A-tract Bend Research Articles

Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes.

A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the Caenorhabditis elegans genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.

FRET studies of a landscape of Lac repressor-mediated DNA loops

DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacI•DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Förster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-β-d-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility.

FRET and Sm-FRET Characterization of a Lac Repressor-Dna Looping Landscape

DNA looping is fundamental to transcriptional regulation, and the archetypal loop is formed by the Lac repressor protein, LacI. Thermodynamic characterization of looping energetics with and without induction is a necessary input for quantitative modeling of gene expression, and the LacI system is also a test case for statistical mechanics and rod mechanics models of protein/DNA flexibility. We have systematically constructed a landscape of LacI-DNA looping variants patterned on previously developed molecules in which lac operators bracket a sequence-directed A-tract bend. The hyperstability of these loops enables study of LacI protein geometries that would otherwise be unstable with respect to unlooped single- or double-bound DNA. FRET donors and acceptors (Alexa 555 and 647) positioned on each side of both operators allow detection of all possible looping topologies (two parallel and two antiparallel trajectories). Comparisons among these fluorophore position variants allow measurement of the population of each loop state. Finally, we have studied the effect of IPTG on loop geometry and stability. Bulk and single-molecule FRET studies show that: (1) Remarkably, all the constructs on the landscape form stable loops. (2) Parallel and antiparallel topologies have different LacI geometries detected by different FRET efficiencies, probably due to differential effects of DNA stiffness in the two contexts. (3) Some constructs form an extended loop that can be identified by loss of FRET. (4) Induction affords a mixture of the initial loop, a state with decreased FRET, and additional FRET-silent states that we tentatively ascribe to specific-nonspecific loops. (5) States on the borders of the landscape regions comprising uniform loops are maximally sensitive to IPTG. The overall results do not agree with rod mechanics models that consider only DNA bending and flexibility, confirming that protein flexibility must be considered in modeling DNA loops.

A LacI-DNA Looping Landscape and Allosteric Effects on the Loop Shapes

The lac operon and its repressor (LacI) are the prototype model for gene regulation. LacI negatively regulates lacZYA by binding a primary DNA operator site overlapping the promoter, and repression is enhanced by secondary operators that deliver LacI via looping intervening DNA. LacI's ability to form stable loops with a variety of DNA lengths has been attributed to protein flexibility and/or to multiple loop topologies. Previously developed DNA constructs in which looping is hyperstabilized by an A-tract bend placed between two operators provide different loop shapes depending on the operator/bend helical phasing. Here, FRET is used to characterize the sequence/structure landscape of a set of related constructs with systematically varied operator/bend phasings. Donor and acceptor fluorophores positioned on either side of the operator provide multiple distance constraints on the orientations of the LacI-DNA loop. The results suggest that LacI can form many different looped states whose relative energetics can be measured. Also, IPTG addition demonstrates that inducer-bound LacI still forms stable loops, probably with different geometries relative to the repressed state. This comprehensive looping landscape should allow determination of whether protein flexibility is necessary to explain the results.

Computational Analysis of Looping of a Large Family of Highly Bent DNA by LacI

Sequence-dependent intrinsic curvature of DNA influences looping by regulatory proteins such as LacI and NtrC. Curvature can enhance stability and control shape, as observed in LacI loops formed with three designed sequences with operators bracketing an A-tract bend. We explore geometric, topological, and energetic effects of curvature with an analysis of a family of highly bent sequences, using the elastic rod model from previous work. A unifying straight-helical-straight representation uses two phasing parameters to describe sequences composed of two straight segments that flank a common helically supercoiled segment. We exercise the rod model over this two-dimensional space of phasing parameters to evaluate looping behaviors. This design space is found to comprise two subspaces that prefer parallel versus anti-parallel binding topologies. The energetic cost of looping varies from 4 to 12 kT. Molecules can be designed to yield distinct binding topologies as well as hyperstable or hypostable loops and potentially loops that can switch conformations. Loop switching could be a mechanism for control of gene expression. Model predictions for linking numbers and sizes of LacI-DNA loops can be tested using multiple experimental approaches, which coupled with theory could address whether proteins or DNA provide the observed flexibility of protein-DNA loops.

Topological measurement of an A-tract bend angle: effect of magnesium.

Sequences of four to six adenine residues, termed A-tracts, have been shown to produce curvature in the DNA double helix. A-tracts have been used extensively as reference standards to quantify bending induced by other sequences as well as by DNA binding proteins when they bind to their sites. However, the ability of an A-tract to serve as such a standard is hampered by the wide variation of values reported for the amount of bend conferred by an A-tract. One experimental condition that differs in these studies is the presence of divalent cation. To evaluate this effect, a new application of a topological method, termed rotational variant analysis, is used here to measure for the first time the effect of the presence of magnesium ion on the bend angle conferred by an A-tract. This method, which has the unique ability to measure a bend angle in the presence or absence of magnesium ion, demonstrates that magnesium ion markedly increases the bend angle. For example, when measured in a commonly used gel electrophoretic buffer, the bend angle conferred by a tract of six adenine residues increases from about 7 degrees in the absence of magnesium ion to 19 degrees in the presence of 3.9 mM magnesium ion. This quantitative demonstration of substantial magnesium ion dependence has several important implications. First, it explains discrepancies among bend values reported in various previous studies, particularly those employing gel electrophoretic versus other solution methods. In addition, these findings necessitate substantial revisions of the conclusions in a large number of studies that have used A-tract DNA as the bend angle reference standard in comparison measurements. Finally, any such future studies employing this comparison methodology will need to use the same sequence analyzed in the original measurements as well as replicate the original measurement conditions (e.g. ionic composition and temperature).

Gapped DNA is anisotropically bent.

Ionizing radiation damages DNA in several ways, including through formation of a single-nucleoside gap in one DNA strand. We have developed a two-dimensional gel electrophoresis method to investigate the effect of a strand gap on DNA structure. We generate a library of gapped DNA molecules by treating a DNA restriction fragment with the hydroxyl radical, generated by the reaction of Fe(II) EDTA with hydrogen peroxide. The DNA molecule studied contains a fixed bend produced by a set of phased adenine tracts. The A-tract bend serves as a reference bend for investigating the conformational nature of a strand gap. In the first electrophoretic dimension, a bent DNA molecule that has been treated with the hydroxyl radical is electrophoresed on a native gel. Smearing of the band on the native gel indicates that the library of gapped DNA molecules contains a variety of DNA conformations. In the second electrophoretic dimension, gapped DNA molecules having different native gel mobilities are electrophoresed on separate lanes of a denaturing gel to reveal how each strand gap affects the native gel mobility (and thus shape) of the DNA. Our results demonstrate that a single-nucleoside gap in a DNA duplex leads to an anisotropic, directional bend in the DNA helix axis. The implications of our findings for recognition of this lesion by DNA repair proteins are discussed.

Topological Measurement of an A-tract Bend Angle: Comparison of the Bent and Straightened States

It is well established that an A-tract imparts curvature to the DNA double helix. Constructs of such A-tracts have been used as bend standards in a large number of both structural and functional studies, and A-tracts can confer significant activation in transcription. An accurate value for the bend angle induced by an A-tract is centrally important to all such studies, but the estimates reported for the bend angle of an A-tract differ by greater than threefold. To address this problem, we have used the rotational variant method to measure the angle of DNA curvature conferred by a tract of six adenine bases (A6 tract). The original version of the method measured a protein-induced bend angle independent of external standards. It compared the effect of bent and straight forms of the sequence on the topology of a DNA plasmid in which the sequence is cloned as a series of tandem repeats. To adapt the approach to the measurement of an intrinsic bend, high temperature was used to generate the straightened reference state, with the required topological relaxation being performed by a hyperthermophile topoisomerase. Appropriate plasmids containing tandem repeats of A-tracts were constructed and topologically analyzed in this manner. The bend value measured at 4 °C was 26(±2), and decreased linearly to 17(±2) at 37 °C. The relationship to other estimates and the application of these values are discussed.

Site-specific labeling of supercoiled DNA at the A+T rich sequences.

Progress in structural biology studies of supercoiled DNA and its complexes with regulatory proteins depends on the availability of reliable and routine procedures for site-specific labeling of circular molecules. For this, we made use of oligonucleotide uptake by plasmid DNA under negative superhelical tension. Subsequent circularization of the oligonucleotide label facilitated by an oligonucleotide scaffold results in its threading between the two strands of duplex DNA. Several lines of evidence, including direct AFM mapping of the label, show that the circular oligonucleotide is stably localized at its target, an A+T rich region. The specific binding mode when the oligonucleotide threads the double helix results in a DNA kink that tends to occupy an apical position in a plectonemically wound supercoiled DNA, similar to the positioning of an A-tract bend. Site-specific labels may allow visualization techniques, such as electron and atomic force microscopies, to reliably map protein binding sites, identify local alternative structures in supercoiled DNA, and monitor structural dynamics of DNA molecules in real time. Site-specific oligonucleotide reactions with DNA may also have application in biotechnology and gene therapy.

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.

(+)-CC-1065 produces bending of DNA that appears to resemble adenine/thymine tracts.

In this communication we demonstrate, using hydroxyl-radical footprinting and high-field proton NMR, that the bend in DNA, which is entrapped or induced by (+)-CC-1065, appears to resemble in overall respects a naturally occurring A-tract bend. The possible relationship of this bend to sequence selectivity and biological consequences of such a feature associated with a potent DNA-reactive drug are discussed