Endonuclease G preferentially cleaves 5-hydroxymethylcytosine-modified DNA creating a substrate for recombination

Nucleic Acids Res. 2014 Dec 1;42(21):13280-93. doi: 10.1093/nar/gku1032. Epub 2014 Oct 29.


5-hydroxymethylcytosine (5hmC) has been suggested to be involved in various nucleic acid transactions and cellular processes, including transcriptional regulation, demethylation of 5-methylcytosine and stem cell pluripotency. We have identified an activity that preferentially catalyzes the cleavage of double-stranded 5hmC-modified DNA. Using biochemical methods we purified this activity from mouse liver extracts and demonstrate that the enzyme responsible for the cleavage of 5hmC-modified DNA is Endonuclease G (EndoG). We show that recombinant EndoG preferentially recognizes and cleaves a core sequence when one specific cytosine within that core sequence is hydroxymethylated. Additionally, we provide in vivo evidence that EndoG catalyzes the formation of double-stranded DNA breaks and that this cleavage is dependent upon the core sequence, EndoG and 5hmC. Finally, we demonstrate that the 5hmC modification can promote conservative recombination in an EndoG-dependent manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5-Methylcytosine / analogs & derivatives
  • Animals
  • Catalytic Domain / genetics
  • Cell Nucleus / enzymology
  • Cytosine / analogs & derivatives*
  • Cytosine / metabolism
  • DNA / chemistry
  • DNA Cleavage*
  • Dioxygenases / genetics
  • Endodeoxyribonucleases / metabolism*
  • HeLa Cells
  • Histones / analysis
  • Humans
  • Intracellular Signaling Peptides and Proteins / analysis
  • Mice
  • Mice, Inbred C57BL
  • Recombination, Genetic*
  • Tumor Suppressor p53-Binding Protein 1


  • H2AX protein, human
  • Histones
  • Intracellular Signaling Peptides and Proteins
  • TP53BP1 protein, human
  • Tumor Suppressor p53-Binding Protein 1
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine
  • Cytosine
  • DNA
  • Dioxygenases
  • Endodeoxyribonucleases
  • endonuclease G