DNA sequences required for the in vitro replication of adenovirus DNA.

Initiation of adenovirus (Ad) DNA replication occurs on viral DNA containing a 55-kilodalton (kDa) protein at the 5' terminus of each viral DNA strand and on plasmid DNAs containing the origin of Ad replication but lacking the 55-kDa terminal protein (TP). Initiation of replication proceeds via the synthesis of a covalent complex between an 80-kDa precursor to the TP (pTP) and the 5'-terminal deoxynucleotide, dCMP. Formation of the covalent pTP-dCMP initiation complex with Ad DNA as the template requires the viral-encoded pTP and DNA polymerase and, in the presence of the Ad DNA binding protein, is dependent upon a 47-kDa host protein, nuclear factor I. Initiation of replication with recombinant plasmid templates requires the aforementioned proteins and an additional host protein, factor pL. Deletion mutants of the Ad DNA replication origin contained within the 6.6-kilobase plasmid pLA1 were used to analyze the nucleotide sequences required for the formation and subsequent elongation of the pTP-dCMP initiation complex. The existence of two domains within the first 50 base pairs of the Ad genome, both of which are required for the efficient use of recombinant DNA molecules as templates in an in vitro DNA replication system, was demonstrated. The first domain, consisting of a 10-base-pair "core" sequence located at nucleotide positions 9-18, has been identified tentatively as a binding site for the pTP [ Rijinders , A. W. M., van Bergen, B. G. M., van der Vliet , P. C. & Sussenbach , J. S. (1983) Nucleic Acids Res. 11, 8777-8789]. The second domain, consisting of a 32-base-pair region spanning nucleotides 17-48, was shown to be essential for the binding of nuclear factor I.     

Isolation of human DNA sequences that bind to nuclear factor I, a host protein involved in adenovirus DNA replication.

Nuclear factor I is a 47,000-dalton protein isolated from human HeLa cells that is required for the in vitro replication of adenovirus DNA. This protein was previously shown to bind specifically to nucleotides 17-48 of the left-hand terminus of cloned adenovirus serotype 5 DNA. An in vitro assay for DNA sequences that compete with adenovirus DNA for the binding of nuclear factor I has been developed. With this assay, we have shown specific binding of human DNA sequences to nuclear factor I. Using the DNA binding activity of nuclear factor I, we have isolated and cloned segments of human DNA that bind tightly to this protein. One nuclear factor I binding site is present about every 100,000 base pairs in the HeLa cell genome. The binding of these DNA molecules to nuclear factor I resembles the binding of cloned adenovirus DNA to the protein and is resistant to high ionic strength. The isolation of DNA sequences from HeLa cells that bind specifically to nuclear factor I suggests that this protein interacts with host DNA in vivo.     

Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex.

A protein factor that participates in the formation of a covalent complex between the 80,000-dalton precursor of the adenovirus (Ad) terminal protein (pTP) and 5'-dCMP has been isolated and characterized. This 47,000-dalton protein, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor I. It is free of detectable DNA polymerase alpha, beta, and gamma activities. In the presence of Ad DNA-prot, the Ad-protein fraction (containing the pTP and the Ad-associated DNA polymerase), ATP, Mg2+, and dCTP, nuclear factor I stimulates formation of the pTP-dCMP complex. Addition of the Ad DNA binding protein (Ad DBP) renders the formation of the pTP-dCMP complex completely dependent on the addition of nuclear factor I. When Ad DNA-prot is replaced with phi X174 single-stranded circular DNA, pTP-dCMP complex formation requires only the Ad-protein fraction; Ad DBP and ATP are inhibitory and nuclear factor I has no effect on this reaction. This suggests that the initiation reaction observed with Ad DNA-prot in the absence of Ad DBP occurs at single-stranded DNA sites. In the presence of Ad DBP, these sites are blocked thus creating a requirement for nuclear factor I in pTP-dCMP complex formation.     

Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins.

A protein required for the elongation of replicating intermediates of adenovirus (Ad) DNA to full length has been isolated and characterized. This factor, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor II. In the presence of Ad DNA with proteins at each 5' end (Ad DNA-protein) and three proteins coded for by the Ad genome [the preterminal protein (pTP), the DNA polymerase (Ad Pol), and the DNA binding protein (Ad DBP)], nuclear factor II complementing activity is detected only in the presence of host nuclear factor I. Highly purified preparations of nuclear factor II that are free of detectable DNA polymerase alpha, beta, and gamma activities contain a DNA topoisomerase activity. Furthermore, type I DNA topoisomerases purified from HeLa cells and calf thymus substitute for nuclear factor II complementing activity in the in vitro Ad DNA replication system. These results indicate that a protein that is involved in higher order DNA structure is required for Ad replication. This protein plus the purified proteins described above carry out the initiation and synthesis of full-length 36,000-base-pair Ad DNA.     

