Double-strand breaks (DSBs) can be efficiently removed from the DNA of higher eukaryotes by nonhomologous end-joining (NHEJ). Genetic studies implicate the DNA-dependent protein kinase (DNA-PK) in NHEJ, but the exact function of this protein complex in the rejoining reaction remains to be elucidated. We compared rejoining of DNA DSBs in a human glioma cell line, M059-J, lacking the catalytic subunit of DNA-PK(DNA-PKcs), and their isogenic but DNA-PK-proficient counterpart,M059-K. In both cell lines, rejoining of DNA DSBs was biphasic, with a fast and a slow component operating with a half-life of approximately 22 min and 12 h, respectively. Deficiency in DNA-PK activity did not alter the half-times of either of these components of rejoining but increased from 17 to 72% the proportion of DNA DSB rejoining with slow kinetics. DNA DSB rejoining was nearly complete in both cell lines, and there was only a small increase in the number of unrejoined breaks in M059-J as compared with M059-K cells after 30 h of incubation. Wortmannin radiosensitized to killing M059-K cells and strongly inhibited DNA DSB rejoining. Wortmannin did not affect the radiosensitivity to killing and produced only a modest inhibition in DNA DSB rejoining in M059-J cells, suggesting that, for these end points, DNA-PK is the principal target of the drug. These observations demonstrate that DNA-PK deficiency profoundly decreases the proportion of DNA DSB rejoining with fast kinetics but has only a small effect on the fraction remaining unrejoined. We propose that in higher eukaryotes, an evolutionarily conserved, independently active, but inherently slow NHEJ pathway is stimulated 30-fold by DNA-PKcs to rapidly remove DNA DSBs from the genome. The stimulation is expected to be of local nature and the presence of DNA-PKcs in the vicinity of the DNA DSB determines whether rejoining will follow fast or slow kinetics. Structural and regulatory functions of DNA-PKcs may mediate this impressive acceleration of DNA DSB rejoining, and regions of chromatin within a certain range from this large protein may benefit from these activities. We propose the term DNA-PK surveillance domains to describe these regions.

There is evidence for a cause-effect relationship between induction of DNA DSBs3and cell killing or mutation induction in mammalian cells exposed to ionizing radiation and other genotoxic agents (1, 2, 3, 4, 5). Therefore, elucidation of the mechanisms used to repair this type of lesion is critical to our understanding of cellular responses to genotoxic stress. Unlike yeast or bacteria that predominantly use homologous recombination to remove DSBs from their genome, mammalian cells use NHEJ (5, 6, 7, 8, 9, 10, 11, 12). Although the exact mechanism and the regulation of NHEJ remain to be elucidated, genetic and biochemical evidence implicate DNA-PK in the process. DNA-PK is a serine-threonine protein kinase that requires ends of double-stranded DNA or transitions from single-stranded to double-stranded DNA for activity(13, 14, 15, 16, 17, 18). It is composed of a kinase element known as DNA-PKcs and a DNA binding element known as Ku, which is itself a heterodimer comprising Ku80 and Ku70. Cells lacking DNA-PK activity as a result of a mutation in any of the subunits are radiosensitive to killing and deficient in the rejoining of radiation-induced DNA DSBs(reviewed in Refs. 5, 16, and 19)). Other factors implicated in NHEJ include DNA ligase IV (20, 21, 22, 23, 24)and the DNA ligase IV accessory factor, XRCC4 (25, 26, 27, 28, 29, 30). It is thought that DNA ligase IV and XRCC4 are members of the DNA-PK-dependent pathway for DNA DSB rejoining (5, 16, 31).

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 scidmutation (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.

Cell Culture

The cell lines M059-J and M059-K were derived from different regions of a human malignant glioma, as described previously (38, 46, 47), and were grown in DMEM supplemented with 10% bovine calf serum, 1% nonessential amino acids, and 1% l-glutamine,at 37°C in a humidified incubator in an atmosphere of 5%CO2 and 95% air. Before beginning with repair experiments, the growth characteristics of the cells were investigated in detail, and the results were used to establish procedures for continuous maintenance of the cells in culture, as well as for the preparation of cultures for experiments. Cells were maintained in a phase of nearly logarithmic growth by subculturing every 4 days at an initial concentration of 106 cells/100-mm tissue culture dish. The same cells were also used to prepare cultures for experiments. For this purpose, 105 cells were plated per 60-mm dish and allowed to grow for 3 days. At this point,cells reached a density of ∼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 × 106cells/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 × 5-mm cylindrical blocks containing ∼1 × 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 0.5× 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 Aand C describe the amplitudes, and parameters band 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.

