How Cations Can Assist DNase I in DNA Binding and Hydrolysis

DNase I requires Ca2+ and Mg2+ for hydrolyzing double-stranded DNA. However, the number and the location of DNase I ion-binding sites remain unclear, as well as the role of these counter-ions. Using molecular dynamics simulations, we show that bovine pancreatic (bp) DNase I contains four ion-binding pockets. Two of them strongly bind Ca2+ while the other two sites coordinate Mg2+. These theoretical results are strongly supported by revisiting crystallographic structures that contain bpDNase I. One Ca2+ stabilizes the functional DNase I structure. The presence of Mg2+ in close vicinity to the catalytic pocket of bpDNase I reinforces the idea of a cation-assisted hydrolytic mechanism. Importantly, Poisson-Boltzmann-type electrostatic potential calculations demonstrate that the divalent cations collectively control the electrostatic fit between bpDNase I and DNA. These results improve our understanding of the essential role of cations in the biological function of bpDNase I. The high degree of conservation of the amino acids involved in the identified cation-binding sites across DNase I and DNase I-like proteins from various species suggests that our findings generally apply to all DNase I-DNA interactions

DNA structures from phosphate chemical shifts

For B-DNA, the strong linear correlation observed by nuclear magnetic resonance (NMR) between the 31P chemical shifts (δP) and three recurrent internucleotide distances demonstrates the tight coupling between phosphate motions and helicoidal parameters. It allows to translate δP into distance restraints directly exploitable in structural refinement. It even provides a new method for refining DNA oligomers with restraints exclusively inferred from δP. Combined with molecular dynamics in explicit solvent, these restraints lead to a structural and dynamical view of the DNA as detailed as that obtained with conventional and more extensive restraints. Tests with the Jun-Fos oligomer show that this δP-based strategy can provide a simple and straightforward method to capture DNA properties in solution, from routine NMR experiments on unlabeled samples

Compaction of RNA Hairpins and Their Kissing Complexes in Native Electrospray Mass Spectrometry

When electrosprayed from typical native MS solution conditions, RNA hairpins and kissing complexes acquire charge states at which they get significantly more compact in the gas phase than their initial structure in solution. Here we show the limits of using force field molecular dynamics to interpret the gas-phase structures of nucleic acid complexes in the gas phase, and we suggest that higher-level calculation levels should be used in the future.<br /

Cation binding to nucleic acids

International audienceIntroductionDetecting cation and metal adducts to nucleic acids by mass spectrometry is easy. Too easy. Cations present in solution (intentionally or not) indeed stick very well to nucleic acid multiply charged ions. In the negative ion mode, metal cations cannot be removed by collisional activation. What is difficult is therefore not to detect cation binding, but to distinguish specific cation binding at peculiar coordination sides (which are often relevant on the structural biology point of view), from nonspecific cation “adducts” at randomly distributed sites. Here we discuss strategies to distinguish specific from nonspecific binding, to quantify specifically bound cations. We will also discuss the origin of nonspecific adduct formation.MethodsDNA sequences forming single-stranded or G-quadruplex structures (which require specifically bound potassium ions to form), and RNA sequences forming single strands, hairpins, duplexes or kissing loop complexes (which require specifically bound magnesium ions to form) were purchased from Eurogentec or IDT. Magnesium or manganese acetate, KCl, ammonium acetate or trimethylammonium acetate were used to fix ionic strength and cation concentration. Samples were analyzed by an Agilent 6560 electrospray-IMS-Q-TOF, which allows to record drift tube ion mobility data from each m/z. The nonspecific adducts distributions were derived from control sequences, as a function of the charge state (including in supercharging conditions obtained with sulfolane). Then, the specific adducts distributions were quantified after subtraction of the nonspecific adducts contribution.Preliminary Data Here nonspecific adducts are defined as metal adducts formed upon binding to groups or structures that are not the specific structural motif under investigation. It does not mean that these adducts (or ion pairs) do not exist in solution. For example, for G-quadruplexes, the specific adducts are the potassium adducts bound to the specific inter-quartet locations, while the nonspecific adducts are those that would form in any single stranded sequence of the same length and base content. In the first part of the presentation, we will discuss the charge state dependence of the nonspecific adducts. If the distribution of adducts in the mass spectra depends on the charge state, we know that adducts formed during the electrospray process (and not just those formed in solution) do contribute to the overall distribution. In purely aqueous solvents, the number of nonspecific adducts decreases when z increases. But surprisingly, upon supercharging with sulfolane, more nonspecific adducts (of cations!) are observed on the extra highly (negatively!) charged ions. This counterintuitive observation actually gives insight on the mechanism of multiply charged ion production. In the second part of the presentation, we will discuss how to subtract the contribution of nonspecific adducts to deduce the fraction of metal ions specifically bound to particular structures such as the G-quadruplexes or RNA kissing loop motifs, and deduce the number of specific binding sites and the equilibrium binding constants, or metal binding rate constants. Finally, we will show how ion mobility spectrometry contributes discerning specific structures formed upon metal cation binding. These techniques were used to decipher the complex potassium-induced folding pathways of telomeric DNA G-quadruplex structures. Novel Aspect:We clarify here the origin of nonspecific adducts formation, and explain how to account for them and contribute suppressing them