Dissection and reconstitution of the adenovirus DNA replication machinery has led to the discovery of two HeLa nuclear proteins which are required in conjunction with three viral proteins. One of these, nuclear factor I (NF-I), recognizes an internal region of the origin between nucleotides 25 and 40 and by binding to one side of the helix stimulates the initiation reaction up to 30-fold. NFI-binding sites have been observed upstream of several cellular genes, such as chicken lysozyme, human IgM and human c-myc, and coincide in most cases with DNase I hypersensitive regions. Here we report the identification of a novel DNA-binding protein from HeLa nuclei, designated NF-III, that recognizes a sequence in the adenovirus origin very close to the NFI-binding site, between nucleotides 36 and 54. This sequence includes the partially conserved nucleotides TATGATAATGAG. NF-III stimulates DNA replication four- to sixfold by increasing the initiation efficiency. Potential cellular binding sites include promoter elements of the histone H2B gene, the human interferon beta gene, the human and mouse immunoglobulin VK and VH genes and the mammal/chicken/Xenopus laevis U1 and U2 small nuclear RNA genes. Furthermore, a subset of the herpes simplex virus immediate early promoter specific TAATGARAT elements is homologous with the adenovirus 2 (Ad-2) NFIII-binding site.

Nuclear factor I (NFI) is a HeLa sequence‐specific DNA‐binding protein that is required for initiation of adenovirus (Ad) DNA replication and may be involved in the expression of several cellular genes. The interaction between NFI and its binding site on the Ad2 origin has been studied. Methylation interference and protection, u.v. irradiation of 5‐BrdU substituted DNA and ethylation interference revealed major groove contacts with G and T, and phosphate backbone contacts. Computer stereographics show that the contacts are located in two blocks showing dyad symmetry to each other and 22 out of 23 contacts are accessible from one side of the helix. Inversion of the NFI binding site did not change the NFI dependent stimulation of Ad2 DNA replication in a reconstituted system. All data are compatible with NFI binding as a dimer at one side of the DNA helix.

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 Ad12 DNA indicate that nucleotide 24 can be mutated. Fragments containing the first 40 bp are bound normally 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.

Nuclear factor HI (NFI) is a protein from HeLa cells that stimulates the initiation of adenovirus type 2 (Ad2) DNA replication by binding to a specific nucleotide sequence in the orign, adjacent to the nuclear factor I recognition site. DNA sequences sharing a high degree of homology to the NFm binding site in Ad2 were found in a number of transcription regulatory elements, all containing the octanucleotide sequence ATGCAAAT. We have analysed the interaction between NF[II and the octamer-containing sequences in a histone H2B promoter, immunoglobulin light and heavy chain promoters, an immunoglobulin heavy chain enhancer, a U2 snRNA enhancer and the SV40 enhancer as well as Ad4. All sequences were recognized by NlI as indicated by gel retardation assays, DNase I footprinting and methylation protection experiments. A comparison of the relative binding affinities using competition assays indicated that mutations in the octanucleotide sequence reduced the binding affinity considerably. Small but signifit differences in affinity were also observed depending on the sequences bordering the conserved octanucleotide. The methylation protection patterns indicate that both major and minor groove contacts are involved in NFIII binding. The data suggest that NFEL could function both in adenovirus DNA replication and in the transcriptional control of several groups of genes sharing the octanucleotide sequence.

Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When XbaI digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments was observed. The newly synthesized DNA was covalently bound to the 82 kD preterminal protein (pTP). Plasmid DNAs containing the Ad2 origin sequence or the Ad12 origin sequence with small deletions were analyzed for their capacity to support pTP-primed DNA replication. Circular plasmid DNAs were inactive. When plasmids were linearized to expose the adenovirus origin, both Ad2 and Ad12 TP-free fragments could support initiation and elongation similarly as Ad5 DNA-TP, although with lower efficiency. These observations indicate that the parental terminal protein is dispensable for initiation in vitro. The presence of 29 nucleotides ahead of the molecular end or a deletion of 14 base pairs extending into the conserved sequence (9-22) destroyed the template activity. DNA with a large deletion within the first 8 base pairs could still support replication while a small deletion could not. The results suggest that only G residues at a distance of 4-8 nucleotides from the start of the conserved sequence can be used as template during initiation of DNA replication.