Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides

Abstract

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures.It remains unclear how this supercoiling affects the double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance 2 between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition of diverse conformational substrates.

DNA sequence-directed cooperation between nucleoid-associated proteins

“…These coexisting structures are in line with the predominantly repressing and activating roles of H-NS and FIS on transcription, respectively. Since the distribution of the H-NS and FIS binding sites in the E. coli genome is non-random (36,67), we hypothesize that the growthphase-dependent global regulation of transcription (68) involves alterations of chromatin accessibility. This accessibility of chromatin is further modulated by a crosstalk between temporally changing composition of NAPs and a non-random distribution of their cognate genomic binding sites, thus leading to spatiotemporal organisation of the 'open' and 'closed' DNA structures that are crucial to genomic expression.…”

Integration host factor bends and bridges DNA in a multiplicity of binding modes with varying specificity

“…All samples were imaged on a Bruker Bioscope Resolve (Bruker, CA, USA) in tapping mode using TAP-300AI-G probes (BudgetSensors, Bulgaria). Images were then loaded using pySPM (40) and preprocessed by using a 1st order line-by-line flattening and 2nd order fits to flatten the surface before filtering scars (41). The images were then analyzed using custom code using the scikit-image package (42) to threshold the image and segment it into the individual DNA strands.…”

Single-molecule measurements reveal that PARP1 condenses DNA by loop formation

“…Images were recorded at a scan size of 1-2 µm, with 1024 pixels per line and at a line rate of 4 Hz. Images were processed using either Gwyddion 62 or a Python that employs the Gwyddion 'pygwy' module, as described in greater detail elsewhere 63 . All images were initially subjected to correction by first-order line-by-line flattening, median line-by-line flattening and zeroth-order plane fitting to remove background offset and tilt.…”