From the Institut de Pharmacologie et de Biologie Structurale, CNRS UPR 9062, 205 route de Narbonne, F-31077 Toulouse Cedex, France and the ** DNA Damage and Repair Group, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
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ABSTRACT |
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The DNA-dependent protein kinase
(DNA-PK) is required for double-strand break repair in mammalian cells.
DNA-PK contains the heterodimer Ku and a 460-kDa serine/threonine
kinase catalytic subunit (p460). Ku binds in vitro to DNA
termini or other discontinuities in the DNA helix and is able to enter
the DNA molecule by an ATP-independent process. It is clear from
in vitro experiments that Ku stimulates the recruitment to
DNA of p460 and activates the kinase activity toward DNA-binding
protein substrates in the vicinity. Here, we have examined in human
nuclear cell extracts the influence of the kinase catalytic activity on
Ku binding to DNA. We demonstrate that, although Ku can enter DNA from
free ends in the absence of p460 subunit, the kinase activity is
required for Ku translocation along the DNA helix when the whole
Ku/p460 assembles on DNA termini. When the kinase activity is impaired,
DNA-PK including Ku and p460 is blocked at DNA ends and prevents their
processing by either DNA polymerization, degradation, or ligation. The
control of Ku entry into DNA by DNA-PK catalytic activity potentially
represents an important regulation of DNA transactions at DNA termini.
DNA double strand breaks
(DSBs)1 are generated by
agents such as ionizing radiation (IR) and also occur as intermediates
in certain recombination reactions. In response to the deleterious consequences associated with DSBs, highly efficient mechanisms have
evolved for their recognition and repair. Mammalian cells appear to
rejoin DBSs primarily by a mechanism of non-homologous recombination
(reviewed in Refs. 1-4). Insights into the proteins involved in DBSs
break repair have been gained from analysis of the molecular defects in
mutant rodent cells that are hypersensitive to IR and unable to carry
out the V(D)J recombination process in the immunoglobulin and T-cell
receptor genes. Recent evidence show that a multiprotein complex, the
DNA-dependent protein kinase (DNA-PK) plays a central role
in DBS repair in mammalian cells (reviewed in Refs. 5 and 6).
The DNA-PK is a heterotrimeric enzyme composed of a large catalytic
subunit of ~460 kDa (DNA-PKcs, p460), a
serine/threonine kinase that belongs to the phosphatidylinositol
3-kinase (p110) family (7), and a regulatory component consisting of
the Ku80 and Ku70 proteins (8, 9).
The unique property of DNA-PK is to exhibit a protein kinase activity
that depends on the interaction of the protein complex with DNA. Both
DNA-PK subunits can interact with nucleic acids (reviewed in Ref. 10).
The Ku80 and Ku70 proteins form a heterodimer (11, 12) that binds with
high affinity to DNA ends of dsDNA and more generally to transitions
from double- to single-stranded DNA (12-14). Ku represents the major
dsDNA end-binding protein as detected by band shift experiments
(15). Both Ku subunits are required for stable binding to
DNA (16-19). Ku is able to translocate along the DNA in an
ATP-independent manner, allowing several Ku dimers to bind to a single
DNA molecule and form a multimeric complex (15, 20). A single-stranded
DNA-dependent ATPase and helicase activities have also been
reported for Ku (19, 21-23). The binding of Ku molecules to one
another leading to DNA loop formation has been reported (24). Ku can
transfer between DNA fragments with compatible ends (25). In addition,
Ku bridges DNA ends (26, 27) and can stimulate DNA end joining by
mammalian DNA ligases (27).
Recently, a direct binding of p460 to DNA leading to kinase activation
has been reported under in vitro conditions with purified preparations of the catalytic subunit (28-30). However, this property of p460 is lost in the presence of Ku on very short dsDNA fragments <26 bp (29, 30). For longer DNA, Ku greatly stabilizes the DNA-PK/DNA
complex and leads to optimal kinase activity (29, 30), in agreement
with the common view that Ku is required to target the catalytic
subunit to DBSs under physiological conditions (9).
Once bound at DNA termini, the active DNA-PK complex acquires the
capacity, at least in vitro, to phosphorylate many DNA-bound proteins in the vicinity (31). For example, DNA-PK
phosphorylates many transcription factors including p53 (32,
33), Sp1 (34), and also the carboxyl-terminal domain of RNA
polymerase II (8).
Ku protein DNA binding properties have been well characterized with
purified native or recombinant proteins. However, the question of the
influence of the catalytic subunit on Ku interaction with DNA is poorly
documented and remains unanswered. DNA-PK complex does not appear to
form stably in the absence of DNA (35, 36). Once the complex has
formed, Ku70, Ku80, and DNA-PKcs can be phosphorylated by
DNA-PK. DNA-PKcs phosphorylation inactivates the kinase
(33) that dissociates from Ku (36). Phosphorylated Ku70 binds to DNA in
Southwestern analysis (36), but whether phosphorylated Ku dimer still
binds DNA is not known. It has been shown that the intrinsic Ku
DNA-dependent ATPase activity is greatly stimulated when Ku
is phosphorylated by DNA-PK (23), but the subsequent possible changes
in the association of Ku with DNA have not been determined. On the
other hand, extracts from DNA-PKcs-deficient cells exhibit
normal DNA end binding activity (37, 38) and mutations in the DNA-PK
phosphorylation sites in Ku70 do not modify IR sensitivity in
vivo (39).
The most common assay for Ku DNA end binding activity is
electrophoretic mobility shift assay (EMSA), but it is performed without ATP, which is a nonpermissive condition for the kinase activity. Moreover, of the two modes of Ku binding to DNA,
i.e. DNA end-binding and translocation into the DNA helix,
EMSA does not allow precise characterization of the second owing to
length limitation of the DNA probe and complexity of the retarded bands pattern. We have examined the possible involvement of DNA-PK catalytic activity on the regulation of Ku binding to DNA in a competition EMSA.
The protein extracts were first preincubated with various potential
competitor DNAs for Ku DNA binding activity under kinase preventive or
permissive conditions; varying the length of linear competitor DNA
allowed to take both modes of Ku binding to DNA into account. Then, the
free remaining Ku DNA binding activity was assessed with a 25-bp
radiolabeled DNA probe under standard EMSA conditions. Nuclear human
cell extracts were used in order to closely mimic the physiological
conditions of molar ratios of the DNA-PK components and of protein environment.
Here, we demonstrate that, although Ku can enter DNA from free
double-stranded ends in the absence of p460 subunit, the kinase activity is required for Ku translocation along the DNA helix when the
whole Ku/DNA-PKcs assembles on DNA ends. When the kinase activity is impaired, DNA-PK including Ku and DNA-PKcs is
blocked at DNA ends and prevents end processing by either DNA
polymerization, degradation, or ligation.
Cell Extracts and Proteins
The HeLa S3 cell line was obtained from the stock of European
Molecular Biology Laboratories (Heidelberg, Germany). Cells were
grown in RPMI 1640 medium (Life Technologies, Inc.), supplemented with 10% fetal calf serum, 2 mM glutamine, 125 units/ml
penicillin, and 125 µg/ml streptomycin at 37 °C, in a humidified
atmosphere containing 5% CO2.
Nuclear protein extracts were prepared from HeLa cells as described
previously (40) except that the final dialysis was performed for 3 h at 4 °C in 25 mM Hepes-KOH, pH 7.9, 17% glycerol, 100 mM potassium glutamate, 2 mM EDTA, 1 mM EGTA, and 2 mM dithiothreitol. After
preparation, extracts were immediately frozen and stored at
The purified Ku complex is baculoviral expressed recombinant proteins
purified as already reported (24).
Plasmids and DNA Fragments
Closed circular 2959-bp pBluescript KS+ (pBS,
Stratagene) was prepared by the standard alkaline lysis method and
cesium chloride gradient centrifugation from Escherichia
coli JM109 (relevant genotype: recA1, endA1, gyrA96,
hsdR17) followed by two neutral sucrose gradient centrifugations
as described (41). When necessary, pBS plasmid was linearized by
digestion at a unique site with HindIII.
End radiolabeled pBS was produced by incubation of linearized pBS with
E. coli Klenow fragment (0.02 unit) (Life Technologies, Inc.) together with 5 µM dNTP and 0.5 µCi of
[ The 173- and 710-bp fragments were prepared by digestion with both
EcoRI and HindIII of pGf1 and pCMV plasmids,
respectively (Ref. 42; kind gifts of Dr M. S. Satoh,
Université Laval, Quebec, Canada), and a mean 1400-bp length
fragment was obtained by digestion of pBS with BglI
producing two fragments (1691 and 1268 bp). The bands corresponding to
the fragments were excised from agarose gel, and the DNA was purified
by phenol extraction and ethanol precipitation.
EMSA
A radiolabeled double-stranded 25-mer DNA probe was prepared
according to Han et al. (43). The two oligonucleotides
(5'-ACTTGATTAGTTACGTAACGTTATG-3' and 5'-CATAACGTTACGTAACTAATCAAGT-3')
were end-labeled with T4 polynucleotide kinase in the presence of
[ Standard EMSA--
EMSA was performed as described previously
(15). Briefly, radiolabeled DNA (4 ng, 100,000 cpm) was incubated with
nuclear extracts (2 µg) in 10 µl of buffer (20 mM
Tris-HCl, pH 7.5, 100 mM KCl, 0.3 mM
dithiothreitol, 2 mM MgCl2, 0.1 mM
EDTA, 5% glycerol) in the presence of closed circular pBS plasmid as a
nonspecific competitor (0.5 µg) at 30 °C for 15 min. The samples
were electrophoresed on a 5% polyacrylamide gel at 4 °C, for 2 h at 100 V. The gel was dried on Whatman 3MM paper and exposed to a
storage phosphor screen (Molecular Dynamics), followed by processing
with a PhosphorImager (Molecular Dynamics, Storm SystemTM).
The regions of the gel containing the free probe and the retardation complexes were quantitated by the ImageQuant software, version 4.2A
(Molecular Dynamics). In the supershift experiments, antibodies were
added to the mixture following the binding incubation and the mixture
was incubated for an additional 5 min prior to gel electrophoresis. The
antibodies used were purified on protein A-Sepharose from human
autoimmune antiserum AF (a generous gift from Dr. E. M. Tan,
Scripps Research Institute, La Jolla, CA), which recognizes Ku70 and
Ku80 (11) and, as a control, from the human serum Sa that contains
anti-ribonucleoprotein antibodies (provided by Dr. Y. Takeda, Medical
College of Georgia, Augusta, GA).
Competition EMSA--
Nuclear protein extracts (20 µg) or
purified Ku protein as indicated in 10 µl of buffer (45 mM Hepes-KOH, pH 7.8, 50 mM KCl, 7 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EDTA, 40 mM phosphocreatine, 2.5 µg of
creatine phosphokinase (type I, Sigma), 3.4% glycerol, and 18 µg of
bovine serum albumin) were first preincubated in the presence of closed
circular pBS plasmid as a nonspecific competitor (1 µg). When
necessary, ATP (from 1 M stock solution in water, potassium
salt; Sigma) and wortmannin (from 10 mM stock solution in
dimethyl sulfoxide; Sigma) were included in the mixture. When indicated, linear DNA was included in the mixture before the
preincubation (sample thereafter named + competitor linear DNA) and in
some experiments, an equivalent amount of closed circular pBS was added in control reactions (sample named Immunoprecipitation
DNA-PKcs
Immunodepletion--
Anti-DNA-PKcs mouse monoclonal
antibody mAb 25-4 (kind gift of Dr. T. Carter, Dept. of Biological
Sciences, St. John's University, Jamaica, NY) was reported previously
(44). The mAb in hybridoma supernatant was coupled to magnetic
anti-mouse IgG beads (Dynabeads M-450, Dynal), according to the
manufacturer recommendations. Under a 20-µl final volume, 250 µg of
HeLa nuclear protein extracts were incubated at 4 °C for 60 min with
20 µl of wet anti-DNAPK beads (equivalent to DNA Fragment Pull-down Assay--
Nuclear protein extracts (20 µg) were incubated in a 10-µl volume reaction under competition
EMSA preincubation conditions (see above) in the presence of 50 ng of
end-radiolabeled HindIII-linearized pBS, with 5 µM ATP and with or without 3 µM wortmannin.
After 60 min at 30 °C, an excess of unlabeled 25-bp dsDNA probe (2.5 pmol) was added to the mixture. This amount was estimated from EMSA
experiments to be sufficient to titrate out any Ku that would not have
bound linear pBS during the preincubation period (data not shown; see
Fig. 5). Then, half of the reaction (5 µl) was incubated in a 20-µl
final reaction volume with SacI enzyme (20 units, Amersham
Pharmacia Biotech) in restriction buffer according to the manufacturer.
After 60 min at 37 °C, 1/4 of the reaction mixture was incubated at
4 °C for 60 min with 10 µl of wet anti-DNAPK (mAb 25.4) or control
anti-IgG magnetic beads under gentle agitation. Then, the supernatant
was removed, the beads were washed twice with 500 µl of buffer (6 mM Hepes-KOH, pH 7.9, 12 mM KCl, 2, 5 mM MgCl2, 0.25 mM dithiothreitol,
0.02% Nonidet P-40, and 5% glycerol) and resuspended in 15 µl of
the same buffer. DNA fragments in both beads and supernatant fractions
were purified by 60 min incubation at 37 °C with 0.5% SDS and
proteinase K (200 µg/ml final concentration) followed by
phenol-chloroform extraction and were electrophoresed on a 5%
polyacrylamide gel at 4 °C, for 2 h at 100 V.
Enzymatic Reactions at DNA Termini
Exonuclease III Assay--
Nuclear protein extracts (20 µg)
were incubated in a 10-µl volume reaction under competition EMSA
preincubation conditions (see above) in the presence of 50 ng of
end-radiolabeled HindIII-linearized pBS, with 5 µM ATP and with or without 3 µM wortmannin
as indicated. After 60 min at 30 °C, 1/3 of the reaction mixture was
further incubated with E. coli exonuclease III (Life
Technologies, Inc.) for 15 min at 37 °C. Reaction was terminated by
addition of 10 mM EDTA and 0.5% SDS. The DNA was recovered
from the reaction mixture by proteinase K digestion (200 µg/ml, 15 min at 37 °C), followed by phenol extraction and ethanol
precipitation, and then separated on TAE-0.8% agarose gels. After
visualization under short wavelength UV light, the gel was dried and
radioactive DNA was revealed by exposure to a storage phosphor screen
(Molecular Dynamics) followed by processing with a PhosphorImager
(Molecular Dynamics, Storm SystemTM).
Klenow Assay--
This assay follows the same procedure as
described above except that the HindIII-linearized plasmid
pBS substrate was unlabeled, and the exo III enzyme was replaced by
E. coli Klenow fragment (0.02 unit) (Life Technologies,
Inc.) together with 5 µM dNTP and 0.5 µCi of
[ Ligase Assay--
Nuclear protein extract (40 µg) was
incubated under conditions as described above but with the following
modifications: 200 ng of unlabeled HindIII-linearized pBS
plasmid without competitor DNA, 1 mM ATP, and with or
without 30 µM wortmannin as indicated, for 60 min at
37 °C. DNA was purified as above. The ligation products were
analyzed by agarose gel electrophoresis and ethidium bromide staining.
ATP Stimulates Nuclear Ku DNA Binding Activity on Large DNA
Fragments--
Ku DNA binding activity can be easily detected by using
dsDNA fragments in an EMSA (15).
When a 25-bp labeled DNA was mixed with nuclear extracts from HeLa
cells, a single slow migrating band appeared in the gel (Fig.
1A). This band was
supershifted in the presence of antibodies purified from a human serum
able to recognize both Ku subunits, while the control antiserum (that
contained anti-ribonucleoprotein autoantibodies) had no effect. Thus,
this retarded band can be confidently ascribed to a Ku DNA complex.
Under these standard EMSA conditions, Ku binds to the DNA probe
independently from the p460 subunit since it has been demonstrated that
a stable Ku-p460 complex fails to form on short fragments (<26 bp)
(28-30) and, moreover, that Ku competes for p460 binding to such short DNA probes (29, 30).
We have tentatively analyzed the co-binding of Ku and p460 on probes
>100 bp under standard EMSA conditions (data not shown). However, the
appearance of multiple retarded bands including material in the wells
of the gel prevents a precise analysis of the protein-DNA complexes
involved. Since the EMSA is performed in the presence of excess
supercoiled plasmid DNA, which competes for unspecific DNA binding
activity in the extracts, we reasoned that it might be possible to
characterize a potential p460-dependent Ku DNA binding
activity by preincubating the extracts with unlabeled linear plasmid
DNA allowing the co-binding of both DNA-PK subunits. For this purpose,
nuclear protein extracts were suitable since, as already observed (33,
44), we found that p460 was concentrated in the nucleus and hardly
detectable in the corresponding cytoplasmic extracts (data not shown).
Indeed, only the nuclear fraction exhibited a significant DNA-PK
activity as measured by the conventional pull-down assay on
DNA-cellulose beads in the presence of a peptide kinase substrate (46)
(data not shown). In addition, since the molar ratio of both DNA-PK
components may be important for interaction with DNA, nuclear extracts
were preferred over whole cell extracts, since in the latter,
cytoplasmic Ku may change the physiologic molar ratio of Ku toward p460
in the nucleus.
