©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of the DNA-dependent Protein Kinase by RNA Polymerase II Transcriptional Activator Proteins (*)

(Received for publication, March 21, 1994; and in revised form, November 9, 1994)

Scott R. Peterson (1)(§) Stephen A. Jesch (1) Thomas N. Chamberlin (1) Arik Dvir (1)(¶) Sridhar K. Rabindran (2)(**) Carl Wu (2) William S. Dynan (1)(§§)

From the  (1)From the Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215 and the (2)Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The DNA-dependent protein kinase (DNA-PK) phosphorylates RNA polymerase II and a number of transcription factors. We now show that the activity of DNA-PK is directly stimulated by certain transcriptional activator proteins, including the human heat shock transcription factor 1 (HSF1) and a transcriptionally active N-terminal 147 amino acid GAL4 derivative. Stimulation of DNA-PK activity required specific sequences in the activator proteins outside the minimal DNA binding domains. The stimulation of DNA-PK activity also required DNA and was greater with DNA containing relevant activator binding sites. Comparison of different HSF binding fragments showed that optimal stimulation occurred when two HSF binding sites were present. Stimulation with HSF and GAL4 was synergistic with Ku protein, another regulator of DNA-PK activity. DNA-PK is tightly associated with the transcriptional template, and an increase in its activity could potentially influence transcription through the phosphorylation of proteins associated with the transcription complex.


INTRODUCTION

Protein phosphorylation is a widespread mechanism for regulation of messenger RNA synthesis(1, 2) . Phosphorylation can affect transcription factors by changing the oligomeric state, the subcellular distribution, the DNA binding activity, or the ability to interact with other components of the transcription apparatus(3, 4, 5, 6, 7) . In addition, phosphorylation of RNAP (^1)II at multiple sites within the C-terminal domain of its largest subunit may facilitate the transition from the initiation to the elongation phase of the transcription reaction(8, 9) .

Among the many protein kinases that phosphorylate the transcriptional apparatus in vitro, the DNA-dependent protein kinase is of particular interest because of its tight association with the transcriptional template and its ability to phosphorylate many proteins that are relevant to transcription, including RNAP II, Sp1, p53, SV40 large T antigen, Myc, Oct-1, Fos, Jun, SRF, and histone H1(10, 11, 12, 13, 14, 15, 16, 17, 18) . The activity of DNA-PK is regulated in a unique way. It is strongly dependent on double stranded DNA, and there is a marked preference for substrates that are colocalized to the same DNA fragment(19, 20, 21, 22, 23) . DNA-PK phosphorylates RNAP II, but only in the presence of transcription factors TBP, TFIIB, and TFIIF(16) , presumably because these factors are required for binding of RNAP II to promoter DNA. DNA-PK has a 350-kDa catalytic component (11, 15) and a separate regulatory component, identical to Ku autoantigen, which recruits p350 into a stable complex on the DNA(16, 21, 22) .

In the current study, we report an additional property of DNA-PK, which is that activity is directly stimulated by certain transcriptional activators in vitro. We find that DNA-PK exhibits behavior consistent with that of a signal transduction kinase, responding to certain proteins by phosphorylating others. This suggests that DNA-PK could be part of a signaling pathway which is initiated by certain transcriptional activators and results in the phosphorylation of other nuclear proteins.


