Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites.

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.     

Metal-dependent binding of a nuclear factor to the rat metallothionein-I promoter.

Genomic footprinting studies in vivo and experiments using synthetic metal regulatory elements (MREs) in vitro suggest protein binding to the MREs of the mouse and rat metallothionein I (MT-I) genes. Using gel-retardation assays of promoter fragments, we observe a cadmium-dependent binding factor for the rat MT-I promoter in rat hepatoma cells. This factor is present in extracts from both uninduced and cadmium-induced cells, but requires the presence of cadmium to bind to the promoter. The formation of a cadmium-dependent complex is competed by an oligonucleotide containing two MREs. This competition is lost when when one of the MREs is mutated, indicating a requirement for at least two MREs for binding of this factor. The cadmium-dependent factor dissociates more rapidly from the MT-I promoter than does a factor that binds to a consensus Sp1 site present on the same DNA fragment. UV crosslinking analysis using nuclear extracts from cadmium induced cells, in the presence of an oligonucleotide probe containing both 5-bromodeoxyuridine and 32P-deoxycytidine, identifies a 39 kDalton protein associated with the metal inducible complex.     

In vivo detection of regulatory factor binding sites of Arabidopsis thaliana Adh

In vivo footprinting experiments have been used to analyze the binding of trans-acting regulatory factors in the 5 flanking region upstream of the alcohol dehydrogenase (Adh) gene from Arabidopsis thaliana. Protein-DNA interactions were detected by dimethyl sulfate footprinting and genomic sequencing, using an A. thaliana cell suspension culture that constitutively expressed the Adh gene. Several distinct footprinting domains have been characterized, and the potential effects of the corresponding trans-acting factors have been inferred from a comparison with data from the maize alcohol dehydrogenase-1 (Adh1) gene. One binding site is similar in sequence to one of the anaerobic response elements (ARE) of the maize gene, which has also been shown to bind to a trans-acting factor. Several of the remaining binding sites apparently represent a class of elements sharing the sequence 5-GTGG-3 within their footprint.Comparisons with maize Adh1 in vivo protein interactions reveal that the elements of Adh promoter structure are highly conserved, but the relative and absolute positions of the elements are variable.     

In vivo selection of protease cleavage sites by using chimeric Sindbis virus libraries

Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.     

High affinity binding site for nuclear factor I next to the hepatitis B virus S gene promoter.

The hepatitis B virus (HBV) surface antigen (HBsAG) is encoded by the S gene under the regulation of a promoter in the pre-S1 region. The S gene promoter does not contain a 'TATA' box-like sequence, but there is a sequence resembling, in part, the late promoter of Simian virus 40 (SV40). In an attempt to study the regulation of the S gene promoter we looked for cellular proteins which bind to this region. We report here that a nuclear protein is tightly bound to the HBV genome at a position approximately 190 bases upstream from the S gene promoter. Evidence is provided to show that (a) this nuclear protein is the nuclear factor I (NF-I) that was previously found to be bound to the inverted terminal repeat of the adenovirus (Ad) DNA and to enhance Ad DNA replication in vitro and (b) this NF-I binding site is required for optimal activity of the S gene promoter.     

Mapping rheumatoid factor binding sites using genetically engineered, chimeric IgG antibodies.

We are using chimeric IgG antibodies consisting of murine variable regions joined to human constant regions as rheumatoid factor (RF) binding substrates to localize and map IgM RF binding sites on IgG. Using chimeric antibodies in a modified RF ELISA, we showed that RFs from rheumatoid arthritis (RA) and Waldenstrom's macroglobulinemia (WMac) patients differ in their binding specificities for IgG3, although some of these RFs share common specificity for IgG1, IgG2, and IgG4. By shuffling constant region domains between IgG3 and IgG4, we showed that sequence variation in the CH3 domain is responsible for WMac-derived RF differentiation of IgG3 and IgG4. By making site-directed mutations in the wild-type IgG3 or IgG4 human gamma constant genes, we showed that His-435 is an essential residue in RF binding to IgG for most WMac RFs. The allotypic polymorphism in IgG3 at 436 is not responsible for differences in previous reports of high-frequency IgG3 binding by WMac RFs. A amino acid loop in the CH2 domain of IgG4 proximal to the CH2-CH3 interface is important in WMac RF binding to IgG; a more distal CH2 loop in CH2 has a more variable effect on WMac RF binding. To evaluate the contribution of the N-linked carbohydrate moiety at Asn-297 to RF binding sites on IgG, we measured RF binding to aglycosylated IgG antibodies produced by mutating the glycosylation signal Asn-297 to another amino acid. Of all four IgG subclasses, only aglycosylated IgG3 was a better RF binding substrate than its glycosylated subclass counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell.     

Origin of adenovirus DNA replication. Role of the nuclear factor I binding site in vivo

An assay is described that detects in vivo a single round of initiation and DNA synthesis directed by a linear molecule containing an exposed single copy of an adenovirus (Ad) origin of replication. This and a previously described assay, which measures multiple rounds of DNA replication, were used to identify DNA sequences within the Ad2 and Ad4 origins of replication that are important for ori function. Linear DNA molecules containing sequences from the Ad2 or Ad4 genome termini were cotransfected with homologous and heterologous helper virus, and net amounts of DNA synthesis were compared. Linear molecules containing the Ad4 inverted terminal repeats were replicated 20-fold better in the presence of the homologous helper, whereas both Ad2 and Ad4 inverted terminal repeats were utilized efficiently by Ad4. DNA sequence analysis of the Ad2 ori and the corresponding region in Ad4 indicated that, although there are only ten variant base-pairs, eight are located within the Ad2 DNA sequence recognized by the cellular protein nuclear factor I. This protein is required to achieve the maximal rate of Ad2 DNA replication in vitro, and these differences therefore identify DNA sequences that are crucial to Ad2 ori function. The Ad4 ITR does not contain a functional nuclear factor I binding site, and deletion analysis has demonstrated that this region of the Ad4 genome is not required for ori function. In contrast to Ad2, the DNA sequences required for the initiation of Ad4 DNA replication were shown to reside entirely within the terminal 18 base-pairs of the Ad4 inverted terminal repeat.