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INTRODUCTION |
The DNA-dependent protein kinase is the only protein
kinase that is known to be directly regulated by double-stranded DNA (1). It is composed of a 470-kDa catalytic subunit and a 70-/80-kDa heterodimeric regulatory protein, Ku (2-4). Mammalian cells deficient in either the catalytic subunit of
DNA-PK1 (5-8) or Ku80
(9-13) exhibit severe defects in both DNA double strand break repair
and V(D)J recombination.
Previous studies have shown that several DNA repair proteins also play
a role in RNAP II-mediated transcription. For example, the general
transcription factor TFIIH provides a direct link between nucleotide
excision repair and transcription mediated by RNA polymerase II. TFIIH
is a multifunctional, multiprotein complex whose subunits play critical
roles in repair, transcription, and progression through the cell cycle
(14). In addition, the Cockayne Syndrome group B protein, one of two
proteins critical for transcription-coupled repair, is known to enhance
elongation by RNAP II (15) and has been found physically associated
with RNAP II elongation complexes (16).
There is also some evidence to suggest that the repair enzyme, DNA-PK,
may be involved in regulation of transcription by RNAP II. Both the Ku
protein and DNA-PKcs have been found associated with the mammalian RNAP
II holoenzyme (17) and with isolated preinitiation complexes (18).
Certain transcription factors, including heat shock factor 1, have been
shown to stimulate the activity of DNA-PK in an in vitro
reaction (19, 20). Also, DNA-PK is known to phosphorylate the
carboxyl-terminal domain of the large subunit of RNAP II in
vitro (18). Progression through the transcription cycle is coupled
to changes in phosphorylation of the carboxyl-terminal domain of the
largest subunit of RNAP II (reviewed in Ref. 21), although the specific
role of DNA-PK in this process has not been established.
To investigate directly the involvement of DNA-PKcs and/or Ku protein
in RNAP II-mediated transcription, we have measured in vitro
transcription levels in nuclear extracts prepared from DNA-PK-deficient
CHO cell lines. We found that cells deficient in either DNA-PKcs (V30T)
or Ku80 (xrs-6cvec) consistently show a 2-7-fold decrease in
transcription with a number of promoters. Surprisingly, in
vitro transcription levels cannot be restored to the deficient
nuclear extracts by supplementing with purified catalytic subunit or Ku
protein. Rather, restoration of activity requires a positively acting
factor or complex derived from a DNA-PK-containing cell line. In
addition, we found that the decreased levels of transcription in
DNA-PK-deficient cells lie in a decreased ability to carry out
secondary initiation subsequent to the initial round of transcriptional initiation.
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MATERIALS AND METHODS |
Template DNAs--
Supercoiled plasmids were used as templates
in all experiments. The AdMLP template consisted of the adenovirus
major late core promoter upstream of a 380-base pair G-less cassette.
The hsp70 template consisted of two idealized heat shock elements joined to the hsp70 core promoter, upstream of a 190-base pair G-less
cassette (22). The HTLV-I template consisted of four Tax-responsive
elements joined to the HTLV-I core promoter, upstream of a 380-base
pair G-less cassette.
Nuclear Extract Preparation--
CHO cells were grown as
confluent monolayers in
-minimum Eagle's medium supplemented with
10% fetal bovine serum and 150 mg/liter L-glutamine in 5%
CO2, 95% air at 37 °C. Cells were harvested from 50, 150 × 25-mm culture dishes by scraping into phosphate-buffered saline-containing 5 mM MgCl2. Cells were washed
twice in phosphate-buffered saline/MgCl2 and resuspended in
4 packed cell volumes of lysis buffer (10 mM KCl, 10 mM Tris-Cl, pH 7.9, 1 mM DTT, 60 µM PMSF). Subsequent steps were carried out at 4 °C.
Cells were incubated 20 min on ice with swirling and then lysed by 20 strokes in a Dounce homogenizer. The homogenate was centrifuged at
3000 × g for 5 min, and the supernatant was discarded,
and the pellet was washed in 2 packed cell volumes of lysis buffer. The
washed pellet was resuspended in 4 packed cell volumes of extraction
buffer (50 mM Tris-Cl, pH 7.9, 0.42 M KCl, 5 mM MgCl2, 0.1 mM EDTA, 20% glycerol, 10% sucrose, 2 mM DTT, 60 µM
PMSF), stirred for 30 min, and then centrifuged for 30 min at
17,000 × g.
