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a reconstituted in vitro system, using a plasmid containing the first 40 bp of the ..... serted between the EcoRI and Hindu sites of pBR327. The plasmid has an AluI ...
The EMBO Journal vol.4 no.6 pp.1515-1521, 1985

Recognition site of nuclear factor I, a sequence-specific DNAbinding protein from HeLa cells that stimulates adenovirus DNA replication

Peter A.J.Leegwater, Wim van Driel and Peter C.van der Vliet Laboratory for Physiological Chemistry, State University of Utrecht, Vondellaan 24 a, 3521 GG Utrecht, The Netherlands

Communicated by H.S.Jansz

Nuclear factor I is a 47-kd protein, isolated from nuclei of HeLa cells, that binds specifically to the inverted terminal repeat of the adenovirus (Ad) DNA and enhances Ad DNA replication in vitro. We have studied the DNA sequence specificity of nuclear factor I binding using cloned terminal fragments of the Ad2 genome and a set of deletion mutants. Binding of nuclear factor I protects nucleotides 19-42 of Ad2 DNA against DNase I digestion. Filter binding assays show that deletion of the first 23 nucleotides does not impair binding while a deletion of 24 nucleotides reduces binding severely. However, binding studies on Adl2 DNA indicate that nucleotide 24 can be mutated. Fragments containing the first 40 bp are bound normaily while the first 38 bp are insufficient to sustain binding. Taken together, these results indicate that the minimal recognition site of nuclear factor I contains 15 or 16 nucleotides, located from nucleotide 25 to nucleotide 39 or 40 of the Ad2 DNA. This site contains two of the four conserved nucleotide sequences in this region. Sequences flanking the minimal recognition site may reduce the binding affinity of nuclear factor I. In accordance with these binding studies, DNA replication of a fragment that carries the sequence of the terminal 40 nucleotides of Ad2 at one molecular end is enhanced by nuclear factor I in an in vitro replication system. Key words: adenovirus/deletion mutants/DNA-binding protein/ DNA replication/nuclear factor I

Introduction The adenovirus (Ad) genome is a 33-36 kbp linear DNA duplex with a 55-kd terminal protein (TP) covalently attached to both 5' ends. The terminal DNA sequences form an inverted repeat (ITR) varying between 103 bp (AdS) and 164 bp (Adl2) in length and contain the origins of DNA replication. The mechanism of DNA replication has been studied extensively since the development of in vitro assays for initiation and elongation (for reviews, see Challberg and Kelly, 1982; Stillman, 1983; Sussenbach and Van der Vliet, 1983). DNA replication initiates at either molecular end by the formation of a covalent complex between the precursor of the TP (pTP) and dCMP, the 5'-terminal nucleotide of all human Ad DNA sequences. A virus-encoded DNA polymerase (pol) catalyzes the initiation reaction and elongates the 3'-OH group of the protein-bound dCMP by a displacement mechanism. Evidence has been presented that the pTP binds to nucleotides 9-18 of the ITR, a sequence that has been conserved in the various human serotypes (Rijnders et al., 1983). Of particular interest are the factors isolated from uninfected © IRL Press Limited, Oxford, England.

HeLa cells that stimulate Ad DNA replication in vitro. So far, two nuclear proteins with such a property have been described (Nagata et al., 1982, 1983a; Rawlins et al., 1984), while stimulation by a cytosolic RNA fraction also has been observed (Van der Vliet et al., 1984). One of the proteins, nuclear factor I, is a 47-kd protein that enhances the initiation reaction significantly (Nagata et al., 1982). Nuclear factor I is a sequence-specific DNA-binding protein that has high affinity for a region of the Ad2 ITR (Nagata et al., 1983b). A similar protein has been described by Rawlins et al. (1984). Footprint analysis has shown that a region between nucleotides 17 and 48 (Nagata et al., 1983b) or 19 and 43 (Rawlins et al., 1984) of the ITR is protected against DNase I attack by nuclear factor I. Studies with deletion mutants have indicated that the region from 15 to 48, containing three blocks of conserved sequences, is necessary for binding (Guggenheimer et al., 1984), but the exact borders of the binding site have not yet been determined. It has been suggested that binding of nuclear factor I facilitates the interaction between the pTP and the conserved site at position 9- 18. We have studied the DNA sequence requirements for binding of nuclear factor I to the Ad ITR in more detail. Using a variety of mutants we show that the recognition sequence of nuclear factor I lies between nucleotides 24 and 40 or 41 and thus constitutes 15-16 bp. This sequence includes two of the four conserved blocks in this region and contains an axis of approximate 2-fold symmetry. In accordance with this result, DNA replication in a reconstituted in vitro system, using a plasmid containing the first 40 bp of the Ad2 genome, is stimulated by nuclear factor I.

