In contrast to 5-methylcytosine (5-mC), which has been studied extensively1–3, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types4,5. Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC–containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized4. We also find a gene expression level–dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.

Escherichia coli AlkB and its human homologues ABH2 and ABH3 repair DNA/RNA base lesions by using a direct oxidative dealkylation mechanism. ABH2 has the primary role of guarding mammalian genomes against 1-meA damage by repairing this lesion in double-stranded DNA (dsDNA), whereas AlkB and ABH3 preferentially repair single-stranded DNA (ssDNA) lesions and can repair damaged bases in RNA. Here we show the first crystal structures of AlkB-dsDNA and ABH2-dsDNA complexes, stabilized by a chemical cross-linking strategy. This study reveals that AlkB uses an unprecedented base-flipping mechanism to access the damaged base: it squeezes together the two bases flanking the flipped-out one to maintain the base stack, explaining the preference of AlkB for repairing ssDNA lesions over dsDNA ones. In addition, the first crystal structure of ABH2, presented here, provides a structural basis for designing inhibitors of this human DNA repair protein.Cellular DNA is constantly subjected to modifications by environmental and endogenous chemicals, which can result in covalent changes 1,2 . Methylating (or alkylating) agents are a common group of DNA modifiers that introduce damage primarily to the heterocyclic bases of DNA, with mutagenic and/or cytotoxic consequences. Alkylating agents are also widely used in cancer therapy and exert anticancer effects by creating cytotoxic DNA lesions in tumour cells. Many of these alkylation DNA damages are detected and repaired by proteins that are conserved across kingdoms.The E. coli AlkB protein is a direct dealkylation DNA repair protein 3-5 . It uses a mononuclear iron(II) site and cofactors 2-ketoglutarate (2KG) and dioxygen to perform an unprecedented oxidative demethylation of DNA base lesions 1-meA, 3-meC, 1-meG and 3-meT ( Supplementary Fig. 2) 6-11 . AlkB also removes etheno DNA lesions by using a similar oxidation mechanism 12,13 . There are nine potential human homologues of AlkB. Two of Correspondence and requests for materials should be addressed to C.H. (chuanhe@uchicago.edu). * These authors contributed equally to this work. Reprints and permissions information is available at www.nature.com/reprints. Author Contributions NIH Public Access Cross-linking to stabilize protein-DNA complexesWe report here the first crystal structures of AlkB-dsDNA and ABH2-dsDNA complexes. The AlkB family proteins bind DNA weakly 21 and form labile complexes with damagecontaining DNA 22 , which makes crystallization of their protein-DNA complexes challenging.To overcome this difficulty we used chemical cross-linking methods 23,24 ; initially using an active site disulphide cross-linking strategy that we developed previously (Fig. 1a) 25,26 . Baserepair proteins flip damaged bases and insert them into the active site for processing. Therefore, we reasoned, a cysteine residue engineered into the active site of AlkB may form a disulphide cross-link, at equilibrium, with a disulphide-modified cytosine (C* in a C*:A base pair) flipped into the active site of the repair protein ( Fig. 1a) 27 ....

The human obesity susceptibility gene, FTO, encodes a protein that is homologous to the DNA repair AlkB protein.The AlkB family proteins utilize iron(II), a-ketoglutarate (a-KG) and dioxygen to perform oxidative repair of alkylated nucleobases in DNA and RNA. We demonstrate here the oxidative demethylation of 3-methylthymine (3-meT) in singlestranded DNA (ssDNA) and 3-methyluracil (3-meU) in singlestranded RNA (ssRNA) by recombinant human FTO protein in vitro. Both human and mouse FTO proteins preferentially repair 3-meT in ssDNA over other base lesions tested. They showed negligible activities against 3-meT in double-stranded DNA (dsDNA). In addition, these two proteins can catalyze the demethylation of 3-meU in ssRNA with a slightly higher efficiency over that of 3-meT in ssDNA, suggesting that methylated RNAs are the preferred substrates for FTO.

Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology1,2, including the oxidative demethylation of methylated nucleic acids and histones3,4. E. coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids (Fig. 1)5,6, which are otherwise cytotoxic and/or mutagenic. AlkB human homologues are known to play pivotal roles in various processes7–11. Presented here is the first structural characterization of oxidation intermediates for these demethylases. Employing a chemical cross-linking strategy12,13, complexes of AlkB-dsDNA containing 1,N6-etheno adenine (εA), N3-methyl thymine (3-meT), and N3-methyl cytosine (3-meC) were stabilized and crystallized, respectively. Exposing these crystals, grown under anaerobic conditions containing iron(II) and α-ketoglutarate (αKG), to dioxygen initiates oxidation in crystallo (Supplementary Fig. 1). A glycol (from εA) and a hemiaminal (from 3-meT) intermediates are captured; a zwitterionic intermediate (from 3-2 meC) is also proposed, based on crystallographic observations and computational analysis. The observation of these unprecedented intermediates provides direct support for the oxidative demethylation mechanism for these demethylases. This study also depicts a general mechanistic view of how a methyl group is oxidatively removed from different biological substrates.

Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.