DNA-binding domains of Fos and Jun do not induce DNA curvature: An investigation with solution and gel methods

AUTOR(ES)

Sitlani, Ayesha

FONTE

The National Academy of Sciences

RESUMO

We demonstrate the use of a DNA minicircle competition binding assay, together with DNA cyclization kinetics and gel-phasing methods, to show that the DNA-binding domains (dbd) of the heterodimeric leucine zipper protein Fos–Jun do not bend the AP-1 target site. Our DNA constructs contain an AP-1 site phased by 1–4 helical turns against an A-tract-directed bend. Competition binding experiments reveal that (dbd)Fos–Jun has a slight preference for binding to linear over circular AP-1 DNAs, independent of whether the site faces in or out on the circle. This result suggests that (dbd)Fos–Jun slightly stiffens rather than bends its DNA target site. A single A-tract bend replacing the AP-1 site is readily detected by its effect on cyclization kinetics, in contrast to the observations for Fos–Jun bound at the AP-1 locus. In contrast, comparative electrophoresis reveals that Fos–Jun–DNA complexes, in which the A-tract bend is positioned close (1–2 helical turns) to the AP-1 site, show phase-dependent variations in gel mobilities that are comparable with those observed when a single A-tract bend replaces the AP-1 site. Whereas gel mobility variations of Fos–Jun–DNA complexes decrease linearly with increasing Mg2+ contained in the gel, the solution binding preference of (dbd)Fos–Jun for linear over circular DNAs is independent of Mg2+ concentration. Hence, gel mobility variations of Fos–Jun–DNA complexes are not indicative of (dbd)Fos–Jun-induced DNA bending (upper limit 5°) in the low salt conditions of gel electrophoresis. Instead, we propose that the gel anomalies depend on the steric relationship of the leucine zipper region with respect to a DNA bend.

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