Thus, we performed a new EMSA (referred to as competition EMSA under
"Experimental Procedures") in which nuclear cell extracts were
preincubated with or without linear plasmid DNA, in the presence or
absence of ATP. Then, the remaining Ku binding activity was assessed on
the labeled 25-bp probe that allows to visualize the DNA binding
activity specifically devoted to the Ku subunit of DNA-PK independently
from p460 (Fig. 1B). Although under these conditions, 10 ng
of linear plasmid slightly competed for Ku (>95% remaining binding to
the probe), ATP dramatically increased this effect (<20% remaining
binding to the probe). A preincubation in the presence of ATP but with
only the standard supercoiled competitor DNA had no competitor effect
(Fig. 1B, lanes
It has been reported that Ku dimer could exhibit
DNA-dependent ATPase and ATP-dependent helicase
activities (19, 21-23). It was of interest to test the effect of ATP
under the same competition EMSA conditions with purified recombinant Ku
heterodimer. Fig. 2 shows that, although
linear plasmid DNA efficiently competes for Ku in a
concentration-dependent manner, ATP has no effect on this
competition.
p460 Kinase Activity Is Necessary for ATP-dependent
Nuclear Ku DNA Binding Activity--
The previous data imply that
there exists an ATP-dependent DNA binding mode that might
be mediated by proteins in the nuclear extracts. DNA-PKcs
was a strong candidate for the protein regulating Ku DNA binding
activity in the presence of ATP. First, at the ends of large DNA
fragments, Ku interacts with the catalytic p460 kinase subunit of
DNA-PK (8, 9). Second, the ATP-dependent Ku DNA binding
relied on ATP hydrolysis, since under the competition EMSA conditions,
we found that ATP could not be replaced by the non-hydrolyzable analog
AMP-PNP (data not shown). Third, we observed no effect of ATP when the
unlabeled 25-bp DNA probe was used as competitor (data not shown),
which correlates well with the impairment of Ku-p460 complex assembly
on short DNA fragments (28, 29).
One of the first inhibitor of DNA-PK identified is the fungal
metabolite wortmannin, which irreversibly inhibits the kinase activity
in vitro (7). The nuclear Ku titration by linear DNA was
assessed in the presence of ATP and increasing concentrations of
wortmannin. As shown in Fig.
3A, the
ATP-dependent competitor effect of linear plasmid DNA was
reversed by wortmannin in a dose-dependent manner and
completely abolished for concentrations between 1 and 3 µM wortmannin (Fig. 3B) (the slight
competition observed was equivalent to the effect of 50 ng of linear
DNA without ATP). Notably, in the absence of linear DNA (Fig. 3,
A and B), wortmannin had no effect on Ku binding
to the 25-bp probe, in agreement with the ATP- and p460-independent Ku
binding to this short DNA fragment. DNA-PK activity was assessed in
parallel using a standard peptide phosphorylation assay (46) and was
found to be inhibited in the same range of wortmannin concentrations
(data not shown).
Wortmannin is a general inhibitor of enzymes of the
phosphatidylinositol 3-kinase (p110) family (47). In order to
demonstrate that p460 was involved in the ATP-dependent Ku
binding to linear DNA, we used monoclonal antibodies to immunodeplete
HeLa nuclear extracts of DNA-PK. We have already reported that two
successive immunoprecipitations with mAb 25-4 removed more than 90%
of the DNA-PK activity (45). A control immunodepletion with anti-XPB magnetic beads (against one of the subunits of the basal transcription factor TFIIH) was performed in parallel. Mock-treated nuclear extracts
and the two immunodepleted extracts were assayed in the competition
EMSA with a fixed amount of linear plasmid DNA (50 ng) and under
preincubation conditions without ATP, with ATP, or with both ATP and
wortmannin (Fig. 4A). The
control and XPB-depleted extracts show the expected profile that is an
ATP-dependent stimulation of Ku titration by linear DNA,
which is reversed by wortmannin. In sharp contrast, no Ku binding to
the labeled probe appeared under the three preincubation conditions
with the DNA-PKcs-depleted extracts.
A simple explanation could rely on a co-depletion of Ku along p460 in
these extracts. However, this possibility was ruled out because in the
absence of linear plasmid, p460-depleted extracts showed <20%
reduction of Ku DNA binding when compared with control-depleted extracts (Fig. 4B, lanes 0 competitor linear DNA). Another
explanation for the above result could be that Ku bound better to the
linear competitor in the absence of DNA-PKcs,
irrespectively of whether ATP or wortmannin were present. Indeed, the
competitor effect of a range of linear DNA concentrations was compared
between the p460-depleted and the control-depleted extracts in the
absence of ATP (Fig. 4B). Linear DNA was much more
competitor for Ku in p460-depleted extracts than in control extracts;
this effect was ATP-independent. In addition, this increased competitor
effect of linear DNA for Ku in p460-depleted extracts did not change in
the presence of ATP or wortmannin (Fig. 4A and data not shown).
Taken together, these results indicate that p460 depletion facilitates
Ku binding to the competitor DNA in an ATP-independent mode. In other
words, when present in nuclear extracts, DNA-PKcs somehow
impairs Ku binding to linear plasmid DNA, unless ATP is provided and
the kinase is active.
DNA-PK Catalytic Activity Regulates Ku Entry into Linear
DNA--
The kinase activity of p460 could modulate Ku binding to DNA
by changing either Ku affinity for DNA or the mode of Ku binding. Indeed, Ku interacts with DNA by a two-step mechanism where it first
recognizes DNA ends and then translocates along the DNA helix, allowing
the binding of several Ku dimers to a single DNA molecule (15, 20, 48).
Both modes of Ku interaction with DNA can operate in nuclear extracts
for Ku titration by the linear competitor plasmid DNA. We postulated
that the competition EMSA would allow to dissociate these two modes by
varying independently two parameters regarding the competitor DNA,
i.e. the number of free ends and the length of the DNA
molecule. If Ku can just bind to the DNA ends (mode 1), the titration
would be only dependent on the number of ends whatever the length of
the linear competitor. On the opposite, if Ku can invade DNA (mode 2),
the titration would be dependent on both parameters.
Fig. 5A shows the result of a
typical experiment, which compares the nuclear Ku competitor effect of
a fixed molarity of DNA ends for three linear DNAs of increasing length
(ranging from 171 bp to 1400 bp), without or with ATP. Due to the
stimulating effect of ATP on the competition and in order to still
visualize a band shift in each case, the amount of DNA ends for the
three fragments was adjusted at 200 fmol or 30 fmol per assay, in the absence or presence of ATP, respectively. In addition, the experiment was repeated for various molarities of DNA ends and the results are
shown in Fig. 5B. The data in Fig. 5 (A and
B) clearly show that, in nuclear extracts in which both Ku
and DNA-PKcs are present, Ku binding to the competitor
without ATP is independent of the DNA length and only increases with
the number of DNA ends while, in the presence of ATP, the extent of Ku
titration is correlated to both the number of ends and the DNA
length.
In the competition EMSA experiments, an alternative explanation for the
apparent ATP-stimulated binding of Ku to the linear competitor would be
that, under kinase permissive conditions, phosphorylated Ku molecules
no longer bind to the 25-bp probe. However, the present data allow to
rule out this simple explanation since, in that case, the extent of Ku
titration in the presence of ATP would solely depend on the number of
kinase-stimulating DNA ends and in any case on the DNA length.
The data above support the interpretation that, in the presence of
DNA-PKcs, the kinase activity can regulate the mode of Ku
binding to linear plasmid DNA. If ATP is available and the kinase
functional, Ku can both bind to the ends and slide along DNA (mode 2),
resulting in high Ku titration rate by the linear competitor. Without
ATP or with non-hydrolyzable ATP, or when the kinase activity is
impaired by wortmannin, Ku can still bind to DNA ends but is unable to
enter the DNA molecule (mode 1), resulting in a poor titration effect.
In the absence of kinase subunit, however (immunodepleted extracts), Ku
can bind to DNA ends and freely enter DNA as shown by the high Ku
titration rate of linear DNA with p460-depleted extracts, with or
without ATP (Fig. 4).
Wortmannin Blocks DNA-PK at DNA Ends--
In nuclear extracts, Ku
cannot enter DNA when the kinase activity is impaired, suggesting that
Ku and probably the whole DNA-PK complex may stay bound at the DNA
ends. In order to test this hypothesis, we have designed an experiment
aimed at determining the proteins bound at the DNA ends under kinase
permissive or preventive conditions. An end-labeled linear plasmid DNA
was incubated with nuclear extracts under the conditions used in the
preincubation step of the competition EMSA, with ATP ± wortmannin. Then, after dilution, the DNA was restricted at a unique
site close to one labeled end. This second incubation was carried out
in the presence of an excess of cold DNA probe (25 bp) calibrated in
order to titrate out the remaining Ku that might bind to the labeled
oligonucleotide released (data not shown). Then, the mixture was
immunoprecipitated in the presence of control or
anti-DNA-PKcs magnetic beads. The DNA retained on the beads
or remaining in the supernatant was purified, run on polyacrylamide gel
electrophoresis, and analyzed by autoradiography.
As shown in Fig. 6, DNA restriction by
SacI released a 66-bp oligonucleotide and a 2893-bp fragment
that were easily separated in the gel (compare lanes
1 and 2, without and with restriction). The
control magnetic beads retained ~25% of the large fragment and <2%
of the small fragment (Fig. 6, A and B,
lanes 2 and 3). The
anti-DNA-PKcs beads revealed a very distinct profile of DNA retention with the control nuclear extracts without or with wortmannin; while ~65% of the large fragment was immunoprecipitated in both cases, up to 90% of the small DNA end fragment was retained with wortmannin (kinase preventive condition), in sharp contrast with ~25%, without wortmannin (kinase permissive condition) (Fig. 6, A and B, compare lanes 4 and 5). A mirror profile was observed in the supernatant. In
order to validate these results, the same experiment was performed with
the p460-depleted nuclear extracts. Strikingly, although wortmannin was
present, only ~25% of the small DNA piece was immunoprecipitated by
the anti-DNA-PKcs beads (Fig. 6, A and
B, lane 6). In addition, these beads
retained only 27% of the large fragment under these conditions, close
to the nonspecific binding on the control beads (Fig. 6, A
and B, lane 6). The same experiment
was performed with anti-Ku beads and gave broadly the same result
regarding the small fragment (data not shown), although the yield of
immunoprecipitation was lower, probably due to steric hindrance by the
large catalytic subunit at the DNA end.
These results demonstrate that in the presence of both Ku and p460
subunits, the kinase activity can regulate Ku entry into DNA. Indeed,
when the kinase activity is impaired, DNA-PK as a whole complex stays
bound at DNA ends and under certain conditions, almost all the ends may
bear a DNA-PK complex.
Regulation of DNA Transactions at DNA Ends by DNA-PK Catalytic
Activity--
Having demonstrated that a defective kinase activity
blocked DNA-PK complexes at DNA ends, it was then of interest to
determine the potential enzymatic consequences of such protein/DNA
structures on DNA transactions at DNA ends.
Therefore, nuclear extracts were incubated with linear plasmid DNA and
an excess of closed circular competitor under kinase permissive (+ATP)
or preventive (+ATP, +wortmannin) conditions. After dilution, the
mixture was assayed for enzymatic reactions involving DNA ends.
First, we tested various concentrations of the bacterial exonuclease
III (exo III) on a reaction performed with a labeled linear plasmid,
under mild nuclease conditions. Fig. 7
shows the yield of DNA recovery (upper panel) and
the extent of DNA end degradation (lower panel).
While wortmannin had no effect on exo III activity in the absence of
extracts (data not shown), it partially prevented the
dose-dependent degradation of DNA ends that was observed
with increasing exo III units in the presence of ATP (~3-fold
inhibition of degradation for 0.1 unit with wortmannin).
Second, the polymerizing activity of Klenow fragment of E. coli DNA polymerase I at the ends was assessed in a similar assay by incubating unlabeled linear plasmid with control or
p460-immunodepleted extracts. As shown in Fig.
8A, DNA end filling by Klenow
was drastically impaired in the presence of wortmannin and control
extracts, while this inhibition was partially released by
DNA-PKcs immunodepletion. The unchanged background
incorporation in the open circular species indicates that wortmannin
has no effect on Klenow activity per se.
Finally, the intrinsic DNA ligase activity of the extracts on the
linear plasmid substrate was tested under similar conditions (Fig.