To validate the pulsed-field gel electrophoresis conditions used in the present study and to obtain information for the quantitative analysis of the repair experiments, we evaluated induction of DNA DSBs in M059-K and M059-J cells. The results in Fig. 1 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).

It is well documented that M059-J cells are radiosensitive when compared with their isogenic counterpart, M059-K cells, and that this increased radiosensitivity is caused by a defect in DNA-PK activity(36, 38, 46, 47). We studied systematically rejoining of DNA DSBs in M059-K and M059-J cells after exposure to 40 Gy X-rays under optimal conditions for growth. The experiments allowed prolonged postirradiation incubation times to assess the ultimate fate of radiation-induced DNA DSBs in the genome of DNA-PK-deficient cells. Fig. 2 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.

As expected, rejoining of DNA DSBs is impaired in M059-J cells, with fewer breaks being rejoined at any time as compared with M059-K cells. Despite this deficiency, nearly 60% of the DNA DSBs rejoined within the 6 h of follow-up. As with M059-K cells, the kinetics of rejoining are biphasic. It is striking that the results of M059-J cells can be fitted using half-times similar to those calculated for M059-K cells, i.e., 22 min (95% CI, 17–31) and 12 h (95%CI, 11–15) for the fast and slow component, respectively. The solid line through the data points reflects this fitting, which also allows us to estimate that in M059-J cells, only 28% of the DNA DSBs are removed with fast kinetics, a value significantly lower than the 83%calculated in M059-K cells. Thus, in M059-J cells, DNA-PK deficiency reduces the fraction of DNA DSBs removed with fast kinetics but does not confer a total deficiency in DNA DSBs rejoining or a significant alteration in the half-times of rejoining.

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.

One could argue that the above observations are a special characteristic of M059-J cells and reflect the loss of DNA-PK activity,together with other compensatory processes activated as a result of this defect, and for which cells may have been selected. If this were the case, the rejoining characteristics observed will not reflect exclusively the lack of DNA-PK activity. To examine this possibility,we evaluated DNA DSB rejoining in cells treated with wortmannin. Wortmannin is a potent inhibitor of PI 3-K, but it also inhibits at higher concentrations other members of the family, such as DNA-PK (see“Discussion”) and has a pronounced effect on DNA DSB rejoining(51, 52, 53, 54, 55).

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.

In M059-K cells, wortmannin strongly inhibits DNA DSB rejoining (Fig. 4, 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.

As a last step in our investigations, we examined the effect of wortmannin on the radiosensitivity to killing in M059-J and M059-K cells, and the results obtained are shown in Fig. 6. 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.

The deficiency in DNA DSB rejoining of cells with compromised DNA-PK activity has been documented in several investigations(36, 37, 38, 39, 40, 41), and the results in their majority suggest that these cells only rejoin a fraction (∼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).4Furthermore, homologous recombination-deficient mutants generated from DT40 cells by gene disruption (Refs. 62 and63; 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.

A stimulatory role of DNA-PK on a preexisting, independently active,and evolutionarily conserved NHEJ apparatus allows for a simple interpretation not only of the in vivo results presented here but also of results obtained by evaluating end-rejoining of plasmid or genomic DNA in vitro(83, 84, 85, 86). These studies frequently suggest the operation of a DNA-PK-independent end-joining process, which has been interpreted as evidence for multiple pathways of NHEJ. In the model developed here, this observation is the mere reflection of the fact that the end-joining apparatus functions efficiently in the absence of DNA-PK under the conditions used in the above in vitro assays. Because DNA-PK has also been implicated in the regulation (and inhibition) of DNA replication after DNA damage (87), it is possible that this protein acts as a local switch to divert activities from DNA replication to DNA repair in the presence of DNA DSBs(88). Finally, the rather mild phenotype of DNA-PKcs, or Ku, deficiency in mouse development and cell growth and viability(42, 43) is more compatible with a stimulatory role for an existing and independently active DNA repair pathway rather than with the activation of a dedicated pathway that remains inactive in the absence of DNA-PK.

Effect of Wortmannin on DNA DSB Rejoining

Wortmannin is a fungal metabolite originally characterized as an irreversible inhibitor of PI 3-K acting at nmconcentrations 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 20μ m 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.

Fig. 1.

Induction of DNA DSBs as a function of radiation dose in logarithmically growing M059-J and M059-K cells. Cells were trypsinized, embedded in agarose blocks, and exposed to various doses of X-rays while kept on ice. The amount of DNA DSBs present was measured by AFIGE and is expressed as FAR. Upper panel,typical gel; lower panel, results obtained by quantitating gels from three experiments. The means are plotted; bars, SE. The value of FAR measured in nonirradiated cells was between 2 and 6% and has been subtracted from the results shown. Line, best linear fit of results up to 40 Gy.

