ABSTRACT
Methylation of cytosine to 5-methylcytosine (mC) is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the methylation mark can be actively erased via a multi-step demethylation mechanism involving oxidation by Ten-eleven translocation (TET) enzyme family dioxygenases, excision of the latter oxidation products by thymine DNA (TDG) or Nei-like 1 (NEIL1) glycosylases followed by base excision repair to restore the unmodified state. Here we probed the activity of the mouse TET1 (mTET1) and Naegleria gruberi TET (nTET) oxygenases with DNA substrates containing extended derivatives of the 5-methylcytosine carrying linear carbon chains and adjacent unsaturated CC bonds. We found that the nTET and mTET1 enzymes were active on modified mC residues in single-stranded and double-stranded DNA in vitro, while the extent of the reactions diminished with the size of the extended group. Iterative rounds of nTET hydroxylations of ssDNA proceeded with high stereo specificity and included not only the natural alpha position but also the adjoining carbon atom in the extended side chain. The regioselectivity of hydroxylation was broken when the reactive carbon was adjoined with an sp1 or sp2 system. We also found that NEIL1 but not TDG was active with bulky TET-oxidation products. These findings provide important insights into the mechanism of these biologically important enzymatic reactions.
Subject(s)
DNA Glycosylases/genetics , DNA Methylation/genetics , DNA-Binding Proteins/genetics , DNA/genetics , Proto-Oncogene Proteins/genetics , 5-Methylcytosine/metabolism , Animals , Cytosine/metabolism , DNA/metabolism , DNA Repair/genetics , Humans , Hydroxylation , Mice , Naegleria/genetics , Oxidation-ReductionABSTRACT
EcoKMcrA from Escherichia coli restricts CpG methylated or hydroxymethylated DNA, and may act as a barrier against host DNA. The enzyme consists of a novel N-terminal specificity domain that we term NEco, and a C-terminal catalytic HNH domain. Here, we report that NEco and full-length EcoKMcrA specificities are consistent. NEco affinity to DNA increases more from hemi- to full-methylation than from non- to hemi-methylation, indicating cooperative binding of the methyl groups. We determined the crystal structures of NEco in complex with fully modified DNA containing three variants of the Y5mCGR EcoKMcrA target sequence: C5mCGG, T5mCGA and T5hmCGA. The structures explain the specificity for the two central base pairs and one of the flanking pairs. As predicted based on earlier biochemical experiments, NEco does not flip any DNA bases. The proximal and distal methyl groups are accommodated in separate pockets. Changes to either pocket reduce DNA binding by NEco and restriction by EcoKMcrA, confirming the relevance of the crystallographically observed binding mode in solution.
Subject(s)
Cytosine/metabolism , DNA Methylation , DNA Restriction Enzymes/chemistry , DNA Restriction Enzymes/metabolism , DNA/metabolism , Escherichia coli/enzymology , 5-Methylcytosine/chemistry , 5-Methylcytosine/metabolism , Binding Sites , Catalytic Domain , CpG Islands/genetics , Crystallography, X-Ray , Cytosine/chemistry , DNA/chemistry , Models, Molecular , Protein Binding , Protein Structure, Tertiary , StereoisomerismABSTRACT
Escherichia coli McrA (EcoKMcrA) acts as a methylcytosine and hydroxymethylcytosine dependent restriction endonuclease. We present a biochemical characterization of EcoKMcrA that includes the first demonstration of its endonuclease activity, small angle X-ray scattering (SAXS) data, and a crystal structure of the enzyme in the absence of DNA. Our data indicate that EcoKMcrA dimerizes via the anticipated C-terminal HNH domains, which together form a single DNA binding site. The N-terminal domains are not homologous to SRA domains, do not interact with each other, and have separate DNA binding sites. Electrophoretic mobility shift assay (EMSA) and footprinting experiments suggest that the N-terminal domains can sense the presence and sequence context of modified cytosines. Pyrrolocytosine fluorescence data indicate no base flipping. In vitro, EcoKMcrA DNA endonuclease activity requires Mn2+ ions, is not strictly methyl dependent, and is not observed when active site variants of the enzyme are used. In cells, EcoKMcrA specifically restricts DNA that is modified in the correct sequence context. This activity is impaired by mutations of the nuclease active site, unless the enzyme is highly overexpressed.
Subject(s)
DNA Restriction Enzymes/chemistry , DNA-Binding Proteins/chemistry , Protein Structure, Tertiary , Amino Acid Sequence/genetics , Binding Sites/genetics , Catalytic Domain/genetics , Cytosine/chemistry , DNA Restriction Enzymes/genetics , DNA-Binding Proteins/genetics , Escherichia coli/chemistry , Escherichia coli/genetics , Gene Expression Regulation, Enzymologic , Protein Binding , Scattering, Small AngleABSTRACT
Thymine DNA glycosylase (TDG) and Nei-like 1 (NEIL1) have both been implicated in the base excision repair step of active DNA demethylation. The robust glycosylase activity of TDG on DNA substrates containing 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC) is universally accepted, but the mode of action of NEIL1 is still debated. Based on genetic experiments, it has been suggested that NEIL1 acts redundantly with TDG and excises 5fC and 5caC directly. However, this result has been disputed, and it was suggested instead that NEIL1 is recruited by the monofunctional TDG for the 2'-deoxyribose excision step. Using purified human NEIL1 and its catalytically impaired P2T and E3Q variants as controls, we detect NEIL1 activity on 5caC, but not a 5fC containing dsDNA substrate. We confirm direct NEIL1 TDG binding and NEIL1 mediated 2'-deoxyribose excision downstream of TDG glycosylase activity. NEIL1 acts not only downstream of TDG, but also enhances TDG activity on 5fC or 5caC containing DNA. NEIL1 mediated enhancement of the TDG glycosylase activity is substrate specific and does not occur for dsDNA with a T/G mismatch.