Results Nuclear factor I was extracted from uninfected HeLa cell nuclei with 0.35 M NaCl and purified by DEAE-cellulose, phosphocellulose, single-stranded DNA cellulose and hydroxylapatite column chromatography (Nagata et al., 1982; Rawlins et al., 1984). Fractions from the hydroxylapatite column were used to investigate sequence requirements for the binding of nuclear factor I to the adenovirus ITR. Footprint analysis For the determination of the DNase I footprint of nuclear factor I on the Ad2 ITR, the plasmid XD-7 was used. XD-7 is a pBR322 derivative with the left terminal XbaI fragment of Ad2 inserted in the EcoRI site (Pearson et al., 1983, see Figure lA). XD-7 was linearized by digestion with ClaI, 5' end-labeled with 32p and subsequently digested with PvuII. The 0.48 kbp ClaI-PvuII fragment that contains the Ad2 terminal sequence was isolated and partially digested with DNase I in the presence or absence of nuclear factor I. The products were separated by electrophoresis on a denaturing polyacrylamide gel and detected by autoradiography. Without nuclear factor I, a large number of labeled degradation products is observed differing by one nucleotide from each other (Figure 2, lanes 2). Binding of nuclear factor I protected part of the DNA against DNase digestion (Figure 2, lanes 1). From the comparison of the protection pattern with external 1515

P.A.J.Leegwater, W.van Driel and P.C.van der Vliet

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20 30 40 50 10 Xl) -7 aattCATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGG pXdel22 aattCC----------------------------pXde123 aattCC---------------------------pXdc124 aattCC--------------------------pXdel25 aattCC-------------------------p P 1.40 aatt----------------------------------------ATCGTCCATTC pT D)38 aatt--------------------------------------GCTTTAATGCGGT pRICI aattCATGCTATCTA-ATAATATACCTTATACTGGACTAGTGCCAATATTAAAATGAAG

Fig. 1. Plasmids used to investigate the binding of nuclear factor I to the adenovirus ITR. (A) Physical map of XD-7. The left terminal 1343 bp of Ad2 (box) are inserted into the EcoRI site of pBR322 (Pearson et al., 1983). The black box marks the position of the ITR. E = EcoRI, P = Pvull, C = ClaI. (B) Sequences of XD-7, deletion mutants and pRlCl after digestion with EcoRI. Regions that are conserved in human adenoviruses are underlined in the sequence of XD-7. The sequence of pRiCI (Adl2) is from J.L.Bos (personal communication). Small letters indicate the EcoRI protruding ends. ----, Region homologous to XD-7. Nucleotide numbers indicate the positions in the Ad2 DNA sequence.

and internal markers, we conclude that binding of nuclear factor I protects the area from nucleotide 19 to nucleotide 42 of the labeled strand of the Ad2 ITR against nicking by DNase I. This region comprises two blocks of conserved sequences (Figure 7). In all experiments an increase in intensity of the bands at positions 44, 45, 50, 51 was observed (Figure 2, arrows), indicating an increased DNase sensitivity of the region bordering the protected area. The 5' border of the nuclear factor I recognition site Sequence requirements for the binding of nuclear factor I were determined by testing various mutants of the Ad2 origin in filter binding assays with nuclear factor I. In such an assay, in which protein-bound DNA is separated from protein-free DNA by filtration, specific binding of nuclear factor I to the Ad2 origin is easily detected. A set of mutants, with deletions that extend from the Ad2 terminus towards the footprint region of nuclear factor I, was constructed by incubating an EcoRI digest of XD-7 with the exonuclease Bal31 and re-insertion of the shortened fragments into the EcoRI site of pBR322. The deletions obtained by this procedure range from the first 16 bp to the first 55 bp of the Ad2 origin. Relevant sequences of mutants used in this study are shown in Figure lB. XD-7 and the mutants pXdel22, 23, 24 and 25 were prepared for the filter binding assay by digestion with EcoRI and PvuIl and 5' end-labeling of the fragments with 32p. After incubation with nuclear factor I, the fragments were filtered through nitrocellulose filters. The DNA fragments bound by nuclear factor I and retained on the filter were eluted and size-fractionated on a polyacrylamide gel. In Figure 3A the autoradiogram of the gel is shown. As expected, nuclear factor I binds specifically to the 454-bp fragment of XD-7 which contains the Ad2 origin (lane 2). The corresponding fragments of pXdel22 and pXdel23 are also bound by nuclear factor I (lanes 3 and 4), whereas those of pXdel24 and pXdel25 are not detectable (lanes 5 and 6). Similar results were obtained when the plasmids were cut with PstI and Sall (unpublished data). This excludes that the negative binding results with pXdel24 and pXdel25 are due to the absence of duplex DNA flanking the nuclear factor binding site after digestion with EcoRI. The previous results indicate that the dT-dA base pair at position 24 of the Ad2 origin is essential for the binding of nuclear factor I, and cannot be replaced by a dC-dG base pair as in pXdel24 (Figure lB). Such a prerequisite for nuclear factor I 1516