Fig. 1.

Induction of DNA DSBs as a function of radiation dose in logarithmically growing M059-J and M059-K cells. Cells were trypsinized, embedded in agarose blocks, and exposed to various doses of X-rays while kept on ice. The amount of DNA DSBs present was measured by AFIGE and is expressed as FAR. Upper panel,typical gel; lower panel, results obtained by quantitating gels from three experiments. The means are plotted; bars, SE. The value of FAR measured in nonirradiated cells was between 2 and 6% and has been subtracted from the results shown. Line, best linear fit of results up to 40 Gy.

Close modal
Fig. 2.

Rejoining of DNA DSBs in logarithmically growing M059-J and M059-K cells. Cells were exposed to 40 Gy X-rays as described in“Materials and Methods” and returned to 37°C. At various times thereafter, cells were trypsinized and prepared for AFIGE. Upper panel, typical gel; lower panel, results obtained by quantitating gels from three experiments. The means are plotted; bars, SE. The value of FAR measured in nonirradiated cells has been subtracted from all data points. Lines, fitting to a double exponential as outlined in detail in the text.

Fig. 2.

Rejoining of DNA DSBs in logarithmically growing M059-J and M059-K cells. Cells were exposed to 40 Gy X-rays as described in“Materials and Methods” and returned to 37°C. At various times thereafter, cells were trypsinized and prepared for AFIGE. Upper panel, typical gel; lower panel, results obtained by quantitating gels from three experiments. The means are plotted; bars, SE. The value of FAR measured in nonirradiated cells has been subtracted from all data points. Lines, fitting to a double exponential as outlined in detail in the text.

Close modal
Fig. 3.

Results similar to those shown in Fig. 2 but after allowing up to 30 h periods of incubation for repair.

Fig. 3.

Results similar to those shown in Fig. 2 but after allowing up to 30 h periods of incubation for repair.

Close modal
Fig. 4.

Rejoining of DNA DSBs in logarithmically growing M059-J and M059-K cells after treatment with 20 μm wortmannin. Cells were treated with wortmannin for 1 h, irradiated, and returned to 37°C. At various times thereafter, cells were trypsinized and prepared for AFIGE. Upper panel, typical gel; lower panel, results obtained by quantitating gels from three independent experiments. The means are plotted; bars, SE. The value of FAR measured in nonirradiated cells has been subtracted from all data points. Solid lines, fitting to a double exponential, as outlined in detail in the text. Broken and dotted lines,fitted curves for the M059-J and M059-K cells, respectively, redrawn from Fig. 2.

Fig. 4.

Rejoining of DNA DSBs in logarithmically growing M059-J and M059-K cells after treatment with 20 μm wortmannin. Cells were treated with wortmannin for 1 h, irradiated, and returned to 37°C. At various times thereafter, cells were trypsinized and prepared for AFIGE. Upper panel, typical gel; lower panel, results obtained by quantitating gels from three independent experiments. The means are plotted; bars, SE. The value of FAR measured in nonirradiated cells has been subtracted from all data points. Solid lines, fitting to a double exponential, as outlined in detail in the text. Broken and dotted lines,fitted curves for the M059-J and M059-K cells, respectively, redrawn from Fig. 2.

Close modal
Fig. 5.

Half-times and amplitudes of the fast and slow components of DNA DSB rejoining estimated by fitting the rejoining data to a double exponential equation, as described in detail in the text. The values shown have been calculated for M059-J or M059-K cells, with or without treatment with wortmannin (wrt).

Fig. 5.

Half-times and amplitudes of the fast and slow components of DNA DSB rejoining estimated by fitting the rejoining data to a double exponential equation, as described in detail in the text. The values shown have been calculated for M059-J or M059-K cells, with or without treatment with wortmannin (wrt).

Close modal
Fig. 6.

Survival of logarithmically growing M059-J and M059-K cells, as measured by colony formation, after exposure to various doses of X-rays. Cells were either exposed to radiation and plated immediately thereafter or were pretreated with 20 μmwortmannin for 45 min, irradiated, and returned to the incubator for 6 h before plating. The results shown are the means from three independent experiments; bars, SE.

Fig. 6.

Survival of logarithmically growing M059-J and M059-K cells, as measured by colony formation, after exposure to various doses of X-rays. Cells were either exposed to radiation and plated immediately thereafter or were pretreated with 20 μmwortmannin for 45 min, irradiated, and returned to the incubator for 6 h before plating. The results shown are the means from three independent experiments; bars, SE.