(NH4)2SO4 (0.33 g/ml) was then
added to the supernatant. The mixture was stirred for 30 min and then
centrifuged for 10 min at 15,000 × g. The pellet was
resuspended in 0.5 original packed cell volumes of TM 0.1 M
(50 mM Tris-Cl, pH 7.9, 0.1 M KCl, 12.5 mM MgCl2, 1 mM EDTA, 20% glycerol,
1 mM DTT, 60 µM PMSF), dialyzed twice against TM 0.1 M buffer, and centrifuged for 10 min at 15,000 × g. The supernatant was collected and protein
concentration determined by Bradford assay (Bio-Rad), using bovine
serum albumin as a standard. HeLa and LTR
3 cells were grown in
suspension in S-minimum Eagle's medium (Joklik-modified) supplemented
with 1% fetal bovine serum, 5% newborn calf serum, 1% nonessential
amino acid supplement, and 100 mM sodium pyruvate in 5%
CO2, 95% air at 37 °C. Cells were harvested and nuclear
extract prepared as described.
In Vitro Transcription--
500 ng of DNA template was incubated
for 30 min at 30 °C with 30-50 µg of the appropriate nuclear
extract in 25 mM Tris-Cl, pH 7.9, 0.05 M KCl,
6.25 mM MgCl2, 0.5 mM EDTA, 10%
glycerol, 0.5 mM DTT, and 20 units of RNasin in a final
volume of 46.5 µl. 3.5 µl of nucleotides was then added to final
concentrations of 250 µM ATP, 250 µM CTP,
12.5 µM CTP, 12.5 µM
3'-O-methyl-GTP and 10 µCi of [
-32P]CTP
and the mixture incubated for another 45 min at 30 °C. In some
reactions, 10 µg/ml heparin was added 2-5 min after the initiation of transcription to prevent further initiation events (23). In some
cases, endogenous GTP permitted extension of transcripts beyond the
G-less cassette. To digest these to a uniform size, 40 units of
ribonuclease T1 was added, and the reactions were incubated for 10 min
at 37 °C. All reactions were stopped by the addition of 150 µl of
0.2 M NaCl, 200 µg/ml tRNA, 0.02 M EDTA, and
1% SDS and 100 µl of 10 mM Tris-Cl, pH 7.9, 1 mM EDTA, and 0.1 M NaCl. The reactions were
extracted with 300 µl of a 1:1 (v:v) mixture of phenol and 96%
chloroform, 4% isoamyl alcohol, and the RNA was precipitated by the
addition of 750 µl of cold ethanol-containing 0.5 M
NH4OAc. The resulting pellets were washed with 500 µl of
70% cold ethanol, dried, and resuspended in 20 µl of 7 M
urea dye. Samples were loaded onto a prerun 5% urea polyacrylamide gel
and electrophoresed at 700 V for 45 min. Incorporated radiolabel was
visualized and quantitated by PhosphorImage analysis (Molecular Dynamics).
Protein Purification--
Recombinant Ku protein was purified as
described previously (20). Native DNA-PKcs was purified from HeLa cell
nuclear extracts as described previously, except that the
phenyl-Superose and Mono S steps were omitted (24). The
/
and
subunits of TFIIA were purified individually using
Ni+-nitrilotriacetic acid-agarose chromatography (25).
