Effect of Hydroxymethylcytosine on the Structure and Stability of Holliday Junctions

5-Hydroxymethylcytosine (^5hmC) is an epigenetic marker that has recently been shown to promote homologous recombination (HR). In this study, we determine the effects of ^5hmC 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 G^xCC trinucleotide core sequence (where ^xC is C, ^5hmC, or the methylated ^5mC), which is part of a sequence also recognized by endonuclease G to promote HR. The hydroxymethyl group of the ^5hmC 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 ^5hmC or from a bridging water in the ^5mC 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 G^5hmCC and G^5mCC 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 ^5hmC and ^5mC are shown to open the structure to make the junction core more accessible. The overall consequences of incorporating ^5hmC 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.