Abstract
Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high-throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. By carefully modeling the TPM geometry, we propose a simple formula based on a kinked Worm-Like Chain model to extract the bend angle from TPM measurements. Using constructs made of 575 base-pair DNAs with in-phase assemblies of one to seven 6A-tracts, we find that the sequence CA6CGG induces a bend angle of 19° ± 4°. Our method is successfully compared to more theoretically complex or experimentally invasive ones such as cyclization, NMR, FRET or AFM. We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well.
Highlights
DNA bending was first revealed in the mid-80s on the mitochondrial DNA of trypanosomatid parasites, the kinetoplast DNA [1] and attributed to the intrinsic bending property of the A-tracts sequences present in kDNA [2,3]
To extract the bend angles from HT-Tethered Particle Motion (TPM) data, we developed a simple analytical formula based on a kinked WormLike Chain (WLC) model that we validated on simulated data
We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well
Summary
DNA bending was first revealed in the mid-80s on the mitochondrial DNA of trypanosomatid parasites, the kinetoplast DNA (kDNA) [1] and attributed to the intrinsic bending property of the A-tracts sequences present in kDNA [2,3] These A-tracts were abundantly found in other prokaryotic and eukaryotic organisms but they were shown to have a biological role, for example, by participating in the regulation of transcription [4,5,6,7,8,9,10,11,12,13]. It is commonly accepted that the bendability of specific DNA sequences relates to their capacity to be bent under the action of DNAbinding proteins. Characterizing rigorously the local bending of DNA molecules is a crucial issue that remains highly challenging [13,19,20]
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