Close modal
Fig. 7.

Model for DNA DSB rejoining in human cells developed on the basis of the results presented here. The drawing shows a chromatin loop attached to the NM. DNA-PKcs is shown attached at the NM in proximity with the NHEJ apparatus (NHEJ App). Formation of a DNA DSB within the chromatin loop leads to Ku binding on the free DNA ends. The DNA-Ku complex is assumed to be subsequently recruited by DNA-PKcs, which is activated and phosphorylates Ku and proteins in the NHEJ apparatus, thus effecting end-joining. According to this model, DNA-PKcs contributes to DNA DSB rejoining in two ways:(a) by bringing the DNA ends to the NHEJ apparatus; and(b) by activating the NHEJ apparatus. In the absence of DNA-PKcs, either because of a mutation that affects the entire genome or because of its absence from that particular site in the genome, the recruitment to and the activation of the NHEJ apparatus will be less effective, and as a result DNA DSB, rejoining significantly slower.

Fig. 7.

Model for DNA DSB rejoining in human cells developed on the basis of the results presented here. The drawing shows a chromatin loop attached to the NM. DNA-PKcs is shown attached at the NM in proximity with the NHEJ apparatus (NHEJ App). Formation of a DNA DSB within the chromatin loop leads to Ku binding on the free DNA ends. The DNA-Ku complex is assumed to be subsequently recruited by DNA-PKcs, which is activated and phosphorylates Ku and proteins in the NHEJ apparatus, thus effecting end-joining. According to this model, DNA-PKcs contributes to DNA DSB rejoining in two ways:(a) by bringing the DNA ends to the NHEJ apparatus; and(b) by activating the NHEJ apparatus. In the absence of DNA-PKcs, either because of a mutation that affects the entire genome or because of its absence from that particular site in the genome, the recruitment to and the activation of the NHEJ apparatus will be less effective, and as a result DNA DSB, rejoining significantly slower.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by National Cancer Institute Grants 2RO1 CA42026 and P30 CA56036 awarded from the NIH, Department of Health and Human Services.

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.

4

Unpublished results.

We are indebted to Dr. Joan Allalunis-Turner for the M059-J and M059-K cells. Special thanks go to Nancy Mott for help in the preparation of the manuscript.

