Dataset Information

Tet2 Catalyzes Stepwise 5-Methylcytosine Oxidation by an Iterative and de novo Mechanism.

ABSTRACT: Modification of cytosine-guanine dinucleotides (CpGs) is a key part of mammalian epigenetic regulation and helps shape cellular identity. Tet enzymes catalyze stepwise oxidation of 5-methylcytosine (mC) in CpGs to 5-hydroxymethylcytosine (hmC), or onward to 5-formylcytosine (fC) or 5-carboxylcytosine (caC). The multiple mC oxidation products, while intricately linked, are postulated to play independent epigenetic roles, making it critical to understand how the products of stepwise oxidation are established and maintained. Using highly sensitive isotope-based studies, we newly show that Tet2 can yield fC and caC by iteratively acting in a single encounter with mC-containing DNA, without release of the hmC intermediate, and that the modification state of the complementary CpG has little impact on Tet2 activity. By revealing Tet2 as an iterative, de novo mC oxygenase, our study provides insight into how features intrinsic to Tet2 shape the epigenetic landscape.

SUBMITTER: Crawford DJ

PROVIDER: S-EPMC4762542 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Tet2 Catalyzes Stepwise 5-Methylcytosine Oxidation by an Iterative and de novo Mechanism.

Crawford Daniel J DJ Liu Monica Yun MY Nabel Christopher S CS Cao Xing-Jun XJ Garcia Benjamin A BA Kohli Rahul M RM

Journal of the American Chemical Society 20160113 3

Modification of cytosine-guanine dinucleotides (CpGs) is a key part of mammalian epigenetic regulation and helps shape cellular identity. Tet enzymes catalyze stepwise oxidation of 5-methylcytosine (mC) in CpGs to 5-hydroxymethylcytosine (hmC), or onward to 5-formylcytosine (fC) or 5-carboxylcytosine (caC). The multiple mC oxidation products, while intricately linked, are postulated to play independent epigenetic roles, making it critical to understand how the products of stepwise oxidation are ...[more]

PMID: 26734843

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Project description:Methylation of cytosine bases is strongly linked to gene expression, imprinting, aging, and carcinogenesis. The Ten-eleven translocation (TET) family of enzymes, which are Fe(II)/2-oxoglutarate (2OG)-dependent enzymes, employ Fe(IV)=O species to dealkylate the lesioned bases to an unmodified cytosine. Recently, it has been shown that the TET2 enzyme can catalyze promiscuously DNA substrates containing unnatural alkylated cytosine. Such unnatural substrates of TET can be used as direct probes for measuring the TET activity or capturing TET from cellular samples. Herein, we studied the catalytic mechanisms during the oxidation of the unnatural C5-position modifications (5-ethylcytosine (5eC), 5-vinylcytosine (5vC) and 5-ethynylcytosine (5eyC)) and the demethylation of N4-methylated lesions (4-methylcytosine (4mC) and 4,4-dimethylcytosine(4dmC)) of the cytosine base by the TET2 enzyme using molecular dynamics (MD) and combined quantum mechanics and molecular mechanics (QM/MM) computational approaches. The results reveal that the chemical nature of the alkylation of the double-stranded (ds) DNA substrates induces distinct changes in the interactions in the binding site, the second coordination sphere, and long-range correlated motions of the ES complexes. The rate-determining hydrogen atom transfer (HAT) is faster in N4-methyl substituent substrates than in the C5-alkylations. Importantly, the calculations show the preference of hydroxylation over desaturation in both 5eC and 5vC substrates. The studies elucidate the post-hydroxylation rearrangements of the hydroxylated intermediates of 5eyC and 5vC to ketene and 5-formylmethylcytosine (5fmC), respectively, and hydrolysis of hemiaminal intermediate of 4mC to formaldehyde and unmodified cytosine proceed exclusively in aqueous solution outside of the enzyme environment. Overall, the studies show that the chemical nature of the unnatural alkylated cytosine substrates exercises distinct effects on the binding interactions, reaction mechanism, and dynamics of TET2.

Dataset Information

Tet2 Catalyzes Stepwise 5-Methylcytosine Oxidation by an Iterative and de novo Mechanism.

Publications

Tet2 Catalyzes Stepwise 5-Methylcytosine Oxidation by an Iterative and de novo Mechanism.

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