Template Activating Factor I, a Novel Host Factor Required to ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 14,Issue of May 15, pp. 10582-10587.1993 Printed in U S.A.

Template Activating Factor I, a Novel Host Factor Required to Stimulate the Adenovirus Core DNA Replication* (Received for publication, July 17, 1992)

Ken MatsumotoSBIl,Kyosuke Nagatall**, MichioVis, and Fumio HanaokaS From the $Cellular Physiology Laboratory, The Institute of Physical and Chemical Research (RZKEN), Wako, Saitama 351-01, the §Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo 113, the llMitsubishi Kmei Institute of Life Sciences,Machida, Tokyo 194,and the JJDepartmentof Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Kanagawa 227, Japan

The adenovirus (Ad) genome in virion exists in the form of the Ad core, which is a complex of Ad DNA and viral basic proteins. The Ad core supported only initiation and limited elongation in DNA replication in a cell-free system which was developed for DNA replication of naked Ad DNA. We found that addition of a cytoplasmic fraction from uninfected HeLa cells on the top of the system stimulated Ad core DNA replication, although it had no effect on DNA replication of naked Ad DNA. The factor stimulating Ad core DNA replication, designated as template activating factor I (TAF-I),was purified by successive steps of ammonium sulfate precipitation, column chromatographies on phosphocellulose and Q-Sepharose, and glycerol density gradient centrifugation. SDS-polyacrylamide gel electrophoresis of the purified fraction revealed the presence of proteins with molecular masses of 41 and 39 kDa. TAF-I stimulated both initiation and elongation in Ad core DNA replication. Judging from its behavior in purification steps, TAF-I could be acidic. These findings suggest that TAF-I stimulates Ad core DNA replication by interacting with viral basic core proteins.

Virus genomes are associated with proteins encoded by virus or host cells in order to be packed invirions and protected from nuclease attack in infected cells. The adenovirus (Ad)’ genome is a linear double-strandedDNA of about 36,000 base pairs and in the virion is associated with viruscoded basic proteins VI1 and V and a minor viral protein IVa2 (1, 2). This protein-DNA complex is called the Ad core. It is not clarified whether the viral core proteins remain associated with the Ad genome or are displaced by cellular histones when Ad gene expression or DNA replication occurs in the host cell nucleus (3-5). Ad DNA replication has been studied extensively in both *This workwas supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan and by a grant for “Biodesign Research Program” from RIKEN. Thecosts of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence should be addressed. Tel.: 45-922-1111 (ext. 2566); Fax: 45-923-0372. The abbreviations used are: Ad, adenovirus; TP, terminal protein; Ad DNA-protein, adenovirus DNA-terminal proteincomplex; Ad pol, adenovirus DNA polymerase; pTP, precursor of terminal protein; Ad DBP, adenovirus DNA binding protein; NFI, nuclear factor I; NFII, nuclear factor 11; NFIII, nuclear factor 111; DTT, dithiothreitol; TAFI, template activating factor I; top0 I, topoisomerase I.

infected cells and cell-free systems (6-15). A viral protein, called the terminal protein (TP), is linked covalently to the 5’ terminus o f Ad DNA. Initiation ofAd DNA replication occurs by a protein-priming mechanism at the inverted terminal repetition which defines the origin of replication. A cell-free DNA replication system has been developed and well characterized using the TP. DNA complex (Ad DNA-protein) of human adenovirus type 2 and type 5 (8-13). This system is composed of three virus-coded proteins, Ad DNA polymerase (Ad pol), the precursor of TP (pTP) and Ad DNA binding protein (Ad DBP) andnuclear extracts of uninfected HeLa cells. Three host factors have been identified in the nuclear extracts; nuclear factorI (NFI) is a sequence-specific DNA binding protein that binds to a part of the origin of replication and is essential for initiation of Ad DNA replication (10, 13); nuclear factor I1 (NFII), which has type I topoisomerase activity, is required for elongation in replication (11);nuclear factor 111 (NFIII) is also a sequence-specific DNA binding proteinthat binds to a site in the origin adjacent to the NFI binding siteand stimulatesinitiation of replication (14). Recently, Hay and co-workers (16, 17) developed a cellfree DNA replication system using Ad type 4 and showed that NFI and NFIII are dispensable to Ad type 4 DNA replication. Russell and co-workers (18, 19) reported that the Ad core prepared from Ad virions functioned as templatefor initiation and limited elongation of DNA replication in the celI-free system described above. Here we report a novel host factor present in the cytoplasmic fraction of uninfected HeLa cells that significantly stimulates cell-free Ad core DNA replication. The molecular mechanism by which this host factor stimulates Ad core DNA replication is discussed. MATERIALSANDMETHODS

