. Thus, chemical
inactivation of DNA-PK by wortmannin causes changes in DNA DSB
rejoining that are similar to those observed after genetic inactivation
of the protein, i.e., a drastic reduction in the proportion
of DNA DSBs rejoining with fast kinetics with no effect on the
half-times of rejoining. We conclude, therefore, that long-term
adaptation and possibly selection can be ruled out as possible causes
for the DNA DSB rejoining characteristics of M059-J cells.
A striking feature of the results shown above, deduced even by a simple
visual inspection, is that the fast and the slow rejoining components
are operating with similar half-times in M059-K and M059-J cells,
irrespectively of whether cells were treated with wortmannin. Because
this feature has important implications for the mechanistic
understanding of the role of DNA-PK in DNA DSB rejoining, we wished to
rigorously assess these trends through statistical analysis of the
results obtained. For this purpose, all data obtained with untreated
M059-K and M059-J cells were pooled and used for curve fitting and
parameter calculation. For this purpose the dual exponential equation
described in "Materials and Methods" was used, but the assumption
was made that the half-times for the individual components of rejoining
were the same in all sets of data from the two cell lines. This
approach gave satisfactory fitting for all data and is reflected in the
lines drawn in Figs. 2
3
4
.
To statistically test the validity of the assumption of common
half-times, we repeated the fitting, this time allowing for different
half-times for each cell line. The parameters from this fitting were
compared with those obtained under the assumption of similar time
constants, and the statistical significance of the calculated
differences was assessed. There was no statistically significant
difference in the values of the half-times calculated for the fast
component for each cell line by the two methods. Statistically
significant differences between the two cell lines were found, on the
other hand, for the slow component of rejoining when the two sets of
data were fitted independently. However, the difference lost
significance when the assumption was made that a variation by <2 h in
the half-time of the slow component is not biologically relevant,
i.e., it does not describe a distinct process. It is not
difficult to justify this assumption, given the 30-fold difference
in the half-times between the fast and the slow component of DNA DSB
rejoining, as well as the calculated CIs for the slow component of
rejoining, 12 h (95% CI, 11–15 h).
Thus, rigorous statistical analysis justifies fitting the results of
M059-J and M059-K cells using the same values for the half-times of the
two components of DNA DSB rejoining. Similar conclusions can also be
drawn when the results obtained after treatment with wortmannin are
considered. In Fig. 5
, a summary of the rejoining half-times and the proportion of DNA DSBs
rejoined by either the fast or the slow component is given. These
results confirm that genetic or wortmannin-induced deficiency in DNA-PK
severely decreases the fraction of DNA DSBs rejoined with fast kinetics
but leaves the half-times of both components of rejoining unchanged.
Whereas the fast, DNA-PK-dependent component of DSB rejoining can be
attributed to NHEJ, the slow component could reflect homologous
recombination or a pathway of NHEJ. In the following section, we
discuss evidence suggesting that the slow component of DNA DSB
rejoining reflects a mode of NHEJ.
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. M059-J cells are significantly more radiosensitive than M059-K cells,
and the survival values obtained are in excellent agreement with
previous reports with these cells (38
, 46)
. Treatment with
wortmannin causes a large increase in the radiosensitivity to killing
of M059-K cells (54
, 55)
but has practically no effect on
M059-J cells. Similar to results on DNA DSB rejoining, the survival
curves of both cell lines are practically indistinguishable after
treatment with wortmannin. Thus, treatment with wortmannin is
functionally equivalent to the genetic inactivation of DNA-PK,
suggesting that DNA-PK is the only target of the drug with regard to
cell killing.
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DISCUSSION |
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50%) of the radiation-induced
DNA DSBs. As pointed out in the "Introduction," it is difficult to
explain survival data and the nearly normal growth characteristics of
cells deficient in DNA-PK when assuming high levels of unrejoined DNA
DSBs, unless the difficult-to-justify assumption is made that
DNA-PK-deficient cells can tolerate much higher levels of unrejoined
DNA DSBs than wild-type cells. The results presented here avoid this
complication by suggesting that DNA-PK-deficient cells are capable of
rejoining radiation-induced DNA DSBs nearly completely, albeit with
slow kinetics. It is particularly interesting that DNA-PK deficiency does not add new components to the kinetics of DNA DSB rejoining but only increases the contribution of the preexisting, slow component. These observations give important clues on the function of DNA-PK in DNA DSB rejoining and are discussed next.