binding to Ad origins is somewhat unexpected, because the Adl2 origin has a dC-dG base pair at 24, while the conserved blocks that are contained within the nuclear factor I footprint have the same position in Ad2 and Adl2 (Figure 1B). Therefore we tested the Adl2 origin in the filter binding assay to further investigate the influence of a dC-dG base pair at 24. The plasmid pRlCl contains the left terminal EcoRI fragment of Adl2 inserted in the EcoRI site of pAT153 (Bos et al., 1981). The plasmid was cut with EcoRI and KpnI, generating fragments of 0.7 kb, 3.7 kb (pATI53) and 4.8 kb. The Adl2 origin is located on the 0.7-kb fragment. The results of the filter binding assays with these fragments and nuclear factor I are shown in Figure 3B. From the comparison of Adl2 (lane 2) with Ad2 (lane 4) it seems that the Adl2 origin fragment binds less effectively than the Ad2 origin fragment. Besides the Adl2 origin fragment, the Adl2 fragment of 4.8 kb is also bound preferentially compared with the vector DNA. This could be due to the presence of two regions (nucleotides 1703 - 1717 and 2332-2346) (Sussenbach, 1984) on the 4.8-kb fragment, that show strong homology to the nuclear factor I binding area of the origin. Such additional nuclear factor I recognition sites on the plasmid might compete with the origin for binding of nuclear factor I, and thereby reduce the binding to the origin fragment. Therefore we compared the binding affinities of nuclear factor I for various DNA sequences in a competition experiment using a purified 0.4-kb EcoRI-PvuH fragment containing the Adl2 origin. Mixtures of a fixed quantity of labeled EcoRI-PvuH fragments of XD-7 and increasing amounts of unlabeled competitor DNA were tested in filter binding assays with nuclear factor I. The amount of label in the bound

fragments was quantitated by scanning the intensity of the bands autoradiogram, and plotted against the amount of competitor DNA. The result (Figure 4) shows that nuclear factor I has an equal affinity for the Adl2 ITR and for the Ad2 ITR. pXdel23 competes like wild-type, while the competition of pXdel24 is only slightly higher than that of pBR322. These data indicate that the dC-dG base pair at position 24 of pXdel24 cannot account for the low affinity of nuclear factor I for this mutant and that other aspects of its sequence prevent binding. Taken together, the results, presented in Figures 3 and 4, locate the 5' border of the nuclear factor I recognition sequence between position 24 and 25 of the ITR. However, sequences flanking this border can modulate the binding, as is the case with pXdel24. on an

Recognition site of nuclear factor I

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Fig. 2. Determination of the DNase I footprint of nuclear factor I on the Ad2 inverted terminal repeat. The 0.48-kb CIaI-PvuH fragment of XD-7, 5' end-labeled with 32P at the ClaI end, was incubated with DNase I in the presence (lanes 1) or absence (lanes 2) of nuclear factor I. The degradation products were denatured and size-fractionated on a 10% polyacrylamide gel with 8.3 M urea. A and B show autoradiograms of the gel with different exposure times. Numbers at the left indicate the distance from the molecular end of the Ad2 ITR sequence. As markers denatured ClaI-MnlI fragments of XD-7 as well as the cleavage products of the G-specific Maxam and Gilbert sequence reaction (Maxam and Gilbert, 1980) were used. Arrows indicate bands with a relatively increased intensity in lanes 1.

3' border of the nuclear factor I recognition site To investigate the right border of the nuclear factor I recognition site we made use of the plasmid pTD38 and of the presence of an MnlI site in the Ad2 ITR at position 40. pTD38 (Lally et al., 1984) contains the first 38 nucleotides of the Ad2 ITR, inserted between the EcoRI and Hindu sites of pBR327. The plasmid has an AluI site at position 39. pTD38 and XD-7 were digested with EcoRI, and the fragments were 5' end-labeled. pTD38 was subsequently digested with AluI and XD-7 with MnlI. Figure 5 shows the results of the binding of nuclear factor I to these digests. The fragment that consists of the first 40 bp of the Ad2 origin is preferentially bound by nuclear factor I (lane 2), whereas the fragment that contains the first 38 bp is not bound detectably (lane 3). The larger 0.4-kb EcoRI-BamHI fragment of pTD38 containing the first 38 bp of Ad2 is also negative for The

Fig. 3. (A) fFilter binding assays with nuclear factor I and Ad2 terminal deletion mutaants. Plasmids XD-7, pXdel22, pXdel23, pXdel24 and pXdel25 were digested with EcoRI and PvulI and the fragments were end-labeled with 32p. The fragments that were retained on nitrocellulose filters after incubation with nuclear factor I were analysed by gel electrophoresis and autoradiography. Lane 1: marker lane with XD-7 fragments; lanes 2-6: filter binding assays with fragments of XD-7 (lane 2), pXdel22 (lane 3), pXdel23 (lane 4), pXdel24 (lane 5) and pXdel25 (lane 6). (B) Binding of nuclear factor I to left terminal DNA fragments of the Adl2 genome. The plasmid pRICI was digested with EcoRI and KpnI. The fragments were end-labeled with 32P and tested in the filter binding assay with nuclear factor I. As a control, the assay was performed in parallel with XD-7 as in A. Lane 1: marker lane with pRICl fragments; lane 2: filter binding assay with pRICl fragments; lane 3: XD-7 marker; lane 4: filter binding assay with XD-7. Numbers at the left indicate the size of the fragments in kb.

