Mapping the intrinsic curvature and flexibility along the DNA chain

Protein structural features are usually determined by defining regularities in a large population of homogeneous molecules. However, irregular features such as structural variation and flexibility are likely to be missed, despite their vital role for their biological function. In this paper, we report the observation of striking irregularities in the flexibility of the coiled-coil region of the human Rad50 DNA repair protein. Existing methods to quantitatively analyze flexibility are applicable to homogeneous polymers only. Because protein coiled-coils cannot be assumed to be homogeneous, we develop a method to quantify the local flexibility from high-resolution atomic force microscopy images. Indeed, in Rad50 coiled-coils, two positions of increased flexibility are observed. We discuss how this dynamic structural feature is integral to Rad50 function.

Section: Methodsmentioning

confidence: 99%

“…A slightly different method has been used to identify subtle sequence-dependent differences in flexibility in DNA molecules (14). In our analysis, the local curvature of a specific polymer at a certain position is defined as the inverse radius of a circle following the trajectory along the molecule at that position.…”

mentioning

confidence: 99%

The coiled-coil of the human Rad50 DNA repair protein contains specific segments of increased flexibility

Noort

Heijden

Jager

et al. 2003

“…The main alternative is provided by single-molecule methods, for example the work of De Santis and colleagues (34). Because cyclization allows intense focus on small sequence elements, and singlemolecule visualization provides an overall view of molecular shape, the methods should be viewed as complementary.…”

Section: 85mentioning

confidence: 99%

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

Zhang

Crothers

2003

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...

“…Thus, a quantitative knowledge of the curvature and the flexibility of doublestranded DNA has become important to understand the determinants of DNA-protein recognition. We have recently described how SFM makes it possible to map sequencedependent curvature and flexibility along the DNA chain (2,3). In these previous investigations, indications of preferential adsorption involving curved DNA tracts on the surface of mica were obtained.…”

mentioning

confidence: 99%

“…The sequence of the two curved tracts in a palindromic construct is characterized by periodical recurrence of repeated AA in the first followed by TT steps in the other, and, conversely, TT followed by AA steps in the other palindromic construct. The use of a palindrome allows the unequivocal correspondence of a position along the DNA trace in the microscope images with a position in the base sequence, without the need to use external chemical labels to define the direction of the sequence (2). The choice to use a palindromic DNA gives way to a dyad symmetry relating the two chemically equivalent halves.…”

mentioning

confidence: 99%

Recognition of the DNA sequence by an inorganic crystal surface

Sampaolese¹,

Bergia²,

Scipioni³

et al. 2002

Self Cite

The sequence-dependent curvature is generally recognized as an important and biologically relevant property of DNA because it is involved in the formation and stability of association complexes with proteins. When a DNA tract, intrinsically curved for the periodical recurrence on the same strand of A-tracts phased with the B-DNA periodicity, is deposited on a flat surface, it exposes to that surface either a T-or an A-rich face. The surface of a freshly cleaved mica crystal recognizes those two faces and preferentially interacts with the former one. Statistical analysis of scanning force microscopy (SFM) images provides evidence of this recognition between an inorganic crystal surface and nanoscale structures of double-stranded DNA. This finding could open the way toward the use of the sequence-dependent adhesion to specific crystal faces for nanotechnological purposes.I t is widely accepted that the local, sequence-dependent curvature and dynamics of the double-stranded DNA chain segments play a crucial and active role in DNA packaging, transcription, replication, recombination, and repair processes, and in nucleosome stability and positioning (1). Thus, a quantitative knowledge of the curvature and the flexibility of doublestranded DNA has become important to understand the determinants of DNA-protein recognition. We have recently described how SFM makes it possible to map sequencedependent curvature and flexibility along the DNA chain (2, 3). In these previous investigations, indications of preferential adsorption involving curved DNA tracts on the surface of mica were obtained. To reach definite evidence of such recognition we have investigated a highly curved DNA fragment to amplify this effect.A periodic recurrence of A-tracts phased with the B-DNA periodicity, like tracts of three to six adenine steps centered approximately every 10.5 base pairs, drives extended intrinsic curvatures along a DNA chain (4). These curved DNA tracts give rise to planar or quasi planar superstructures. It is worth noting that, when these short stretches of adenine steps are positioned on the same strand, the adenine bases tend to be preferentially positioned on one side of the curvature plane, while the complementary thymines will be found on the other side. When deposited on a flat surface, these curved DNA tracts will thus interact with that surface, on average, with either an A-or a T-rich face. A tract of DNA with very extensive phasing of repeated A-tracts is readily available in the kinetoplast DNA of the Trypanosomatidae protozoan Crithidia fasciculata. This DNA segment is the most highly curved DNA we know of at present (5). The profiles assumed by these curved DNA molecules on deposition on a flat surface can be observed by scanning force microscopy (SFM). Only if the sequence orientation for each of the imaged molecules could be known would it be possible to determine the face with which the molecules adsorb on the surface (on average) through the study of the average chain-curvature of the molecule ensemble. To remo...