Structural diversity of DNA

Since its discovery, supercoiled DNA has been extensively studied by biologists and by physicists. The former were interested in its implication in biological processes, while the latter investigated its structure and studied its topology and conformation. DNA supercoiling plays key roles in genetic processes in the cell such as replication, transcription, recombination as well as DNA packaging (Sinden 1994). A lot of studies was performed in order to clarify the structural properties of supercoiled DNA and large amounts of experimental data are available in literature. In the last decade, Atomic Force Microscopy (AFM) has proven to be one of most valuable techniques for the studies of the biological structure and dynamic processes of DNA. Despite its great potential, all

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studies performed until now used as substrate linear or negatively supercoiled DNA molecules while no studies on positively supercoiled DNA molecules have been performed by AFM.

In my work I analyzed, for the first time the positively supercoiled DNA in comparison with the negatively one in order to investigate structural peculiarities of both topologies. Moreover I analyzed both conformations also by classical biochemical techniques.

For the first the biochemical analyses revealed the presence of exposed bases when the DNA was negatively supercoiled but not in positively supercoiled DNA, supporting their biological roles. As already known, the exposed bases create local helix distortions that allow DNA to assume conformations that would be energetically unfavourable if the bases remained paired. The presence of single strand regions in negatively supercoiled DNA is in line with its biological role during replication and transcription. Conversely the absence of exposed bases in positively supercoiled molecules suggests that the positive conformation could play a key role in DNA stability maintenance preventing the local denaturation and protecting the DNA structure.

AFM data showed different conformations for negatively and positively DNA in almost all the conditions used. In particular I observed that in presence of a low concentration of divalent cations (3 mM MgCl2), negatively supercoiled DNA molecules (with a superhelical density of -0.04) tend to adopt loosely interwound conformations (as already reported in Schmatko et al. 2014) while positively supercoiled DNA (with a superhelical density of +0.05) showed a typical plectonemic conformation. It could be hypothesized that the different electrostatic repulsion at crossings causes a different immobilization of supercoiled molecules on mica substrate: in the case of negatively supercoiled plasmids the repulsion exceeds bending and twisting costs. Therefore, a DNA molecule would prefer to be under high twist rather than having crossings. In the case of positively supercoiled, the electrostatic repulsion at crossings is probably reduced because of the different orientation of supercoiling and as a consequence we observe plectonemes. These results represent a starting point for further studies to identify possible differences between negative and positive supercoiling and to elucidate their biological roles in vivo.

Determining how the compacted genetic material can still remain accessible to replication/transcription machinery is of great importance for understanding the basic principles of genetic regulation. For this purpose AFM analyses in liquid represent a tool with enormous potential to investigate the accessibility and binding of proteins to both negatively and positively supercoiled DNA molecules. This technique permits nanometric-scale imaging of single molecules under near-physiological conditions and this advantage can be also used to study the dynamics of different biological processes involving negative and positive supercoiling (Alonso-Sarduy et al. 2011).

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Recently it was reported that negatively circular supercoiled DNA could dynamically fold in particular higher-order structures called hyperplectonemes. Hyperplectonemic coiling could provide substantial compaction. This is especially relevant for prokaryotes, in which plectonemic coiling is thought to be the major mode of DNA packaging. The presence of different molecules in living organisms that have DNA condensing function in combination with high level of DNA negative supercoiling, suggests that hyperplectonemic compaction could be found in vivo. These studies illustrate how the bacterial nucleoid can be effectively compacted and organized, while remaining dynamic in nature and readily accessible to DNA binding proteins and processing enzymes (Japaridze et al. 2017).

Hyperthermophilic organisms require specific mechanisms inducing positive supercoiling to counteract the denaturing effect of their growth temperature (Valenti et al. 2011). Indeed, several lines of evidence suggest that DNA is more positive in (hyper)thermophilic archaea as compared with mesophiles: several plasmids from different hyperthermophilic strains were found in relaxed to positively supercoiled form (Forterre et al. 1996; Charbonnier and Forterre 1994). In addition, all hyperthermophiles invariably possess the DNA topoisomerase reverse gyrase, which is the only hyperthermophilic specific able to introduce positive supercoils in DNA (Brochier-Armanet and Forterre 2006). In addition to reverse gyrase, it is reported the presence in hyperthermophiles of DNA-binding proteins able to induce both positive and negative supercoiling (Napoli et al. 2001;Xue et al. 2000; Bell et al.2002). These evidences are bringing out a complex picture concerning the genome topology of these organisms. In addition, (hyper)thermophilic archaea belonging to the subdomain Euryarchaeota contain tetrameric eukaryoticlike histones, which wrap DNA into positive supercoils at high salt concentrations, but into negative supercoils in low salt conditions (Musgrave 1991, 2000).

In this context the possibility to perform AFM analyses on positively supercoiled DNA at the larger scales (several tens of kilo base pairs) can give elucidation on the complex genomic situation in hyperthermophiles and can highlight the link between the nanomechanical properties of nucleoprotein complexes and the protein regulatory function as already seen for negatively supercoiled DNA.