Epigenetic modifiers play vital roles in directing DNA structure, regulating gene expression, and determining disease-states. 5-hydroxymethylcytosine (5hmC) has recently gained interest in the scientific community because little is currently known about this epigenetic marker despite its presence in up to one percent of cytosines in the mammalian genome. Initially, 5hmC was thought primarily to be an intermediate in 5-methylcytosine metabolism, but now the field is beginning to recognize 5hmC’s involvement in a variety of cellular functions, even calling it the “sixth base” of the genome. 5hmC has recently been linked to DNA recombination, with evidence that suggests recombination is promoted via interaction between 5hmC and the protein Endonuclease G (Endo G). Our research question is how are DNA cutting proteins, resolvases, able to identify a specific target when scanning across whole genes of sequence? We aim to explore the mechanism behind 5hmC as a marker for target recombination sequences. Our structural studies reveal that 5hmC induces a unique hydrogen bond between the 5hmC’s hydroxyl group and an oxygen on the DNA backbone when incorporated into the 4-stranded DNA intermediates for homologous recombination (Holliday junctions). 5hmC further impacts the global Holliday junction shape to make a uniquely recognizable structure for recognition by the protein Endo G. Thermodynamic analysis confirms that 5hmC incorporation is not destabilizing to the Holliday junction. Determination of Endo G binding affinity to various DNA constructs has shown preferential binding for Endo G to Holliday junction DNA, suggesting the protein is a resolvase in homologous recombination. This work is helping to decode the role of 5hmC in promoting DNA recombination and further revealing a mechanism describing how specific DNA targets are recognized for recombination.

5-Hydroxymethylcytosine impacts Holliday junction structure to promote recombination via recognition by Endonuclease G

253rd National Meeting (2017)

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