Purification of nuclear factor I by DNA recognition site affinity chromatography

Nuclear factor I (NF-I) is a cellular protein that enhances the initiation of adenovirus DNA replication in vitro. The enhancement of initiation correlates with the ability of NF-I to bind a specific nucleotide sequence within the viral origin of replication. We have developed a method for the purification of NF-I which is based upon the high affinity interaction between the protein and its recognition site. This approach may be generally applicable to the purification of other site-specific DNA binding proteins. The essential feature of the method is a two-step column chromatographic procedure in which proteins are first fractionated on an affinity matrix consisting of nonspecific (Escherichia coli) DNA and then on a matrix that is highly enriched in the specific DNA sequence that is recognized by NF-I. During the first step NF-I coelutes with proteins that have similar general affinity for DNA. During the second step NF-I elutes at a much higher ionic strength than the contaminating nonspecific DNA binding proteins. The DNA recognition site affinity matrix used in the second step is prepared from a plasmid (pKB67-88) that contains 88 copies of the NF-I binding site. This plasmid was constructed by means of a novel cloning strategy that generates concatenated NF-I binding sites arranged exclusively in a direct head to tail configuration. Using this purification scheme, we have obtained a 2400-fold purification of NF-I from crude HeLa nuclear extract with a 57% recovery of specific DNA binding activity. Throughout the purification procedure NF-I retained the ability to enhance the efficiency of initiation of adenovirus DNA replication in vitro. Electrophoresis of the purified fraction on sodium dodecyl sulfate-polyacrylamide gels revealed a population of related polypeptides that ranged in apparent molecular weight from 66,000 to 52,000. The native molecular weight of NF-I deduced from gel filtration and glycerol sedimentation studies is 55,000 and the frictional ratio is 1.3. These results suggest that NF-I exists as a globular monomer in solution.     

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.     

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

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.     

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.     

Analysis of nuclear factor I binding to DNA using degenerate oligonucleotides.

Nuclear factor I (NFI) binds tightly to DNA containing the consensus sequence TGG(N)6-7GCCAA. To study the role of the spacing between the TGG and GCCAA motifs, oligonucleotides homologous to the NFI binding site FIB-2 were synthesized and used for binding assays in vitro. The wild-type site (FIB-2.6) has a 6bp spacer region and binds tightly to NFI. When the size of this spacer was altered by +/- 1 or 2bp the binding to NFI was abolished. To further assess the role of the spacer and bases flanking the motifs, two oligonucleotide libraries were synthesized. Each member of these libraries had intact TGG and GCCAA motifs, but the sequence of the spacer and the 3bp next to each motif was degenerate. The library with a 6bp spacer bound to NFI to 40-50% the level of FIB-2.6. The library with a 7bp spacer bound to NFI to only 4% the level of FIB-2.6 and some of this binding was weaker than that of FIB-2.6 DNA. This novel use of degenerate DNA libraries has shown that: 1) the structural requirements for FIB sites with a 7bp spacer are more stringent than for sites with a 6bp spacer and 2) a limited number of DNA structural features can prevent the binding of NFI to sites with intact motifs and a 6bp spacer region.     

Adenovirus DNA replication in vitro: site-directed mutagenesis of the nuclear factor I binding site of the Ad2 origin.

The template requirements for efficient adenovirus DNA replication were studied in vitro in a reconstituted system with cloned DNA fragments, containing the Ad2 origin region, as templates. Replication is enhanced by nuclear factor I, a cellular protein that binds specifically to the Ad2 origin. This stimulation is shown to be strongly dependent on the concentration of the adenovirus DNA binding protein. Using synthetic oligonucleotides we have constructed plasmids with base substitutions in the nuclear factor I binding region. Footprint analysis and competition filter binding studies show that two of the three small blocks of conserved nucleotides in this region are involved in the binding of nuclear factor I. The binding affinity can be influenced by the base composition of the degenerate region just outside these two blocks. In vitro initiation and DNA chain elongation experiments with the mutants demonstrate that binding of nuclear factor I to the Ad2 origin is necessary for stimulation. However, binding alone is not always sufficient since a mutation which only slightly disturbs binding is strongly impaired in stimulation of DNA replication by nuclear factor I.     

Site-specific DNA binding of nuclear factor I: effect of the spacer region.

