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Biochemical characterization of two evolutionary distant ten‐eleven translocation enzymes and their utility in 5‐methylcytosine sequencing in the genomes at single‐base resolution
Author(s) -
Saleh Lana,
Tamanaha Esta,
Vaisvila Romualdas,
Pais June E.,
Dai Nan,
Guan Shengxi,
Corrêa Ivan R,
Zheng Yu
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.834.2
Subject(s) - 5 methylcytosine , enzyme , dna , dna methylation , epigenetics , biology , biochemistry , cpg site , 5 hydroxymethylcytosine , mutagenesis , genome , genetics , chemistry , gene , mutation , gene expression
The ten‐eleven translocation (TET) enzymes iteratively oxidize 5‐methylcytosine ( 5m C) on DNA to 5‐hydroxymethylcytosine, 5‐formylcytosine, and 5‐carboxycytosine. Here, we examine the in vitro biochemical activity of two evolutionary distant TETs, mTET1 from mouse and NgTET1 from the single‐celled protist Naegleria gruberi . We show that both of these enzymes are 5‐methylpyrimidine oxygenases with activity on both 5m C (major activity) and thymidine (T) (minor activity) and preference to 5m CpG and TpG dinucleotide sites. Intriguingly, NgTET1 displays higher T oxidation activity in vitro than mTET1 supporting a closer evolutionary relationship between NgTET1 and the base J binding proteins from trypanosomes. In fact, unprecedented evidence for the formation of two new bases, 5‐formyluridine and 5‐carboxyuridine, is shown for NgTET1 activity in vitro . Mutagenesis studies performed in NgTET1 reveal a delicate balance between choice of 5m C or T as the preferred substrate. Steady‐state kinetic analysis show that both mTET1 and NgTET1 are distributive in their oxidative chemistry with each oxidized species released from the enzyme upon formation. Furthermore, both enzymes are physically distributive in the recognition of their substrates on template DNA with no apparent preference to a specific 5m C oxidation intermediate. These data indicate a role for the TET enzymes in the maintenance of the three oxidized forms of 5m C and support the in vivo data implicating that these bases are not simply intermediates in a methylation cycle but represent additional epigenetic states in genomic DNA with regulatory functions to be explored. Finally, we demonstrate the utility of both enzymes in 5m C sequencing technologies and present examples for the mapping of 5m C in different genomes at single base resolution.