©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of DNA Transcription by the Replication Factor from the Adenovirus Genome in a Chromatin-like Structure (*)

Ken Matsumoto (1) (2), Mitsuru Okuwaki (3), Hiroyuki Kawase (3), Hiroshi Handa (3), Fumio Hanaoka (1), Kyosuke Nagata (3)(§)

From the (1) Cellular Physiology Laboratory, the Institute of Physical and Chemical Research (RIKEN), Saitama 351-01, (2) Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194, and the (3) Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Kanagawa 227, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Adenovirus (Ad) genome DNA is complexed with viral core proteins in the virus particle and in host cells during the early stages of infection. This DNA protein complex, called Ad core, is thought to be the template for transcription and DNA replication in infected cells. The Ad core functioned as template for DNA replication in the cell-free system consisting of viral replication proteins, uninfected HeLa nuclear extracts, and a novel factor, template activating factor-I (TAF-I) that we have isolated from uninfected HeLa cytoplasmic fractions. The Ad core did not function as an efficient template in the cell-free transcription system with nuclear extracts of uninfected HeLa cells. The addition of TAF-I resulted in the stimulation of transcription from E1A and ML promoters on the Ad core. TAF-I was required, at least, for the formation of preinitiation complexes. These observations suggest that, in addition to factors essential for transcription on naked DNA template, the factor such as TAF-I needed for replication of the Ad core is also required for transcription from the Ad genome in a chromatin-like structure.


INTRODUCTION

In general, the transcription level of an individual gene is regulated by the quantity and the combination of a variety of transcription factors. However, the kinetical alteration of transcription pattern, for instance, in the early development or in the cell cycle progression, is thought to be dependent on changes of not only transcription factors but also the template DNA structure. Namely, the amount of active transcription factors in nucleus and the accessibility of the transcription factors to the regulatory regions on DNA play important roles in gene regulation. It has not been well known how the genes that are transcriptionally repressed presumably by forming tight chromatin structure are activated. Recently, the existence of mechanisms of the direct access of the transcription factor to the repressed chromatin by nucleosome displacement is suggested (reviewed by Adams and Workman (1) ). Accordingly, the recent suggestion that acetylation of core histones exerts an influence on the accessibility of chromatin to the transcription factor (2) is of interest.

Adenovirus (Ad)() is a double-stranded DNA virus of which DNA replication and transcription have been studied extensively as a good model of the eukaryotic system. The Ad genome DNA in the virion associates closely with viral basic proteins VII and V in nucleosome-like structure (3, 4) . It is reported that the Ad genome DNA is associated with protein VII in the nuclei of infected cells (5) , suggesting that the Ad core is the bona fide template for replication and transcription in infected cells. Russell and co-workers revealed that the Ad core can act as template for the initiation of Ad DNA replication in a cell-free system, although the elongation of DNA replication is quite limited to only to 0.1 the length of the genome (6, 7) . In the course of experiments to test the possibility that the Ad core is the functional template for early transcription and DNA replication in cell-free systems, we found that a novel host factor, template activating factor I (TAF-I), greatly stimulates the elongation of DNA replication using the Ad core as template in the cell-free system consisting of viral replication proteins and uninfected HeLa nuclear extracts (8) .

We have been studying the correlation between the regulations of Ad DNA replication and transcription in static ways (9, 10, 11) . Here we developed a cell-free transcription system using the Ad core as template and examined the effect of TAF-I on transcription from promoters in the Ad core. TAF-I, which is essential for DNA replication of the Ad core, also stimulated transcription from Ad E1A and ML promoters on the Ad core in the cell-free transcription system. Furthermore, transcription from the ML promoter in the Ad core was further stimulated when DNA replication of the Ad core proceeded prior to the transcription reaction. Ad transcription units can be divided into early and late genes, based on their timing of expression after infection. Thomas and Mathews (12) have revealed that the Ad late gene products appear in the infected cells only after Ad DNA replication. Therefore, this system is promising for the analysis of the molecular mechanism for activation of transcription of the genes in the Ad core, as one of models for the activation of repressed genes.