MATERIALS AND METHODS

Protein Purification

DNA-PK was purified from HeLa cells (21) , with the phenyl-Superose step sometimes omitted. GAL4 derivatives were expressed in Escherichia coli and purified as described (24) with minor modifications. Cells were lysed in Buffer A (20 mM Hepes, pH 7.5, 10 µM ZnCl(2), 20 mM 2-mercaptoethanol) containing 0.2 M NaCl and 0.5% Nonidet P-40. After polyethyleneimine fractionation and (NH(4))(2)SO(4) precipitation, material was sequentially chromatographed on DE52 (flowed through in Buffer A with 0.1 M NaCl), heparin-agarose (elution in Buffer A with 0.2-1.0 M NaCl gradient) and Superdex 75 HR16/60 (elution in TZ buffer (0.1 M KCl, 50 mM Tris-HCl, pH 7.9, 12.5 mM MgCl(2), 1 mM EDTA, 10 µM ZnCl(2), 20% glycerol, 20 mM 2-mercaptoethanol)). HSF1 was expressed in E. coli, and lysates were prepared in HEMGN buffer as described(25) . Crude HSF1 was sequentially chromatographed on heparin-agarose (elution in HEMGN with 0.15-0.5 M KCl step), Mono Q HR5/5 (elution in HEMGN with 0.1-0.5 M KCl gradient), and Superdex 200 HR 16/60 (elution in TZ buffer). CTD fusion proteins were expressed in E. coli and purified as described(17) . Sp1CTD contained Sp1 amino acids 527-638 inserted between the GST and CTD domains of GCTD.

DNA Fragments

Experiments with GAL4 used DNA fragments derived from pG(5)E4T, which has five tandem GAL4 binding sites(26) . Experiments with HSF1 used fragments from pHSPDelta50HSE(2), which has two tandem heat shock elements (HSEs). Each of the two HSEs was composed of three nGAAn elements and is expected to bind a single HSF1 trimer(27) . Some experiments with HSF1 also used pHSPDelta50HSE(0), pHSPDelta50HSE(1), pHSPDelta50HSE(4), pHSPDelta50HSE(5), and pHSPDelta50HSE(7), which are similar to pHSPDelta50HSE(2) except that they contain zero, one, four, five, and seven tandem HSEs, respectively. The plasmid pHSPDelta50HSE(0) contained a 52-bp ApaI/SmaI fragment from the pBluescript polylinker in place of the HSF1 binding sites. To prepare other pHSPDelta50HSE(2) derivatives, a BamHI site downstream of the hsp70 coding sequence was first ablated. The plasmid pHSPDelta50HSE(1) was then created by digesting pHSPDelta50HSE(2) with ApaI and BamHI to excise the distal HSE. The resulting ends were filled in using the Klenow fragment of DNA polymerase I, and the plasmid was religated. The plasmids pHSPDelta50HSE(4), pHSPDelta50HSE(5), and pHSPDelta50HSE(7) were constructed by digesting pHSPDelta50HSE(2) with BamHI, which cleaves between the two HSEs, and inserting synthetic oligonucleotides containing additional HSEs. The orientation and the number of inserts were determined by DNA sequencing.

Phosphorylation Assays

Standard phosphorylation reactions contained 50 nM GCTD fusion protein(17) , 0.2 nM of a 440-bp HindIII/EcoRI DNA fragment from pG(5)E4T (26) or a 453-bp KpnI/XbaI fragment of pHSPDelta50HSE(2)(27) , 0.125 µl of the p350 subunit of DNA-PK(16) , 12.5 µM [-P] ATP (8 Ci/mmol), 0.5 times TZ buffer, and activator protein as described in the figure legends, in a volume of 20 µl. Reactions were performed at 30 °C for 30 min.

DNA Binding Assays

GAL4-DNA binding reactions contained 0.5 nMP-labeled 115-bp HindIII/BamHI fragment from pG(5)E4T, 0.5 times TZ buffer, and GAL4 derivatives as described in figure legends, in a volume of 10 µl. Reactions were incubated for 20 min at 25 °C and analyzed by 5% PAGE.

HSF1-DNA binding reactions contained 0.1 nM of a singly endlabeled DNA fragment, as indicated in figure legends. Reactions also contained HSF1 derivatives, as described in the figure legends, and 0.5 times TZ buffer, in a volume of 50 µl. DNase I footprinting was performed as described(28) .


RESULTS

HSF1 and a GAL4 Derivative Stimulate Purified DNA-PK

Initial studies were performed with a 147-amino acid N-terminal GAL4 derivative, GAL4-(1-147). This protein contains a DNA binding domain and adjacent sequences that activate transcription in vitro(24) . We discovered its effect on DNA-PK serendipitously, in the course of studies using GAL4 fusion proteins.

