Fos-Jun heterodimers and Jun homodimers bend DNA in opposite orientations: implications for transcription factor cooperativity

Association of Fos and Jun with the AP-1 site results in a conformational change in the basic amino acid regions that constitute the DNA-binding domain. We show that Fos and Jun induce a corresponding alteration in the conformation of the DNA helix. Circular permutation analysis indicated that both Fos-Jun heterodimers and Jun homodimers induce flexure at the AP-1 site. Phasing analysis demonstrated that Fos-Jun heterodimers and Jun homodimers induce DNA bends that are directed in opposite orientations. Fos-Jun heterodimers bend DNA toward the major groove, whereas Jun homodimers bend DNA toward the minor groove. Fos and Jun peptides encompassing the dimerization and DNA-binding domains bend DNA in the same orientations as the full-length proteins. However, additional regions of both proteins influence the magnitude of the DNA bend angle. Thus, despite the amino acid sequence similarity in the basic region Fos-Jun heterodimers and Jun homodimers form topologically distinct DNA-protein complexes.     

DNA binding activities of three murine Jun proteins: stimulation by Fos

Three members of the Jun/AP-1 family have been identified in mouse cDNA libraries: c-Jun, Jun-B, and Jun-D. We have compared the DNA binding properties of the Jun proteins by using in vitro translation products in gel retardation assays. Each protein was able to bind to the consensus AP-1 site (TGACTCA) and, with lower affinity, to related sequences, including the cyclic AMP response element TGACGTCA. The relative binding to the oligonucleotides tested was similar for the different proteins. The Jun proteins formed homodimers and heterodimers with other members of the family, and they were bound to the AP-1 site as dimers. When Fos translation product was present, DNA binding by Jun increased markedly, and the DNA complex contained Fos. The C-terminal homology region of Jun was sufficient for DNA binding, dimer formation, and interaction with Fos. Our general conclusion is that c-Jun, Jun-B, and Jun-D are similar in their DNA binding properties and in their interaction with Fos. If there are functional differences between them, they are likely to involve other activities of the Jun proteins.     

Anisotropic flexibility of DNA depends on the base sequence. Conformation calculations of double-stranded tetranucleotides AAAA:TTTT, (AATT)2, (TTAA)2, GGGG:CCCC, (GGCC)2, (CCGG)2

The bending flexibility of six tetramers was studied in an assumption that they were extended in the both directions by regular double helices. The bends of B-DNA in different directions were considered. The stiffness of the B-DNA double helix when bent into the both grooves proved to be less pronounced than in the perpendicular direction by the order of magnitude. Such an anisotropy is a feature of the sugar-phosphate backbone structure. The calculated fluctuations of the DNA bending along the dyad axis, 5-7 degrees, are in agreement with the experimental value of DNA persistence length. Anisotropy of the double helix is sequence-dependent: most easily bent into the minor groove are the tetramers with purine-pyrimidine dimer (RY) in the middle. In contrast, YR dinucleotides prefer bending into the major groove, moreover, they have an equilibrium bend of 6-12 degrees into this groove. The above inequality is caused by the stacking interaction of the bases. The bend in the central dimers is distributed to some extent between the adjacent links, though the main fraction of the bend remains within the central link. Variation of the sugar-phosphate geometry in the bent helix is unessential, so that DNA remains within the limits of the B-family of forms: namely, when the helical axis is bent by 20 degrees the backbone dihedral angles vary by no more than 15 degrees. The obtained results are in accord with the X-ray structure of B-DNA dodecamer; they further substantiate our earlier model of DNA wrapping in the nucleosome by means of "mini-kinks" separated by a half-pitch of the double helix, i.e. by 5-6 b. p. Sequence-dependent anisotropy of DNA presumably dictates the three-dimensional structure of DNA in solution as well. We have found that nonrandom allocation of YR dimers leads to the systematic bends in the equilibrium structure of certain DNA fragments. To the four "Calladine rules" two more can be added: the minor-groove steric clash of purines in the YR sequences are avoided by: (1) bending of the helix into the major groove; (2) increasing the distance between the base pairs (stretching the double helix).     