MATERIALS AND METHODS

Templates

The Ad core was prepared by heating Ad type 5 virions in the presence of 0.5% sodium deoxycholate and purified as described elsewhere (8) . Ad DNA-prot was prepared from Ad type 2 virions as previously described (13) . Plasmid pLA1 (6572 base pairs) contains 3290 base pairs of Ad type 5 DNA left terminus (14, 15) . pSmaF is a plasmid-containing SmaIF fragment of Ad type 2 (16) . Plasmid DNAs were prepared from transformants by the alkali-SDS method and purified by CsCl centrifugation (17) .

Denaturation-Renaturation Protocol of TAF-I

TAF-I fraction eluted from MonoQ column chromatography (8) was electrophoresed in 12.5% SDS-polyacrylamide gel. The gel was excised, and the proteins were eluted and renatured under conditions as described elsewhere (18) . To remove guanidine HCl, the proteins were dialyzed against 10 m M Tris-HCl, pH 7.8, 0.1 m M EDTA, 10% glycerol, and 0.1 m M dithiothreitol. Renatured proteins were assayed for TAF-I activity using the cell-free Ad core DNA replication system (8) . The Ad core (60 ng of DNA) was incubated with Ad pol/pTP, Ad DBP, uninfected HeLa nuclear extracts, and the renatured fractions in 12.5 µl of reaction mixture containing 25 m M Hepes, pH 7.5, 4 m M dithiothreitol, 5 m M MgCl, 5 m M creatine phosphate, 20 µg/ml phosphocreatinekinase, 200 µg/ml bovine serum albumin, 20 m M NaCl, 40 µ M each dATP, dGTP, and dTTP, 4 µ M dCTP, 5 µCi of [-P]dCTP, and 3 m M ATP at 30 °C for 2 h. The products were digested with KpnI and electrophoresed on 0.8% agarose gel. The gel was dried and subjected to autoradiography.

Cell-free Transcription

Nuclear extracts were prepared from uninfected HeLa cells according to the method described by Dignam et al. (19) . Cell-free transcription using HeLa nuclear extracts was performed essentially as previously described (9) . After the transcription mixture was incubated at 30 °C for 1 h, the transcripts were analyzed by primer extension. Primers PE1A (5`-GGCAGATAATATGTCTC-3`, nucleotide sequence at positions 577-561 of Ad type 2) for E1A transcript, and PML (5`-AATATCAAATCCTCCTCGT-3`, 6158-6140) or PML2 (5`-GCATCACCGCGGGCCAGGTGAATATCAAAT-3`, 6178-6149) for transcript from ML promoter were end-labeled. The purified RNAs were incubated at 65 °C for 10 min with the primers in 10 µl of reaction mixture containing 50 m M Tris-HCl, pH 8.3, 75 m M KCl, 3 m M MgCl, 10 m M dithiothreitol, and 1 m M each 4 dNTPs, followed by gradual cooling to room temperature. The primer annealed with transcript was extended by 80 units of Moloney murine leukemia virus RNase H free reverse transcriptase (Life Technologies Inc.) at 42 °C for 1 h. The products were precipitated with ethanol and electrophoresed on 8% polyacrylamide gel in the presence of urea. The gels were dried and subjected to autoradiography.

Coupled Cell-free System

For replication prior to the transcription reaction, the Ad core (480 ng of DNA) was incubated with Ad pol/pTP, Ad DBP, TAF-I, and HeLa nuclear extracts (7 µg of protein) in 40 µl of reaction mixture as above at 30 °C for 2 h, except that [-P]dCTP was omitted and the concentration of dCTP was raised to 40 µ M. For transcription, the Ad core which had been subjected to the replication reaction was mixed with HeLa nuclear extracts (50 µg of protein) in 80 µl of reaction mixture under the conditions suitable for transcription by the addition of MgCl(final concentration, 7.5 m M), creatine phosphate (final concentration, 7.5 m M), RNase inhibitor, GTP, CTP, and UTP. The final concentration of ATP was lowered to 1.5 m M, since higher concentration of ATP inhibits transcription (10) . After the mixtures were incubated at 30 °C for 1 h, the transcripts were analyzed by primer extension as the section above.