TFIIA was further purified by gel filtration on a Superdex 200 column
to isolate assembled complexes from free subunits. Recombinant TFIIB (26) and recombinant TFIIF 30- and 74-kDa subunits (27) were purified
individually by phosphocellulose and gel filtration chromatography. Recombinant TFIIE 34- and 56-kDa subunits were purified as described (28). RNAP II (29) was purified from a HeLa cell nuclear pellet by
DE52, heparin-agarose, and DEAE hydrocell 1000 (Rainin) chromatography. TFIIH was purified from HeLa cell nuclear extracts by phosphocellulose and DE52 chromatography, followed by GST-VP16 affinity chromatography as described previously (30). TFIIH was also purified from HeLa nuclear
extracts using phosphocellulose, DE52, Superdex 200 (in 0.1 M KCl buffer), DEAE hydrocell, phenyl-Superose, and
Superdex 200 (in 0.3 M KCl buffer) chromatography. For both
purification schemes, TFIIH fractions were identified by Western blots
using anti-TFIIH p62 antibody (Santa Cruz) and anti-TFIIH p40 antibody (Zymed Laboratories Inc.). Native TFIID was purified
as described previously (31). The peak from the Mono S column was
further purified on a PureGel strong cation exchange resin. Highly
purified preparations of epitope-tagged affinity purified TFIID (eIID) were obtained from a HeLa cell line expressing an influenza
hemagglutinin peptide-TBP fusion protein. The eIID was purified as
described previously (32) except that the 1.0 M KCl
phosphocellulose fractions containing the eIID complexes were passed
over a column (1 ml of beads per 60 liters of starting volume of cell
culture) of anti-epitope antibody coupled to protein A. After washing,
eIID was eluted with 3 ml of 1 mg/ml influenza epitope peptide.
Fractions containing eIID were identified by Western blots probed with
anti-TBP antibody (Santa Cruz Biotechnology). Individual factors were
assayed for activity by comparing levels of in vitro
transcription in their absence to levels seen in reactions containing
the complete complement of general transcription factors.
DNA-PK Assay--
The kinase activity of DNA-PK was determined
using the SignaTECT DNA-dependent Protein Kinase Assay
System from Promega. In brief, 300 ng of a purified mixture containing
both Ku and DNA-PKcs was incubated with activator DNA, a biotinylated
p53-derived peptide substrate, and [
-32P]ATP in the
presence or absence of the DNA-PK inhibitor, wortmannin. The
biotinylated substrate was then isolated using a streptavidin-cellulose filter. Incorporation of phosphate into the peptide was analyzed by
liquid scintillation counting.
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RESULTS |
In Vitro Transcription--
To investigate the possible
involvement of DNA-PKcs, Ku protein, or both in RNA polymerase
II-mediated transcription, we measured in vitro
transcription levels in nuclear extracts prepared from matched pairs of
DNA-PK-containing and DNA-PK-deficient CHO cell lines. The V30T cell
line is radiation-sensitive and DNA-PKcs-deficient, and AA8 is a
matched wild-type, parental CHO cell line (5, 33). The xrs-6cvec cell
line is radiation-sensitive and -deficient in the 80-kDa subunit of Ku
protein, and xrs-6cKu80 is a matched cell line rescued with the
cDNA encoding the human Ku80 protein (34).
We performed transcription assays using extracts from these cell lines
and a supercoiled DNA template containing either the AdMLP, hsp70, or
HTLV-I promoter incorporated upstream of a G-less cassette. Nuclear
extracts were prepared and stored under identical conditions as
described under "Materials and Methods." Equal amounts of total
nuclear extract protein were added to each transcription reaction.
Representative transcription assays are shown in Fig. 1. In each case, the extracts from
DNA-PK-deficient cells (xrs-6cvec and V30T) showed less transcription
than the matched, wild-type counterparts (xrs-6cKu80 and AA8). Results
from this and four similar, independent experiments are summarized in
Table I. These data show a clear defect
in the transcriptional activity of DNA-PK-deficient nuclear extracts,
ranging from 2.4- to 7.7-fold, when compared with their matched
controls. The defect appears to be general, in that it is observed with
all promoters tested.

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Fig. 1.
A general transcription defect in nuclear
extracts from DNA-PK-deficient cell lines. A, transcription
reactions were performed as described under "Materials and Methods"
using supercoiled DNA templates containing the AdMLP, hsp70, or HTLV-I
promoter linked to a G-less cassette. Reactions contained equal amounts
of protein (30 µg) from nuclear extracts of xrs-6cvec or xrs6c-Ku80
cell lines as indicated. Transcription was performed under basal
conditions without the addition of exogenous heat shock factor 1 or
other activator proteins. Lower panel shows a quantitation
of results with xrs-6cvec and xrs-6c Ku80 nuclear extracts.
