title: Endonuclease G preferentially cleaves 5-hydroxymethylcytosine-modified DNA creating a substrate for recombination.
abstract: 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.
authors.affiliation: Institute of Medical Microbiology, Oslo University Hospital, Rikshospitalet, Norway adam.robertson@rr-research.no.
authors.forename: Adam B
authors.initials: AB
authors.lastname: Robertson
authors.affiliation: Institute of Medical Microbiology, Oslo University Hospital, Rikshospitalet, Norway.
authors.forename: Julia
authors.initials: J
authors.lastname: Robertson
authors.affiliation: Institute of Medical Microbiology, Oslo University Hospital, Rikshospitalet, Norway.
authors.forename: Markus
authors.initials: M
authors.lastname: Fusser
authors.affiliation: Institute of Medical Microbiology, Oslo University Hospital, Rikshospitalet, Norway Institute of Basic Medical Sciences, University of Oslo, PO Box 1018 Blindern, N-0315 Oslo, Norway.
authors.forename: Arne
authors.initials: A
authors.lastname: Klungland
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002467:Cell Nucleus / Q000201:enzymology; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D053837:DNA Cleavage; D049308:Dioxygenases / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D006367:HeLa Cells; D006657:Histones / Q000032:analysis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D011995:Recombination, Genetic; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D049308:Dioxygenases; D004706:Endodeoxyribonucleases; C052391:endonuclease G