A DNA binding protein specific for an origin of replication of herpes simplex virus type 1.

We have identified a protein that binds specifically to an origin of replication (oris) of the herpes simplex virus type 1 genome. The oris binding protein, detectable only in nuclear extracts of infected cells, shows the same time course of appearance as the herpesvirus-induced DNA polymerase and the DNA binding protein ICP8. The partially purified oris binding protein generates a DNase I "footprint" that spans 18- of the 90-base-pair minimal oris sequence. The oris binding protein may, therefore, be analogous to other origin-specific binding proteins that are required for the initiation of viral and chromosomal DNA replication.     

The subunits of activator 1 (replication factor C) carry out multiple functions essential for proliferating-cell nuclear antigen-dependent DNA synthesis.

p37 and p40 are two cloned gene products of the five-subunit human cellular DNA replication factor activator 1 (A1) protein complex (also called replication factor C). Here, we describe the solubilization, purification, and characterization of these two proteins that were overproduced in Escherichia coli. Using a nitrocellulose filter binding assay, we demonstrated that the purified A1 p37 protein associated with DNA preferentially at the primer terminus, a property resembling that of the A1 complex. We also show that in the presence of relatively high levels of salt, the recombinant p37 protein alone activated DNA polymerase epsilon but not polymerase delta in catalyzing the elongation of DNA chains. The p40 protein specifically associated with cellular p37 and proliferating-cell nuclear antigen (PCNA) present in HeLa cell cytosolic extract. The addition of purified p40 protein abolished the in vitro polymerase delta-catalyzed DNA elongation reaction dependent on both PCNA and A1. However, this inhibition was reversed by excess polymerase delta, suggesting a specific interaction between the polymerase and the p40 protein. Thus, while p37 binds DNA at the primer end and has a specific affinity for pol epsilon, p40, which binds ATP, interacts with PCNA and pol delta. These activities are essential for the DNA elongation reactions that lead to the synthesis of leading-strand DNA and the maturation of Okazaki fragments.     

DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication.

Efficient initiation of DNA replication in the absence of nonspecific DNA repair synthesis was obtained by using a modification of the system developed by J.J. Li and T.J. Kelly [(1984) Proc. Natl. Acad. Sci. USA 81, 6973-6977]. Circular double-stranded DNA plasmids replicated in extracts of CV-1 cells only when the plasmids contained the cis-acting origin sequence for simian virus 40 DNA replication (ori) and the extract contained simian virus 40 large tumor antigen. Competition between plasmids containing ori and plasmids carrying deletions in and about ori served to identify a sequence that binds the rate-limiting factor(s) required to initiate DNA replication. The minimum binding site (nucleotides 72-5243) encompassed one-half of the simian virus 40 ori sequence that is required for initiation of replication (ori-core) plus the contiguous sequence on the late gene side of ori-core containing G + C-rich repeats that facilitates initiation (ori-auxiliary). This initiation factor binding site was specific for the simian virus 40 ori region, even though it excluded the high-affinity large tumor antigen DNA binding sites.     

Upstream DNA sequences required for tissue-specific expression of the HLA-DR alpha gene

We have used in vitro deletion mutagenesis in combination with DNA transfection to search for cis-acting regulatory elements involved in the tissue-specific expression of a human class II major histocompatibility complex gene. A 140-base-pair 5' flanking fragment that contains the class II box consensus sequences and an octamer sequence (ATTTGCAT) confers tissue specificity on the promoter of the HLA-DR alpha gene. Recombinant DNA plasmids containing this DR alpha gene segment fused to the coding sequence of the bacterial chloramphenicol acetyltransferase gene are expressed at higher levels in human B-cell lines than in human T-cell lines. We have demonstrated that the most 5' of the class II boxes is essential for tissue-specific DR alpha promoter function. In addition, using an electrophoretic mobility shift assay to identify DNA binding proteins, we have detected binding of nuclear proteins to DNA probes containing the class II boxes and the octamer sequence. A protein that binds to the octamer is present at higher levels in nuclear extracts of B-cell lines than in other cell lines examined. This protein may be important for the tissue-specific expression of the HLA-DR alpha gene.     