B, transcription reactions were performed as in A
using nuclear extracts of V30T and AA8 cell lines as indicated.
Txn, transcription; arb, arbitrary.
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Table I
Fold difference in in vitro transcription levels in extracts of DNA-PK
positive and negative cells
Values are means ± S.D. n = 5 in all cases.
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Overall levels of transcription were generally higher with the hsp70
promoter than with the others, and this promoter also showed the
greatest fold difference between the paired nuclear extracts. Similar
results were obtained with a template containing only the hsp70 core
promoter and lacking the heat shock elements (data not shown).
Nuclear Extract Mixing Experiments--
To determine if the
in vitro transcriptional activity of the DNA-PK-deficient
nuclear extracts could be rescued, a series of mixing experiments was
performed. In these experiments, 7.5, 15, 22.5, or 30 µg of a test
nuclear extract was mixed with 30 µg of the deficient nuclear
extract, and in vitro transcription assays were carried out
using the AdMLP template as described under "Materials and
Methods." Transcription levels of the mixed nuclear extracts were
compared with the appropriate DNA-PK-positive control. Results of these
experiments are shown in Fig. 2. As can
be seen in A and D, addition of xrs-6cKu80
nuclear extract resulted in the complete rescue of in vitro
transcription levels of both xrs-6cvec and V30T nuclear extracts. The
decrease in transcription levels observed in reactions containing
greater than 45 µg of total nuclear extract protein may be due to an
excess of protein. Similarly, addition of AA8 nuclear extract resulted
in the complete rescue of transcription levels in the
DNA-PKcs-deficient V30T nuclear extracts (Fig. 2B). However,
AA8 gave less than maximal restoration in the Ku80-deficient xrs-6cvec
extracts (Fig. 2C). The xrs-6cKu80 cells express a human
Ku80 gene under control of the strong cytomegalovirus
promoter. It may be that expression of this gene at higher than
endogenous levels makes the extracts even more active than would
otherwise be the case.

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Fig. 2.
In vitro transcription levels of
DNA-PK-deficient nuclear extracts can be restored to control levels in
mixing experiments. Transcription reactions were performed in
which variable quantities (7.5-30 µg) of one nuclear extract were
added to a fixed quantity (30 µg) of another nuclear extract. The
template was supercoiled plasmid-containing the AdMLP promoter.
A, titration of xrs-6cKu80 extract added to xrs-6cvec
extract. B, titration of AA8 extract added to V30T extract.
C, titration of AA8 extract added to xrs-6cvec extract.
D, titration of xrs-6cKu80 extract added to V30T extract.
E, titration of V30T extract added to xrs-6cvec extract.
F, titration of xrs-6cvec extract added toV30T extract.
Txn, transcription; arb, arbitrary.
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We have also seen that V30T extracts failed to rescue xrs-6cvec nuclear
extracts (Fig. 2E), despite the fact that these cells represent different genetic complementation groups (XRCC5
versus XRCC7) (35). There was greater, but still incomplete,
rescue when the experiment was performed in the opposite direction,
with xrs-6cvec extracts added to V30T. These results suggest that both mutant cell lines lack a common factor, not DNA-PK itself, the absence
of which results in decreased levels of in vitro
transcription. This was confirmed by the experiments described in the
next section.
In Vitro Transcription Assays Supplemented with Purified
DNA-PK--
Initially, we attempted to restore transcription levels to
the DNA-PK-deficient nuclear extracts by the addition of purified recombinant Ku and DNA-PKcs purified from HeLa cells. However, we
observed no increase in transcription levels in the presence of these
purified proteins (Fig. 3). Similar
results were obtained when proteins were added individually over a wide
range of concentrations (data not shown). It is possible that the human
Ku70 subunit or DNA-PKcs subunit is not capable of functioning in the
hamster cell lines, explaining the lack of complementation between
species. However, the amino acid sequences of DNA-PK components are
relatively conserved between species (reviewed in Ref. 36). Also,
previous studies have shown that cross-species complementation occurs
between human and yeast Ku70 (37) and between human and mouse DNA-PKcs (38). Thus it is likely that human DNA-PK components can function in
hamster cells.

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Fig. 3.