The phosphorylation reactions shown in Fig. 1contained purified DNA-PK catalytic subunit, a DNA fragment with five GAL4 sites and a core promoter, and a model substrate with sequences from the CTD of the large subunit of RNAP II. The basal level of DNA-PK activity in this system was very low, and phosphorylation increased 20-fold in the presence of GAL4-(1-147). Input substrate (CTD(A)) was shifted to a lower mobility form (CTD(0)), indicative of multiple phosphorylation. Although the substrate used here contained a DNA binding domain from the transcription factor Sp1, separate experiments showed that its DNA binding activity was very weak (not shown), and the same substrate without Sp1 sequences responded similarly to activators (see Fig. 3).


Figure 1: Stimulation of DNA-PK by GAL4 derivatives. A, effect of GAL4-(1-147). Phosphorylation reactions contained Sp1CTD fusion protein, purified DNA-PK catalytic subunit, a DNA fragment containing five GAL4 binding sites, and variable amounts (µM) of GAL4-(1-147) activator protein. Radiolabeled products were analyzed by 8% SDS-PAGE and visualized by autoradiography. Arrows indicate the position of the input CTD(A) and multiply phosphorylated CTD(0) forms of the Sp1CTD substrate. B, comparison of GAL4-(1-147) and GAL4-(1-94). Reactions in A and parallel reactions containing GAL4-(1-94) were analyzed using a Molecular Dynamics PhosphorImager. Data were normalized to the amount of radiolabeling in the absence of GAL4 derivatives. C, DNA binding by GAL4-(1-94) and GAL4-(1-147). Radiolabeled DNA probe containing five GAL4 binding sites were incubated with various amounts (µM) of GAL4-(1-147) or GAL4-(1-94) as indicated. Protein-DNA complexes were analyzed by 5% native PAGE and visualized by autoradiography. Arrows indicate probe with five GAL4 binding sites occupied.




Figure 3: Transcriptional activators promote phosphorylation of multiple DNA-PK substrates. A, effect of GAL4-(1-147). Phosphorylation was assayed as described in the legend to Fig. 1except with 20 ng of GCTD fusion protein, 400 ng of HSP 90, or 200 ng of alpha-casein as substrate. Concentrations of GAL4-(1-147) are indicated in µM. Products were analyzed by 10% SDS-PAGE and visualized by autoradiography. Arrows indicate the position of the phosphorylated substrates. B, effect of HSF1. Phosphorylation was assayed as in A, except with concentrations of HSF1 as indicated in nM.



DNA binding assays were performed using GAL4-(1-147) and a subfragment of the G(5)E4T DNA used in the phosphorylation assays. The amount of GAL4-(1-147) required to stimulate the kinase approximately correlated with the amount required to saturate the binding sites in the probe (compare Fig. 1, B and C).

Specific GAL4 sequences outside the DNA binding domain influenced the ability to stimulate the kinase. GAL4-(1-94), which contains only a minimal DNA binding and dimerization domain, showed a reduced ability to stimulate the kinase (Fig. 1B). The difference between the two GAL4 derivatives could not be attributed to differences in DNA binding affinity, as binding approached saturation with the same amount of each GAL4 derivative (Fig. 1C).

Separate experiments confirmed previous reports that GAL4-(1-147) strongly activates transcription in vitro, and GAL4-(1-94) does not(24, 29) . Thus, in vitro transcription and kinase stimulatory activities are both influenced by sequences within a 53-amino acid region of GAL4. The correlation between transcription and kinase stimulatory activities is not perfect; however, since GAL4-(1-94) showed some residual activity in the kinase assay at the highest concentration tested, whereas no activity was detected in an in vitro transcription assay (data not shown).

We next tested the effect of a different factor, the human heat shock transcription factor, HSF1, in similar experiments. Whereas GAL4 had been identified serendipitously, HSF1 was chosen for study because of prior evidence that protein phosphorylation plays a role in the heat shock transcription response. Heat shock increases total CTD kinase activity in cell extracts(30) , and it has been proposed that CTD phosphorylation may be involved in release of RNAP II arrested at heat shock promoters(30, 31) . Recent cytologic and biochemical studies show that phosphorylation of Drosophila RNAP II accompanies heat shock transcriptional induction in vivo(32, 33) .