Nuclear factor I (NFI) is a site-specific DNA binding protein required for the replication of adenovirus type 2 DNA in vitro and in vivo. To study sequence requirements for the interaction of NFI with DNA, we have measured the binding of the protein to a variety of synthetic sites. Binding sites for NFI (FIB sites) were previously shown to contain a consensus sequence composed of 2 motifs, TGG (Motif 1), and GCCAA (Motif 2), separated by a 6 or 7bp spacer region. To assess conserved sequences in the spacer region and flanking sequences which affect NFI binding, we have isolated clones from oligonucleotide libraries that contain the two motifs flanked by 3 degenerate nucleotides and separated by degenerate spacer regions of 6 or 7 nucleotides. With a 6bp spacer region, a strong bias exists for a C or A residue in the first position of the spacer. Sites with a 7bp spacer region contain a G and C or A residue at the first and second positions, respectively, of the spacer, but also possess conserved residues at other positions of the site.     

A quantitative analysis of nuclear factor I/DNA interactions.

Nuclear factor I (NFI) was purified to homogeneity from porcine liver by DNA-affinity chromatography and displays a single band with a molecular weight of 36 kDa in SDS-polyacrylamide gels. The purified protein was used to determine absolute equilibrium binding constants by gel retardation techniques for a variety of DNA fragments with genuine or mutated NFI binding sites and a number of DNA fragments derived from various eukaryotic promoters carrying the CCAAT-box as a half-site for NFI binding. We present a model which allows prediction of the functional significance of mutated NFI binding-sites from sequence data. The data suggest that the single molecular species of NFI from porcine liver may not be able to recognize and activate the -CCAAT- promoter element in vivo without additional interactions, e.g. with other proteins.     

Mutant analysis of protein interactions with a nuclear factor I binding site in the SL3-3 virus enhancer.

Nuclear factor I (NFI) is shown to be of importance for the activity of the enhancer element of a T-cell leukemogenic murine retrovirus, SL3-3, and for the regulation of this element by glucocorticoid. Each nucleotide of the binding site of the NFI proteins was mutated, and the effects of the mutations were quantitated with an electrophoretic mobility shift assay. Mutations in the inverted repeat of the binding site have symmetric effects which strongly support the notion that NFI proteins preferentially bind to dyad symmetry sites. Such binding sites were shown to be more than 100 fold stronger than the corresponding single binding sites. We find dyad symmetry sequences which are much stronger NFI binding sites than NFI sites identified in different genes and also stronger than previously proposed consensus binding sequences for NFI.     

A strained DNA binding helix is conserved for site recognition,folding nucleation,and conformational modulation

Nucleic acid recognition is often mediated by α‐helices or disordered regions that fold into α‐helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical α‐helix is stabilized at the N‐terminus, while the PII forms at the C‐terminus half of the peptide. Re‐examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N‐terminus half is a canonical α‐helix, while the C‐terminal half adopts a 3₁₀ helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 10⁴‐fold lower affinity, and 500‐fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained α‐helix conformation, “presented” by this unique architecture. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 432–443, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Reconstruction of adenovirus replication origins with a human nuclear factor I binding site

Nuclear factor I is a host-coded DNA-binding protein that stimulates initiation of adenovirus DNA replication. To understand the mechanism of action of nuclear factor I, we have constructed, by recombinant DNA techniques, origins of replication in which the adenovirus type 5 nuclear factor I binding site (FIB site) has been replaced by a FIB site isolated from human genomic DNA (Gronostajski, R. M., Nagata, K., and Hurwitz, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4013-4017). Assays of such recombinants for initiation and elongation in vitro showed that nuclear factor I was active only when the FIB site was relatively close to the DNA terminus, i.e. the FIB site was centered at nucleotides 30-36 from the end of the DNA. Nuclear factor I was active in either orientation within this distance range. The presence of one or two additional FIB sites in the downstream region had no effect. The implications of these results for the mechanism of nuclear factor I action are discussed.     

High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site.

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.     

DNA binding site of the helix destabilizing protein gp32 from bacteriophage T4.

A helix destabilizing protein, the product of gene 32 (gp32) of bacteriophage T4, was subjected to limited proteolysis to produce three types of products with differing affinities for DNA. Previous work has suggested that the 18 amino acids at the N-terminus are required for tight binding to single-stranded DNA (Hosoda &; Moise, 1978). This paper reports the sequence of the N-terminal region and predicts the amino acid residues responsible for DNA binding.