Cell Extracts and Enzymes-HeLa S3 cells were maintained at 37 “C in suspension culture in RPMI 1640 media supplemented with 5% calf serum. A cytoplasmic fraction and nuclear extracts were prepared as described by Dignam et al. (20). Briefly, uninfected cells were harvested, suspended in hypotonic buffer, and homogenized by 10 strokes of a Dounce homogenizer. After removal of nuclei by lowspeed centrifugation, the cytoplasmic fraction was clarified by recentrifugation at 100,000 X g for 1 h. Protein concentration was determined by the method of Bradford (21) with reagents from Bio-Rad. NFI was purified from uninfected HeLa cells as described previously (10). The single-stranded DNA cellulose fraction of NFI was used in this study. Ad pol, pTP, andAd DBP were prepared from Ad-infected HeLa cells as described (8, 9). Preparation of Templates-HeLa S3 cells at the stage of logarithmic growth were infected with Ad type 5 at a multiplicity of 0.5 and then incubated at 37 “C under 5% CO, in air for 3 or 4 days. When cytopathic effects appeared, the infected cells were harvested and disrupted by 10 cycles of freeze-thawing. Ad virions were purified with centrifugation in CsCl solution and dialyzed against 10 mM Tris. HCI (pH 7.5), 1 mM EDTA. The Ad core was prepared as

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described (18, 22). Briefly, Ad type 5 virions were heated at 56 "C for 45 s in the presence of 0.5% sodium deoxycholate and immediately chilled on ice. The Ad core was then purified by centrifugation in a 15-60%glycerol density gradient in 5 mM Tris.HCI (pH 7.6). Ad DNA-protein was prepared from Ad type 2 virions as described (7). Initiation of Cell-freeAd DNA Replication-Formation of the pTP. dCMP initiation complex was assayed essentially as described (10). Template DNA (2.4 pg/ml) was incubated at 30 "C for 1 h with Ad pol, pTP, Ad DBP, and HeLa nuclear extracts in reaction mixture containing 25 mM Hepes (pH 7.5), 4 mM DTT, 5 mM MgC12, 3 mM ATP, 200 pg/ml bovine serum albumin, 10 pCiof [w3'P]dCTP (3,000 Ci/mmol, Amersham), and 30 mM NaCI. The products were precipitated with trichloroacetic acid and separated by electrophoresis in 10% SDS-polyacrylamide gel.The pTP.dCMP complex was located by autoradiography of the dried gel. Elongation inCell-freeAd DNA Replication-In standard reactions, template DNA (60 ng) was incubated at 30 "C for 2 h with Ad pol, pTP. Ad DBP,andHeLanuclear extracts in 12.5 p1 of reaction mixture containing 25 mM Hepes (pH 7.5). 4 mM DTT, 5 mM MgCI,, 3 mM ATP, 2.5 pg of BSA, 5 mM creatinine phosphate, 20 pg/ml phosphocreatine kinase, 40 p~ each of dATP, dGTP, and dTTP, 4 p~ [cx-~'P]~CTP (5 pCi), and30 mM NaCl(11). Aliquots were assayed for acid-insoluble radioactivity. For analysis of the products by gel electrophoresis,the reactions were terminated by the addition of 100 mM Tris.HC1 (pH 7.5), 12.5 mM EDTA, 150 mM NaCI, and 1%SDS, and the mixture was digested with 100 pg/ml proteinase K at 37 "C for 30 min. The products were extracted with phenol-chloroform and precipitated with ethanol. DNA was digested with 10 units of KpnI at 37 "C for 2 h and separated by electrophoresis in 0.8% agarose gel. The gel was dried and autoradiographed. GlycerolDensityGradientCentrifugation-The TAF-I Mono Q fraction (200 p l ) was loaded on a 15-35% glyceroldensity gradient in buffer H containing 50 mM NaCI andcentrifuged a t 50,000 rpm (Beckman,TLS 55 rotor) for 20 h at 4 "C. The gradient was collected in 20 fractions from the top, and3 pi of each fractionwas assayed for activity to stimulate cell-freeAd core DNA replication. RESULTS