binding by nuclear factor I (unpublished data). The relative affinities of nuclear factor I for the first 40 bp and for the intact ITR were compared in a competition experiment. The 40-bp EcoRI-MnlI fragment of XD-7 was subcloned by insertion between the EcoRI and EcoRV sites of pBR322. The plasmid thus constructed, named pPL40, was tested as competitor for binding by nuclear factor I, as outlined above. The result (Figure 4) shows that the affinity of nuclear factor I for fragments that only contain the terminal 40 bp of Ad2 is as high as for fragments containing the complete ITR sequence. This indicates that the 3' border of the nuclear factor I recognition site is located between base pairs 39 and 41 of the Ad2 ITR. Correlation between binding of nuclear factor I and stimulation of adenovirus DNA replication in vitro Plasmid DNAs containing the intact inverted terminal repeat or small deletions can function as templates for DNA replication, provided that they are linearized near the molecular end of the inserted Ad origin (Tamanoi and Stillman, 1982; Van Bergen et al., 1983; Guggenheimer et al., 1984). We have investigated the template properties of XD-7, pTD38 and pPL40 in a reconstituted DNA replication system consisting of the purified viral replication proteins (DNA-binding protein, pre-terminal protein and DNA polymerase), nuclear factor I and cytosol RNA from HeLa cells which stimulates the reaction (Van der Vliet et al., 1984). XD-7 DNA was digested with EcoRI, pTD38 and pPL40 with EcoRI and AvaI, generating fragments of 1.4 kb, 1.4 kb, and 1517

P.A.J.Leegwater, W.van Driel and P.C.van der Vliet 1

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Fig. 4. Analysis of the competition of mutant DNA with plasmid XD-7 for binding of nuclear factor I. The fragments of an EcoRI-PvuH digest of XD-7 were end-labeled with 32P and assayed in the filter binding assay with increasing amounts of EcoRI digests of pBR322 (0), XD-7 (A), pXdel23 (A), pXdel24 (X), pPL40 (l) and the 0.4-kb EcoRI-Pvull fragment of pRlC1 that contains the Adl2 ITR (0). After gel electrophoresis and autoradiography, the amount of the 0.45-kb XD-7 fragment bound by nuclear factor I was quantitated by spectrophotometric scanning of the bands and normalized to the amount that was bound in the absence of competitor DNA.

Fig. 5. Binding of nuclear factor I to fragments containing terminal sequences of the Ad2 genome. DNA fragments of EcoRI-Mnll digests of XD-7 and EcoRI-AluI digests of pTD38, labeled with 32p at the EcoRI ends, were tested in filter binding assays with nuclear factor I. Lane 1: input of XD-7 fragments; lane 2: filter binding assay with XD-7 fragments; lane 3: filter binding assay with pTD38 fragments; lane 4: input of pTD38 fragments. The length of the fragments is indicated in base pairs. Only part of the autoradiogram is shown.

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1.3 kb, respectively, containing the origin at the molecular end. The digests were incubated with the purified replication components in the presence of [32P]dCTP as the labeled nucleotide triphosphate and the reaction products were analyzed by SDSagarose gel electrophoresis and autoradiography (Figure 6). Ad DNA replication can be observed by the appearance of a pTPcontaining labeled fragment which migrates more slowly than the protein-free DNA fragment (Figure 6, see arrow). Some labeling of the vector and the insert is also observed. This is presumably due to repair synthesis caused by the associated 3'-5' exonuclease activity of the Ad DNA polymerase (Field et al., 1984). However, this repair process does not significantly contribute to the label in the replication product. Figure 6 shows that replication of XD-7 DNA containing the intact ITR is stimulated 10-fold by nuclear factor I. The same stimulation is observed with pPL40 while replication of the pTD38 fragment is not stimulated at all. Thus, the minimal recognition sequence is required both for binding of nuclear factor I and for its stimulating effect on DNA replication.

Discussion Nuclear factor I, first described by Nagata et al. (1982) is one of the few sequence-specific DNA-binding proteins from higher eukaryotes detected so far. The experiments presented here define a minimal sequence of 15-16 nucleotides for binding of nuclear factor I to double-stranded Ad2 DNA. This recognition com25 28 34 38 prises two conserved blocks, TGGA and GCCAA, separated by 5 bp. It contains an axis of approximate 2-fold symmetry passing through the GC base pair at position 31 (see Figure 7). 1518

Fig. 6. The effect of nuclear factor I on the replication of DNA fragments containing different terminal sequences of the Ad2 genome. The fragments of an EcoRI digest of XD-7 and of EcoRI-AvaI digests of pPL40 and pTD38 were tested as templates for replication with purified components in the presence and absence of nuclear factor I. Lanes 1 and 2: replication of fragments of XD-7; lanes 3 and 4: pPL40; lanes 5 and 6: pTD38. Incubations of lanes 1, 3 and 5 were without nuclear factor I, of lanes 2, 4 and 6 with nuclear factor I. Bands indicated by arrows represent the replicated, pTP-containing DNA fragments.

Recognition site of nuclear factor I protected against DNase I

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5'- C A T C A T C A A T A A T A T A C C T T A T T T T G G A T T G A A G C C A A T'A T G A T A A T G A G G G G G T A G T A G T T A T T A T A T G G A A T A A A A C C T A A C T T C G G T T A,T A CTATTACTCCCC minimal binding sequence Fig. 7. The binding site of nuclear factor I on the terminal sequence of the Ad2 genome. Nuclear factor I recognizes sequences between positions 24 and 40 or 41 (boxed area). This area contains two of four regions that are conserved in human adenoviruses (underlined sequences). Six out of the nine conserved base pairs of the binding site form a 2-fold symmetrical sequence. The axis of symmetry is indicated by an arrow. Binding of nuclear factor I protects the region from position 19 to 42 against DNase I digestion as indicated.