The phosphorylation reactions in Fig. 2contained bacterially expressed HSF1, which is constitutively active for DNA binding and transcription, a DNA fragment containing two HSF binding sites, and the same CTD model substrate as before. HSF1 increased phosphorylation about 6-fold. The concentration of HSF1 required for maximal stimulation was at least 15-fold lower than with GAL4-(1-147), probably reflecting the higher affinity of HSF1 for DNA. In this experiment, somewhat higher concentrations of HSF1 were required to activate the kinase than to saturate the binding sites in the probe. However, this result appears to be anomalous, as later experiments demonstrated a closer correlation (see Fig. 4). The variability in activation at low concentrations of HSF1 has not been explained, but could reflect nonspecific loss of HSF1 at low protein concentrations, as no carrier protein was present in the assay.


Figure 2: Stimulation of DNA-PK by HSF1. A, effect of wild-type HSF1. Phosphorylation of Sp1CTD fusion protein was analyzed as described in the legend to Fig. 1with variable amounts (nM) of HSF1. Arrows indicate the position at CTD(A), CTD(0), and HSF1. B, comparison of wild type and M3 mutant HSF1. Phosphorylation reactions were performed as in A except with GCTD substrate. Data were analyzed as described in the legend to Fig. 1. C, DNA binding by wild type and mutant HSF1. Radiolabeled probe containing two idealized HSEs was incubated with variable amounts (nM) of HSF1 and HSF1 M3 as indicated. DNase I footprinting was performed as described under ``Materials and Methods.'' Probe DNA was a Kas/DdeI fragment of pUCHSEhsp70 (created by insertion of the KpnI/XbaI fragment of pHSPDelta50HSE(2) into pUC19), singly end-labeled at the DdeI site, downstream of the HSEs.




Figure 4: Dependence of DNA-PK stimulation on HSF1 binding sites. A, schematic diagram of HSF1 binding DNA fragments, constructed as described under ``Materials and Methods.'' Arrows indicate the orientation of HSEs, relative to pHSPDelta50HSE(2). B, phosphorylation of GCTD fusion protein was assayed in the presence of 439-565-bp KpnI/XbaI fragments derived from constructs shown in A. Reactions were performed with variable amounts of HSF1 as indicated. Radiolabeled products were analyzed as described in the legend to Fig. 3. C, binding of HSF1 to DNA fragments derived from constructs used in A. DNase footprinting was as described under ``Materials and Methods.'' Probe DNAs were a BglI/EspI fragments singly end-labeled at the EspI site in the hsp70 gene, downstream of the HSEs.



HSF1 was itself phosphorylated by DNA-PK (Fig. 2A). HSF1 is phosphorylated during the heat shock response in vivo(34, 35) , although the sites of phosphorylation have not been mapped and it is not known if they are the same as are used by DNA-PK in vitro.

As with GAL4, specific sequences in HSF1 were required for stimulation of the kinase. A 202-amino acid derivative, HSF1 M3, contains only the minimal DNA binding and trimerization domains (25) and lacks transcriptional activity(36) . (^2)This mutant failed to stimulate the kinase (Fig. 2B). Although wild type HSF1 had a 2-fold greater affinity for DNA than HSF1 M3 (Fig. 2C), this could not account for the difference in activity, since HSF1 M3 worked poorly even at concentrations sufficient to drive DNA binding to completion.

Additional experiments showed that stimulation of DNA-PK activity is not a universal property of transcriptional activators(36) . It is now recognized that activators fall into a number of classes, which may work by different mechanisms. The acidic class is the best studied. We tested several fusion proteins containing GAL4 sequences joined to the prototypical acidic activation domain from herpes virus VP16. The kinase stimulatory activity of the GAL4-VP16 fusion proteins was no greater than that of GAL4-(1-147) and in some constructs was less, suggesting that the VP16 sequences hindered the ability of the GAL4 domain to activate the kinase(36) . VP16 binds a number of transcription factors in vitro(37, 38) ; its inability to stimulate the kinase underscores the specificity of the activator-kinase interaction.