1
Frankenberg-Schwager M. Review of repair kinetics for DNA damage induced in eukaryotic cells in vitro by ionizing radiation.
Radiother. Oncol.
,
14
:
307
-320,  
1989
.
2
Weaver D. T. Regulation and repair of double-strand DNA breaks.
Crit. Rev. Eukaryotic Gene Expression
,
6
:
345
-375,  
1996
.
3
Iliakis G. The role of DNA double strand breaks in ionizing radiation induced killing of eukaryotic cells.
BioEssays
,
13
:
641
-648,  
1991
.
4
Ward J. F. Biochemistry of DNA lesions.
Radiat. Res.
,
104
:
S103
-S111,  
1985
.
5
Jeggo P. A. DNA breakage and repair.
Adv. Genet.
,
38
:
186
-218,  
1998
.
6
Haber J. E. Exploring the pathways of homologous recombination.
Curr. Opin. Cell Biol.
,
4
:
401
-412,  
1992
.
7
Haber J. E. In vivo biochemistry: physical monitoring of recombination induced by site-specific endonucleases.
BioEssays
,
17
:
609
-620,  
1995
.
8
Chu G. Double strand break repair.
J. Biol. Chem.
,
272
:
24097
-24100,  
1997
.
9
Kanaar R., Hoeijmakers J. H. J., van Gent D. C. Molecular mechanisms of DNA double-strand break repair.
Cell Biol.
,
8
:
483
-489,  
1998
.
10
Friedberg, E. C., Walker, G. C., and Siede, W. DNA Repair and Mutagenesis. Washington DC: ASM Press, 1995.
11
Petrini J. H. J., Bressan D. A., Yao M. S. The RAD52 epistasis group in mammalian double strand break repair.
Semin. Immunol.
,
9
:
181
-188,  
1997
.
12
Dynan W. S., Yoo S. Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids.
Nucleic Acids Res.
,
26
:
1551
-1559,  
1998
.
13
Anderson C. W. DNA damage and the DNA-activated protein kinase.
Trends Biochem. Sci.
,
18
:
433
-437,  
1993
.
14
Anderson C. W., Carter T. H. The DNA-activated protein kinase–DNA-PK.
Curr. Top. Microbiol. Immunol.
,
217
:
91
-111,  
1996
.
15
Jeggo P. A., Taccioli G. E., Jackson S. P. Menage á trois: double strand break repair. V(D)J recombination and DNA-PK.
BioEssays
,
17
:
949
-957,  
1995
.
16
Smith G. C. M., Jackson S. P. The DNA-dependent protein kinase.
Genes Dev.
,
13
:
916
-934,  
1999
.
17
Leuther K. K., Hammarsten O., Kornberg R. D., Chu G. Structure of DNA-dependent protein kinase: implications for its regulation by DNA.
EMBO J.
,
18
:
1114
-1123,  
1999
.
18
Smider V., Rathmell W. K., Brown G., Lewis S., Chu G. Failure of hairpin-ended and nicked DNA to activate DNA-dependent protein kinase: implications for V(D)J recombination.
Mol. Cell. Biol.
,
18
:
6853
-6858,  
1998
.
19
Hammarsten O., Chu G. DNA-dependent protein kinase: DNA binding and activation in the absence of Ku.
Proc. Natl. Acad. Sci. USA
,
95
:
525
-530,  
1998
.
20
Wilson T. E., Grawunder U., Lieber M. R. Yeast DNA ligase IV mediates non-homologous DNA end joining.
Nature (Lond.)
,
388
:
495
-498,  
1997
.
21
Grawunder U., Zimmer D., Fugmann S., Schwarz K., Lieber M. R. DNA ligase IV is essential for V(D)J recombination and DNA double-strand break repair in human precursor lymphocytes.
Mol. Cell
,
2
:
477
-484,  
1998
.
22
Frank K. M., Sekiguchi J. M., Seidl K. J., Swat W., Rathbun G. A., Cheng H-L., Davidson L., Kangaloo L., Alt F. W. Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV.
Nature (Lond.)
,
396
:
173
-177,  
1998
.
23
Schaer P., Herrmann G., Daly G., Lindahl T. A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks.
Genes Dev.
,
11
:
1912
-1924,  
1997
.
24
Riballo E., Critchlow S. E., Teo S-H., Doherty A. J., Priestley A., Broughton B., Kysela B., Beamish H., Plowman N., Arlett C. F., Lehmann A. R., Jackson S. P., Jeggo P. A. Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient.
Curr. Biol.
,
9
:
699
-702,  
1999
.
25
Stamato T. D., Weinstein R., Giaccia A., Mackenzie L. Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell.
Somatic Cell Genet.
,
9
:
165
-173,  
1983
.
26
Leber R., Wise T. W., Mizuta R., Meek K. The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase.
J. Biol. Chem.
,
273
:
1794
-1801,  
1998
.
27
Li Z., Otevrel T., Gao Y., Cheng W-L., Seed B., Stamato T. D., Taccioli G. E., Alt F. W. The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination.
Cell
,
83
:
1079
-1089,  
1995
.
28
Grawunder U., Zimmer D., Kulesza P., Lieber M. R. Requirement for an interaction of XRCC4 with DNA ligase IV for wild-type V(D)J recombination and DNA double-strand break repair in vivo.
