The genome-wide function of supercoiling and alternative DNA conformations

Division Of Basic Sciences - Nci

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Abstract

Genetic processes - transcription, replication, recombination, DNA repair, and packing and unpacking of DNA into chromatin imparts high levels of mechanical force onto the genome. While bending and compressing and stretching forces are obviously required to force 6 meters of DNA into a 10 micron nucleus, more problematic is the twisting and untwisting demanded. In fact, the synthesis of new nucleic acids involves the daily unwinding of 10 billion turns of DNA. The torque and kinks generated requires topoisomerases 1 and 2 to relieve the stress to accommodate these genetic transactions. The torque generated by these genetic transactions is transmitted through twisting force or as writhe (supercoils) that have the potential to remodel DNA structure converting the right handed B-DNA double-helix into other structures such as G4 quadruplex, left-handed Z-DNA duplex, triplex H-DNA, i-motif, R-loops, cruciforms and single-stranded bubbles. These structures have the potential to alter the regulation of gene expression by creating or destroying binding sites for regulatory proteins or by changing the flexibility of DNA to twisting and bending, allowing regulatory sites (cis-elements) to become juxtaposed or held rigidly apart. To probe DNA structures across the genome we have previously used a method we developed called ssDNA-seq to identify sites with unpaired bases that have become susceptible to modification by potassium permanganate. Permanganate freely penetrates cells and so is an in vivo probe of DNA structure. However, ssDNA-seq identifies structures present in a population of cells but cannot reveal the coupling between elements in a single molecule to distinguish the state of individual cells. To accomplish this, we have been developing Nanopore as means to interrogate DNA structure and its coupling with other genetic and chromatin processes at the single molecule level. With Nanopore individual DNA molecules are driven by an electric potential through a narrow bore. Because the current through the pore varies along with the particular sequence of bases transiting through the channel, the sequence can be directly read. Base-modification by permanganate disturbs the base-calling and alters the electrical signal. We have used this approach to demonstrate communication between DNA elements through the DNA fiber. These interactions between remote sites do not require looping nor any physical contact between the elements. We validated our method in vitro using plasmid DNAs. We have now improved the biochemical, biophysical, and computational infrastructure necessary for this approach and are interrogating genes in Drosophila melanogaster and human cell lines and genetic transactions using Nanopore. Our method will reveal regulatory elements and structures as sites of modification which are characteristically enriched in particular patterns associated with different DNA structures. For example, we can see the melted bubble created by RNA polymerase early in the promoters of highly expressed genes. Also, because individual bases flip in and out of the double helix differently whether the DNA is negatively or positively supercoiled or torsionally relaxed, we attempting to develop high resolution full genome maps of supercoiling. The development of this approach is dependent on using artificial intelligence and neural networks to analyze the electrical traces emanating from the pores. We expect that these studies will lead to much greater understanding of the role of DNA mechanics and topology in the regulation of gene expression.