An unusual symmetric recombinant between adenovirus type 12 DNA and human cell DNA

On purification of human adenovirus type 12 (Ad12) by equilibrium sedimentation in CsCl density gradients, two bands of particles, Ad12-3 and Ad12-3a, are observed. The particles from band Ad12-3a contain a recombinant of human host cell DNA and of Ad12 DNA. The human cell DNA sequences contain repetitive DNA recurring 200 to 500 times in cellular DNA. Ad12 DNA and the recombinant genomes exhibit the same or similar lengths. This finding suggests that a constant amount of DNA is packaged into complete Ad12 particles. On cleavage of KB cellular DNA with EcoRI, BamHI, HinfI, Msp I, Mbo I Pst I, or Bgl II, the (32)P-labeled cellular DNA from Ad12-3a particles hybridizes on Southern blots to distinct bands of KB DNA. There is also less-specific background hybridization that is not observed in the control. The cellular DNA from Ad12-3a particles is not methylated, whereas the same cellular sequences in KB cell DNA appear to be extensively methylated. On denaturation and renaturation, the recombinant DNA molecules are converted to molecules half as long as Ad12 DNA, as determined by gel electrophoresis and electron microscopy. The recombinant DNA molecules were terminally labeled by exonuclease III treatment and subsequent refilling of the depleted segments with [(32)P]dNTPs by using DNA polymerase I (Klenow fragment). When these molecules were cleaved with EcoRI, BamHI, Msp I, or Pst I, only one terminal DNA fragment was found to be labeled. The results of partial digestion experiments using Msp I, HinfI, or Mbo I are consistent with a model in which 700-1150 base pairs from the left terminus of Ad12 DNA are linked to host cell DNA containing repetitious sequences, and this structure is symmetrically duplicated as a large inverted repeat of the type ABCDD'C'B'A'. The Ad12 DNA sequences are flanking the entire molecule, which consists mainly of human KB cell DNA. The recombinants appear to be stable on serial passage of the virus preparation for many years, although variations in the sequence of the recombinants occur. These symmetric recombinant (SYREC) molecules suggest a way to use adenovirus DNA as a eukaryotic vector. Their occurrence provides further evidence for the generation of virus-host DNA recombinants and may help elucidate the role this interaction may have in adenovirus replication and oncogenesis.     

RepA and DnaA proteins are required for initiation of R1 plasmid replication in vitro and interact with the oriR sequence.

RepA, an initiation protein of R1 plasmid replication, was purified from an Escherichia coli strain overproducing the protein. The purified RepA protein specifically initiated replication in vitro of plasmid DNA bearing the replication origin of R1 plasmid (oriR). The replication, strictly dependent on added RepA protein, was independent of host RNA polymerase but required other host replication functions (DnaB and DnaC proteins, the single-stranded-DNA-binding protein SSB, and DNA gyrase). The replication was also completely dependent on the host DnaA function. In filter binding assays in high salt (0.5 M KCl) conditions, RepA specifically binds to both supercoiled and linear plasmid DNA containing the oriR sequence, whereas it binds to nonspecific DNA in low salt. DNase I-protection studies on a linearized DNA fragment revealed that DnaA protein specifically binds to a 9-base-pair DnaA-recognition sequence ("DnaA box") within oriR only when RepA is bound to the sequence immediately downstream of the DnaA box. These results indicate that initiation of R1 plasmid replication is triggered by interaction of RepA and DnaA proteins with the oriR sequence.     

Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication

The herpes simplex virus type 1 origin of DNA replication, oriS, contains three copies of the recognition sequence for the viral initiator protein, origin binding protein (OBP), arranged in two palindromes. The central box I forms a short palindrome with box III and a long palindrome with box II. Single-stranded oriS adopts a conformation, oriS*, that is tightly bound by OBP. Here we demonstrate that OBP binds to a box III-box I hairpin with a 3' single-stranded tail in oriS*. Mutations designed to destabilize the hairpin abolish the binding of OBP to oriS*. The same mutations also inhibit DNA replication. Second site complementary mutations restore binding of OBP to oriS* as well as the ability of mutated oriS to support DNA replication. OriS* is also an efficient activator of the hydrolysis of ATP by OBP. Sequence analyses show that a box III-box I palindrome is an evolutionarily conserved feature of origins of DNA replication from human, equine, bovine, and gallid alpha herpes viruses. We propose that oriS facilitates initiation of DNA synthesis in two steps and that OBP exhibits exquisite specificity for the different conformations oriS adopts at these stages. Our model suggests that distance-dependent cooperative binding of OBP to boxes I and II in duplex DNA is succeeded by specific recognition of a box III-box I hairpin in partially unwound DNA.     

Epstein-Barr nuclear antigen 1 mediates a DNA loop within the latent replication origin of Epstein-Barr virus.