Methylation protection experiments (unpublished data) suggest that nuclear factor I has several intensive contacts with G residues in the major groove of the DNA. Taken together, these binding properties show strong resemblance to the interaction of several prokaryotic DNA-binding proteins, like the bacteriophage X, cro repressor and the CAP protein, with their recognition sites (Takeda et al., 1983). Based on the binding of nuclear factor I to deletion mutants it has been suggested that the region spanning nucleotides 15-48 is essential for binding. The region of 17-51 has the potential to form a hairpin structure (Nagata et al., 1983b; Guggenheimer et al., 1984). In our study (see Figure 2) only protection of nucleotides 19-42 could be observed, in agreement with Rawlins et al. (1984). Furthermore, nuclear factor I binds to the terminal 40 bp with the same affinity as to the intact ITR. This indicates that the sequence between 40 and 50, containing a conserved pentanucleotide (45 -49, see Figure 7), is not essential for binding of nuclear factor I. This notion is supported by studies with a point mutation at position 46 which does not influence nuclear factor I binding (E. de Vries et al., in preparation). We therefore consider formation of a hairpin by nucleotides 17-51 as a prerequisite for nuclear factor I binding less likely. The region protected from DNase I attack is larger than the minimal recognition site. This could indicate that certain sequences bordering the recognition site are not available for the enzyme due to steric hindrance. Also, nuclear factor I might make some weak contacts outside the minimal recognition sequence which could influence both binding and nuclease accessibility. Evidence for this latter possibility comes from the studies using 23

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Adl2 DNA. The Adl2 ITR, containing ACTG and pXdel23 26 23 ( CTTG) are bound considerably more strongly than pXdel24 26 23 ( CCTG). Thus, only the combination of two dC-dG base pairs at positions 23 and 24 seems to block the binding of nuclear factor I. It is possible that binding can only occur when the DNA flanking the recognition site can be unwound easily locally. The presence of two dC-dG base pairs at positions 23 and 24 could prevent such a structural change. Some evidence for such a distortion is obtained from the increased DNase sensitivity just outside the protected area. An exposed area bordering the nuclear factor I site was also suggested from footprint analysis of the combination of DBP and nuclear factor I (Nagata et al., 1983b). However, in the case of Adl2 DNA, we cannot exclude that sequences at the center of the recognition site (29-33), which differ from those of Ad2, also influence the binding. Nuclear factor I is isolated based upon its ability to stimulate adenovirus DNA in vitro. With all mutants studied so far, binding of nuclear factor I to the ITR correlates with stimulation of DNA synthesis (Guggenheimer et al., 1984; Rawlins et al., 1984). Our results indicate that a plasmid containing the first 40

nucleotides replicates as efficiently as a plasmid containing the intact ITR. These experiments were performed in a reconstituted system consisting of the purified viral proteins and nuclear factor I. Although it is tempting to assume that the first 40 nucleotides constitute an intact functional origin of DNA replication, we cannot exclude that other factors, present in crude nuclear extracts or in intact cells, require interaction with other parts of the ITR. Also, these experiments were performed with plasmid DNA devoid of the terminal protein, a situation which differs from that in vivo. It has been reported (Lally et al., 1983) that pTD38, when studied in crude nuclear extracts, supports DNA replication equally well as a plasmid containing the first 1003 bp. The apparent discrepancy between these results and the results described here may be accounted for by the presence of limiting amounts of nuclear factor I in crude extracts, which could weaken the response of the replication system to the enhancing effect of a nuclear factor I binding site. Alternatively, the higher concentration of template DNA used in the study of Lally et al. (1984) could obscure the effect of nuclear factor I. Such a dependence of nuclear factor I stimulation on template concentration was earlier observed by others (Guggenheimer et al., 1984; Tamanoi and Stillman, 1983). At present the mechanism of action of nuclear factor I in DNA replication is unclear. The protein might help to unwind the DNA at the origin or change the double-stranded DNA structure to increase access of other replication proteins to origin sequences. Alternatively, a nuclear factor I-DNA complex might attract or stabilize other replication proteins by direct protein-protein interaction. In this respect, it is interesting that stimulation of DNA replication by nuclear factor I is dependent on the DBP concentration (E.de Vries et al., in preparation). Nuclear factor I binding sites are not confined to the adenovirus genome. Several human DNA sequences which bind nuclear factor I have been cloned and it is estimated that one binding site is present about every 100 000 bp (Gronostajski et al., 1984). In particular, sites very similar to the Ad2 nuclear factor I recognition site have been detected upstream of the myc gene, the human IgH locus (Siebenlist et al., 1984) and the chicken lysozyme gene (Borgmeyer et al., 1984). Some of these sites lie close to, or coincide with, DNase I-hypersensitive sites. It is not known whether these sites are recognized by nuclear factor I or a related protein. Recently, Borgmeyer et al. (1984) isolated a protein, called TGGCA-protein, from chicken oviduct nuclei, which also binds to the Adl2 ITR. Its consensus binding sequence contains 2-fold rotational symmetry. In collaboration with these authors, we have recently observed that this protein has properties very similar to nuclear factor I from HeLa cells and can replace nuclear factor I in DNA replication (Leegwater, van der Vliet, Rupp, Nowock and Sippel, in preparation). This would support the notion that nuclear factor I is well conserved 1519

P.A.J.Leegwater, W.van Driel and P.C.van der Vlet

in evolution (Rawlins et al., 1984) and could perform an important function, either in cellular DNA replication or in gene activation, or both.