DNA-PK-independent rejoining of DNA DSBs
A crucial conclusion from the observation that M059-J cells ultimately
rejoin DNA DSBs is that human cells, and probably cells of higher
eukaryotes in general, are equipped with a DNA DSB rejoining apparatus
that remains active in the absence of DNA-PK and which can remove
nearly all DNA DSBs from the genome. Although the molecular
characteristics of such a mechanism are not presently known, the
results suggest that it operates with slow kinetics, coinciding with
the slow component frequently observed in the kinetics of rejoining of
DNA DSBs in mammalian cells. The slow rejoining of DNA DSBs in
mammalian cells has been attributed by some investigators to homologous
recombination (56)
, in line with the slower kinetics
observed in organisms using such processes for DNA DSB rejoining
(1
, 10)
.
Despite the obvious attractiveness, experimental support for this hypothesis is presently lacking, and available evidence suggests that slow rejoining of DNA DSBs does not require homologous recombination. Thus, radiosensitive mutants shown to be defective in genes involved in homologous recombination (57, 58, 59) show no obvious defects in DNA DSB rejoining (60 , 61) .4 Furthermore, homologous recombination-deficient mutants generated from DT40 cells by gene disruption (Refs. 62 and 63 ; RAD54-/- and a conditional RAD51-/- cell line) show kinetics of DNA DSB rejoining identical to those of wild-type cells (results not shown). More importantly, a RAD54-/-/KU70-/- double mutant shows kinetics of DNA DSB rejoining similar to those of KU70-/- cells, suggesting that even on a NHEJ-deficient genetic background, a significant contribution by homologous recombination cannot be confirmed (results not shown).
The view that not only the fast but also the slow component of DNA DSB rejoining reflects a NHEJ process is also supported by studies on the fidelity of DNA DSB repair, suggesting that the slow component of rejoining is largely error prone (64) . DNA DSB rejoining by homologous recombination in yeast or bacteria is an essentially error-free process (10) . Finally, studies at the chromosome level show an increase in dicentric formation in scid cells (65) , suggesting higher levels of illegitimate recombination in the absence of DNA-PK, which is again incompatible with a significant contribution by homologous recombination and points to an error-prone NHEJ process.
The lack of complete rejoining and the substantial increase in the
fraction of unrejoined breaks observed in DNA-PK-deficient cells by
other investigators (36, 37, 38, 39, 40, 41)
is partly attributable to the
use of relatively short postirradiation incubation periods. As
indicated by the results shown in Fig. 3
, as well as by results
published by Nevaldine et al. (44)
, more than
24 h of postirradiation incubation may be required to achieve
complete rejoining of DNA DSBs, a significantly longer period than the
4–8 h typically allowed in the former studies. Furthermore, DNA DSB
rejoining may have been occasionally masked by the inception of
apoptosis or other processes causing DNA degradation. It is relevant
that the experiments reported by Nevaldine et al.
(44)
were carried out using a pulsed-field gel
electrophoresis method, allowing the separation of DNA degradation from
DNA repair, and the cells used in the present study did not show
evidence for apoptosis (expressed as DNA degradation) in the interval
of observation (see Fig. 3
). We have reported on the difficulty in
measuring DNA DSB rejoining in mouse cells using pulsed-field gel
electrophoresis conditions that do not allow separation of apoptotic
from unrepaired DNA (50)
. Furthermore, DNA degradation may
be dose dependent and may explain the apparent increase in residual
damage with increasing dose in cells deficient either in DNA-PKcs or Ku
(40
, 66)
.
Role of DNA-PK in DNA DSB Rejoining
The results presented in Figs. 2
and 3
suggest that DNA-PK enables fast
rejoining for a large fraction (80%) of radiation-induced DNA DSBs.
