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

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

Author information

1
Institute of Medical Microbiology, Oslo University Hospital, Rikshospitalet, Norway adam.robertson@rr-research.no.
2
Institute of Medical Microbiology, Oslo University Hospital, Rikshospitalet, Norway.
3
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.

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.

PMID:
25355512
PMCID:
PMC4245937
DOI:
10.1093/nar/gku1032
[Indexed for MEDLINE]
Free PMC Article

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