Opening dynamics of 8‐oxoguanine in DNA

Abstract

8-oxoguanine is a major lesion of genomic DNA that results from oxidation of guanine by reactive oxygen species. The repair of this lesion is initiated by 8-oxoguanine glycosylases, which excise the damaged base by "flipping" it outside the DNA double helix. The molecular mechanisms involved in the specific recognition of the damaged base by the enzyme are not yet fully understood. Several models have proposed that, in DNA, the base pair between 8-oxoguanine and cytosine may possess altered dynamic properties that could help the enzyme locate the lesion and could favor the selective extra-helical flipping of the damaged base. To test this proposal, we have characterized the spontaneous opening of the base pair between 8-oxoguanine and cytosine in a DNA double helix using NMR spectroscopy and proton exchange. The results show that the rate of spontaneous opening of 8-oxoguanine and the lifetime of the base in the extra-helical state are the same as those of a canonical guanine-cytosine base pair, in the same base sequence context. This finding suggests that the opening dynamics of 8-oxoguanine, when paired with cytosine in DNA, does not play a significant role in the recognition of the lesion by glycosylases.

Global deformation facilitates flipping of damaged 8-oxo-guanine and guanine in DNA

“…These simulations predicted a significant free energy penalty in good agreement with experiment and in qualitative agreement with previous simulations using different force fields, flipping restraints and sampling protocols (21,38–40,57–59). …”

“…Proton exchange of the base polar hydrogens at the WC interface can be used to determine the equilibrium between intrahelical and extrahelical of damaged or undamaged bases (16,21,50–52). In order to define the onset of the transition we calculated the solvent accessibility (SASA) of the bases central polar hydrogen (H1) versus reaction coordinate.…”

“…Given the low abundance of oxidized damaged guanine relative to undamaged DNA (e.g. in the order of 10 −6 ) the stability of the 8oxoG:C base pair further lowers the probability for forming an extrahelical conformation relative to the total concentration by a factor of 10 −6 –10 −7 according to estimates of the relative life times of the intrahelical and extrahelical states based on nuclear magnetic resonance (NMR) spectroscopy (21,22). Hence, in the passive recognition model, the effective concentration of accessible extrahelical damaged 8oxoG relative to undamaged bases at any given time point is only ∼10 −12 –10 −13 .…”

NMR Analysis of Base‐Pair Opening Kinetics in DNA

“…Such imino proton exchange experiments and determination of base pair opening kinetics have been conducted previously on modified DNA (Leroy, Gao et al 1992, Moe and Russu 1992, Bohon and Carlos 2005, Parker and Stivers 2010, Friedman, Jiang et al 2011, Every and Russu 2013, Szulik, Voehler et al 2013). …”

“…NMR on the other hand provides a powerful tool to locally monitor the kinetics of each base pair opening under physiological conditions. The most common NMR approach to determine these local opening/closing rates relies on the 1 H NMR measurement of the proton exchange between water and the imino protons after magnetization transfer (Gueron, Kochoyan et al 1987, Gueron, Charretier et al 1990, Moe and Russu 1992, Folta-Stogniew and Russu 1996, Dhavan, Lapham et al 1999, Dornberger, Leijon et al 1999, Chen and Russu 2004, Every and Russu 2007, Every and Russu 2008, Lee, Lee et al 2009, Parker and Stivers 2010, Crenshaw, Wade et al 2011, Friedman, Jiang et al 2011, Every and Russu 2013, Szulik, Voehler et al 2013). Depending on the base pair stability, base pair opening occurs more or less frequently, allowing for the imino proton to exchange with water.…”