Purified DNA-PK does not restore the in
vitro transcription levels of DNA-PK-deficient nuclear
extracts. Transcription reactions were performed using nuclear
extract from DNA-PK-deficient (xrs-6cvec, V30T) or DNA-PK-containing
(xrs-6cKu80, AA8) cell lines. Amount of protein from each extract is
indicated in µg. Additional proteins, purified as described under
"Materials and Methods," were added as indicated. Template was
supercoiled plasmid-containing the AdMLP promoter. A,
addition of recombinant Ku protein and purified PKcs amounts to
xrs-6cvec nuclear extract. B, addition of recombinant Ku
protein and purified PKcs amounts to V30T nuclear extract.
Txn, transcription; arb, arbitrary.
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Assuming that human DNA-PK can, in principle, function in hamster
cells, then the failure to rescue transcription by the addition of
purified DNA-PK suggests that the defect may not be directly attributable to the absence of Ku or DNA-PKcs from the extract but
rather to the absence of some other common factor. This could be an
individual protein that lies downstream of DNA-PK in a signaling pathway or a complex of proteins that fails to assemble in the absence
of Ku and DNA-PKcs. When this positively acting factor or complex is
supplied to the DNA-PK-deficient cell lines, there is a complete
restoration of in vitro transcription levels.
Transcription in the Presence of the DNA-PK Inhibitor
Wortmannin--
Since DNA-PK has intrinsic protein kinase activity, it
was of interest to know whether ongoing phosphorylation activity of endogenous DNA-PK contributed to the higher level of transcription seen
with extracts from DNA-PK-containing cells. The catalytic subunit of
DNA-PK falls into the phosphatidylinositol 3-kinase family, and its
activity has been shown to be effectively inhibited by approximately
250 nM of the phosphatidylinositol 3-kinase inhibitor wortmannin (39). If the enzymatic activity of DNA-PK is responsible for
the higher levels of in vitro transcription observed in the DNA-PK-containing nuclear extracts, one would expect that the difference in transcription levels between xrs-6cKu80 and xrs-6cvec nuclear extracts would be eliminated in the presence of wortmannin. Transcription reactions were performed in the presence of various concentrations of wortmannin, with results shown in Fig.
4. The approximately 2-fold difference in
transcription levels between the paired nuclear extracts was maintained
throughout a range of 0 to 1000 nM wortmannin. In control
experiments, we show that the enzymatic activity of purified DNA-PK is
almost completely abolished at a concentration of 500 nM
wortmannin (Fig. 4C).

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Fig. 4.
Effects of the DNA-PK kinase inhibitor,
wortmannin, on transcription by xrs-6cvec and xrs-6cKu80 nuclear
extracts. A, transcription reactions were performed
using 50 µg of either xrs-6cvec or xrs-Ku80 nuclear extracts, the
hsp70 G-less cassette template, and wortmannin as indicated.
B, quantitation of data in A. C,
DNA-PK kinase assays were performed as described under "Materials and
Methods." Reactions contained 300 ng of a purified mixture containing
both DNA-PKcs and Ku as well as the indicated concentrations of
wortmannin. Txn, transcription; arb,
arbitrary.
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We conclude from these experiments that ongoing DNA-PK enzymatic
activity is not required for transcription. Consistent with this, we
have compared linear and circular templates under the conditions of our
assays, and we find that the difference between extracts from
DNA-PK-positive and -negative cells is maintained in both cases (data
not shown). It is established that linear DNAs are much more effective
than circular DNAs in stimulating DNA-PK phosphorylation activity (40,
41). The finding that both types of templates give equivalent results
is one more line of evidence that ongoing enzymatic activity of DNA-PK
does not contribute to the difference between extracts.
In Vitro Transcription Assays Supplemented with Purified
Transcription Factors--
Because the transcriptional defect of the
DNA-PK-deficient cell lines occurred at all promoters tested, one
possibility was that the missing component was a general transcription
factor. We attempted to rescue transcription by the addition of
purified transcription factors to our in vitro transcription
assays. Recombinant TFIIA, recombinant TFIIB, recombinant TBP,
epitope-tagged TFIID, recombinant TFIIE, recombinant TFIIF, RNAP II,
and TFIIH were purified and assayed for activity as described under
"Materials and Methods." No single factor, when added in the
amounts indicated (Fig. 5A),
was capable of restoring transcription levels in xrs-6cvec nuclear
extract to the levels seen in the xrs-6cKu80 control. Transcription was
also not restored by addition of a combination of purified
transcription factors (Fig. 5B). Thus, the observed decrease
in transcription levels of DNA-PK-deficient nuclear extracts cannot be
attributed to limiting amounts of a general transcription factor.