J. Biol. Chem.
,
273
:
24708
-24714,  
1998
.
29
Critchlow S. E., Bowater R. P., Jackson S. P. Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV.
Curr. Biol.
,
7
:
588
-598,  
1997
.
30
Grawunder U., Wilm M., Wu X., Kulesza P., Wilson T. E., Mann M., Lieber M. R. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells.
Nature (Lond.)
,
388
:
492
-495,  
1997
.
31
Baumann P., West S. C. DNA end-joining catalyzed by human cell-free extracts.
Proc. Natl. Acad. Sci. USA
,
95
:
14066
-14070,  
1998
.
32
Haber J. E. The many interfaces of Mre11.
Cell
,
95
:
583
-586,  
1998
.
33
Paull T. T., Gellert M. The 3′ to 5′ exonuclease activity of Mre11 facilitates repair of DNA double-strand breaks.
Mol. Cell
,
1
:
969
-979,  
1998
.
34
Trujillo K. M., Yuan S-S., Lee E. Y-H., Sung P. Nuclease activities in a complex of human recombination and DNA repair factors Rad50, Mre11, and p95.
J. Biol. Chem.
,
273
:
21447
-21450,  
1998
.
35
Carney J. P., Maser R. S., Olivares H., Davis E. M., Le Beau M., Yates J. R., Hays L., Morgan W. F., Petrini J. H. J. The hMre 11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair.
Cell
,
93
:
477
-486,  
1998
.
36
Lees-Miller S. P., Godbout R., Chan D. W., Weinfeld M., Day R. S., III, Barron G. M., Allalunis-Turner J. Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line.
Science (Washington DC)
,
267
:
1183
-1185,  
1995
.
37
Biedermann K. A., Sung J., Giaccia A. J., Tosto L. M., Brown J. M. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair.
Proc. Natl. Acad. Sci. USA
,
88
:
1394
-1397,  
1991
.
38
Allalunis-Turner M. J., Zia P. K. Y., Barron G. M., Mirzayans R., Day R. S., III. Radiation-induced DNA damage and repair in cells of a radiosensitive human malignant glioma cell line.
Radiat. Res.
,
144
:
288
-293,  
1995
.
39
Hendrickson E. A., Qin X-Q., Bump E. A., Schatz D. G., Oettinger M., Weaver D. T. A link between double-strand break-related repair and V(D)J recombination: the scid mutation.
Proc. Natl. Acad. Sci. USA
,
88
:
4061
-4065,  
1991
.
40
Chang C., Biedermann K. A., Mezzina M., Brown J. M. Characterization of the DNA double strand break repair defect in scid mice.
Cancer Res.
,
53
:
1244
-1248,  
1993
.
41
Stackhouse M. A., Bedford J. S. An ionizing radiation-sensitive mutant of CHO cells: irs-20. III. Chromosome aberrations. DNA breaks and mitotic delay.
Int. J. Radiat. Biol.
,
65
:
571
-582,  
1994
.
42
Taccioli G. E., Amatucci A. G., Beamish H. J., Gell D., Xiang X. H., Arzayus M. I. T., Priestley A., Jackson S. P., Rothstein A. M., Jeggo P. A., Herrera V. L. M. Targeted disruption of the catalytic subunit of the DNA-PK gene in mice confers severe combined immunodeficiency and radiosensitivity.
Immunity
,
9
:
355
-366,  
1998
.
43
Kurimasa A., Ouyang H., Dong, L-j., Wang S., Li X., Cordon-Cardo, C., Chen D. J., Li G. C. Catalytic subunit of DNA-dependent protein kinase: impact on lymphocyte development and tumorigenesis.
Proc. Natl. Acad. Sci. USA
,
96
:
1403
-1408,  
1999
.
44
Nevaldine B., Longo J. A., Hahn P. J. The scid defect results in much slower repair of DNA double-strand breaks but not high levels of residual breaks.
Radiat. Res.
,
147
:
535
-540,  
1997
.
45
Woo R. A., McLure K. G., Lees-Miller S. P., Rancourt D. E., Lee P. W. K. DNA-dependent protein kinase acts upstream of p53 in response to DNA damage.
Nature (Lond.)
,
394
:
700
-704,  
1998
.
46
Allalunis-Turner M. J., Barron G. M., Day R. S., Dobler K. D., Mirzayans R. Isolation of two cell lines from a human malignant glioma specimen differing in sensitivity to radiation and chemotherapeutic drugs.
Radiat. Res.
,
134
:
349
-354,  
1993
.
47
Galloway A. M., Spencer C. A., Anderson C. W., Allalunis-Turner M. J. Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells.
Oncogene
,
18
:
1361
-1368,  
1999
.
48
Iliakis G., Metzger L., Denko N., Stamato T. D. Detection of DNA double strand breaks in synchronous cultures of CHO cells by means of asymmetric field inversion gel electrophoresis.
Int. J. Radiat. Biol.
,
59
:
321
-341,  
1991
.
49
Metzger L., Iliakis G. Kinetics of DNA double strand breaks throughout the cell cycle as assayed by pulsed field gel electrophoresis in CHO cells.