Epstein-Barr virus-encoded nuclear antigen 1 (EBNA-1) binds and activates the viral latent origin of DNA replication, oriP. We have used electron microscopy to examine the assembly of EBNA-1 onto oriP. The oriP region consists of two essential elements separated by approximately 1 kilobase pair of DNA. One element contains 20 tandom EBNA-1 binding sites [called the family of repeats (FR)] and serves to activate initiation of replication at the dyad symmetry (DS) element, which contains 4 EBNA-1 binding sites. Titration of homogeneous EBNA-1 produced in baculovirus (bEBNA-1) onto oriP DNA showed an order to the assembly of bEBNA-1 onto oriP. At low concentrations, bEBNA-1 was located exclusively on the FR element. As the level of bEBNA-1 was raised, a loop between the FR and DS elements became the most prevalent DNA-protein complex. These data suggest protein-mediated DNA looping may play a role in activating latent-phase replication of the Epstein-Barr virus.     

Sequence-specific interactions of nuclear factors with conserved sequences of human class II major histocompatibility complex genes

All class II major histocompatibility complex genes contain two highly conserved sequences, termed X and Y, within the promoter regions(s), which may have a role in regulation of expression. To study trans-acting factors that interact with these sequences, sequence-specific DNA binding activity has been examined by the gel electrophoresis retardation assay using the HLA-DQ2 beta gene 5' flanking DNA and nuclear extracts derived from various cell types. Several specific protein-binding activities were found using a 45-base-pair (bp) HinfI/Sau96I (-142 to -98 bp) and a 38-bp Sau96I/Sau96I (-97 to -60 bp) fragment, which include conserved sequence X (-113 to -100 bp) and conserved sequence Y (-80 to -71 bp), respectively. Competition experiments, methylation interference analysis, and DNase I foot-printing demonstrated that distinct proteins in a nuclear extract of Raji cells (a human B lymphoma line) bind to sequence X, to sequence Y, and to DNA 5' of the X sequence (termed sequence W). The factor binding site in the W sequence is also found to be conserved among beta-chain genes and is suggested to be a gamma-interferon control region.     

The homeo domain of a murine protein binds 5'' to its own homeo box.

Nuclear protein extracts from day 12.5 mouse embryos were used to study protein binding to DNA sequences 5' of the Hox 1.5 homeo box. Embryos of this developmental stage are known to express this gene. DNA binding protein blotting and retardation gel techniques show that murine embryonic nuclear proteins specifically bind a 753-base pair (bp) DNA fragment from the region upstream of the Hox 1.5 homeo box. A fusion protein containing the Hox 1.5 homeo domain constructed in lambda gt11 also binds the same 753-bp DNA fragment. Specific binding of the fusion protein to the upstream DNA fragment shows that the homeo box contains the sequences required for specific protein-DNA interactions, and the 753-bp fragment contains a homeo domain binding site. These results support the hypothesis that murine homeo boxes are DNA binding domains of proteins involved in the regulation of embryonic development.     

Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter.

Escherichia coli RNA polymerase (EC 2.7.7.6), bound in a tight complex at an early T7 promoter, protects 41 to 43 base pairs of DNA from digestion by DNase. I. The protected DNA fragment contains both the binding site for RNA polymerase and the mRNA initiation point for the promoter. The sequence of the DNA fragment and the sequence of the mRNA that it codes for are presented here. A seven-base-pair sequence, apparently common to all promoters, is implicated in the formation of a tight binary complex with RNA polymerase.     

Sequence recognition protein for the 17-base-pair A + T-rich tract in the replication origin of simian virus 40 DNA.

A DNA-binding protein has been identified that recognizes runs of deoxyadenines and/or deoxythymines (dA/dT sequences) and purified from a chromatographic fraction containing the multiprotein DNA polymerase alpha-primase complex of HeLa cells by successive steps of chromatography on oligo(dT)-cellulose and Q-Sepharose. Polyacrylamide gel electrophoresis of the purified dA/dT sequence-binding protein in the presence of NaDodSO4 showed a single protein band of 62 kDa. Nitrocellulose filter binding assays using homopolydeoxynucleotides indicated that the purified protein preferentially binds to dA/dT sequences in single-stranded or duplex DNAs. Gel mobility shift assays with a variety of DNAs showed that the purified protein specifically binds to a fragment of simian virus 40 DNA containing the minimal (core) origin for replication. The binding occurred in a protein-dependent manner and in the presence of a vast excess of competing DNAs lacking the simian virus replication origin. The origin binding was reduced, however, when DNA fragments from simian virus 40 deletion mutants containing deletions within the 17-base-pair A + T-rich tract in the core DNA replication origin were used in the assays. These results indicate that the dA/dT sequence-binding protein preferentially binds to the 17-base-pair A + T-rich tract and suggest a possible role for the protein in the initiation of DNA replication.     

