Solution structures of a duplex containing an adenine opposite a gap (absence of one nucleotide)

Abstract

We investigated the behaviour of a 15mer DNA duplex, [5 H d(CAGAGTCACTGGCTC)3 H ].[5 H d(GAGCCAG)3 H + 5 H d(GACTCTG)3 H ] which contained an adenine opposite the gap. Analysis of the NMR data showed the existence of one major species, which was in equilibrium with two minor species. Their relative concentrations varied as a function of pH with a pK a of <4.5. For the major species, the duplex was globally in B conformation with the central adenine stacked in the helix. The two G´C base pairs adjacent to the central adenine were well formed and a gap was present in front of this adenine. For the minor species, major structural perturbations occurred in the centre of the duplex. At neutral pH, the central adenine was involved in a G´A mismatch with G23 adjacent to the gap. Cytosine C7 was then extrahelical and no gap was observed. Under these conditions, the major neutral species corresponded to 70% of the total and the minor species to 30%. At acidic pH, the central adenine of the minor species was protonated and was involved in a G(syn)´A + (anti) mismatch. The difference is that C9 is now extrahelical and G22 is implicated in the mispair. Three-dimensional models were built to initiate molecular dynamic simulations, which were in good agreement with the NMR data. Their structural stability in terms of hydrogen bonding and their flexibility are discussed and the biological significance for the interaction with DNA polymerase is evoked.Keywords: DNA; gap; molecular dynamics; NMR; repair.DNA duplexes containing gaps of one nucleotide may occur as a result of damaging processes, but also as an intermediate during a DNA repair mechanism. The damage product is the result of an oxidative attack on DNA, which has two main origins: normal aerobic cellular metabolism and ionizing radiation [1]. Oxidative damage to DNA is a frequent event: it occurs up to 150 000 times per day per cell (105 times per min per cell; [2]). In aerobic metabolism and after ionizing radiation, the hydroxyl radical is the most important species to reacts with and modify the DNA structure [3,4]. The nature of the damage product depends on the reaction site of the hydroxyl radical on the DNA [5,6]. Hydroxyl attack on the sugars at the C1 H atom leads to base release (abasic sites); attack at the C4 H atom or on the phosphate backbone may lead to single-strand breaks (nicks) or loss of a single nucleotide (gap). Finally, hydroxyl attack on the bases leads to base damage or, ultimately, base release. In these different cases, O-H, phosphate groups or modified sugars are adjacent to the gap The proteins or protein±DNA complexes involved in this mechanism have been the subject of several recent structural or biochemical reports [16±18]. These reports showed that a particular structure of the DNA duplex during the complexation with DNA polymerase b is required for nucleotide insertion. The base facing the gap must be stacked over its neighbouring base in the 5 H direction. This position of DNA in the active site of DNA polymerase is necessary for plac...

Self‐Assembly of Single‐Stranded RNA on Carbon Nanotube: Polyadenylic Acid to Form a Duplex Structure

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Intrinsic Conformational Energetics Associated with the Glycosyl Torsion in DNA: A Quantum Mechanical Study

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