Effect of Hydroxymethylcytosine on the Structure and Stability of Holliday Junctions.

5-Hydroxymethylcytosine (C) is an epigenetic marker that has recently been shown to promote homologous recombination (HR). In this study, we determine the effects of C on the structure, thermodynamics, and conformational dynamics of the Holliday junction (the four-stranded DNA intermediate associated with HR) in its native stacked-X form. The hydroxymethyl and the control methyl substituents are placed in the context of an amphimorphic GCC trinucleotide core sequence (where C is C, C, or the methylated C), which is part of a sequence also recognized by endonuclease G to promote HR. The hydroxymethyl group of the C junction adopts two distinct rotational conformations, with an in-base-plane form being dominant over the competing out-of-plane rotamer that has typically been seen in duplex structures. The in-plane rotamer is seen to be stabilized by a more stable intramolecular hydrogen bond to the junction backbone. Stabilizing hydrogen bonds (H-bonds) formed by the hydroxyl substituent in C or from a bridging water in the C structure provide approximately 1.5-2 kcal/mol per interaction of stability to the junction, which is mostly offset by entropy compensation, thereby leaving the overall stability of the GCC and GCC constructs similar to that of the GCC core. Thus, both methyl and hydroxymethyl modifications are accommodated without disrupting the structure or stability of the Holliday junction. Both C and C are shown to open the structure to make the junction core more accessible. The overall consequences of incorporating C into a DNA junction are thus discussed in the context of the specificity in protein recognition of the hydroxymethyl substituent through direct and indirect readout mechanisms.