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Fig. 5.
Purified general transcription factors do not
restore the in vitro transcription levels of
DNA-PK-deficient nuclear extracts. Transcription reactions were
performed using nuclear extract from DNA-PK-deficient (xrs-6cvec, V30T)
or DNA-PK-containing (xrs-6cKu80, AA8) cell lines. Amount of protein
from each extract is indicated in micrograms. Additional proteins,
purified as described under "Materials and Methods," were added as
indicated. Template was supercoiled plasmid containing the AdMLP
promoter. A, addition of either 10-15 ng of recombinant
TFIIA, 7 ng of recombinant TFIIB, 3 ng of recombinant TBP, 1-2 ng of
epitope-tagged TFIID, 50 ng of the 56-kDa subunit plus 30 ng of the
36-kDa subunit of TFIIE, 30 ng of the 58-kDa subunit plus 15 ng of the
26-kDa subunit of TFIIF, 50-100 ng of RNA polymerase II, or 0.5-1
µl of TFIIH. B, addition of same purified transcription
factors as in A in combination. Txn,
transcription; arb, arbitrary.
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Most of the transcription factors used in this experiment were from
recombinant sources and may lack post-translational modifications. Therefore, we cannot at present rule out the possibility that DNA-PK
induces some post-translational modification in vivo
(e.g. phosphorylation) that is responsible for the higher
levels of transcriptional activity seen in the DNA-PK-containing cell lines.
Primary Versus Secondary Initiation--
The RNAs that are
synthesized in vitro can be divided into two categories:
those that result from "primary" initiation events that occur
immediately upon addition of ribonucleoside triphosphates and those
that result from "secondary" initiation events that occur later in
the course of the reaction. These secondary events are sometimes
referred to as reinitiation events because they may reflect reuse of
templates or RNAP II. However, secondary initiation events can also
occur for other reasons, for example, if there is template or RNAP II
that is sequestered in nonproductive complexes at the time of
ribonucleoside triphosphate addition.
Experimentally, primary and secondary initiation events can be
distinguished by the addition of the sulfated polysaccharide heparin
immediately following the initiation of transcription. Heparin, in low
concentrations, has been shown to prevent the formation of new
initiation complexes without disrupting transcriptionally active
complexes already present on the template (23).
In order to determine whether the xrs-6cvec nuclear extracts are
defective in primary or secondary transcriptional initiation, the
transcription assay illustrated in Fig. 6
was performed. Both the rate of PIC formation and the number of
productive PICs formed were measured by varying the length of time that
the nuclear extract was incubated with template prior to the addition
of ribonucleoside triphosphates. The ability of the extracts to undergo
secondary initiation events was monitored by comparing the level of RNA synthesis in the presence and absence of heparin.

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Fig. 6.
Differences in transcription levels between
xrs-6cvec and xrs-6cKu80 extracts occur at the level of multiple round
transcription. Transcription reactions were performed using 30 µg of nuclear extract prepared from xrs-6cvec or xrs6c-Ku80 cell
lines. Extract was preincubated (Preinc.) with 500 ng of the
hsp70 supercoiled template at 30 °C for the indicated times.
Ribonucleoside triphosphates were added, followed after 5 min by 10 µg/ml heparin. This allowed the initiation rate for a single round of
transcription to be measured. Transcription was also carried out in the
absence of heparin to allow comparison of single versus
multiple round transcription.
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These experiments produced a clear but unanticipated result. When
transcription was limited to primary initiation events by the addition
of heparin, little or no difference was observed in the ability of
either nuclear extract to initiate transcription (Fig. 6; compare
intensity of bands at 30 min preincubation in presence of heparin). The
rate of preinitiation complex formation and the number of productive
complexes formed were similar in both extracts. In fact, preinitiation
complex formation was actually higher in the xrs-6cvec extracts when
compared with xrs-6cKu80 extracts. In contrast, the two extracts
differed considerably in levels of transcription in the absence of
heparin. This difference could be solely attributed to the
approximately 4-fold greater ability of the xrs-6cKu80 extracts to
carry out secondary initiation.