Stimulation of Ad Core DNA Replication-The Ad core was prepared from Ad type 5 virions disrupted by heating in the presence of sodium deoxycholate. The purified Ad core was a complex composedof viral proteinsV and VI1 and Ad genomic DNA (data not shown). First we examined the activityof the Ad core as a template for initiation in the cell-free Ad DNA replication system (Fig. 1). Ad DNA replication is initiated by covalent transfer of the 5'-extreme nucleotide, dCMP, to the OH moiety of the serine residue of pTP. Initiation of replication in the cell-free system can be measured as formation of the pTP.dCMP complex separated by electrophoresis in SDS-polyacrylamide gel. The Ad virion had no activity as template for formation of the pTP. dCMPcomplex, whereas the Ad core functioned efficiently as a template in the cellfree system with Ad pol, pTP, Ad DBP, and nuclear extracts of uninfected HeLa cells. This result is consistent with the reports of Russell and co-workers (18, 19). Next, elongation in Ad core DNA replicationwas examined (Fig. 2). The productsof the DNA replication reaction in our cell-free system were digestedwith KpnI and subjected to agarose gel electrophoresis. With Ad type 5, KpnI-G and -H fragments are located at the endsof the genome DNA where Ad DNA replication starts. As shown in lane 4 of Fig. 2, in this cell-free DNA replicationsystemonlyfaintbands of KpnI-G and -H fragmentswere detected, indicating that the Ad core supported a low level of limited elongation in DNA replication. This result is consistent with thatof Leith et al. (19). Inthissystem,naked Ad genomicDNA, Ad DNAprotein prepared from Ad type 2 virions, functioned as an efficient template for elongation in DNA replication (Fig. 2, lane 1). These results suggest that core proteins inhibit the progression of DNA polymerase with Ad core as a template and may be consistent with the observation by Korn and

10583 E

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FIG. 1. Template activity for initiation of the cell-free Ad DNA replication. Ad virions (lane I ) and the Ad core ( l a n e 2) were assayed for template activityin pTP. dCMP complex formation in 50 p1 of reaction mixture containing 0.003 unit of Ad pol/pTP, 1.05 pg of Ad DBP, and5 pg of protein of HeLa nuclear extracts as described under "Materials and Methods." The products were analyzed by electrophoresis in 10% SDS-polyacrylamide gel. The position of the pTP.dCMP complex (80 kDa) is indicated. Horwitz (23) that the addition of core protein VI1 inhibited cell-free DNA replication of Ad DNA-protein. The cell-free system used here could lack a factor(s) involved in Ad core DNA replication. Possibly in the presence of Ad pol, pTP, and Ad DBP, an early gene product(s) such as E1A proteinis requiredfor Ad core DNA replication. Therefore, we tested nuclear extracts preparedfrom 293 cells, which express Ad type 5 E1A and E1B proteins, in place of those from HeLa cells in thecell-free DNA replication system. Like HeLa nuclear extracts, however, nuclear extracts from 293 cells supported only limited elongation in Ad core DNA replication (data not shown). Therefore, we supposed that some otherhostfactor(s) was required for Ad core DNA replication. This putative factor could be present in the cytoplasmic fraction, and in fact,we found that addition of a cytoplasmic fraction preparedfrom uninfected HeLacells stimulated elongation in Ad core DNA replication (Fig. 2, lanes 5-8). The extent of elongation of replication was proportional to the amount of cytoplasmic fraction added. The band between KpnI-I and-J in lanes5-7 could be a replicative intermediate, because it disappeared in the presence of excess amounts of cytoplasmic fraction. Additionof the cytoplasmic fraction had no effect on DNA replication of Ad DNA-protein, indicating that the stimulatory activity of the cytoplasmic fraction acted in an Ad core-specific manner (Fig. 2, lanes 2 and 3 ) . Purification of the Factor with Stimulatory Activity-We attempted to purify the factor that stimulated Ad core DNA replication from the uninfected HeLa cytoplasmic fraction. Its purification was monitored by measuring stimulation of [32P]dCMP incorporation intoacid-insoluble materials in 1 h in cell-free DNA replication with the Ad core as a template. The cytoplasmic fraction prepared from 2.6 X IO9 uninfected HeLa cells was separated by precipitation with ammonium sulfate. The precipitate with 55-80% of ammonium sulfate was dissolved in buffer H (30 mM Hepes (pH 7.8), 0.5 mM EDTA, 10% glycerol, 1 mM DTT, 0.25 mM phenylmethylsul-