RESULTS

Stimulation of the Ad Core DNA Replication by TAF-I in the Cell-free System

Recently we found that the elongation of DNA replication in a cell-free system using the Ad core as template was greatly stimulated by the addition of cytoplasmic fractions prepared from uninfected HeLa cells. We purified the stimulatory factor of the Ad core DNA replication from HeLa cytoplasmic fractions (8) . The SDS-polyacrylamide gel of the fraction of the final purification step revealed proteins with molecular mass of 41, 39, and 32 kDa (Fig. 1 A). In order to identify the protein(s) that carries the stimulatory activity of the Ad core DNA replication, the proteins that were eluted from the gel and renatured were assayed for the complementing activity for the cell-free Ad core DNA replication system (Fig. 1 B). The stimulatory activity was detected in the fractions 1, 2, and 3 and the maximal activity existed in the fraction 2. The SDS-polyacrylamide gel of the eluted proteins revealed that the 39-kDa protein is commonly present in the fractions 1, 2, and 3 (Fig. 1 C). Therefore, we concluded that the activity is mainly associated to the 39-kDa protein and designated this activity as TAF-I, although it is possible that the 39-kDa protein is a product of proteolysis from the 41-kDa protein. Fig. 1 E shows the elution profile of the purified TAF-I through a Superose 2PC column. Proteins with molecular mass of 41 and 39 kDa formed a peak corresponding to an apparent molecular mass of about 240 kDa. This observation suggests that TAF-I exists as either hetero- or homohexamer of 41- and 39-kDa polypeptides.


Figure 1: The stimulatory activity (TAF-I) for the Ad core DNA replication purified from HeLa cytoplasmic fractions. A, TAF-I fraction eluted from Mono Q column chromatography (8) was electrophoresed in 12.5% SDS-polyacrylamide gel. The molecular mass of the detected proteins were 41, 39, and 32 kDa from left to right. The gel was excised into five fragments shown by brackets ( 1-5) and the proteins were eluted and renatured. B, the Ad core DNA replication assay was performed in the absence ( lane -) or presence ( lanes 1-5) of the eluted and renatured proteins. The products were digested with KpnI and separated in a 0.8% agarose gel. The fragments of KpnI digestion of Ad type 5 are indicated on the left of the gel. The G and H fragments are located at the ends of the genome DNA in which replication starts (see D). C, the SDS-polyacrylamide gel pattern of the TAF-I fraction ( lane T) and the eluted proteins ( lanes 1-5). Lane M indicates molecular mass markers corresponding to proteins with molecular masses of 43 and 31 kDa. The gel was stained with silver. The common band between 41 and 39 kDa seen in lanes 1-5 is probably derived from the bovine serum albumin fraction added to the all fractions in the renaturation step. D, KpnI fragments of Ad type 5 DNA. E, elution profile of the TAF-I protein through gel filtration. One microgram of the Mono-Q fraction of the TAF-I protein was loaded onto Pharmacia Superose 2PC column equilibrated with a buffer containing 30 m M Hepes-NaOH, pH 7.8, 0.5 m M EDTA, 10% glycerol, and 50 m M NaCl. Aliquots of the indicated fractions ( 7-25) were analyzed by electrophoresis on 10% polyacrylamide gel in the presence of SDS. The gel was stained with a silver stain kit (Bio-Rad). Lane TAF-I indicates the sample before gel filtration. For calibration of the molecular mass, thyrogloblin (670 kDa), -globin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa) were analyzed under the same conditions, and their positions are indicated by arrowheads.



