The novel Mrf-2 DNA-binding domain recognizes a five-base core sequence through major and minor-groove contacts.

Recent NMR studies of the purified Mrf-2 DNA-binding domain peptide have shown that its structure differs significantly from previously characterized classes of DNA-binding domains. Here we report biochemical studies of the DNA-binding properties of this peptide. Binding interference and binding site selection assays indicated that Mrf-2 requires the core sequence AATA(C/T) for high affinity binding. Kinetic analyses of several selected sequences indicated that the core sequence alone is not sufficient for high affinity binding, however. Kinetic analyses were also performed using a series of synthetic oligonucleotides with single base analogues at each position in the core sequence. Base analogues that altered the major groove structure reduced or eliminated Mrf-2 binding when present in the second, third, and fourth base-pairs of the core sequence, but had little or no effect in the first and fifth positions. These results suggest that Mrf-2 contacts both the major and minor grooves of its target sequences.     

Activation of nuclear protein binding to the antioxidant/electrophile response element in vascular smooth muscle cells by benzo(a)pyrene

This laboratory has previously shown that binding of nuclear proteins to the antioxidant/electrophile response element (ARE/EpRE) participates in deregulation of vascular gene expression by benzo(a)pyrene (BaP), a suspected atherogen. In the present study, oligonucleotides representing ARE/EpREs within the c-Ha-ras and glutathione-S-transferase (GST-Ya) promoters were employed to evaluate the role of flanking sequences in stabilizing protein:DNA interactions in BaP-treated vascular smooth muscle cells (vSMCs). We also wanted to define promoter-specific patterns of protein recognition to ARE/EpREs in this cell system. In electrophoretic mobility shift assays (EMSA), optimal protein binding to a human Ha-ras ARE/EpRE variant sequence fitted to match the extended mouse(m) GST-Ya ARE/EpRE core (5'-TMAnnRTGAYnnnGCR-3') was dependent on 5' nucleic acid sequence. Using immobilized DNA affinity chromatography (IDAC), we identified four nuclear proteins of M(r) 62, 60, 50, and 30 kDa that associated specifically with the mGSTYa ARE/EpRE. Photo crosslinking to a BrdU-substituted hHa-ras or mGST ARE/EpRE probe identified specific proteins of M(r) 80, 60, 55, 25, 23 kDa or 80, 60, 55, 27, 25, 23 kDa, respectively. Protein:DNA complexes detected using IDAC eluate overlapped with those observed in crude nuclear extracts. Chemical treatments known to modulate ARE/EpRE protein binding in vSMCs did not alter overall protein:DNA affinity and/or sequence recognition to either hHa-ras or mGST-Ya elements. We conclude that nucleotide sequences 5' to the core ARE/EpRE influence specific binding of nuclear proteins and that multiple proteins bind to ARE/EpREs in a promoter-specific manner in vSMCs.     

Determination of recognition-sequences for DNA-binding proteins by a polymerase chain reaction assisted binding site selection method (BSS) using nitrocellulose immobilized DNA binding protein.

We have developed a simple procedure for rapid determination of a DNA sequence recognized by a DNA binding protein based on immobilization of the protein on nitrocellulose filters. The procedure consists of the following steps: A recombinant protein with a functional DNA binding domain is expressed in E. coli. The protein is purified to homogeneity, immobilized on nitrocellulose paper, and exposed to a pool of double stranded oligonucleotides carrying in the central part a 20 bp random sequence, which is flanked by conserved sequences with restriction endonuclease recognition sites for analytical and subcloning purposes and sequences complementary to polymerase chain reaction primers. Oligonucleotides retained by the DNA-binding protein are liberated by increasing the ionic strength and used in a new binding process after amplification by the polymerase chain reaction technique. Finally the amplified product is cloned for determination of the DNA sequence selected by the DNA-binding protein. Murine Zn-finger and basic helix-loop-helix DNA binding proteins were used to demonstrate the efficiency of the method. We show that the yield of oligonucleotides binding to the protein was increased by several consecutive rounds of filter binding and amplification, and that the protein extracted a specific sequence from the pool of random oligonucleotides.     