for Adenovirus DNA Replication

Novel Host Factor Required

10584

Ad2 Ad5 core DNA-mot

FIG. 2. Stimulation of elongation of Ad core DNA replication by the addition of HeLa cytoplasmic fraction. Left, Ad type 2 DNA-protein (lanes 1-3) or Ad type 5 core (lanes 4-8) was assayed for the template activity in elongation in DNA replication in 12.5 pl of reaction mixture containing 0.0005 unit of Ad pol/pTP, 0.175 pg of Ad DBP, and 5 pg of protein of HeLa nuclear extracts. The cell-free system was supplemented with 7.5 pg of protein (lane 5 ) . 15 pg of protein (lane 6 ) , 30 pg of protein (lanes 2 and 7), and 60 pg of protein (lanes 3 and 8 ) of uninfected HeLa cytoplasmic fraction. KpnI fragments of Ad type 2 and Ad type 5 DNA are indicated on the left and right of the gel, respectively. Bottom, positions of KpnI fragments in Ad DNA. Right, assay of acid-insoluble radioactivity in aliquots of the reaction mixtures with Ad DNA-protein (open circle) or Ad core (closed circle).

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The purified fractions were subjected to electrophoresis in SDS-polyacrylamide gel and stained with silver (Fig. 4). The FIG. 3. Separation of TAF-I activity by Q-Sepharose col- fractions elutedfrom Q-Sepharose andMono Q columns gave umn chromatography. Flow-through materials froma phosphocel- three bands of proteins with molecular masses of 41, 39, and lulose column were applied to a Q-Sepharose column and eluted as described in the text. The fractions were monitored for Am (broken 32 kDa. Characterization of TAF-I-The TAF-I Mono Q fraction line), concentration of NaCl (solid line),and activity to stimulate Ad was subjected to glycerol density gradientcentrifugation. The core DNA replication (closed circle). material stimulating Ad core DNA replication sedimented at fonyl fluoride) containing 50 mM NaCl, dialyzed against the 4.4 S (Fig. 4, top panel). On SDS-polyacrylamide gel electro41- and 39same buffer, and applied to a phosphocellulose column (5 ml). phoresis of glycerol density gradient fractions, the (Fig. 4). These Material thatwas not absorbed by the columnwas applied to kDa proteinscosedimented with TAF-I activity a Q-Sepharose column (5 ml). The column was washed with two proteins could not be separated by additional chromatowere eluted from 50 mM NaCl and 200 mM NaCl in buffer H and developed graphic steps (data not shown). The proteins SDS-polyacrylamide gel and subjected to a denaturationwith a linear gradient of 200-700 mM NaCl in buffer H. A activmain peakof activity was eluted with400-500 mM NaCl (Fig. renaturation protocol. Results indicated that the TAF-I 3). We named the material in this main peak template acti- ity was associated with a protein(s) having a molecular mass vating factor I (TAF-I). The purification of TAF-I is sum- of about 39 kDa.* When the cytoplasmic fraction was submarized in Table I. The finalyield was about 10% with about jected toglycerol density gradient centrifugationin a low-salt 300-fold increase in specific activity. Peak fractions from the condition (50 mM NaCl), the TAF-I activity sedimented at 7 Q-Sepharose column were combined, dialyzed against buffer S, whereas it sedimentedat 4.4 S in a high-salt condition (500 H containing 50 mM NaCl, andconcentratedon ahigh mM NaCl containing 0.01%Nonidet P-40) (10) like the TAFperformance liquid chromatograph Mono Q HR5/5 column. I Mono Q fraction in the low-salt condition (data not shown The column was washed with 50 mM NaCl and300 mM NaCl and Fig. 4). These results suggest that the protein(s) carrying in buffer H and developed with a linear gradient of 300-700 TAF-I activity in thecytoplasmic fraction is associated with mM NaCl in buffer H. TAF-I activity was eluted with 400- another component(s). 500 mM NaCl. The peak fractions werepooled (Mono Q fraction) and dialyzed against buffer H containing 50 mM * K. Matsumoto, F. Hanaoka, M. Ui, and K. Nagata, manuscript NaCl. in preparation. fractlonnumber