Activation of the Kinase Is Seen with a Variety of Substrates

It was of interest to determine whether the stimulation of CTD phosphorylation by GAL4-(1-147) and HSF1 was a special case or whether stimulation occurred with other kinase substrates as well. We compared the phosphorylation of GCTD, a model substrate similar to Sp1CTD but lacking the Sp1 sequences, and the weak substrates hsp90 and casein (Fig. 3). Although higher concentrations of hsp90 and casein were needed to obtain equivalent labeling, the effect of the activators was the same as with the CTD substrates. The ability of the activators to stimulate phosphorylation of unrelated substrates suggests that they influence the kinase itself, rather than the conformation or DNA binding properties of a particular substrate.

Activation Is Dependent on DNA Containing Activator Binding Sites

The dependence on activator binding sites was investigated in detail for HSF1. Derivatives of the original two-HSE plasmid were prepared with zero, one, four, five, and seven tandem HSEs binding sites, as diagrammed in Fig. 4A. Fragments from each derivative were tested in a phosphorylation assay (Fig. 4B). In addition, the ability of each fragment to bind HSF1 was determined in parallel DNase footprinting assays, which were performed separately using the same batch of HSF1 (Fig. 4C).

Neither the HSE(0) nor the HSE(1) derivative was effective in promoting DNA-PK activity, despite the fact that HSF1 bound relatively well to the HSE(1) construct. By contrast, HSE(2) was effective in promoting DNA-PK activity. Maximal activation of the kinase appeared to correlate with occupancy of the second HSE (compare Fig. 4, B and C). Together, these data suggest that two DNA-bound HSF molecules are required for a high level of stimulation and therefore that the interaction of HSF and DNA-PK may be cooperative.

Interestingly, HSE(4), HSE(5), and HSE(7) were less able to promote DNA-PK activity than HSE(2). This was unexpected, and the explanation has not yet been determined. It could be that in some instances a long array of tandem sites rendered the DNA less able to assume a conformation required for interaction with the bulky 350-kDa DNA-PK catalytic subunit. The ability to support activation was not critically dependent on the orientation of the HSEs. A construct similar to HSE(4) with the central two HSEs inverted behaved similarly to HSE(4) in a phosphorylation assay (data not shown).

The dependence of GAL4-(1-147) activation on GAL4 DNA binding sites was also investigated, although not in as much detail as for HSF1. Stimulation was 5-fold greater when DNA containing GAL4 sites was used in the reaction, compared with DNA without GAL4 sites. There was also virtually no stimulation when DNA was omitted (data not shown). Taken together, the dependence on factor binding sites, seen with both HSF1 and GAL4, implies that binding of the activators to DNA is necessary to activate the kinase.

Synergistic Effect of Activators and Ku Protein

Previous work has shown that the activity of DNA-PK is regulated by the Ku autoantigen(16, 22) . It was of interest to test the effect of Ku protein in combination with activators. Under the conditions of these experiments, we found that Ku protein stimulated the kinase in a concentration-dependent manner by up to 5-fold. GAL4-(1-147) alone stimulated 20-fold, and Ku and GAL4-(1-147) together stimulated up to 100-fold (Fig. 5). Similar synergy was seen between Ku protein and HSF1 (data not shown). This synergy suggests that Ku protein and the activators may work by different mechanisms. Consistent with this, Ku protein has been shown to work by recruiting the p350 catalytic subunit of DNA-PK to DNA(16, 22) . In similar experiments, GAL4-(1-147) had no direct effect on p350-DNA binding. (^3)


Figure 5: GAL4-(1-147) protein cooperates with Ku protein to stimulate DNA-PK. Phosphorylation of Sp1CTD fusion protein was assayed as described in the legend to Fig. 1A in the presence of variable amounts of Ku protein in the absence (solid bars) or presence (stippled bars) of 1 µM GAL4-(1-147) protein. Products of the reaction were analyzed as described in the legend to Fig. 1, and incorporation of radiolabel into the CTD(0) form was quantitated by Molecular Dynamics and PhosphorImager analysis.