Pentaprobe: a comprehensive sequence for the one-step detection of DNA-binding activities

The rapid increase in the number of novel proteins identified in genome projects necessitates simple and rapid methods for assigning function. We describe a strategy for determining whether novel proteins possess typical sequence-specific DNA-binding activity. Many proteins bind recognition sequences of 5 bp or less. Given that there are 4⁵ possible 5 bp sites, one might expect the length of sequence required to cover all possibilities would be 4⁵ × 5 or 5120 nt. But by allowing overlaps, utilising both strands and using a computer algorithm to generate the minimum sequence, we find the length required is only 516 base pairs. We generated this sequence as six overlapping double-stranded oligonucleotides, termed pentaprobe, and used it in gel retardation experiments to assess DNA binding by both known and putative DNA-binding proteins from several protein families. We have confirmed binding by the zinc finger proteins BKLF, Eos and Pegasus, the Ets domain protein PU.1 and the treble clef N- and C-terminal fingers of GATA-1. We also showed that the N-terminal zinc finger domain of FOG-1 does not behave as a typical DNA-binding domain. Our results suggest that pentaprobe, and related sequences such as hexaprobe, represent useful tools for probing protein function.     

A consensus DNA-binding site for the androgen receptor.

We have used a DNA-binding site selection assay to determine a consensus binding sequence for the androgen receptor (AR). A purified fusion protein containing the AR DNA-binding domain was incubated with a pool of random sequence oligonucleotides, and complexes were isolated by gel mobility shift assays. Individually selected sites were characterised by nucleotide sequencing and compiled to give a consensus AR-binding element. This sequence is comprised of two 6-basepair (bp) asymmetrical elements separated by a 3-bp spacer, 5'-GGA/TACANNNTGTTCT-3', similar to that described for the glucocorticoid response element. Inspection of the consensus revealed a slight preference for G or A nucleotides at the +1 position in the spacer and for A and T nucleotides in the 3'-flanking region. Therefore, a series of oligonucleotides was designed in which the spacer and flanking nucleotides were changed to the least preferred sequence. Competition experiments with these oligonucleotides and the AR fusion protein indicated that an oligonucleotide with both the spacer and flanking sequences changed had greater than 3-fold less affinity than the consensus sequence. The functional activity of these oligonucleotides was also assessed by placing them up-stream of a reporter gene in a transient transfection assay and correlated with the affinity with which the AR fusion protein bound to DNA. Therefore, sequences surrounding the two 6-bp half-sites influence both the binding affinity for the receptor and the functional activity of the response element.     

Enthalpy/entropy compensation: influence of DNA flanking sequence on the binding of 7-amino actinomycin D to its primary binding site in short DNA duplexes

The effect of the context of the flanking sequence on ligand binding to DNA oligonucleotides that contain consensus binding sites was investigated for the binding of the intercalator 7-amino actinomycin D. Seven self-complementary DNA oligomers each containing a centrally located primary binding site, 5'-A-G-C-T-3', flanked on either side by the sequences (AT)(n) or (AA)(n) (with n = 2, 3, 4) and AA(AT)(2), were studied. For different flanking sequences, (AA)(n)-series or (AT)(n)-series, differential fluorescence enhancements of the ligand due to binding were observed. Thermodynamic studies indicated that the flanking sequences not only affected DNA stability and secondary structure but also modulated ligand binding to the primary binding site. The magnitude of the ligand binding affinity to the primary site was inversely related to the sequence dependent stability. The enthalpy of ligand binding was directly measured by isothermal titration calorimetry, and this made it possible to parse the binding free energy into its energetic and entropic terms. Our results reveal a pronounced enthalpy-entropy compensation for 7-amino actinomycin D binding to this family of oligonucleotides and suggest that the DNA sequences flanking the primary binding site can strongly influence ligand recognition of specific sites on target DNA molecules.