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FIG. 4. Glycerol density gradient centrifugation of TAF-I. Top, TAF-I Mono Q fraction was subjected to glycerol density gradient centrifugation as described under “Materials and Methods.” Fractions were assayed for stimulation of Ad core DNA replication. Marker proteins, phosphorylaseb (8.8 S), bovine serum albumin (4.4 S),and lysozyme (2.1 S)were centrifuged inparallel. Bottom, samples of fractions at each step of TAF-I purification and glycerol density gradient centrifugation weresubjected to electrophoresis in 12.5% SDS-polyacrylamide gel and stained with silver. Molecular weight markers ( M ) were obtained from Bio-Rad (low range).

FIG. 5. Stimulationof the Ad core DNA replicationby TAFI. The Ad core was incubated in the cell-free DNA replication system reconstituted with Ad pol/pTP (0.0025 unit), Ad DBP (0.175 pg), HeLa nuclearextracts (3.5pg of protein), and TAF-I(0.05unit) ( l a n e 1 ). Aphidicolin was added into the reaction tothe final concentration of 10 p~ (lane 2) and 100 p~ (lane 3 ) . The products were digested with KpnI and subjectedto electrophoresis in 0.8% agarose gel. KpnI fragments of Ad type 5 DNA are indicated on the kit of the gel. template in the presenceof TAF-I is replicative. The cell-free system for Ad coreDNAreplication was reconstituted with purified proteins. Using Ad DNA-protein as template, NFI and NFII can replace HeLa nuclear extracts for elongation in DNA replication (11).Moreover, NFII can be replaced by topoisomerase I (topo I) purified from HeLa cells. Thus we reconstituted a cell-free Ad core DNA replication system withAd pol, pTP, Ad DBP, and TAF-I, NFI, and top0 I purified from uninfected HeLa cells (Fig. 6, lune 1). Elongation in DNA replication was observed, although the elongation was limited to about one-third of the Ad DNA from the left and right termini. To determine the extent of elongation, we separated the productsof DNA replication by electrophoresis in alkaline agarose gel. In the complete reaction system, the sizes of the products reached about 10 kilobases (data not shown). Elongation of DNA replication was completely dependent on Ad core as template, Ad pol/pTP, and TAF-I (lams 7, 6, and 2,respectively). Omission of NFI or top0 I resulted in decreases in theefficiencies of initiation and elongation,respectively (lanes 3 and 4 ) . Ad DBP seemed to affect the specificity of initiation (lane 5). These results indicate thatAd core DNA replication is quite similarto well studied Ad DNA-protein DNA replication except for dependence on TAF-I(11). Finally, we examined the effect of TAF-I on initiation of Ad core DNA replication (Fig. 7). Formation of the pTP. dCMP complex was dependent on HeLa nuclear extracts in the cell-free system with the Ad core as a template (lane 1 uersus 2). Theaddition of HeLa cytoplasmic fraction or TAFI at each step of its purification stimulated formation of the p T P .dCMP complex (lanes3 and 5-8). This finding indicates that TAF-I stimulated initiation as well as elongation in Ad core DNA replication.