8B). Control and DNA-PKcs-immunodepleted
extracts exhibited the same ligation profile of the linear DNA in the
absence of wortmannin, leading to high molecular weight DNA species
identified as multimers, as reported previously (49). Strikingly,
wortmannin almost completely abolished this activity in control
extracts while it remained unchanged in the
DNA-PKcs-depleted extracts.
We report here for the first time that the DNA-PK catalytic
activity regulates DNA end processing by mean of Ku entry into the
helix. This conclusion was drawn from several data in experiments with
nuclear extracts from human cells; (i) in the presence of DNA-PKcs, Ku entry into DNA depends on ATP hydrolysis and
is blocked by inhibition of the kinase activity, (ii) inhibition of
DNA-PK catalytic activity results in a stable Ku/DNA-PKcs
complex blocked at DNA ends, (iii) blocked DNA-PK at DNA ends prevents
end processing by either DNA polymerization, degradation, or ligation.
Data of the literature and the present results support a model of the
successive steps for DNA end processing in the presence of DNA-PK (Fig.
9). Ku and p460 assemble at DNA ends (8,
9). Under kinase permissive conditions, interactions between DNA and DNA-PK lead to efficient activation of the kinase, which in turn allows
Ku entry into DNA, assembling of new DNA-PK subunits, and/or further
DNA transactions at the ends. When the kinase is present but not
active, both components of DNA-PK are blocked at the ends, thus
impeding their processing. This model raises several questions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
80 °C.
-32P]dCTP (NEN Life Science Products) for 30 min at
30 °C, followed by chromatography through Sephadex G-50 (Amersham
Pharmacia Biotech) and phenol/chloroform extraction.
-32P]ATP and subsequently annealed together. The
probes were purified by chromatography through Sephadex G-50 (Amersham
Pharmacia Biotech).
competitor linear DNA).
After 1 h at 30 °C, 1/10 of the reaction mixture (1 µl,
equivalent to 2 µg of protein extracts) was incubated in a 10-µl
final reaction volume with the radiolabeled 25-mer DNA probe for 10 min
at 30 °C under standard EMSA conditions as described above. The
samples were electrophoresed and the gel processed as described above.
2 µg of anti-DNA-PK
IgG) in extract dialysis buffer under gentle agitation. The supernatant
was removed over a magnet (Dynal MPC, Dynal). A second depletion was
performed immediately under the same conditions. An aliquot of the
supernatant was assayed for DNA-PK activity and reached <10% of the
initial activity as reported (45). As control, immunodepletions were run in parallel with uncoupled anti-mouse IgG beads (extracts thereafter referred to as mock-treated) or with beads coupled to
anti-XPB mouse monoclonal antibody mAb 1B3 (kind gift from Dr. J. M. Egly, IGBMC, Illkirch, France).
-32P]dCTP (NEN Life Science Products). DNA was
purified as above. The radioactivity incorporation due to the overhang
fill-in was visualized by exposure to a storage phosphor screen
(Molecular Dynamics) followed by processing with a PhosphorImager
(Molecular Dynamics, Storm SystemTM).
RESULTS
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Fig. 1.
Effect of ATP on nuclear Ku DNA binding
activity in a competition EMSA. A, standard EMSA in the
presence of 2 µg of HeLa nuclear extracts and a 25-bp radiolabeled
DNA probe. When indicated, anti-Ku Abs (0.1, 0.3, and 0.6 µg) or
control antibodies (0.6 µg) purified from human sera were added to
the reaction before loading on the gel. The position of the major
retarded band is indicated by the arrow. FP,
position of the free DNA probe. B, competition EMSA under
preincubation conditions with 20 µg of nuclear extracts, with (+) or
without ( ) 10 ng of HindIII-linearized pBS plasmid DNA and
with (+) or without (
) 1 mM ATP as indicated. The
positions of Ku band-shift and of the free probe (FP) are
indicated. C, quantification of competition EMSA in the
presence of increasing amounts of linear pBS DNA, with or without 1 mM ATP as indicated. The percentage of radioactivity in the
Ku retarded band without linear plasmid was set at 100% and was
identical with or without ATP (see also B).
linear DNA). The stimulating effect of ATP on the Ku titration by
linear DNA was detected all over a range of DNA concentration (Fig.
1C) yielding 10-fold for 100 ng of DNA (7% and 65%
remaining binding to the probe in the presence and absence of ATP,
respectively). A concentration of ATP down to 1 µM in the
presence of an ATP-regenerating system was sufficient to promote a
significant stimulation of Ku titration (data not shown).
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Fig. 2.
Effect of ATP on purified Ku DNA binding
activity in a competition EMSA. Competition EMSA in the presence
of 40 ng of recombinant Ku heterodimer, increasing amounts of
HindIII-linearized pBS plasmid and with or without 1 mM ATP as indicated. The positions of Ku band-shift and of
the free probe (FP) are indicated.
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Fig. 3.
Reversion of the ATP-dependent
nuclear Ku DNA binding activity by wortmannin. A,
competition EMSA under preincubation conditions with 20 µg of nuclear
extracts, 5 µM ATP, with (+) or without ( ) 50 ng of
HindIII-linearized pBS plasmid DNA and wortmannin as
indicated. The positions of Ku band-shift and of the free probe
(FP) are indicated. B, quantification of
A. The percentage of radioactivity in the Ku retarded band
without linear plasmid and wortmannin was set at 100%.
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Fig. 4.
Competition EMSA with
DNA-PKcs-depleted nuclear protein extracts.
A, competition EMSA under preincubation conditions with 20 µg of nuclear extracts and 50 ng of HindIII-linearized pBS
plasmid DNA. Reactions containing mock-immunodepleted extracts
(control) or extracts treated under immunoprecipitation
conditions with magnetic beads bearing control (anti-XPB) or 25.4 (anti-DNA-PKcs) monoclonal antibodies were run in parallel.
When indicated, ATP (5 µM) or wortmannin (3 µM) were added to the reaction. The positions of Ku
band-shift and of the free probe (FP) are indicated.