Cell-free Transcription on Cloned DNA in Plasmid

Next, we examined transcription activities from Ad E1A and ML promoters as representatives of early and late promoters in the cell-free transcription system with uninfected HeLa nuclear extracts. Transcription activities on truncated plasmid DNA were compared with those on closed circular plasmid DNA in the cell-free system to examine whether the trans-acting factors which bind to the cis-acting elements in these promoters are active in our system (Fig. 2). The products from the cell-free transcription were analyzed by primer extension.


Figure 2: Transcription activity of the truncated templates. The cell-free transcription using uninfected HeLa nuclear extracts was performed without DNA ( no DNA), with 500 ng of DNA in the form of supercoiled ( ccc) or truncated DNA as shown at the top of the figure as template. Transcripts were analyzed by primer extension using end-labeled primer PE1A ( Panel A) or PML ( Panel B). The products were electrophoresed in 8% polyacrylamide gel in the presence of urea. Lane M indicates HpaII-digested pUC18 DNA as markers. The positions of expected products of accurate transcription from E1A or ML promoter are indicated.



For the Ad E1A promoter, plasmid pLA1 which contains 3290 base pairs of Ad type 5 DNA left terminus was used as the template DNA (Fig. 2 A). When the plasmid DNA was digested with EcoRI which cut at the left end of Ad DNA or with BalI which cut at -231 relative to the transcription initiation site (+1), the transcription activity on these truncated DNAs was similar to that on supercoiled template DNA (Fig. 2 A, lanes 1-3). In contrast, only a slight activity was detected with pLA1 digested with PvuII which cuts at -47 and +124 of E1A promoter ( lane 4). In this case, a TATA box is the only known cis-regulatory element in the upstream of the promoter. These results suggest that the region between the BalI and PvuII sites is essential for the effective transcription from Ad type 5 E1A promoter (20) . Plasmid pSmaF was used for testing the Ad ML promoter activity in the cell-free transcription system (Fig. 2 B). In this case, negatively supercoiled template DNA was the most active among the template structures tested (Fig. 2 B). Linearization by SmaI resulted in decrease of the transcription activity to about a half level of that on supercoiled template DNA (Fig. 2 B, lanes 2 and 3). pSmaF digested with PmaCI showed a faint signal of the transcription from the ML promoter ( lane 4), while ScaI digestion of the plasmid leaded to no signal in primer extension ( lane 5). PmaCI digests at the site recognized by ML transcription factor which is the most important factor involved in transcription of the ML promoter, and ScaI-truncated template does not contain the primer site. These data indicate that nuclear extracts from uninfected HeLa cells contain all the essential transcription factors which support efficient transcription from both E1A and ML promoters, although it has been reported that the Ad infection induces transcription factor(s) which increase(s) the efficiency of the transcription from the ML promoter (21) . Since the primer extension experiment using the primer PML in Fig. 2 resulted in a product even without template DNA ( lane 1), we used another primer PML2 in the latter experiments with which a 140-nucleotide long product is synthesized.

Transcription Activity on Ad DNA Genome

Ad genome is a linear DNA covalently linked to TP at the 5` ends, which is called Ad DNA-prot. It is possible that TPs at the end of genome DNA play roles in transcription from E1A and ML promoters. Thus we next examined the transcription activities from E1A and ML promoters on Ad DNA-prot. As shown in Fig. 3, the transcription on Ad DNA-prot appeared to be as much as that on plasmid DNA, since the apparent level of transcripts in Fig. 3would reflect the molar ratio between the plasmids and Ad DNA-prot used. These results suggest that TP had no effect on transcription activity from Ad DNA.


Figure 3: Transcription activity of Ad DNA-prot. The cell-free transcription was performed with supercoiled plasmid DNAs (300 ng) or Ad type 2 DNA-prot (300 ng DNA) as template. Transcripts were analyzed by primer extension using end-labeled primer PE1A ( Panel A) or PML2 ( Panel B). The positions of expected products of accurate transcription from E1A or ML promoter are indicated.