T o assess that the DNA synthesisobserved in this system is due to the origin-dependent Ad DNA replication, thereaction was performed in the presence of aphidicolin (Fig. 5). Aphidicolin is known as an inhibitor for cellular DNA polymerases a, 6, and e. Aphidicolin does not inhibit the Ad pol itself. However, it inhibits the extensive elongation in the reconstitution system and thereby results in replicating terminal regions containing theorigin of the Ad DNA replication (11). The addition of purified TAF-I to the Ad core DNA replication system composed of Ad pol, pTP, Ad DBP, and HeLa nuclear extractsrevealed the nonrandom incorporation of [32P]dCMP into the Ad type 5 DNA KpnI fragments(lune 1).The KpnI-G and -H fragments which locate a t both ends of Ad DNA were labeled with the highest intensity. Other internal KpnI fragments werelabeled with the intensities depending on the length of the fragments and the distance from the endof Ad DNA. In the presenceof aphidicolin, the DISCUSSION incorporation into the internal fragments was significantly reduced. In contrast,aphidicolin did not inhibit the synthesis Ad DNA replication is the first case among eukaryotic DNA of the terminal fragments (lunes 2 and 3 ) . This agrees with with which a cell-free replication system was established (24, the previous results that the elongation of Ad DNA replication 25). Similar cell-free DNA replication systems have been is limited by the addition of aphidicolin (11).These results developed from several viruses using naked DNA as template. indicate that DNA synthesis induced on the Ad core DNA In thecase of sV40, minichromosomes prepared from infected

Novel Host Factor Required

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A 1 2 3 4 5 6 7

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FIG. 7. Stimulation of initiation of Ad core DNA replication by TAF-I. The Ad core was incubated with Ad pol/pTP (0.00375 Iunit) andAd DBP (0.26 pg) in the absence(lane I ) or presence (lanes 2-8) of HeLa nuclear extracts (3.5 pg of protein) described under “Materials and Methods.” The reaction was supplemented with the JHeLa cytoplasmic fraction (lane 3 ) , the cytoplasmic fraction precipitated with ammonium sulfatea t 55% saturation (lane 4 ) or 55-8075 saturation (lane 5),the TAF-Iphosphocellulose fraction (lane 6 ) ,the TAF-I Q-Sepharose fraction (lane 7 ) , or the TAF-I MonoQ fraction (lane 8).The fractions added in lanes 3 and 5-8 all contained 0.1 unit FIG. 6. Requirements with purified proteins for elongation of TAF-I activity. The autoradiogram in R is part of that in A after in Ad core DNA replication. The Ad core was incubated in the longerexposure. Radioactivities of the bands of the pTP.dCMP cell-freeDNAreplication systemreconstitutedwith Ad pol/pTP complex were quantified using the Bio Image analyzer (Fujix BAS (0.0005 unit), Ad DBP (0.17 pg), and TAF-I (0.05 unit), NFI (0.008 2000). Relative radioactivities normalized by the value of lane 2 as unit), and top0 I (100 units) purified from HeLa cells (lane I ) . The 100% were as follows: 0% (lane I), 100% (lane 2), 518% (lane 3 ) , indicated factors (lanes 2-6) or Ad core (lane 7) were omitted from 43% (lane 4 ) , 572% (lane 5), 617% (lane 6 ) ,511% (lane 7), and 571% the reaction. The products were digested with KpnI and subjected to (lane 8). electrophoresis in 0.8% agarose gel. KpnI fragments ofAd type 5 DNA are indicated on theleft of the gel.

