title: Effect of Hydroxymethylcytosine on the Structure and Stability of Holliday Junctions.
abstract: 5-Hydroxymethylcytosine (5hmC) is an epigenetic marker that has recently been shown to promote homologous recombination (HR). In this study, we determine the effects of5hmC 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 GxCC trinucleotide core sequence (wherexC is C,5hmC, or the methylated5mC), which is part of a sequence also recognized by endonuclease G to promote HR. The hydroxymethyl group of the5hmC 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 in5hmC or from a bridging water in the5mC 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 G5hmCC and G5mCC 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. Both5hmC and5mC are shown to open the structure to make the junction core more accessible. The overall consequences of incorporating5hmC 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.
authors.affiliation: Department of Biochemistry & Molecular Biology, Colorado State University , 1870 Campus Delivery, Fort Collins, Colorado 80523-1870, United States.
authors.forename: Crystal M
authors.initials: CM
authors.lastname: Vander Zanden
authors.affiliation: Department of Biochemistry & Molecular Biology, Colorado State University , 1870 Campus Delivery, Fort Collins, Colorado 80523-1870, United States.
authors.forename: Rhianon K
authors.initials: RK
authors.lastname: Rowe
authors.affiliation: Department of Biochemistry & Molecular Biology, Colorado State University , 1870 Campus Delivery, Fort Collins, Colorado 80523-1870, United States.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Broad
authors.affiliation: Department of Molecular Microbiology, Oslo University Hospital , Sognsvannsveien 20, NO-0027 Oslo, Norway.
authors.forename: Adam B
authors.initials: AB
authors.lastname: Robertson
authors.affiliation: Department of Biochemistry & Molecular Biology, Colorado State University , 1870 Campus Delivery, Fort Collins, Colorado 80523-1870, United States.
authors.forename: P Shing
authors.initials: PS
authors.lastname: Ho
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D018360:Crystallography, X-Ray; D045566:DNA, Cruciform; D009690:Nucleic Acid Conformation; D011789:Quantum Theory; D013816:Thermodynamics
chemical_list: D045566:DNA, Cruciform; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine