Helical Chirality: a Link between Local Interactions and Global Topology in DNA

DNA supercoiling plays a major role in many cellular functions. The global DNA conformation is however intimately linked to local DNA-DNA interactions influencing both the physical properties and the biological functions of the supercoiled molecule. Juxtaposition of DNA double helices in ubiquitous crossover arrangements participates in multiple functions such as recombination, gene regulation and DNA packaging. However, little is currently known about how the structure and stability of direct DNA-DNA interactions influence the topological state of DNA. Here, a crystallographic analysis shows that due to the intrinsic helical chirality of DNA, crossovers of opposite handedness exhibit markedly different geometries. While right-handed crossovers are self-fitted by sequence-specific groove-backbone interaction and bridging Mg2+ sites, left-handed crossovers are juxtaposed by groove-groove interaction. Our previous calculations have shown that the different geometries result in differential stabilisation in solution, in the presence of divalent cations. The present study reveals that the various topological states of the cell are associated with different inter-segmental interactions. While the unstable left-handed crossovers are exclusively formed in negatively supercoiled DNA, stable right-handed crossovers constitute the local signature of an unusual topological state in the cell, such as the positively supercoiled or relaxed DNA. These findings not only provide a simple mechanism for locally sensing the DNA topology but also lead to the prediction that, due to their different tertiary intra-molecular interactions, supercoiled molecules of opposite signs must display markedly different physical properties. Sticky inter-segmental interactions in positively supercoiled or relaxed DNA are expected to greatly slow down the slithering dynamics of DNA. We therefore suggest that the intrinsic helical chirality of DNA may have oriented the early evolutionary choices for DNA topology

Structural diversity of supercoiled DNA

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA min icircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function

Voltage-Gated Proton Channels as Novel Drug Targets: From NADPH Oxidase Regulation to Sperm Biology

Abstract Significance: Voltage-gated proton channels are increasingly implicated in cellular proton homeostasis. Proton currents were originally identified in snail neurons less than 40 years ago, and subsequently shown to play an important auxiliary role in the functioning of reactive oxygen species (ROS)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Molecular identification of voltage-gated proton channels was achieved less than 10 years ago. Interestingly, so far, only one gene coding for voltage-gated proton channels has been identified, namely hydrogen voltage-gated channel 1 (HVCN1), which codes for the HV1 proton channel protein. Over the last years, the first picture of putative physiological functions of HV1 has been emerging. Recent Advances: The best-studied role remains charge and pH compensation during the respiratory burst of the phagocyte NADPH oxidase (NOX). Strong evidence for a role of HV1 is also emerging in sperm biology, but the relationship with the sperm NOX5 remains unclear. Probably in many instances, HV1 functions independently of NOX: for example in snail neurons, basophils, osteoclasts, and cancer cells. Critical Issues: Generally, ion channels are good drug targets; however, this feature has so far not been exploited for HV1, and hitherto no inhibitors compatible with clinical use exist. However, there are emerging indications for HV1 inhibitors, ranging from diseases with a strong activation of the phagocyte NOX (e.g., stroke) to infertility, osteoporosis, and cancer. Future Directions: Clinically useful HV1-active drugs should be developed and might become interesting drugs of the future. Antioxid. Redox Signal. 00, 000-000

Temperature dependence of NADPH oxidase in human eosinophils

The phagocyte NADPH oxidase helps kill pathogens by producing superoxide anion, O2−. This enzyme is electrogenic because it translocates electrons across the membrane, generating an electron current, Ie. Using the permeabilized patch voltage-clamp technique, we studied the temperature dependence of Ie in human eosinophils stimulated by phorbol myristate acetate (PMA) from room temperature to > 37°C. For comparison, NADPH oxidase activity was assessed by cytochrome c reduction. The intrinsic temperature dependence of the assembled, functioning NADPH oxidase complex measured during rapid temperature increases to 37°C was surprisingly weak: the Arrhenius activation energy Ea was only 14 kcal mol−1 (Q10, 2.2). In contrast, steady-state NADPH oxidase activity was strongly temperature dependent at 20–30°C, with Ea 25.1 kcal mol−1 (Q10, 4.2). The maximum Ie measured at 34°C was −30.5 pA. Above 30°C, the temperature dependence of both Ie and O2− production was less pronounced. Above 37°C, Ie was inhibited reversibly. After rapid temperature increases, a secondary increase in Ie ensued, suggesting that high temperature promotes assembly of additional NADPH oxidase complexes. Evidently, about twice as many NADPH oxidase complexes are active near 37°C than at 20°C. Thus, the higher Q10 of steady-state Ie reflects both increased activity of each NADPH oxidase complex and preferential assembly of NADPH oxidase complexes at high temperature. In summary, NADPH oxidase activity in intact human eosinophils is maximal precisely at 37°C