Colorimetric detection of glutamine synthetase-catalyzed transferase activity in glucocorticoid-treated skeletal muscle cells.

Induction of the enzyme glutamine synthetase (GS) by corticosteroids correlates with muscle wasting and gluconeogenesis, characteristic side effects of chronic glucocorticoid treatment. This highlights the importance of developing robust high-throughput assays to measure drug-induced GS in whole cells. We have optimized a colorimetric method to measure GS-catalyzed gamma-glutamyltransferase (GT) activity in rat L6 skeletal muscle cells (96-well-plate format) and human skeletal muscle cells (24-well-plate format). We observe a fourfold increase in GT activity in dexamethasone treated L6 cells, as compared to untreated cells, with good reproducibility in the measurements (errors of less than 5%). This assay can distinguish between partial agonists such as halopredone acetate and complete agonists such as prednisolone and measure the potency of known glucocorticoid receptor (GR) antagonists like mifepristone. Importantly, the ability of corticosteroids to induce GS-catalyzed GT activity correlates well with their whole cell GR binding potency, indicating a GR-specific effect. Interestingly, in general, induction of GT activity by commonly administered anti-inflammatory corticosteroid drugs is comparable in rat and human skeletal muscle cells, which emphasizes the potential of a rat model system to study GS induction and muscle wasting by these drugs in humans.

Global structure and mechanical properties of a 10-bp nucleosome positioning motif.

The method of DNA cyclization kinetics reveals special properties of the TATAAACGCC sequence motif found in DNA sequences that have high affinity for core histones. Replacement of 30 bp of generic DNA by three 10-bp repeats of the motif in small cyclization constructs increases cyclization rates by two orders of magnitude. We document a 13 degrees bend in the motif and characterize the direction of curvature. The bending force constant is smaller by nearly 2-fold and there is a 35% decrease in the twist modulus, relative to generic DNA. These features are the likely source of the high affinity for bending around core histones to form nucleosomes. Our results establish a protocol for determination of the ensemble-averaged global solution structure and mechanical properties of any approximately 10-bp DNA sequence element of interest, providing information complementary to that from NMR and crystallographic structural studies.

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

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 degrees) 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.

Fos and Jun do not bend the AP-1 recognition site.

We have used a solution-based DNA cyclization assay and a gel-phasing method to show that contrary to previous reports [Kerppola, T. K. & Curran, T. (1991) Cell 66, 317-326], basic region leucine zipper proteins Fos and Jun do not significantly bend their AP-1 recognition site. We have constructed two sets of DNA constructs that contain the 7-bp 5'-TGACTCA-3' AP-1 binding site, from either the yeast or the human collagenase gene, which is well separated from and phased by 3-4 helical turns against an A tract-directed bend. The cyclization probabilities of DNAs with altered phasings are not significantly affected by Fos-Jun binding. Similarly, Fos-Jun and Jun-Jun bound to differently phased DNA constructs show insignificant variations in gel mobilities. Both these methods independently indicate that Fos and Jun bend their AP-1 target site by <5 degrees, an observation that has important implications in understanding their mechanism of transcriptional regulation.

Sequence-specific recognition of DNA by phenanthrenequinone diimine complexes of rhodium(III): importance of steric and van der Waals interactions.

The importance of steric and van der Waals interactions in the sequence-specific recognition of DNA by [Rh(phi)]3+ complexes has been explored through the synthesis and application of a series of Rh(phi)3+ (phi: 9,10-phenanthrenequinone diimine) derivatives. [Rh(phi)]3+ complexes intercalate in the major groove of DNA via the phi ligand and promote strand scission in the presence of UV light. The complexes reported here are derivatives of the parent molecules [Rh(phi)2bpy]3+ and [Rh(bpy)2phi]3+ (bpy: 2,2'-bipyridyl). The [Rh(phi)]3+ complexes have comparable photoefficiencies; therefore, their different photocleavage patterns on 32P-end-labeled DNA fragments reflect their unique sequence-specific recognition characteristics. The shapes of the [Rh(phi)]3+ complexes are found to govern DNA recognition and reaction. Importantly and generally, the more sterically bulky complexes, containing methyl or phenyl groups on the ancillary ligands, cleave DNA at a subset of sequences recognized by their parent molecules. [Rh-(diphenylbpy)2phi]3+ specifically targets the site 5'-CTCTAGAG-3'. Furthermore, chiral discrimination in site selectivity is observed; the different isomers target different sites. delta- and lambda-[Rh(5,5'-dimethylbpy)2phi]3+ cleave specifically at sites that are defined by the consensus sequences 5'-C-T-N-G-3' and 5'-A-C/G-T-C/G-3', respectively. The sequence selectivities may be understood on the basis of both negative steric clashes and positive van der Waals interactions between methyl groups on the metal complex and thymine methyl groups in the DNA major groove.