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

Abstract

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.

Identification of a potent adipocyte-specific enhancer: involvement of an NF-1-like factor.

“…We therefore used adipocyte and HeLa nuclear extracts to compare the binding specificity of the ARE with that of a consensus NF-1 oligomet. Figure 6A shows an alignment of the aP2 ARE with two known NF-l-binding sites: the adenovirus 2 origin of replication (NF-1; Leegwater et al 1985), and a sequence from the mouse albumin gene {ALB; Cereghini et al 1987;Lichtsteiner et al 1987). Both of these oligonucleotides compete effectively for the binding of adipocyte extract to the ARE oligomer {Fig.…”

Identification of factors that interact with the E1A-inducible adenovirus E3 promoter.

“…The E3 BS4 sequence contains homology to the binding site for the nuclear factor NF1, which has the consensus TGGN6_TGCCAA (Leegwater et al 1985). A 24-bp oligonucleotide containing the region of the Ad2-inverted terminal repeat that is protected from DNase I digestion by the NF1 factor (Leegwater et al 1985)was synthesized and tested for competition for binding to the BS4 oligonucleotide.…”

“…A 24-bp oligonucleotide containing the region of the Ad2-inverted terminal repeat that is protected from DNase I digestion by the NF1 factor (Leegwater et al 1985)was synthesized and tested for competition for binding to the BS4 oligonucleotide. The NF1 oligonucleotide competed very efficiently (data not shown); therefore, we conclude that the E3.F4 factor is NF1.…”

Adenovirus DNA replication in vitro: Duplication of single-stranded DNA containing a panhandle structure

“…The purification procedures for DBP [33], the complex of pTP and the Ad DNA polymerase (pTP-pol [34]), NFI [35], NFIII [13] and HeLa cytosol [36] were as described. The C-terminal 39 kDa DBP fragment was prepared by digestion with chymotrypsin in 2 M NaC1 [37] followed by DNA-cellulose chromatography.…”