DISCUSSION

In the present study, we have shown that two proteins that activate RNAP II transcription also stimulate the activity of a template-associated protein kinase, DNA-PK. Our findings reveal a previously unrecognized behavior of DNA-PK. DNA-PK acts as a molecular transducer, in that it responds to one kind of signal, the presence of certain DNA-bound activator proteins, by generating another kind of signal, the modification of target proteins by phosphorylation.

Mechanism of DNA-PK Stimulation

Three observations are relevant to the mechanism by which activator proteins stimulate DNA-PK. First, stimulation was seen with all substrates tested (Fig. 3), indicating that the activators interact directly with the kinase, rather than influencing the conformation or DNA binding properties of a single substrate. Second, stimulation is dependent on DNA, and is greatest with DNA containing relevant factor binding sites, suggesting that an activator-DNA complex is required. Finally, the activation is synergistic with Ku protein. As the major function of Ku protein is believed to be to recruit DNA-PK to DNA, it is likely that the kinase is also bound to DNA when the activation occurs. Thus, stimulation of DNA-PK most likely involves a functional interaction between proteins that are each bound to DNA.

Role of DNA-PK in Transcription

The suggestion that DNA-PK may play a role in transcription arises from circumstantial evidence. DNA-PK is associated with the transcriptional template and is regulated by known transcriptional activators. For both GAL4-(1-147) and HSF1, sequences outside the minimal DNA binding domain were required for stimulation of the kinase, although in neither case has a fine scale genetic analysis been performed to identify these sequences precisely or to establish their exact relationship to the transcriptional activation domain.

The effect of HSF1 on DNA-PK is of special interest because of previous suggestions that the heat shock transcription response involves phosphorylation of RNAP II(30, 31, 32, 33) . A protein kinase with the properties of DNA-PK is a strong candidate to be involved in the CTD phosphorylation event known to accompany induction of transcription in vivo in certain organisms(32) . Caution is warranted, however, since several other protein kinases are activated during heat shock, and the situation in vivo may be complex(39, 40, 41) . It is possible that transcription factor-mediated activation of DNA-PK stimulates localized phosphorylation of proteins bound at nearby promoter sites. Alternatively, activation could be a more generalized phenomenon, stimulating phosphorylation of proteins throughout the nucleus. These alternatives have not yet been distinguished experimentally.

There are several plausible mechanisms by which an increase in DNA-PK activity could influence transcription. We emphasize that none of these mechanisms has been definitively tested, and some may operate only in vivo, not with in vitro transcription systems. DNA-PK phosphorylates the CTD of RNAP II, and an increase in CTD phosphorylation could facilitate the transition to the elongation phase of the transcription reaction(42) . A similar model has been invoked to explain the ability of another DNA-bound CTD kinase, c-abl, to enhance GAL4-VP16-mediated transactivation in vivo(43) . Alternatively, DNA-PK phosphorylates a number of transcriptional activator proteins. At certain promoters, one activator may induce phosphorylation of another, perhaps altering the ability of the second activator to interact with the transcriptional machinery. Finally, DNA-PK phosphorylates histone H1(15) , which could affect the ability of histone H1 to repress transcription. To better address which targets are relevant to the physiological function of DNA-PK, it will be helpful to establish systems in which the role of DNA-PK and its different substrates can be studied more directly.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant GM 35866, by an American Cancer Society Faculty Research Award (to W. S. D.), and by the Intramural Research Program of the National Cancer Institute (to C. W. and S. K. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Los Alamos National Laboratory, Los Alamos, NM 87545.

Fellow of the Israel Cancer Research Foundation. Present address: Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.

**
Present address: American Cyanamid Co., Medical Research Division, Pearl River, NY 10965.

§§
To whom correspondence and reprint requests should be addressed: Dept. of Chemistry and Biochemistry, Campus Box 215, University of Colorado, Boulder, CO 80309-0215. Tel.: 303-492-1550; Fax: 303-492-3586.

