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

Sitlani, Ayesha; Crothers, Donald M.

doi:10.1073/pnas.93.8.3248

Cited by 56 publications

(82 citation statements)

References 31 publications

(28 reference statements)

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“…Two strategies have been developed to extract the curvature and mechanical properties from a set of J factor measurements: the total length assay and the phasing assay (13,14,27). In the first assay, the total length of the DNA construct is changed, whereas the phasing between the test sequence and the A tracts is fixed, and vice versa in the phasing assay.…”

Section: Resultsmentioning

confidence: 99%

High-throughput approach for detection of DNA bending and flexibility based on cyclization

Zhang

Crothers

2003

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a high-throughput approach to the laborintensive problems of DNA cyclization, which we use to characterize DNA curvature and mechanical properties. The method includes a combinatorial approach to make the DNA constructs needed and automated real-time measurement of the kinetics using fluorescence. We validated the approach and investigated the flexibility of two kinds of nicked DNA and AT dinucleotide repeats. We found that, although the nicks hardly alter the bending flexibility, they significantly increase the torsional flexibility, and that the AT repeat has 28% (؎12%) lower bending rigidity than a generic DNA sequence.S equence-dependent DNA curvature and flexibility are important characteristics of DNA structure (1-5), which influences almost every aspect of DNA-related processes (6, 7). The idea that DNA sequence information might also be encoded through its 3D shape and mechanical properties may provide a new dimension for functional genomics (8). However, our limited power of mapping DNA tertiary structure from its sequence impedes the testing of this idea, due to lack of a reliable and convenient approach to quantify DNA bending and flexibility (9).Among a variety of approaches that have been used to measure the global curvature and flexibility of DNA, DNA cyclization has the advantage of high sensitivity, a complete theoretical basis, and the capacity to measure almost all interesting features of global geometric and mechanical properties of DNA, including the magnitude and direction of its curvature, helical repeat, and bending and twisting flexibility (10-13). Its principle is illustrated in Fig. 7, which is published as supporting information on the PNAS web site, www.pnas.org. In this method, the J factors of a series of DNA molecules containing the same test sequence are measured and analyzed by using a model that relates them to DNA properties. The J factor is the effective concentration of one end of a DNA molecule at the other and equal to the ratio of the unimolecular ligation rate constant (k 1 ) to the bimolecular ligation rate constant (k 2 ) catalyzed by DNA ligase under certain conditions. Because k 2 is the same for all constructs with the same cohesive ends, the main aim of DNA cyclization is to measure k 1 .The traditional implementation (10, 14) of cyclization involves radioactive labeling of DNA constructs and separation of the ligation mixture by PAGE to obtain data on ligation kinetics. The data are mainly interpreted by Monte Carlo simulation to determine curvature and flexibility parameters. Several aspects limit throughput, including the time needed to prepare the Ϸ15 constructs required for the study of a single sequence. The need for multiple oligonucleotide syntheses, constant optimization of PCR reactions, and the limited lifetime of radioactive labeled constructs make the sample preparation extremely tedious. Second, determining ligation rates from gels is labor-intensive. Finally, data interpretation using Monte Carlo simulation is cumbersome and computat...

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Section: Resultsmentioning

confidence: 99%

High-throughput approach for detection of DNA bending and flexibility based on cyclization

Zhang

Crothers

2003

Proc. Natl. Acad. Sci. U.S.A.

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“…Sitlani and Crothers reported that phasing oligonucleotides containing directly adjacent reference and test bends could lead to erroneous protein-induced bend angles because of interactions in the gel between the bound protein and the reference A-tract (Sitlani & Crothers, 1996). To test whether such interactions existed in our system, which contained directly adjacent reference and test bends, we performed phasing analyses with oligonucleotides containing a CRE site whose center was separated from the A-tract center by between five and six helical turns of DNA.…”

Section: Methodsmentioning

confidence: 99%

“…These experiments showed clear evidence of intrinsic CRE curvature and straightening of this curvature by CRE-BP1-derived peptides. In addition, minicircle ligation experiments were performed with CRE phasing oligonucleotides ( Figure 3A) in the presence and absence of peptides ccc, csg, cgg, and ggg ( Figure 2) (Sitlani & Crothers, 1996). Peptides ccc, csg, and cgg decreased the ratio of circular to linear ligation products formed from CRE-26 in a concentration-dependent manner, whereas peptide ggg did not (M. A. Fabian, unpublished results).…”