Considering that in the absence of DNA-PK, most of these DNA DSBs are
rejoined with slow kinetics, it can be inferred that DNA-PK stimulates
rejoining from 12 h to 22 min, a 32.7-fold change. Such a dramatic
acceleration in the rate of rejoining effectively reduces the half-life
of DNA DSBs in the genome and as a result the probability for
nucleolytic degradation and illegitimate recombination that probably
lead to error-prone rejoining (64
, 65)
. However, it should
be noted that because the DNA ends generated by ionizing radiation are
not ligatable and require modification before rejoining
(67, 68, 69)
, it is likely that NHEJ in general does
not restore the original sequence in the DNA and that other processes,
such as homologous recombination, complete repair.
Because the DNA-PK complex lacks DNA ligase activity, an acceleration of DNA DSB rejoining could be mediated by regulatory and/or structural interactions between the DNA and the rejoining apparatus. One could envision a highly specialized DNA DSB rejoining apparatus activated by DNA-PK or a stimulation by DNA-PK of the preexisting slow rejoining apparatus. Several arguments can be developed in favor of the latter model.
With the exception of DNA-PK, which has not been found in lower eukaryotes, a good conservation across evolution (from yeast to humans) is observed for several putative components of the NHEJ apparatus, such as, for example, Ku, DNA ligase IV, XRCC4, MRE11, p95, and others (5 , 16 , 32 , 70) . Despite the conservation of functional components of the NHEJ apparatus, its relative contribution to DNA DSB rejoining is not conserved between lower and higher eukaryotes, and the increased significance of NHEJ in the latter group of organisms coincides with the appearance of DNA-PK. Because several conserved components of the NHEJ apparatus (Ku, ligase IV, and XRCC4) may also be involved in the DNA-PK-independent rejoining of DNA DSBs (20 , 21 , 28 , 30 , 31 , 71, 72, 73, 74) , it is likely that DNA-PKcs functions by interacting with the preexisting NHEJ apparatus. By stimulating the function of NHEJ, DNA-PKcs may have parsimoniously shifted, in higher eukaryotes, the task of DNA DSB rejoining from homologous recombination to NHEJ.
Biochemical studies will be required to characterize the mechanism of stimulation of DNA DSB rejoining by DNA-PK. It is possible that this stimulation is achieved by the activation of components of the NHEJ apparatus and/or by facilitating the synapsis of DNA ends (8 , 12 , 16 , 75) . Indeed DNA-PK has been found to phosphorylate XRCC4 (29) , an accessory component of DNA ligase IV, and atomic force microscopy studies revealed that Ku can bring DNA ends together (76, 77, 78) .
Considering that DNA-PKcs is an abundant molecule in human cells
present at a number approximately equal to the number of replicons
(14)
, the following model for DNA DSB rejoining can be
developed from the above results (Fig. 7)
. DNA-PKcs may be attached to the NM in proximity with the NHEJ
apparatus. Formation of a DNA DSB within the chromatin loop leads to Ku
binding on the free DNA ends (79
, 80)
. The DNA-Ku complex
may be subsequently recruited by DNA-PKcs (81)
, which is
thus activated and phosphorylates Ku and proteins in the NHEJ apparatus
(8
, 13
, 82)
, effecting end-joining according to mechanisms
suggested previously (5
, 8 , 16)
. In the absence of
DNA-PKcs, the recruitment to the NHEJ apparatus is less effective, and
as a result DNA DSB rejoining is significantly slower. According to
this model, fast rejoining of a DNA DSB in DNA-PKcs-proficient cells is
ensured by the presence of DNA-PKcs in the vicinity of a DNA DSB,
whereas DNA-PK-independent, slow rejoining removes DNA DSBs from areas
in the genome remote from DNA-PKcs. Thus, the fast and slow components
in the kinetics of DNA DSB rejoining in normal cells may reflect
DNA-PK-dependent and DNA-PK-independent rejoining as a consequence of
the local availability of DNA-PKcs rather than of the nature of the
lesion. We propose the term "DNA-PK surveillance domains" to
describe regions in the nucleus benefiting from the presence of
DNA-PKcs.