Materials and methods Enzymes and DNA replication components Restriction enzymes, pancreatic DNase I, Bal3l, calf intestine phosphatase, T4 DNA ligase and T4 polynucleotide kinase were purchased from Boehringer (Mannheim). The purification of DBP, of the complex of pTP-DNA polymerase (pTPpol), of the complex between viral DNA and terminal protein (DNA-TP) from virions and the isolation of cytosol RNA from uninfected HeLa cells have been described (Tsernoglou et al., 1984; Rijnders et al., 1983; Van Bergen et al., 1983; Van der Vliet et al., 1984). Plasmid DNA was isolated according to Bimboim and Doly (1979) and further purified by centrifugation in ethidium bromide/CsCl and gel filtration on Sepharose 4B (Pharmacia). XD-7 was a gift of J.Corden and plasmid pTD38 was kindly provided by E.L.Winnacker. Construction of pPL4O The 454-bp fragment of an EcoRI-Pvul digest of XD-7 was isolated and subsequently digested with Mnl, generating blunt-ended fragments of 248 bp and 166 bp and an EcoRI-MnII fragment that consists of the terminal 40 bp of Ad2. This digest was mixed with an EcoRI-EcoRV digest of pBR322 and ligation was performed under standard conditions. After ligation the mixture was incubated with Hindu to linearize pBR322 molecules that were regenerated, followed by phenol extraction and precipitation ofthe DNA. Escherichia coli HB101 was transformed with the DNA to ampicillin resistance. Plasmids of transformants were isolated and digested with TaqI. pPL40 carries a TaqI fragment of 540 bp instead of the 315-bp and the 368-bp fragments of pBR322. Sequence analysis confirmed the identity of the 40-bp insert. Construction of deletion mutants The 1343-bp EcoRI fragment of XD-7 was isolated and 7 itg of the fragment were incubated with BaM3M (2.4 U) at 12°C in 75 Ad of 20 mM Tris-HCl pH 8.0, 600 mM NaCl, 12 mM CaCl2, 12 mM MgCl2, and 1 mM EDTA. Aliquots were withdrawn at time points varying between 30 s and 7.5 min of incubation. Reactions in these samples were stopped by addition of 0.5 volume of 0.1 M EGTA. The samples were pooled, followed by phenol extraction and precipitation of the DNA with ethanol. Phosphorylated EcoRI linkers (GGAATTCC) were incubated with the DNA fragments for 65 hat 14°C in 15 Al of 66 mM Tris-HCI pH 7.6, 10 mM MgCl2, 1 mM ATP, 15 mM dithiothreitol (DTT), 1 mM spermidine, 0.2 mg/ml BSA and 1 U T4 ligase. The ligation products were cut with EcoRl and remnants of the linkers were removed by gel filtration on Sepharose 4B. 200 ng of EcoRI-linearized and dephosphorylated pBR322 was added to 150 ng of the fragments and ligation was performed in 10 1d for 20 h under the conditions described above. The products of the reaction were used to transform E. coli HB101 to ampicillin resistance. Plasmids from transformants were isolated according to Birnboim and Doly (1979) and the extent of deletions was determined by DNA sequencing according to Smith (1980) or Maxam and Gilbert (1980). Mutants were obtained with deletions of the first 18, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 45, 51, 53 and 55 bp of the Ad2 ITR, and designated as pXdell8 etc. Assay for detection of nuclear factor I activity During purification, nuclear factor I was monitored by its ability to enhance Ad DNA replication in vitro. Reaction mixtures (15 1) contained 0.5 mU pTP-pol, 0.6yg DBP, 0.5 Al cytosol, 40 ng DNA-TP, nuclear fractions at various stages of purification and 100,uM aphidicolin to suppress aspecific, repair-like synthesis, in replication buffer [40 mM Hepes-KOH (pH 7.5); 4 mM MgCI2; 0.4 mM DTT; 1.7 mM ATP, 5 mM creatine phosphate; 0.5 mg/ml creatine kinase; 17 zM each of dATP, dGTP and dTTP, 8 tiM [a-32P]dCTP (6-14 Ci/mmol)]. M EDTA After 60 min at 37°C reactions were stopped by addition of 15 (pH 8.0); 0.5 mg/ml BSA; 10 mM sodium pyrophosphate, and DNA was precipitated by adding trichloroacetic acid (TCA) to a final concentration of 10%. The pellets were washed twice with 1% TCA, once with 96% ethanol and counted One unit is defined as the incorporation of 1 nmol dCMP in acid-insoluble form. Purification of nuclear factor I The procedure described by Nagata et al. (1982) was followed with minor modifications. Nuclei from 0.7 x 1010 HeLa cells were isolated as described (Challberg and Kelly, 1979) and washed three times with 25 mM Tris-HCl (pH 7.5); 10% sucrose. The nuclei were suspended in the same buffer with 1 mM EDTA and 1 mM DTT and extracted by addition of 5 M NaCl to a concentration of 0.35 M NaCl and slow stirring at 4°C for 30 min. Debris was removed by successive centrifugations at 12 000 g for 10 min and 105 000 g for 40 min. The supematant (41 ml) was adjusted to 0.2 M NaCI by addition of buffer A

