Dual base-flipping of cytosines in a CpG dinucleotide sequence

Simultaneous flipped-out conformation of two neighboring bases on opposite strands of DNAs has been observed in several X-ray structures. It has also been detected for two cytosines on opposite strands in different contexts of CpG sites. In this paper, we study by MD simulations the dual base flipping of the two cytosines in hemi-methylated CpG site. We calculate the potential of mean force of flipping-out the unmethylated cytosine in three model systems. The first is for DNA bound to the regulatory protein UHRF1. In this case, the methyl-cytosine on the complementary strand is flipped-out into the binding pocket of the SRA domain of the protein. The other two systems are for unbound DNAs in which the methyl-cytosine is either intra-helical or extra-helical. We find that when the methyl-cytosine is flipped-out it is easier to flip-out the other (unmethylated) cytosine on the opposite strand by about 14-16kJ/mol. This lower penalty for dual-base flipping is observed for both the bound and unbound states of the DNA. Analyses of the hydrogen bond network and stacking interactions within the CpG site indicate that the lower penalty is due to stabilization of the dual-base flipped-out conformation via interactions involving the orphan guanines. The results presented in this paper suggest that the extra-helical conformation of the methyl-cytosine recognized by UHRF1 can facilitate the base-flipping process of the target cytosine to be methylated by Dnmt1.