Int. J. Radiat. Biol.
,
59
:
1325
-1339,  
1991
.
50
Wachsberger P. R., Li W-H., Guo M., Chen D., Cheong N., Ling C. C., Iliakis G. Rejoining of DNA double strand breaks in Ku80-deficient mouse fibroblasts.
Radiat. Res.
,
151
:
398
-407,  
1999
.
51
Boulton S., Kyle S., Yalcintepe L., Durkacz B. W. Wortmannin is a potent inhibitor of DNA double strand break but not single strand break repair in Chinese hamster ovary cells.
Carcinogenesis (Lond.)
,
17
:
2285
-2290,  
1996
.
52
Okayasu R., Suetomi K., Ullrich R. L. Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells.
Radiat. Res.
,
149
:
440
-445,  
1998
.
53
Chernikova S. B., Wells R. L., Elkind M. M. Wortmannin sensitizes mammalian cells to radiation by inhibiting the DNA-dependent protein kinase-mediated rejoining of double-strand breaks.
Radiat. Res.
,
151
:
159
-166,  
1999
.
54
Rosenzweig K. E., Youmell M. B., Palayoor S. T., Price B. D. Radiosensitization of human tumor cells by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G2-M delay.
Clin. Cancer Res.
,
3
:
1149
-1156,  
1997
.
55
Price B. D., Youmell M. B. The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes murine fibroblasts and human tumor cells to radiation and blocks induction of p53 following DNA damage.
Cancer Res.
,
56
:
246
-250,  
1996
.
56
Weibezahn K. F., Coquerelle T. Radiation induced DNA double strand breaks are rejoined by ligation and recombination processes.
Nucleic Acids Res.
,
9
:
3139
-3150,  
1981
.
57
Liu N., Lamerdin J. E., Tebbs R. S., Schild D., Tucker J. D., Shen M. R., Brookman K. W., Siciliano M. J., Walter C. A., Fan W., Narayana L. S., Zhou Z-Q., Adamson A. W., Sorensen K. J., Chen D. J., Jones N. J., Thompson L. H. XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages.
Mol. Cell
,
1
:
783
-793,  
1998
.
58
Thacker J. A surfeit of RAD51-like genes?.
Trends Genet.
,
15
:
166
-168,  
1999
.
59
Thacker J., Tambini C. E., Simpson P. J., Tsui L-C., Scherer S. W. Localization to chromosome 7q36.1 of the human XRCC2 gene, determining sensitivity to DNA-damaging agents.
Hum. Mol. Genet.
,
4
:
113
-120,  
1995
.
60
Thacker J., Ganesh A. N. DNA-break repair, radioresistance of DNA synthesis, and camptothecin sensitivity in the radiation-sensitive irs mutants: comparisons to ataxia-telangiectasia cells.
Mutat. Res.
,
235
:
49
-58,  
1990
.
61
Cheong N., Wang Y., Jackson M., Iliakis G. Radiation-sensitive IRS mutants rejoin DNA double strand breaks with efficiency similar to that of parental V79 cells but show altered response to radiation induced G2-delay.
Mutat. Res.
,
274
:
111
-122,  
1992
.
62
Takata M., Sasaki M. S., Sonoda E., Morrison C., Hashimoto M., Utsumi H., Yamaguchi-Iwai Y., Shinohara A., Takeda S. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break.
EMBO J.
,
17
:
5497
-5508,  
1998
.
63
Sonoda E., Sasaki M. S., Buerstedde J-M., Bezzubova O., Shinohara A., Ogawa H., Takata M., Yamaguchi-Iwai Y., Takeda S. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death.
EMBO J.
,
17
:
598
-608,  
1998
.
64
Loebrich M., Rydberg B., Cooper P. K. Repair of X-ray-induced DNA double-strand breaks in specific NotI restriction fragments in human fibroblasts: joining of correct and incorrect ends.
Proc. Natl. Acad. Sci. USA
,
92
:
12050
-12054,  
1995
.
65
Evans J. W., Liu X. F., Kirchgessner C. U., Brown J. M. Induction and repair of chromosome aberrations in scid cells measured by premature chromosome condensation.
Radiat. Res.
,
145
:
39
-46,  
1996
.
66
Iliakis G., Mehta R., Jackson M. Level of DNA double-strand break rejoining in Chinese hamster xrs-5 cells is dose-dependent: implications for the mechanism of radiosensitivity.
Int. J. Radiat. Biol.
,
61
:
315
-321,  
1992
.
67
Feingold J. M., Masch J., Maio J., Mendez F., Bases R. Base sequence damage in DNA from X-irradiated monkey CV-1 cells.
Int. J. Radiat. Biol.
,
53
:
217
-235,  
1988
.
68
Henner W. D., Rodriguez L. O., Hecht S. M., Haseltine W. A. Gamma-ray induced deoxyribonucleic acid strand breaks.
J. Biol. Chem.
,
258
:
711
-713,  
1983
.
69
Winters T. A., Henner W. D., Russell P. S., McCullough A., Jorgensen T. J. Removal of 3′-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1.
Nucleic Acids Res.