Section: Methodsmentioning

confidence: 99%

Electrostatic Mechanism for DNA Bending by bZIP Proteins

et al. 1997

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Biology is replete with examples of protein-induced DNA bending, yet the forces responsible for bending have been neither established nor quantified. Mirzabekov and Rich proposed in 1979 that asymmetric neutralization of the anionic phosphodiester backbone by basic histone proteins could provide a thermodynamic driving force for DNA bending in the nucleosome core particle [Mirzabekov, A. D., & Rich, A. (1979) A. 93, 9515-9520]. Here it is shown that bZIP proteins bend DNA via a mechanism involving direct contacts between one or two basic side chains and a symmetry-related pair of unique, nonbridging phosphate oxygens. The locations of these phosphates provide direct experimental support for a protein-induced bending mechanism based on asymmetric charge neutralization. This straightforward mechanism is compatible with many DNA-recognition motifs and may represent a general strategy for the assembly of protein-DNA complexes of defined stereochemistries.

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“…In summary, there is a clear need for experimentally based parameters that describe the ensembleaveraged solution curvature and local bending and twist flexibility of specific DNA sequences. In addition to providing a test for parameter sets based on crystallography and theory, such a database would allow more stringent test of the intriguing indications that curved and flexible sequences are organized coherently in promoters and other DNA sequences (20)(21)(22) and would enable analysis of the properties of the noncoding DNA sequences that constitute Ϸ95% of the human genome.We have developed a strategy, based on specialized DNA molecules for measurement of cyclization kinetics, that offers a general solution to this problem (23)(24)(25)(26)(27)(28). A key feature is inclusion of phased A-tracts, whose intrinsic curvature fixes the mean rotational setting of the circle.…”

mentioning

confidence: 99%

“…We have developed a strategy, based on specialized DNA molecules for measurement of cyclization kinetics, that offers a general solution to this problem (23)(24)(25)(26)(27)(28). A key feature is inclusion of phased A-tracts, whose intrinsic curvature fixes the mean rotational setting of the circle.…”

mentioning

confidence: 99%

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

Roychoudhury

Sitlani

Lapham

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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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°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 ensembleaveraged global solution structure and mechanical properties of any Ϸ10-bp DNA sequence element of interest, providing information complementary to that from NMR and crystallographic structural studies.D NA in cells is highly compacted from the unfettered size it would have if free in solution. The first stage of packaging is achieved by wrapping DNA around core histones in a welldefined conformation (1), forming a beads-on-a-string structure that is further compacted by additional proteins into folded chromatin and ultimately into metaphase chromosomes. Interphase chromatin is generally less condensed, but it contains regulatory sites such as promoters at which DNA must be distorted to enable interactions between proteins that bind distant sites on linear DNA. As a relatively stiff polymer, DNA resists the bending required for packaging, which requires free energy assistance from strong interactions with core histones and other proteins.Variations in DNA sequence can, in principle, modulate the energy cost of bending and packaging. For example, intrinsic curvature of the kind provided by A-tracts (2) should, if in the proper direction, lessen the energy required for bending. Increased local bending flexibility could reduce the energetic cost of packaging, as could an increase in twist flexibility (3), assuming that the helical phasing between distal regulatory sequence elements must be altered to form a protein complex.The stiffness of DNA (the inverse of its flexibility) is traditionally described by the persistence length P, defined as the average projection of the end-to-end vector of a very long chain on its initial direction. Classical techniques, such as light scattering (4) and rotational diffusion (5), supplemented by DNA cyclization kinetics (6), have led to estimates of around 140-180 bp for P. However, these methods are generally unable to deconvolute rigorously the contributions of curvature and flexibility to apparent persistence length (7), unless special constructs are used (8).Parameter sets exist for predicting DNA curvature based on roll and tilt values for dinucleotide (9-13) and tetranucleotide (14) steps, but the dinucleotide parameters vary considerably from set to set (15). Nor is there sufficient data to define unambiguously the sequence dependence of the experimental persistence length, ...

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

Cited by 56 publications

References 31 publications

High-throughput approach for detection of DNA bending and flexibility based on cyclization

High-throughput approach for detection of DNA bending and flexibility based on cyclization

Electrostatic Mechanism for DNA Bending by bZIP Proteins

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

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