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Despite the negative charge of the DNA backbone, acidic residues (Asp/Glu) commonly participate in the base readout, with a strong preference for cytosine. In fact, in the solved DNA/protein structures, cytosine is recognized almost exclusively by Asp/Glu through a direct hydrogen bond, while at the same time, adenine, regardless of its amino group, shows no propensity for Asp/Glu. Here, we analyzed the contribution of Asp/Glu to sequence-specific DNA binding using classical and ab initio simulations of selected transcription factors and found that it is governed by a fine balance between the repulsion from backbone phosphates and attractive interactions with cytosine. Specifically, Asp/Glu lower the affinity for noncytosine sites and thus act as negative selectors preventing off-target binding. At cytosine-containing sites, the favorable contribution does not merely rely on the formation of a single H-bond but usually requires the presence of positive potential generated by multiple cytosines, consistently with the observed excess of cytosine in the target sites. Finally, we show that the preference of Asp/Glu for cytosine over adenine is a result of the repulsion from the adenine imidazole ring and a tendency of purine-purine dinucleotides to adopt the BII conformation.

We have used a variety of theoretical and experimental techniques to study the role of four basic amino acids-Arginine, Lysine, Ornithine and L-2,4-Diaminobutyric acid-on the structure, flexibility and sequence-dependent stability of DNA. We found that the presence of organic ions stabilizes the duplexes and significantly reduces the difference in stability between AT- and GC-rich duplexes with respect to the control conditions. This suggests that these amino acids, ingredients of the primordial soup during abiogenesis, could have helped to equalize the stability of AT- and GC-rich DNA oligomers, facilitating a general non-catalysed self-replication of DNA. Experiments and simulations demonstrate that organic ions have an effect that goes beyond the general electrostatic screening, involving specific interactions along the grooves of the double helix. We conclude that organic ions, largely ignored in the DNA world, should be reconsidered as crucial structural elements far from mimics of small inorganic cations., Competing Interests: The authors have declared that no competing interests exist.

Brominated nucleobases sensitize double stranded DNA to hydrated electrons, one of the dominant genotoxic species produced in hypoxic cancer cells during radiotherapy. Such radiosensitizers can therefore be administered locally to enhance treatment efficiency within the solid tumor while protecting the neighboring tissue. When a solvated electron attaches to 8-bromoadenosine, a potential sensitizer, the dissociation of bromide leads to a reactive C8 adenosyl radical known to generate a range of DNA lesions. In the current work, we propose a multiscale computational approach to elucidate the mechanism by which this unstable radical causes further damage in genomic DNA. We employed a combination of classical molecular dynamics conformational sampling and QM/MM metadynamics to study the thermodynamics and kinetics of plausible reaction pathways in a realistic model, bridging between different time scales of the key processes and accounting for the spatial constraints in DNA. The obtained data allowed us to build a kinetic model that correctly predicts the products predominantly observed in experimental settings-cyclopurine and β-elimination (single strand break) lesions-with their ratio and yield dependent on the effective lifetime of the radical species. To date, our study provides the most complete description of purine radical reactivity in double stranded DNA, explaining the radiosensitizing action of electrophilic purines in molecular detail as well as providing a conceptual framework for the computational modeling of competing reaction pathways in biomolecules.

Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein-DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Telomeres are specialized nucleoprotein assemblies that protect the ends of linear chromosomes. In humans and many other species, telomeres consist of tandem TTAGGG repeats bound by a protein complex known as shelterin that remodels telomeric DNA into a protective loop structure and regulates telomere homeostasis. Shelterin recognizes telomeric repeats through its two major components known as Telomere Repeat-Binding Factors, TRF1 and TRF2. These two homologous proteins are therefore essential for the formation and normal function of telomeres. Indeed, TRF1 and TRF2 are implicated in a plethora of different cellular functions and their depletion leads to telomere dysfunction with chromosomal fusions, followed by apoptotic cell death. More specifically, it was found that TRF1 acts as a negative regulator of telomere length, and TRF2 is involved in stabilizing the loop structure. Consequently, these proteins are of great interest, not only because of their key role in telomere maintenance and stability, but also as potential drug targets. In the current study, we investigated the molecular basis of telomeric sequence recognition by TRF1 and TRF2 and their DNA binding mechanism. We used molecular dynamics (MD) to calculate the free energy profiles for binding of TRFs to telomeric DNA. We found that the predicted binding free energies were in good agreement with experimental data. Further, different molecular determinants of binding, such as binding enthalpies and entropies, the hydrogen bonding pattern and changes in surface area, were analyzed to decompose and examine the overall binding free energies at the structural level. With this approach, we were able to draw conclusions regarding the consecutive stages of sequence-specific association, and propose a novel aspartate-dependent mechanism of sequence recognition. Finally, our work demonstrates the applicability of computational MD-based methods to studying protein-DNA interactions.