,
22
:
1866
-1873,  
1994
.
70
Haber J. E. Sir-Ku-itous routes to make ends meet.
Cell
,
97
:
829
-832,  
1999
.
71
Boulton S. J., Jackson S. P. Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone repair pathways.
EMBO J.
,
15
:
5093
-5103,  
1996
.
72
Boulton S. J., Jackson S. P. Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance.
Nucleic Acids Res.
,
24
:
4639
-4648,  
1996
.
73
Boulton S. J., Jackson S. P. Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing.
EMBO J.
,
17
:
1819
-1828,  
1998
.
74
Siede W., Friedl A. A., Dianova I., Eckardt-Schupp F., Friedberg E. C. The Saccharomyces cerevisiae Ku autoantigen homologue affects radiosensitivity only in the absence of homologous recombination.
Genetics
,
142
:
91
-102,  
1996
.
75
Lieber M. R., Grawunder U., Wu X., Yaneva M. Tying loose ends: roles of Ku and DNA-dependent protein kinase in the repair of double-strand breaks.
Curr. Opin. Genet. Dev.
,
7
:
99
-104,  
1997
.
76
Yaneva M., Kowalewski T., Lieber M. R. Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies.
EMBO J.
,
16
:
5098
-5112,  
1997
.
77
Cary R. B., Peterson S. R., Wang J., Bear D. G., Bradbury E. M., Chen D. J. DNA looping by Ku and the DNA-dependent protein kinase.
Proc. Natl. Acad. Sci. USA
,
94
:
4267
-4272,  
1997
.
78
Pang D., Yoo S., Dynan W. S., Jung M., Dritschilo A. Ku proteins join DNA fragments as shown by atomic force microscopy.
Cancer Res.
,
57
:
1412
-1415,  
1997
.
79
Blier P. R., Griffith A. J., Craft J., Hardin J. A. Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks.
J. Biol. Chem.
,
268
:
7594
-7601,  
1993
.
80
Falzon M., Fewell J. W., Kuff E. L. EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA.
J. Biol. Chem.
,
268
:
10546
-10552,  
1993
.
81
Suwa A., Hirakata M., Takeda Y., Jesch S. A., Mimori T., Hardin J. A. DNA-dependent protein kinase (Ku protein-p350 complex) assembles on double-stranded DNA.
Proc. Natl. Acad. Sci. USA
,
91
:
6904
-6908,  
1994
.
82
Anderson C. W., Lees-Miller S. P. The nuclear serine/threonine protein kinase DNA-PK.
Crit. Rev. Eukaryotic Gene Expression
,
2
:
283
-314,  
1992
.
83
Cheong N., Perrault A. R., Wang H., Wachsberger P., Mammen P., Jackson I., Iliakis G. DNA-PK-independent rejoining of DNA double-strand breaks in human cell extracts in vitro.
Int. J. Radiat. Biol.
,
75
:
67
-81,  
1999
.
84
Daza P., Reichenberger S., Goettlich B., Hagmann M., Feldmann E., Pfeiffer P. Mechanisms of nonhomologous DNA end-joining in frogs, mice and men.
J. Biol. Chem.
,
377
:
775
-786,  
1996
.
85
Johnson A. P., Fairman M. P. The identification and characterization of mammalian proteins involved in the rejoining of DNA double-strand breaks in vitro.
Mutat. Res.
,
364
:
103
-116,  
1996
.
86
Mason R. M., Thacker J., Fairman M. P. The joining of non-complementary DNA double-strand breaks by mammalian extracts.
Nucleic Acids Res.
,
24
:
4946
-4953,  
1996
.
87
Wang Y., Zhou X. Y., Wang H-Y., Iliakis G. Roles of replication protein A and DNA-dependent protein kinase in the regulation of DNA replication following DNA damage.
J. Biol. Chem.
,
274
:
22060
-22064,  
1999
.
88
Lees-Miller S. P., Anderson C. W. The DNA-activated protein kinase. DNA-PK: a potential coordinator of nuclear events.
Cancer Cells
,
3
:
341
-345,  
1991
.
89
Powis G., Bonjouklian R., Berggren M. M., Gallegos A., Abraham R., Ashendel C., Zalkow L., Matter W. F., Dodge J., Grindley G., Vlahos C. J. Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase.
Cancer Res.
,
54
:
2419
-2423,  
1994
.
90
Nakanishi S., Kakita S., Takahashi I., Kawahara K., Tsukuda E., Sano T., Yamada K., Yoshida M., Kase H., Matsuda Y. Wortmannin, a microbial product inhibitor of myosin light chain kinase.
J. Biol. Chem.
,
267
:
2157
-2163,  
1992
.
91
Wymann M. P., Bulgarelli-Leva G., Zvelebil M. J., Pirola L., Vanhaesebroeck B., Waterfield M. D., Panayotou G. Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction.
Mol. Cell. Biol.
,
16
:
1722
-1733,  
1996
.
92
Hartley K. O., Gell D., Smith G. C. M., Zhang H., Divecha N., Connelly M. A., Admon A., Lees-Miller S. P., Anderson C. W., Jackson S. P. DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product.
Cell
,
82
:
849
-856,  
1995
.