The SKN-1 amino-terminal arm is a DNA specificity segment

The Caenorhabditis elegans SKN-1 protein binds DNA through a basic region like those of bZIP proteins and through a flexible amino-terminal arm segment similar to those with which numerous helix-turn-helix proteins bind to bases in the minor groove. A recent X-ray crystallographic structure suggests that the SKN-1 amino-terminal arm provides only nonspecific DNA binding. In this study, however, we demonstrate that this segment mediates recognition of an AT-rich element that is part of the preferred SKN-1 binding site and thereby significantly increases the sequence specificity with which SKN-1 binds DNA. Mutagenesis experiments show that multiple amino acid residues within the arm are involved in binding. These residues provide binding affinity through distinct but partially redundant interactions and enhance specificity by discriminating against alternate sites. The AT-rich element minor groove is important for binding of the arm, which appears to affect DNA conformation in this region. This conformational effect does not seem to involve DNA bending, however, because the arm does not appear to affect a modest DNA bend that is induced by SKN-1. The data illustrate an example of how a small, flexible protein segment can make an important contribution to DNA binding specificity through multiple interactions and mechanisms.     

The basic region of Fos mediates specific DNA binding.

The DNA-binding domains of the members of the Fos and Jun families of proteins consist of a basic region followed by a dimerizing segment with heptad repeats of leucine. Fos-Jun heterodimers and Jun alone, but not Fos alone, bind to the symmetrical sequences TGACTCA (AP-1 site) or TGACGTCA (cAMP response element or CRE). We set out to test the hypothesis that in the Fos-Jun heterodimer the basic region of Fos confers specific DNA-binding properties equivalent to the contribution of the basic region of Jun. Fos-Jun chimeric proteins were prepared consisting of the basic region of one protein joined to the leucine repeat of the other. Heterodimers with mixed Fos and Jun leucine repeat segments showed high affinity binding to the AP-1 site or CRE whether they contained two basic regions from Jun, two basic regions from Fos, or one from each source. Heterodimers with two Fos basic regions showed somewhat greater affinity for the CRE and AP-1 site than the heterodimer with two Jun basic regions. The DNA sequence specificity and the purine and phosphate DNA contact sites for each heterodimer were similar. We conclude that in the Fos-Jun heterodimer the basic region of Fos contributes specific DNA-binding properties equivalent to those of Jun. Our results support a model in which the Fos and Jun basic regions of the Fos-Jun heterodimer each interact with symmetrical DNA half sites.     

Helical phasing between DNA bends and the determination of bend direction.

The presence and location of bends in DNA can be inferred from the anomalous mobility of DNA fragments or protein-DNA complexes during electrophoresis in polyacrylamide gels. Direction of bending is not so easily determined. We show here that a protein-induced bend, when linked to a protein-independent DNA bend by a segment of variable length, exhibits an electrophoretic mobility that varies in a sinusoidal manner with the length of the linker. Mobility minima occur once for each addition to the linker of one helical turn of DNA. Since minima should occur when two bends reinforce one another, the direction of one bend relative to the other can be determined from the distances between the two centers of bending at which minima occur. Our results strongly support the idea that the A5-6 tracts in kinetoplast DNA bend towards the minor groove while the bend at the recombination site of the gamma delta resolvase (binding site I of the gamma delta res site) bends towards the major groove.     

Large,sequence-dependent effects on DNA conformation by minor groove binding compounds

To determine what topological changes antiparasitic heterocyclic dications can have on kinetoplast DNA, we have constructed ligation ladders, with phased A5 and ATATA sequences in the same flanking sequence context, as models. Bending by the A5 tract is observed, as expected, while the ATATA sequence bends DNA very little. Complexes of these DNAs with three diamidines containing either furan, thiophene or selenophene groups flanked by phenylamidines were investigated along with netropsin. With the bent A5 ladder the compounds caused either a slight increase or decrease in the bending angle. Surprisingly, however, with ATATA all of the compounds caused significant bending, to values close to or even greater than the A5 bend angle. Results with a mixed cis sequence, which has one A5 and one ATATA, show that the compounds bend ATATA in the same direction as a reference A5 tract, that is, into the minor groove. These results are interpreted in terms of a groove structure for A5 which is largely pre-organized for a fit to the heterocyclic amidines. With ATATA the groove is intrinsically wider and must close to bind the compounds tightly. The conformational change at the binding site then leads to significant bending of the alternating DNA sequence.