the cytoplasmic fraction on biochemical fractionation. cells support DNA replication in a cell-free system composed With the conventional cell-free Ad DNA replication system of the same setof proteins asused for naked DNA replication using Ad type 2 or 5 DNA-protein as template, three host (26). The present resultsprovide the firstevidence that a host factors required for replication have been isolated from unfactor, in addition to the proteins required for replication of infected HeLa nuclear extracts(10, 11, 13, 14). Two of these, naked DNA template,isrequired for DNAreplication of NFI and NFIII, are sequence-specific DNA binding proteins template complexed with basic proteins in a nucleosome-like that bind to the sites in the origin of replication (12, 14). structure. They have also been shown to be transcription factors that We found that the cytoplasmic fraction of uninfected HeLa activate or repress a number of cellular and viral transcripcells stimulated Ad core DNA replication ina cell-free system tional promoters (27-30). The other host factor, NFII, has composed ofAd pol, pTP, Ad DBP, and uninfected HeLa type I topoisomerase activity that is required for elongation nuclear extracts. The factor stimulating cell free DNA repli- in Ad DNA replication (11).Further characterizationof NFII cation of the Ad core was purified from uninfected HeLacells. has not yet been performed, but the involvement of type I The Mono Q fraction at the final step of purification of this topoisomerase in Ad DNA replication in host cells has been factor contained three proteins withmolecular masses of 41, demonstrated recently (31, 32). The cell-free system for Ad 39, and 32 kDa asjudged by SDS-polyacrylamide gel (Fig. 4). core DNA replicationwas reconstituted withpurified proteins We named the stimulatory factor TAF-I. Glycerol density of three viral replication proteins and NFI, top0 I, and TAFgradient centrifugation of the purified fraction showed that I purified from uninfected HeLacells. NFI and top0I stimuthe 41- and 39-kDa proteins cosedimented with the stimula- lated initiation and elongation, respectively, in DNA replicatory activity. We have not been able to separate these two tion. When TAF-I was omitted, no detectable elongation of proteins by any chromatographic procedure tested so far, so DNA replication was observed. In the complete reaction systhese two proteins may be related or associated with each tem, the extent of elongation was limited to about one-third other. Denaturation-renaturation protocols of the proteins of the Ad DNA. Another factor(s) may be required for the recovered from the gel revealed that the TAF-I activity cor- synthesis of full-length DNA with the Ad core as template. responded mainly to protein of 39 kDa.2 These two proteins Alternatively, transcription of early genes may stimulate Ad are relatively abundant; from the results on its purification, DNA replication. With Ad DNA-protein as template, addition the 39-kDa protein is present at 1 X IO6molecules/cell. TAF- of the HeLa cytoplasmic fraction to thecell-free system had I was purified from the cytoplasmic fraction, but it is unclear no effect or reduced the background (Fig. 2, lanes 2 and 3 ) . whether it locates in the cytoplasm or nucleus in HeLa cells, Purified TAF-I had a similar effect (data not shown). These because proteins that function in the nucleus, such as Ad pol results indicate that TAF-I has activity distinct from those of and Ad DBP sometimes (9), are recovered in the three host factors identified already. TAF-I activity was

Novel Host Factor Required

for Adenovirus D N A Replication

not absorbed to phosphocellulose and was eluted from a QSepharosecolumnwith 400-500 m M NaC1. Theseresults suggest that TAF-I is a negatively charged acidic protein(s), thereby interacts with basic core proteins rather than Ad DNA. The Ad core is a protein.DNA complex composed of viral majorbasic proteins V and VI1 and the Ad genome DNA covalently linked with TP. Upon infection, the Ad genome DNA should enter the hostcell and reach the nucleus in the form of the Ad core. Dery et al. (3) concluded from digestion of Ad-infected nuclei with micrococcal nuclease and hybridization with virus-specific probes that Ad DNA is associated with cellular histones during the early phase of infection. In contrast, Corden et al. (4) concluded from micrococcal nuclease digestion that Ad DNA in nuclei of host cells is complexed with core proteins rather than cellular histones. In either case, Ad core proteins should be dissociated from Ad DNApriortoearly gene expressionorDNA replication. Protein VI1 inhibits DNA replication and transcription in cell-free systems with Ad DNA-protein and Ad naked DNA (23, 33). TAF-I is supposed to activate the Ad core for DNA replication by stimulating dissociation of core proteins from Ad DNA, although the significance of TAF-I in infected cells is unclear. The cellular functions of TAF-I also require study. Severalproteinsthatfacilitate nucleosomeassembly have been identified in Xenopus and mammaliancells (34-36), but nucleosome disassembly activity has not yet been studied. It is possible that TAF-I is involved in alteration of the cellular nucleosome structure.Examination of theinvolvement of TAF-I in theAd infection cycle should shed light on a novel mechanism of virus-hostinteractionas well asstructural change of nucleosomes during gene replication and transcription. Acknowledgments-We thank Dr. Yukio Ishimi for providing HeLa topoisomerase I and for helpful discussions. We also thank Drs. Akihiko Kikuchi and Masayuki Seki for encouragement and Yuko Kat0 for preparing the manuscript.

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