(^1)
The abbreviations used are: RNAP, RNA polymerase; CTD, C-terminal domain; DNA-PK, DNA-dependent protein kinase; p350, 350-kDa catalytic subunit of DNA-PK; HSF1, human heat shock transcription factor 1; HSE, heat shock element; PAGE, polyacrylamide gel electrophoresis; hsp90, human heat shock protein 90; bp, base pair(s).

(^2)
S. R. Peterson, S. A. Jesch, T. N. Chamberlin, A. Dvir, S. K. Rabindran, C. Wu, and W. S. Dynan, unpublished results.

(^3)
S. A. Jesch and W. S. Dynan, unpublished observations.


ACKNOWLEDGEMENTS

We thank Lisa Stein for nuclear extract preparation and purification of DNA-PK, Mark Kissinger for cell culture maintenance, Steve Triezenberg for providing the GAL4-(1-147) expression vector and samples of GAL4-(1-147) used in initial experiments, Jim Kadonaga for advice and the expression clone for the GAL4-(1-94) protein, Michael Carey for providing the G(5)E4T plasmid, and Rhea-Beth Markowitz and Matthew Galman for comments on the manuscript.


REFERENCES

  1. Hunter, T., and Karin, M. (1992) Cell 70, 375-387 [Medline] [Order article via Infotrieve]
  2. Jackson, S. P. (1992) Trends Cell Biol. 2, 104-109 [CrossRef]
  3. Amster-Choder, O., and Wright, A. (1992) Science 257, 1395-1398 [Medline] [Order article via Infotrieve]
  4. Luscher, B., Christenson, E., Litchfield, D. W., Krebs, E. G., and Eisenman, R. N. (1990) Nature 344, 517-522 [CrossRef][Medline] [Order article via Infotrieve]
  5. Gonzalez, G. A., and Montminy, M. R. (1989) Cell 59, 675-680 [Medline] [Order article via Infotrieve]
  6. Chrivia, J. C., Kwok, R. P. S., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman, R. H. (1993) Nature 365, 855-858 [CrossRef][Medline] [Order article via Infotrieve]
  7. Baeuerle, P. A., and Baltimore, D. (1988) Cell 53, 211-217 [Medline] [Order article via Infotrieve]
  8. Laybourn, P. J., and Dahmus, M. E. (1990) J. Biol. Chem. 265, 13165-13173 [Abstract/Free Full Text]
  9. Arias, J., Peterson, S. R., and Dynan, W. S. (1991) J. Biol. Chem. 266, 8055-8061 [Abstract/Free Full Text]
  10. Jackson, S. P., MacDonald, J. J., Lees-Miller, S., and Tjian, R. (1990) Cell 63, 155-165 [Medline] [Order article via Infotrieve]
  11. Lees-Miller, S. P., Chen, Y.-R., and Anderson, C. W. (1990) Mol. Cell. Biol. 10, 6472-6481 [Medline] [Order article via Infotrieve]
  12. Iljima, S., Teraoka, H., Date, T., and Tsukada, K. (1992) Eur. J. Biochem. 206, 595-603 [Abstract]
  13. Lees-Miller, S. P., and Anderson, C. W. (1991) Cancer Cells 3, 341-346 [Medline] [Order article via Infotrieve]
  14. Bannister, A. J., Gottlieb, T. M., Kouzarides, T., and Jackson, S. P. (1993) Nucleic Acids Res. 21, 1289-1295 [Abstract]
  15. Carter, T., Vancurova, I., Sun, I., Lou, W., and DeLeon, S. (1990) Mol. Cell. Biol. 10, 6460-6471 [Medline] [Order article via Infotrieve]
  16. Dvir, A., Peterson, S. R., Knuth, M. W., Lu, H., and Dynan, W. S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11920-11924 [Abstract]
  17. Peterson, S. R., Dvir, A., Anderson, C. W., and Dynan, W. S. (1992) Genes & Dev. 6, 426-438
  18. Liu, S.-H., Ma, J.-T., Yueh, A. Y., Lees-Miller, S. P., Anderson, C. W., and Ng, S.-Y. (1993) J. Biol. Chem. 268, 21147-21154 [Abstract/Free Full Text]