In contrast to naked DNAs, Ad core functioned only poorly as the template of transcription (Fig. 4 B). Transcription from E1A promoter was observed to be inefficient, whereas ML promoter was virtually inactive on Ad core. It is possible that TAF-I would have an effect on transcription from Ad core as it does stimulate the replication reaction from the Ad core. This was indeed the case as shown in Fig. 4 B. TAF-I stimulated transcription from E1A promoter effectively, and it also activated transcription from the ML promoter on the Ad core less efficiently than that from E1A promoter. TAF-I had no effect on transcription from naked DNAs, both of plasmids, and Ad genome DNA (Fig. 4 A). The data presented so far suggest that the factor required for DNA replication of Ad core facilitates transcription from transcription promoters on Ad core.


Figure 4: Stimulation of transcription from Ad core DNA by template activating factor-I. The cell-free transcription was performed with supercoiled plasmid DNAs (150 ng of pLA1 and 150 ng of pSmaF, lanes 1-3 in Panel A and lane 1 in Panel B), 300 ng Ad type 2 DNA-prot ( lanes 4-6 in Panel A and lanes 2 and 3 in Panel B) or Ad core (300 ng for lane 4 in Panel B and 600 ng for lanes 5-7 in Panel B) in the absence ( lanes 1 and 4 in Panel A and lanes 1, 2, 4, and 5 in Panel B) or presence (0.04 unit, lanes 2 and 5 in Panel A and lane 6 in Panel B; 0.12 unit, lanes 3 and 6 in Panel A and lanes 3 and 7 in Panel B) of TAF-I. Transcripts were analyzed by primer extension as shown in Fig. 3.



To clarify the step affected by TAF-I, the level of the formation of transcription initiation complexes on the Ad core was measured in the presence or absence of TAF-I (Fig. 5). The Ad core was first incubated with the components indicated, and then the remaining components and Sarkosyl, which inhibits the formation of additional initiation complexes but not the process of RNA synthesis from preformed initiation complexes, were added. TAF-I was required during the formation of initiation complexes ( lanes 2 and 3). This step did not need ATP. Therefore, it is suggested that TAF-I is necessary at least for the formation of initiation complexes, although it is unclear whether or not TAF-I is required for the elongation process.


Figure 5: Effect of TAF-I on the formation of initiation complex. The Ad core (300 ng) was first incubated at 30 °C for 30 min in nuclear extracts with 0.04 unit of TAF-I ( lanes 2 and 3) in the presence ( lanes 3 and 5) or absence ( lanes 1, 2, and 4) of ATP. At the zero time ( 0 min) Sarkosyl at the final concentration of 0.025% and the remaining components indicated in the figure were added and further incubated at 30 °C for 60 min. Transcripts were analyzed by primer extension as described above. Lane M indicates size markers. The positions of transcripts from E1A and ML promoters are indicated.



Stimulation of Transcription from Ad ML Promoter by Ad DNA Replication in the Coupled Cell-free System