Characterization of Drosophila DNA-binding protein DB-2: demonstration of its sequence-specific interaction with DNA.

A DNA-binding protein (DB-2) was isolated from unfertilized Drosophila eggs by DNA-cellulose chromatogrphy. In competition assays with DNA from other species, DB-2 preferentially binds to Drosophila DNA. This binding protein can also be isolated from pupal nuclei and comprises only a small fraction ( < 0.01%) of the total nonhistone chromosomal proteins. In order to investigate the specificity of the interaction between DB-2 and the DNA, we attempted to isolate the DNA sequences to which DB-2 binds. DB-2 was used as a probe to screen our gene bank established by inserting randomly sheared fragments of Drosophila DNA into bacterial plasmids. Groups of plasmids were tested for binding to DB-2 by a filter binding assay. The plasmids bound to the nitrocellulose filter were eluted and used for bacterial transformation. After several cycles of transformation and cloning, two plasmids, A17 and B10, were isolated that bind DB-2 specifically, as measured by filter binding and competition assays. In plasmid A17, binding of DB-2 protects two short DNA segments of approximately 13 and 30 base pairs from digestion by DNase I. By filter hybridization according to Southern, these sequences were mapped to a defined restriction fragment. Further evidence for the binding specificity was obtained by visualizing the protein-DNA complex in the electron microscope. In salivary gland giant chromosomes, A17 DNA hybridizes to a single site (95A/B) on chromosome 3.     

Fractionated nuclear extracts from hamster cells catalyze cell-free recombination at selective sequences between adenovirus DNA and a hamster preinsertion site.

We have explored the mechanism of adenovirus type 12 (Ad12) DNA integration because of its importance for viral oncogenesis and as an example of insertional recombination. We have used a fractionated cell-free system from nuclear extracts of hamster cells and have partly purified nuclear proteins that could catalyze in vitro recombination. As recombination partners, the 20,880- to 24,049-nucleotide Pst I D fragment of Ad12 DNA and the hamster preinsertion sequence p7 from the Ad12-induced tumor CLAC1 have proven to recombine at higher frequencies than randomly selected adenoviral or cellular DNA sequences. A preinsertion sequence might carry elements essential in eliciting recombination. Patch homologies between the recombination partners seem to play a role in the selection of sites for recombination in vivo and in the cell-free system. Nuclear extracts from BHK21 cells were prepared by incubating the nuclei in 0.42 M (NH4)2SO4 and fractionated by Sephacryl S-300 gel filtration, followed by chromatography on Mono S and Mono Q columns. The purified products active in recombination contained a limited number of different protein bands, as determined by polyacrylamide gel electrophoresis and silver staining. The most highly purified fraction IV had helicase and topoisomerase I activities. We used two different methods to assess the in vitro generation of hamster DNA-Ad12 DNA recombinants upon incubation with the purified protein fractions: (i) transfection of the recombination products into recA- strains of Escherichia coli and (ii) the polymerase chain reaction by using amplification primers unique for each of the two recombination partners. In p7 hamster DNA, the nucleotide sequence 5'-CCTCTCCG-3' or similar sequences served repeatedly as a preferred recombination target for Ad12 DNA in the tumor CLAC1 and in five independent cell-free recombination experiments.     

Herpes simplex virus DNA replication: a spacer sequence directs the ATP-dependent formation of a nucleoprotein complex at oriS.

The origin-binding protein (OBP) from herpes simplex virus 1 is a member of the SF2 helicase superfamily and is required for the initiation of DNA synthesis from a viral origin of DNA replication (oriS). The high-affinity binding sites for OBP in oriS, boxes I and II, are separated by an A+T-rich spacer. We used the gel retardation technique to examine the influence of this spacer sequence on the formation of a specific complex, referred to as complex II, between OBP and oriS. The formation of this OBP-oriS complex was greatly promoted by adenosine 5'-[gamma-thio]triphosphate and other nucleotide cofactors. Surprisingly, oriS constructs where the spacer sequence had been altered with approximately half of a helical turn (+4 or -6 base pairs) supported the formation of a more stable complex II than the wild-type origin. DNase I footprinting experiments showed that the cooperative binding of OBP to boxes I and II was affected by the length of the spacer sequence in the same way. In contrast, the ability of oriS-containing plasmids to replicate was most efficient with wild-type oriS. This paradox can be resolved if it is assumed that an ATP-dependent cooperative binding of OBP to properly spaced recognition sequences in oriS is required to induce a conformational change of DNA, thereby facilitating initiation of DNA replication.