Abstract
DNA damage can affect various regulatory elements of the genome, with the consequences for DNA structure, dynamics, and interaction with proteins remaining largely unexplored. We used solution NMR spectroscopy, restrained and free molecular dynamics to obtain the structures and investigate dominant motions for a set of DNA duplexes containing CpG sites permuted with combinations of 5-methylcytosine (mC), the primary epigenetic base, and 8-oxoguanine (oxoG), an abundant DNA lesion. Guanine oxidation significantly changed the motion in both hemimethylated and fully methylated DNA, increased base pair breathing, induced BI→BII transition in the backbone 3′ to the oxoG and reduced the variability of shift and tilt helical parameters. UV melting experiments corroborated the NMR and molecular dynamics results, showing significant destabilization of all methylated contexts by oxoG. Notably, some dynamic and thermodynamic effects were not additive in the fully methylated oxidized CpG, indicating that the introduced modifications interact with each other. Finally, we show that the presence of oxoG biases the recognition of methylated CpG dinucleotides by ROS1, a plant enzyme involved in epigenetic DNA demethylation, in favor of the oxidized DNA strand. Thus, the conformational and dynamic effects of spurious DNA oxidation in the regulatory CpG dinucleotide can have far-reaching biological consequences.
Highlights
Enzymatic methylation of cytosine (C) in the CpG context results in the formation of 5-methylcytosine, a principal epigenetic regulatory base (Figure 1A) [1]
To put the methylated/oxidized CpG dinucleotides into a known structural and dynamic context, we have designed a series of substrates based on the Drew–Dickerson sequence (DDD, Table 1), which had been characterized by solution NMR in the unmodified and oxidized state [42]
To see whether the effects of oxoG on the CpG dinucleotide dynamics may be of biological significance, we addressed the ability of ROS1 DNA glycosylase to remove mC from oxoG-modified CpG dinucleotides
Summary
Enzymatic methylation of cytosine (C) in the CpG context results in the formation of 5-methylcytosine (mC), a principal epigenetic regulatory base (Figure 1A) [1]. Assuming a consensus level of oxoG as 1 per G [15,16] and 2.8 × CpG dinucleotides per haploid human genome [17], oxoG would be expected to be found in ∼100 CpG dinucleotides per diploid human cell. This number has the potential to rise significantly with an increasing guanine oxidation typical of oxidative stress [18,19]. Considering 70–80% of CpG dinucleotides are methylated in the human genome [17,20], possible interference of oxoG with the epigenetic functions of mC may be of biological significance
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