B, competition EMSA under preincubation conditions with 20 µg of immunodepleted nuclear extracts as indicated, without ATP and
increasing amounts of HindIII-linearized pBS plasmid.
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Fig. 5.
Effect of the linear competitor DNA length in
the competition EMSA. A, competition EMSA under
preincubation conditions with 20 µg of nuclear extracts, without or
with 5 µM ATP and in the presence of linear DNA fragments
of various lengths (A, 171 bp; B, 710 bp; C, 1400 bp). The molarity of
DNA ends was adjusted to 200 fmol ( ATP) and 30 fmol
(+ATP). B, quantification of competition EMSA as
in A in the presence of increasing DNA end molarity of the
linear fragments A, B, and C, with or without 5 µM ATP.
The percentage of radioactivity in the Ku retarded band without linear
competitor DNA was set at 100%.
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Fig. 6.
Immunoprecipitation of proteins/DNA end
complexes. A, nuclear extracts (20 µg) were incubated
with 50 ng of end-radiolabeled linear pBS plasmid, 5 µM
ATP, and wortmannin (3 µM) as indicated. Extracts used
were mock-immunodepleted extracts (lanes 1-5)
and DNA-PKcs-immunodepleted extracts (lane
6). Then the reaction mixture was diluted 1/4 in 1×
SacI restriction buffer and incubated without restriction
enzyme (lane 1) or with SacI in excess
(lanes 2-6). The reactions were then incubated
under immunoprecipitation (IP) conditions with magnetic
beads bearing anti-mouse IgG (control) antibodies or 25.4 (anti-DNA-PKcs) monoclonal antibodies. DNA from
the beads and the supernatant was purified and analyzed on 5%
polyacrylamide gel electrophoresis, followed by autoradiography of the
dried gel. Upper panel, DNA fraction retained on the
magnetic beads; lower panel, DNA fraction in the
supernatant. B, quantification of A. The
percentage of DNA retained on the beads versus the total DNA
input was calculated for each lane using the software ImageQuant.
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Fig. 7.
Digestion of DNA ends by E. coli
exo III. After incubation of the end-radiolabeled linear pBS
plasmid (L) with nuclear cell extracts (20 µg) under
competition EMSA conditions, in the presence of ATP, and without or
with wortmannin as indicated, various amount of E. coli exo
III (0.1, 1, or 2 units as indicated) has been added to 1/3 of the
reaction mixtures. Upper panel, visualization of DNA
recovery by agarose gel electrophoresis and ethidium bromide staining
(L, linear pBS plasmid; OC and CC,
open circular and closed circular forms of the competitor plasmid for
unspecific DNA binding activity, respectively). Lower panel,
extent of DNA end degradation by autoradiography of the dried
gel.
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Fig. 8.
Effect of DNA-PKcs on DNA end
processing by DNA polymerization or ligation. A, after
preincubation of the unlabeled linear pBS plasmid (L) under
competition EMSA conditions with 20 µg of mock-immunodepleted
extracts (control) or DNA-PKcs-immunodepleted
extracts, in presence of ATP and without or with wortmannin as
indicated, E. coli Klenow fragment (0.02 unit) was added to
1/3 of the reaction mixtures in presence of
[ -32P]dCTP. Upper panel, DNA recovery by
agarose gel electrophoresis and ethidium bromide staining
(L, linear pBS plasmid; OC and CC,
open circular and closed circular forms of the competitor plasmid for
unspecific DNA binding activity, respectively). Lower panel,
incorporated radioactivity by autoradiography of the dried gel.
B, preincubation was as in A except that the
circular competitor pBS was omitted and linear plasmid DNA, ATP, and
wortmannin concentrations were adjusted as detailed under
"Experimental Procedures." The presence of ligated forms of the
substrate (dimer, trimer, tetramer) were detected by agarose gel
electrophoresis and ethidium bromide staining.
DISCUSSION
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Fig. 9.
Schematic model for the regulation of Ku
entry into DNA by DNA-PK catalytic activity. Parallel
lines figure DNA of variable length. + and refer to
kinase permissive or preventive conditions, respectively. See
"Discussion" for details.
What Might Be The Target of DNA-PK Activity for Ku Entry into DNA?-- Several results argue against a prominent role for Ku phosphorylation; (i) extracts from DNA-PKcs-deficient cells or DNA-PKcs-depleted extracts exhibit normal DNA end binding activity (Refs. 37 and 38, and our results), (ii) mutations in the DNA-PK phosphorylation sites in Ku70 do not modify IR sensitivity in vivo (39), (iii) purified recombinant Ku dimer freely enters DNA as judged by multimerization of Ku on DNA probe in EMSA (50).2 However, Ku cannot be ruled out as a key target of p460 for its entry into DNA since (i) the consequence on IR sensitivity of mutations in Ku80 DNA-PK phosphorylation sites has not been evaluated yet, (ii) the intrinsic Ku DNA-dependent ATPase activity has been reported to be greatly stimulated when Ku was phosphorylated by DNA-PK (23), which could in turn modify its putative helicase activity, (iii) Ku molecules bound to a DNA-PK activating DNA fragment are indeed in a phosphorylated form under kinase permissive conditions.2
Another possible candidate for the target of DNA-PK is p460 itself. Indeed, DNA-PKcs autophosphorylation inactivates the kinase (33), which dissociates from Ku (36). According to our model (Fig. 9), Ku might then enter freely the DNA helix, and other binding, phosphorylation, or processing events could then occur at the free ends.
From our results with nuclear extracts, we cannot exclude another protein as the key target of DNA-PK-catalyzed phosphorylation for DNA end cleaning. Therefore, clarifying the protein phosphorylation events at DNA ends and their consequences deserve further experiments with both purified components of the DNA-PK complex (i.e. Ku and p460). However, the present results with nuclear extracts clearly emphasize an endogenous regulation of DNA end processing by the DNA-PK-dependent control of end blocking or cleaning.