Ad transcription units can be divided into early and late genes, based on their timing of expression after infection. Thomas and Mathews (12) have revealed that the Ad late gene products appear in the infected cells only after Ad DNA replication. Since transcription from ML promoter on the Ad core template by TAF-I was less than that from E1A promoter, we next tried to reproduce the DNA replication-dependent stimulation of transcription from Ad ML promoter in a cell-free system. Because the reaction conditions of Ad DNA replication disagree with those of the transcription in the cell-free systems (data not shown), we used the two-step reaction in which the DNA replication was performed, followed by the transcription. The Ad core was incubated with the DNA replication enzymes in the presence of TAF-I, dNTPs, and ATP for DNA replication reaction. Then, uninfected HeLa nuclear extracts, GTP, CTP, and UTP, were added to the reaction mixture, and the second incubation was carried out for transcription. The transcripts from E1A and ML promoters were analyzed by primer extension (Fig. 6). Without DNA replication E1A promoter was more active than ML promoter in agreement with results shown in Figs. 4 B and 5 ( lanes 5 versus 6). When the transcription reaction was performed after DNA replication, the transcription from ML promoter was activated ( lanes 1-4). The level of the activation was increased as a function of the amount of TAF-I added in the replication reaction. In the standard replication reaction using 60 ng of Ad core, the amount of TAF-I needed for the saturation level of replication was 0.01-0.02 unit (8) . In the coupled system using 480 ng of Ad core, 0.13-0.2 unit ( lanes 3 and 4) of TAF-I was needed for the maximal transcription from ML promoter. These results suggest that the stimulation of DNA replication by TAF-I acts as the primary mediator of the activation of transcription from ML promoter, and TAF-I does not function independently as an activator. The transcription from ML promoter was repressed to the similar level with reaction without replication, when aphidicholin, which has been shown to be an inhibitor of elongation of the cell-free Ad DNA replication (8) , was added to the replication reaction (data not shown). Thus this cell-free system reproduced the DNA replication-dependent stimulation of late transcription.


Figure 6: The DNA replication-dependent activation of late transcription in the cell-free system. For replication prior to the transcription reaction ( lanes 1-4), the Ad core (480 ng) was incubated with Ad pol/pTP, Ad DBP, and HeLa nuclear extracts in the absence ( lane 1) or presence of 0.07 ( lane 2), 0.13 ( lane 3), and 0.2 ( lane 4) unit of TAF-I at 30 °C for 2 h. For transcription, 480 ng of DNA of the Ad core which had been subjected to the replication reaction ( lanes 1-4), or 480 ng of DNA of the unreplicated Ad core ( lanes 5 and 6) were mixed with HeLa nuclear extracts in the absence ( lane 5) or presence ( lane 6) of 0.13 unit of TAF-I under the conditions suitable for transcription. After the mixtures were incubated at 30 °C for 1 h, the transcripts were analyzed by primer extension with end-labeled PE1A and PML2. The products were electrophoresed on 8% polyacrylamide gel in the presence of urea. The gel was dried and subjected to autoradiography.




DISCUSSION

It has been suggested that the Ad genome DNA complexed with basic proteins (Ad core) is a bona fide template for early stage transcription and DNA replication (5) . Therefore we have first established the cell-free replication system with the Ad core as a template (8) and purified a novel host factor, TAF-I, that stimulates DNA replication of Ad core (Fig. 1). The denaturation-renaturation experiment indicates that a major portion of TAF-I complementation activity corresponds to the 39-kDa peptide, while the 41-kDa peptide shows less activity than the 39-kDa peptide. TAF-I would be an acidic protein, judging from its elution profiles on ion exchange chromatographies during purification (8) . It is possible that TAF-I activates the Ad core for DNA replication by interacting with basic core proteins V and/or VII and by inducing structural change(s) of the Ad core. The same could be true for activation of transcription from the Ad core.

Using Ad core as the template (Fig. 4), a quite low level of transcription from E1A promoter was detected. We could not observe the transcription activity from ML promoter on the Ad core. The addition of TAF-I stimulated the transcription activities from both E1A and ML promoters. The level of stimulation by TAF-I was greater for E1A promoter than for ML promoter. The reason for this difference is presently unclear. In the replication reaction, DNA synthesis is found to proceed from the terminus to the inside depending on the increasing amounts of TAF-I (8) . It is, therefore, presumed that TAF-I could interact with the Ad core from the terminus where the E1A promoter resides. Analysis using Sarkosyl (Fig. 5) indicated that TAF-I is required at least for the step of the formation of initiation complexes. ATP was not needed for TAF-I action. Since TAF-I stimulates two different mechanisms on DNA, i.e. replication and transcription, TAF-I could interact directly with core proteins rather than the mechanisms involved in each reaction. Accordingly, it has been reported that the addition of core protein VII repressed the cell-free DNA replication of Ad DNA-prot (22) and the cell-free transcription of the Ad naked DNA (23) . It is possible that treatment by TAF-I induces a structural change of the Ad core or dissociates the core proteins from DNA and thereby makes the means for replication and transcription accessible to its association sites.