  19. Walker, A. I., Hunt, T., Jackson, R. J., and Anderson, C. W. (1985) EMBO J. 4, 139-145 [Abstract]
  20. Lees-Miller, S. P., and Anderson, C. W. (1989) J. Biol. Chem. 264, 17275-17280 [Abstract/Free Full Text]
  21. Dvir, A., Stein, L. Y., Calore, B. L., and Dynan, W. S. (1993) J. Biol. Chem. 268, 10440-10447 [Abstract/Free Full Text]
  22. Gottlieb, T. M., and Jackson, S. P. (1993) Cell 72, 131-142 [Medline] [Order article via Infotrieve]
  23. Lees-Miller, S. P., Sakaguchi, K., Ullrich, S. J., Appella, E., and Anderson, C. W. (1992) Mol. Cell. Biol. 12, 5041-5049 [Abstract]
  24. Lin, Y.-S., Carey, M. F., Ptashe, M., and Green, M. R. (1988) Cell 54, 659-664 [Medline] [Order article via Infotrieve]
  25. Rabindran, S. K., Giorgi, G., Clos, J., and Wu, C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6906-6910 [Abstract]
  26. Carey, M., Lin, Y.-S., Green, M. R., and Ptashne, M. (1990) Nature 345, 361-364 [CrossRef][Medline] [Order article via Infotrieve]
  27. Becker, P. B., Rabindran, S. K., and Wu, C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4109-4113 [Abstract]
  28. Dynan, W. S. (1987) Genet. Eng. 9, 75-87
  29. Workman, J. L., Taylor, I. C., and Kingston, R. E. (1991) Cell 64, 533-544 [Medline] [Order article via Infotrieve]
  30. Legagneux, V., Morange, M., and Bensuade, O. (1990) Eur. J. Biochem. 193, 121-126 [Abstract]
  31. Lis, J., and Wu, C. (1993) Cell 74, 1-4 [Medline] [Order article via Infotrieve]
  32. O'Brien, T., Hardin, S., Greenleaf, A., and Lis, J. T. (1994) Nature 370, 75-77 [CrossRef][Medline] [Order article via Infotrieve]
  33. Weeks, J. R., Hardin, S. E., Shen, J., Lee, J. M., and Greenleaf, A. L. (1993) Genes & Dev. 7, 2329-2344
  34. Sarge, K. D., Murphy, S. P., and Morimoto, R. I. (1993) Mol. Cell. Biol. 13, 1392-1407 [Abstract]
  35. Larson, J. S., Schuetz, T. J., and Kingston, R. E. (1988) Nature 335, 372-375 [CrossRef][Medline] [Order article via Infotrieve]
  36. Peterson, S. R. (1993) Phosphorylation of the Carboxyl-terminal Domain of RNA Polymerase II by a DNA-dependent Protein Kinase. Ph.D. thesis, University of Colorado
  37. Stringer, K. F., Ingles, C. J., and Greenblatt, J. (1990) Nature 345, 783-786 [CrossRef][Medline] [Order article via Infotrieve]
  38. Lin, Y.-S., Ha, I., Maidonado, E., Reinberg, D., and Green, M. R. (1991) Nature 353, 569-571 [CrossRef][Medline] [Order article via Infotrieve]
  39. Dubois, M.-F., and Bensaude, O. (1993) FEBS Lett. 324, 191-193 [CrossRef][Medline] [Order article via Infotrieve]
  40. Derijard, B., Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76, 1025-1037 [Medline] [Order article via Infotrieve]
  41. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Ruble, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156-160 [CrossRef][Medline] [Order article via Infotrieve]
  42. Dahmus, M. E., and Dynan, W. S. (1992) in Transcriptional Regulation (McKnight, S. L., and Yamamoto, K. R., eds) pp. 109-128, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  43. Welch, P. J., and Wang, J. Y. (1993) Cell 75, 779-790 [Medline] [Order article via Infotrieve]

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