What Is the Fate of p460 Subunit after Dissociation of the DNA-PK/DNA End Complex?-- Some conclusions can be drawn from immunoprecipitations of proteins/DNA end complexes by anti-DNA-PKcs Abs (Fig. 6). While p460 is mostly localized at DNA ends when the kinase is inhibited by wortmannin, it is bound to internal DNA positions under permissive conditions for the kinase activity (since the large DNA fragment is preferentially precipitated over the short end fragment; Fig. 6, lane 4). It has been shown that p460 has low selectivity for DNA ends and can bind to internal sites on DNA, but without activation (29). Whether the internal p460 DNA binding that we observe is Ku-dependent or -independent remains to be established. Nevertheless, this might indicate that either the DNA-PKcs subunit can slide inside the helix together with Ku or that it can reassociate at internally localized Ku dimers. The significance of such internal DNA-PK complexes for DNA-PK functions remains to be determined.
Has Ku Entry into the DNA Helix a Function?-- Ku entry into DNA may serve different role per se. For example, Ku rigidifies DNA structure (48), is able to form a DNA loop by contact of distant Ku dimers (24), and is able to transfer from one DNA molecule to another (25). Alternatively, multiple Ku proteins bound to a single dsDNA may serve the recruitment of additional proteins. Recently, the yeast Ku homolog has been shown to interact with Sir4 protein (51), a regulatory factor in telomere transcriptional silencing that is believed to rely on a condensed heterochromatin-like DNA organization. In addition, it has been demonstrated that Sir proteins are also necessary for Ku-dependent non-homologous end joining (51, 52). The spreading of several Ku dimers possibly integrated in multiprotein complexes on DNA may have various consequences on local DNA metabolism. We have shown that Ku is the major determinant of the inhibition of nucleotide excision repair on linear DNA (49, 53). Strikingly, we found that repair inhibition could be released in the presence of wortmannin.2 This may indicate that repair inhibition in the presence of double strand breaks actually relies at least on Ku binding at internal sites in DNA. An identical inhibitory property of Ku has been described since, in EMSA, purified Ku complex was found to strongly compete with transcription factors for their sequence specific binding (50). In addition, in an in vitro transcription assay, Ku was found to inhibit transcription from linear but not from circular template DNA (50).
What Role May Serve a Stable DNA-PK/DNA End Complex for End Processing?-- This reminds of the model established for poly(ADP-ribose) polymerase in the repair of single-strand DNA breaks. In the presence of NAD, the DNA-bound enzyme automodifies and dissociates; if the enzyme is not released, it actually inhibits DNA repair (54, 55). The transient binding of the enzyme may avoid aberrant recombination initiations at sites of DNA breaks (56). Similarly, a transient DNA-PK/DNA end complex may, for example, protect DNA ends from unspecific degradation or prevent aberrant recombination during the time interval required for downstream events to take place. In addition, this complex might favor end bridging by Ku as described recently (26, 27) by transiently stabilizing Ku location at DNA ends. Conversely, blocking the p460 subunit at the ends might help its postulated role as structural framework (5) by attracting protein actors of subsequent steps for end processing or signaling pathways.
Gu et al. reported recently that wortmannin blocked rejoining of double-strand breaks but also repair of oxidatively modified ends by phosphoglycolate removal in Xenopus egg extracts (57). The authors suggest that a specific DNA-PK-catalyzed phosphorylation event might regulate end-joining pathway in Xenopus egg extracts (57). The Xenopus homologue of DNA-PKcs has been identified (58), and DNA-PK activity has been detected in Xenopus egg extracts (59, 60). Then, our results in human nuclear extracts regarding the impairment by DNA-PK of DNA end enzymatic processing in the presence of wortmannin can fully explain the above results. Indeed, the phosphorylation event required for end processing as suggested by Gu et al. may not be specific for end joining and/or repair but may rather allow end cleaning from blocked DNA-PK as a prerequisite for any end-specific biochemical pathway.
Although under our experimental conditions we visualized a stable
DNA-PK/DNA end complex only by inhibiting the kinase activity, such
complex might exist under physiological conditions. The stability of
this complex may then depend on either the DNA structure involved or
the presence of regulators of DNA-PK catalytic activity. Indeed, DNA
structures that bind Ku without DNA-PK activation have been described
(61-63). For example, efficient processing of hairpin coding ends
during V(D)J recombination requires DNA-PKcs (64, 65).
However, hairpin-ended DNA fails to activate DNA-PK even though DNA-PK
binds hairpin ends (63). Stable interaction of DNA-PK with this DNA
structure might be an important step for its processing or for
downstream signaling events, possibly relying on a non-kinase activity
of the p460 subunit. On the other hand, ionizing radiation-sensitive
cell lines have been established that exhibit a defect in DNA end
joining but have normal Ku and DNA-PK activities in vitro
(67, 68). However, these criteria may not be sufficient under the
standard assay conditions used to conclude that DNA-PK interaction with
DNA is normal, since an abnormal regulation of Ku dissociation from DNA
ends would not be detected. Our model allows prediction of the
existence of other potentially important actors like cellular
regulators of DNA-PK that may be necessary for DNA end processing and
that remain to be identified.
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. T. H. Carter, J. M. Egly, E. M. Tan, and Y. Takeda for their generous gift of antibodies and Dr. M. S. Satoh for the gift of plasmids.
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FOOTNOTES |
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* This work was supported in part by grants from the Association pour la Recherche sur le Cancer, the Ligue Nationale Contre le Cancer, and the Action Radiobiologie 98 from the Ministère de l'Education Nationale de la Recherche et de la Technologie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: 33-561-17-59-33;
E-mail: calsou{at}ipbs.fr.
§ The two first authors contributed equally to this work.
¶ Present address: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université Louis Pasteur de Strasbourg, F-67404 Illkirch Cedex, France.
Supported by a postdoctoral fellowship from the Ligue
Nationale Contre le Cancer.
Supported by National Institutes of Health Grant CA50519.
2 P. Calsou, P. Frit, and B. Salles, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: DBS, DNA double-strand break; IR, ionizing radiation; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK catalytic subunit; EMSA, electrophoretic mobility shift assay; Ab, antibody; mAb, monoclonal antibody; bp, base pair(s); exo, exonuclease; dsDNA, double-stranded DNA.
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