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Effect of Wortmannin on DNA DSB Rejoining
Wortmannin is a fungal metabolite originally characterized as an
irreversible inhibitor of PI 3-K acting at nM
concentrations by covalently binding to the lys-802 residue of the
p110a subunit (89, 90, 91)
. At higher concentrations,
wortmannin inhibits other kinases of the PI 3-K-like family including
DNA-PK (52
, 92) . Wortmannin-induced inhibition of DNA-PK
interferes with the binding of C-19 antibody, which recognizes the
COOH-terminal 3491–3511 amino acid region of DNA-PKcs, close to
lys-3752, the predicted binding site of wortmannin (52)
.
Because of its relatively wide spectrum of activity, it is not clear
whether the effect of wortmannin on cell radiosensitivity to killing
and DNA DSB rejoining result only from an inhibition of DNA-PK or are
also attributable to the inhibition of other kinases. Cells deficient
in DNA-PK provide a unique system to test the mechanism of wortmannin
action on cell radiosensitivity and DNA DSB rejoining. The results
shown in Fig. 6
confirm that wortmannin radiosensitizes cells to
killing (54
, 55)
, specifically by inhibiting DNA-PK.
M059-J cells show no radiosensitization after exposure to 20
µM wortmannin, suggesting that inhibition of other
members of the PI 3-K family does not cause radiosensitization. On the
other hand, M059-K cells show a significant radiosensitization, leading
to survival levels practically identical to those of M059-J cells.
Thus, inhibition of DNA-PK by wortmannin has the same effect on cell
survival as genetic inactivation of the protein. Similar conclusions
were also drawn in a study using rodent cells deficient in DNA-PK
(53)
.
The results on the inhibition by wortmannin of DNA DSB rejoining allow similar conclusions, but there are also differences that need to be pointed out. Treatment of M059-J cells with wortmannin reduces the contribution of the fast rejoining component from 28 to 7%. This is a significant reduction in the contribution of the fast component of DNA DSB rejoining and suggests either some residual, yet undetectable, DNA-PK activity in M059-J cells or the contribution of other wortmannin-sensitive kinases in DNA DSB rejoining. Some support for the former comes from recent studies that demonstrated the presence of DNA-PKcs transcripts in M059-J cells, albeit at 20-fold reduced levels, suggesting that residual activity may indeed be present (47) . Despite this difference from survival data, the kinetics of DNA DSB rejoining were identical in M059-J and M059-K cells after treatment with wortmannin. Thus, DNA-PK appears as the main, if not the only, target of wortmannin with regard to cell killing and inhibition of DNA DSB rejoining (53) .
In summary, the results presented here suggest that DNA-PK deficiency does not affect the overall capacity of human cells to rejoin DNA DSBs, although it profoundly decreases the proportion of DNA DSB rejoining with fast kinetics. These results are compatible with the operation of an evolutionarily conserved, independently active, but inherently slow and probably error-prone NHEJ pathway that can be stimulated 30-fold by DNA-PK to rapidly remove DNA DSBs from the genome. Elucidation of the biochemical characteristics of this stimulation should significantly advance our understanding of DNA DSB repair in higher eukaryotes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Cancer Institute Grants
2RO1 CA42026 and P30 CA56036 awarded from the NIH, Department of Health
and Human Services. 
2 To whom requests for reprints should be
addressed, at Thomas Jefferson University, Department of Radiation
Oncology, Thompson Building, Room B-1, Philadelphia, PA 19107. Phone:
(215) 955-6473; Fax: (215) 955-2052; E-mail: George.Iliakis{at}mail.tju.edu
3 The abbreviations used are: DSB, double-strand
break; NHEJ, nonhomologous end-joining; DNA-PKcs, catalytic subunit of
DNA-PK; scid, severe combined immunodeficient; AFIGE, asymmetric field
inversion gel electrophoresis; FAR, fraction of activity reached; CI,
confidence interval; PI 3-K, phosphatidylinositol 3-kinase; NM, nuclear
matrix.
Received 9/23/99. Accepted 1/ 4/00.
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