Trapping of RNA Polymerase II by G-less Cassette Template--
In
order to better understand the mechanistic differences between primary
and secondary initiation events in the xrs-6cvec versus the
xrs-6cKu80 nuclear extracts, we have taken advantage of a technique
first described by Szentirmay and Sawadogo (42), and later refined by
Lei et al. (43) that involves "trapping" of the RNA
polymerase II at the end of a G-less cassette by the addition of high
concentrations of the chain terminator 3'-O-methyl GTP
(O-Me-G). As can be seen in lanes 3 and
8 of Fig. 7A,
addition of O-Me-G to a final concentration of 500 µM is sufficient to compete with endogenous GTP present
in the nuclear extracts, preventing elongation beyond the G-less
cassette, and resulting in the formation of stalled RNAP II complexes.
The majority of these complexes can be chased to yield long RNAs by the
addition of GTP (Fig. 7B).

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Fig. 7.
Direct comparison of xrs-6cvec and xrs6c-Ku80
transcription when RNA Pol II is "trapped" by a G-less cassette
template. A, transcription reactions were performed
using 50 µg of either xrs-6cvec or xrs-6cKu80 nuclear extract and the
hsp70 G-less cassette template in the absence or presence of 500 µM O-Me-G. Readthrough RNAs were trimmed to
the size of the G-less cassette by the addition of RNase T1. Single
round transcription was measured by the addition of 10 µg/ml heparin
to some reactions. B, GTP chase-transcription reactions
using 50 µg of xrs-6cKu80 extract were carried out as described above
in the presence of 500 µM O-Me-G followed by a
30-min chase with 2 mM GTP.
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This approach allows one to identify whether multiple rounds of
transcription are limited by the number of active templates, by the
concentration of active RNAP II, or by some other kinetic phenomenon.
If reinitiation of transcription is occurring via the reuse of
templates, the presence of stalled RNAP II forces subsequent RNAP II
molecules to pile up at the end of the G-less cassette resulting in a
ladder of shortened transcripts at intervals of approximately 30 nucleotides. We do not observe such a ladder in the presence of
O-Me-G in either the xrs-6cvec (lane 3) or xrs-6cKu80 (lane 8) nuclear extracts. Therefore, few, if
any, templates are being reused in our assays. Consequently the
differences in observed transcription levels cannot be directly
attributed to differences in the ability of the two nuclear extracts to
recycle template.
Likewise, if the recycling of RNAP II and/or tightly associated
transcription factors is required for later rounds of transcription, trapping of RNAP II at the end of the G-less cassette should inhibit new initiation. Although there is some inhibition of transcription with
O-Me-GTP (compare lanes 2 and 3 and
lanes 7 and 8), it does not ablate the difference
between xrs-6cKu80 and xrs-6cvec nuclear extracts (lanes 3 and 8) nor does it reduce transcription to the level seen in
the presence of heparin (lanes 5 and 10). This
suggests that whereas RNAP II and/or some tightly associated factor is partially limiting in the reaction, this limitation is not responsible for the difference of in vitro transcription levels between
these two nuclear extracts. This result is consistent with our earlier findings that addition of purified RNAP II does not rescue
transcription in the Ku-negative nuclear extract. Thus the difference
between extracts falls into the category that Lei et al.
(43) described as "kinetic limitation." The Ku80 nuclear extracts
must contain an activity that promotes multiple rounds of transcription
but this activity is not RNAP II itself.
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DISCUSSION |
We present the first definitive evidence that nuclear extracts
prepared from cells that are deficient in DNA-PK, a protein primarily
known for its essential role in repair of DNA double-strand breaks, are
also impaired in their ability to carry out basal transcription
in vitro. The effect was seen with extracts of both Ku80-deficient and DNA-PKcs-deficient cells. The relative decrease in
transcription levels was always greater for the Ku80-negative nuclear
extracts than for the DNA-PKcs-negative extracts. This suggests that Ku
protein may have a greater or perhaps more direct role than DNA-PKcs in
transcriptional regulation.