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(25 mM Tris-HCl, pH 7.5; 1 mM EDTA; 1 mM DTT; 20% glycerol) and applied to a DEAE-Sephacel column (2.3 x 10 cm) that was equilibrated with buffer A/0.2 M NaCl. The flow-through (45 ml) was dialyzed against buffer B (25 mM Tris-HCl, pH 8.5; 1 mM EDTA; 1 mM DTT; 20% glycerol; 10% sucrose; 0.01% Nonidet P-40)/0.03 M NaCl and applied to a second DEAE column (1.0 x 8 cm) equilibrated with buffer B/0.04 M NaCl. The column was washed with 15 ml buffer B/0.04 M NaCl and eluted with a 70 ml linear gradient of 0.04-0.4 M NaCl in buffer B. The activity eluted in fractions of 0.07 -0.15 M NaCl. These fractions were pooled (18 ml) and applied to a phosphocellulose column (1 x 7 cm) that was equilibrated with buffer B/0.15 M NaCl. After washing with 17 ml of buffer B/0.2 M NaCl, the column was eluted with buffer B/0.5 M NaCl. Active fractions were combined (6 ml), dialyzed against bufferB/O.1 M NaCl, and applied to a denatured DNA-ellulose column (1 x 8.5 cm) equilibrated with buffer B/0.1 M NaCl. The column was washed with 15 ml of the same buffer and eluted with a 60 ml linear gradient of 0.15-0.6 M NaCl in buffer B. The activity eluted in fractions of 0.2 -0.35 M NaCl. Peak fractions were pooled (22 ml) dialyzed against buffer B/0.2 M NaCl and applied to a hydroxylapatite column (0.5 x 4 cm) (Rawlins etal, 1984) equilibrated with buffer B/0.2 M NaCl. The column was washed with 4 ml buffer C [1 mM EDTA; 1 mM DTT; 20% glycerol; 0.01% Nonidet P-40)/10 mM sodium phosphate (pH 7.5)] and eluted with a 10 ml linear gradient of 10-750 mM sodium phosphate (pH 7.5) in buffer C. The peak of activity eluted at 200 mM sodium phosphate. Peak fractions were pooled and dialyzed against buffer A with 0.01% Nonidet P-40, resulting in a yield of 2.4 ml (0.2 U/ml) of nuclear fraction I (HAP fraction). DNase Ifootprinting XD-7 DNA was linearized by ClaI digestion and 5' end-labeled with 32P according to Maxam and Gilbert (1980). After digestion with Pvul and agarose gel electrophoresis, the 0.48-kb ClaI-PvuII fragment, that carries the Ad2 ITR (Figure 1) was isolated. The fragment (2 ng, 2.5 x 0I c.p.m.) was incubated with 100 ng pBR322 and 10 Id nuclear factor I (HAP fraction) or 8 ug BSA in 25 ul containing 25 mM Hepes-NaOH (pH 7.5); 5 mM MgCl2; 4 mM DTT and 150 mM NaCl for 60 min at 0°C. DNase I (0.1 U) was added, digestion was allowed for 1 min at 24-C and stopped by addition of 2.5 Id 0.2 M EDTA, 1.5% SDS. The degradation products were phenol extracted, precipitated, denatured in 80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1 % bromophenol blue, 0.1 % xylenecyanol and size-fractionated by electrophoresis on a 10% polyacrylamide gel (120 cm x 40 cm x 0.2 mm) with 8.3 M urea in TBE (90 mM Tris base, 90 mM boric acid, 2 mM EDTA) as the running buffer. The gels were autoradiographed on Fuji RX films. Filter binding assay Plasmids were digested with restriction enzymes as indicated and the fragments were 5' end-labelled with 32P according to Maxam and Gilbert (1980). 50 ng of the fragments were incubated with 1 ul nuclear factor I (HAP fraction) in 50 j1l buffer that contained 25 mM Hepes-NaOH (pH 7.5); 5 mM MgCl2, 4 mM DTT, 150 mM NaCl and after 30-60 min at 0°C the mixture was passed through nitrocellulose filters (Millipore, HA). The filters were washed with 1 ml of the same buffer and the retained DNA fragments were eluted with 400 01 of 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.2% SDS. The fragments were ethanol precipitated with 5 rg tRNA as carrier, and analyzed by polyacrylamide gel electrophoresis and autoradiography of the gel. For the competition experiments the same procedure was followed, except that the indicated amounts of unlabeled competitor DNA were incubated together with a labeled EcoRI-Pvull digest of XD-7 DNA (10 ng) and nuclear factor I. Analysis of retained DNA was by electrophoresis on a 1 % agarose gel. The gel was placed on DE81 paper (Whatman) and dried under vacuum. The combined DE81 paper and gel were autoradiographed. This procedure prevents the loss of small DNA fragments during vacuum drying of the gels. The 0.45-kb XD-7 band of each lane was quantitated by scanning with a Beckman DU-8B spectrophotometer. Assay for replication of plasmid DNA fragments Reaction mixures (15 1d) contained 40 mM Hepes-KOH (pH 7.5), 4 mM MgCl2, 0.4 mM DTT, 1.7 mM ATP, 5 mM creatine phosphate, 0.5 mg/mi creatine kinase, 17 tM each of dATP, dGTP and dTTP, 8 tIM [a1-32P]dCTP (6-14 Ci/mmol), 1 mU pTP-pol, 1.25 rg DBP, 1 lAg cytosol, 100 mM aphidicolin, and 1 yd nuclear factor I when indicated. After 1 h at 37°C reactions were stopped by addition of 1.5 I&l 40% sucrose, 1% SDS, 0.1% bromophenol blue and electrophoresed on a 1 % agarose, 0.1 % SDS gel, with TBE/0.1 % SDS as the running buffer. Replication products were visualized by autoradiography of the dried gel.