Transcription units of many DNA viruses, including papovaviruses (24) , herpesviruses (25) , and adenoviruses (12) can be divided into early and late genes depending on the onset of their expression before or after DNA replication of the genome, respectively. It has been shown that the activation of transcription from Ad ML promoter is dependent on the Ad DNA replication in infected cells. Thomas and Mathews (12) showed by superinfection experiments that late gene products from secondarily infected viruses are not detected before their own DNA replication even when the host cells are in ``the late stage'' by the first infection. This observation suggests that late stage-specific change(s) of environment, i.e. change(s) in activity of transcription factor(s), is not enough for the onset of transcription of Ad ML promoter. We developed the coupled cell-free system that reproduced the DNA replication-dependent stimulation of transcription from ML promoter using the Ad core as template (Fig. 6). This DNA replicational activation of the late gene transcription was not detected using the naked Ad DNA-prot as template (data not shown). The exact mechanism of the DNA replicational activation of Ad ML promoter transcription remains to be resolved. Replication of the Ad genome and transcription of E1A gene, whose cis-acting elements are present in the terminal region of Ad DNA, are readily activated by the addition of TAF-I. It is hypothesized that transcription factors and/or transcription machineries become accessible to the ML promoter region only when DNA polymerase proceeds. Workman et al. (26) have shown that ML transcription factor present during nucleosome assembly enhances the ability of transcription factor IID for binding to the TATA-box in competition with nucleosomes. Toth et al. (27) have shown that in Ad-infected cells transcription factors ML transcription factor and transcription factor IID bind to Ad ML promoter only in the late stage of infection. Although it is unclear whether the core proteins are associated with Ad DNA after the Ad core DNA replication, the DNA replication should provide the opportunity that the structure of the Ad core is perturbed, as previously suggested for disruption of nucleosome structure by DNA replication (28, 29) .

Recently static and dynamic correlations between DNA-mediated mechanisms have been revealed in, for instance, transcription and recombination (30) and transcription and repair (reviewed by Buratowski (31) ). Mechanisms for activation of genes transcriptionally repressed are not yet well known. Access of transcription factors to such the repressed genes directly (32) or by modifying the chromosomal proteins (2) is suggested. Here, we have proven a novel mechanism of transcription regulation in that a host factor, such as TAF-I, could play a role in activation of transcription of repressed genes, and DNA replication would disrupt the repressed chromatin structure and exert the influence of the accessibility of the chromatin to transcription apparatus. Although much remains to be done, the study using this cell-free system must provide a useful cue for the understanding of transcription regulation not only by changes of transcription factors in quantity and quality but also by the structural change of template DNA.


FOOTNOTES

*
This work was supported in part by the Ministry of Education, Science, and Culture of Japan and by RIKEN. 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.

§
To whom correspondence should be addressed. Tel.: 81-45-924-5798; Fax: 81-45-924-5940.

The abbreviations used are: Ad, adenovirus; TAF-I, template activating factor-I; ML, major late; Ad DNA-prot, Ad DNA associated with terminal protein covalently linked to each 5` end; TP, terminal protein; pTP, 80-kDa precursor to terminal protein; Ad DBP, Ad coded DNA binding protein; Ad Pol, 140-kDa Ad DNA polymerase.


ACKNOWLEDGEMENTS

We thank Dr. A. Ishihama for his support at the initial stage of this work and Drs. A. Kikuchi and Y. Ishimi and our colleagues in RIKEN for useful discussions.


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