Mixing experiments show that the impaired transcription is due to the
lack of a positively acting factor or complex of factors in the
deficient extracts. We have yet to identify this positively acting
factor. Our inability to restore transcription by the addition of
purified DNA-PK suggests that the role of DNA-PK in RNAP II-mediated transcription may be either less direct or more complex than we had
initially anticipated.
Interestingly, it has been reported that nuclear extracts from
DNA-PK-deficient cells also show a modest (2-fold) decrease in
nucleotide excision repair activity. As with the transcription defect,
the excision repair defect could not be restored by purified DNA-PK
protein components (44). It remains to be established whether the
transcription and nucleotide excision repair defects have any common
mechanistic basis.
We have shown that the Ku-negative nuclear extracts are not defective
in their ability to carry out single round transcription. Rather, the
observed difference in in vitro transcription levels between
Ku80-positive and Ku80-negative nuclear extracts can be attributed
solely to the ability of the Ku80-containing nuclear extracts to
undergo multiple rounds of transcription.
The role of secondary initiation, or reinitiation, in the overall
levels of RNA synthesis has been overshadowed by the tremendous effort
required to obtain our basic understanding of how primary initiation
takes place. However, there is every reason to expect that the process
of reinitiation is also a highly regulated step. Recently Oelgeschlager
et al. (45) have shown that in HeLa cell nuclear extracts,
TAF(II)s (general transcription co-factors that associate with the
TATA-binding protein of TFIID and play a central role in RNAP II
transcriptional regulation) (reviewed in Ref. 46) actually impair
functional PIC assembly but elevate absolute levels of transcription by
facilitating secondary initiation events. Activators of transcription
such as heat shock factor and the estrogen receptor have also been
shown to selectively enhance reinitiation (45, 46).
Reinitiation may be enhanced by recycling promoter and/or RNAP II and
other transcription related proteins more efficiently (47). For
example, a promoter might be "marked" as competent for reinitiation
during the primary initiation event. Our RNAP II trapping experiments
indicate, however, that under our conditions templates are in
functional excess and are not being recycled. Similarly, whereas we did
find that the concentration of RNAP II may be partially limiting in
both extracts, recycling of RNAP II was not required in order to
observe the difference in transcription levels between Ku80-negative
and Ku80-positive nuclear extracts.
Our results thus fall into the category of "kinetic limitation"
described by Lei et al. (43). In this respect, it is
interesting that the multiple round transcription defect in
DNA-PK-deficient extracts resembles that observed by these workers with
carboxyl-terminally truncated forms of the 74-kDa subunit of TFIIF
(43). One possibility is that the effect of DNA-PK on multiple round
transcription is mediated through phosphorylation of TFIIF in the
carboxyl-terminal region. The activity of the 74-kDa subunit of TFIIF
is known to be regulated by phosphorylation (48). TFIIF is
phosphorylated by the TAFII 250-associated protein kinase (49), but it
is not known if other kinases also contribute to its phosphorylation. It will be of interest to determine whether the pattern of TFIIF phosphorylation changes in the presence and absence of DNA-PK in
vivo.
Alternatively, DNA-PK could affect transcription by phosphorylation of
a different general transcription or through interactions with an
unknown or novel protein. This protein could facilitate active
reloading of transcription factors at new promoters or releases
nonproductively loaded factors from DNA. The extraordinarily large
DNA-PK holoenzyme could serve as a nucleus for assembly of a
multiprotein complex that carries out these functions.
One of the important questions that remains to be answered, in addition
to the identity of the missing factor, is whether DNA-PK-deficient
cells are impaired in their ability to carry out transcription in
vivo. In this respect, it is interesting to note that certain
transcriptional activators are capable of binding to and stimulating
the activity of DNA-PK (19). This interaction could provide a mechanism
for increasing the level of reinitiation at specific promoters
regulated by these factors. Moreover, mice lacking Ku 80 (50) or Ku 70 (51) genes are proportional dwarfs. A defect in the general
transcription machinery is one possible explanation for this phenotype.