Acknowledgements The authors gratefullly acknowledge gifts of plasmids by E.L.Winnacker (pTD38), J.Corden (XD-7) and J.L.Bos (pRICI). We also thank B.G.M.van Bergen for the construction of the pXdel plasmids, E.de Vries for useful suggestions and discussions and J.S.Sussenbach for critical reading of the manuscript. This work

Recognition site of nuclear factor I was supported in part by the Netherlands Foundation for Chemical Research (SON), with financial aid from the Netherlands Organization for the Advancement of Pure Research.

References Bimboim,H.C. and Doly,J. (1979) Nucleic Acids Res., 7, 1513-1523. Borgmeyer,U., Nowock,J. and Sippel,A.E. (1984) Nucleic Acids Res., 12, 42954311. Bos,J.L., Polder,L.J., Bernards,R., Schrier,P.I., Van den Elsen,P.J., Van der Eb, A.J. and Van Ormondt,H. (1981) Cell, 27, 121-131. Challberg,M.D. and Kelly,T.J., Jr. (1979) Proc. Natl. Acad. Sci. USA, 76, 655-659. Challberg,M.D. and Kelly,T.J.,Jr. (1982) Annu. Rev. Biochem., 51, 901-934. Field,J., Gronostajski,R.M. and Hurwitz,J. (1984) J. Biol. Chem., 259, 9487-9495. Gronostajski,R.M., Nagata,K. and Hurwitz,J. (1984) Proc. Natl. Acad. Sci. USA, 81, 40134017. Guggenheimer,R.A., Stillman,B.W,. Nagata,K., Tamanoi,F. and Hurwitz,J. (1984) Proc. Natl. Acad. Sci. USA, 81, 3069-3073. Lally,C., Dorper,T., Groger,W., Antoine,G. and Winnacker,E.L. (1984) EMBO J., 3, 333-337. Maxam,A.M. and Gilbert,W. (1980) Methods Enzymol., 651, 499-560. Nagata,K., Guggenheimer,R.A., Enomoto,T., Lichy,J.H. and Hurwitz,J. (1982) Proc. Natl. Acad. Sci. USA, 79, 6438-6442. Nagata,K., Guggenheimer,R.A. and Hurwitz,J. (1983a) Proc. Natl. Acad. Sci. USA, 80, 42664270. Nagata,K., Guggenheimer,R.A. and Hurwitz,J. (1983b) Proc. Natl. Acad. Sci. USA, 80, 6177-6181. Pearson,G.D., Chow,K.-C., Enns,R.E., Ahern,K.G., Corden,J.L. and Harpst,J.A. (1983) Gene, 23, 293-305. Rawlins,D.R., Rosenfeld,P.J., Wides,R.J., Challberg,M.D. and Kelly,T.J.,Jr. (1984) Cell, 37, 309-319. Rijnders,A.W.M., Van Bergen,B.G.M., Van der Vliet,P.C. and Sussenbach,J.S. (1983) Nucleic Acids Res., 11, 8777-8789. Siebenlist,U., Hennighausen,L., Battey,J. and Leder,P. (1984) Cell, 37, 381-391. Smith,A.J.H. (1980) Methods Enzymol., 65, 560-580. Stillman,B.W. (1983) Cell, 35, 7-9. Sussenbach,J.S. and Van der Vliet,P.C. (1983) Curr. Top. Microbiol. Irnnunol., 109, 53-73. Sussenbach,J.S. (1984) in Ginsberg,H.S. (ed.), 7he Adenoviruses, Plenum Press, NY, pp. 35-124. Takeda,Y., Ohlendorf,D.H., Anderson,W.F. and Matthews,B.W. (1983) Science (Wash.), 221, 1020-1026. Tamanoi,F. and Stillman,B.W. (1982) Proc. Natl. Acad. Sci. USA, 79, 2221-2225. Tamanoi,F. and Stillman,B.W. (1983) Proc. Natl. Acad. Sci. USA, 80, 6446-6450. Tsemoglou,D., Tucker,A.D. and Van der Vliet,P.C. (1984) J. Mol. Biol., 172, 237-239. Van Bergen,B.G.M., Van der Ley,P.A., Van Driel,W., Van Mansfeld,A.D.M. and Van der Vliet,P.C. (1983) Nucleic Acids Res., 11, 1975-1989. Van der Vliet,P.C., Van Dam,D. and Kwant,M. (1984) FEBS Lett., 171, 5-8.

Received on 15 March 1985

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