A Non-Heme Iron-Mediated Chemical Demethylation in DNA and RNA

Abstract

DNA methylation is arguably one of the most important chemical signals in biology. However, aberrant DNA methylation can lead to cytotoxic or mutagenic consequences. A DNA repair protein in Escherichia coli, AlkB, corrects some of the unwanted methylations of DNA bases by a unique oxidative demethylation in which the methyl carbon is liberated as formaldehyde. The enzyme also repairs exocyclic DNA lesions-that is, derivatives in which the base is augmented with an additional heterocyclic subunit-by a similar mechanism. Two proteins in humans that are homologous to AlkB, ABH2 and ABH3, repair the same spectrum of lesions; another human homologue of AlkB, FTO, is linked to obesity. In this Account, we describe our studies of AlkB, ABH2, and ABH3, including our development of a general strategy to trap homogeneous protein-DNA complexes through active-site disulfide cross-linking.AlkB uses a non-heme mononuclear iron(II) and the cofactors 2-ketoglutarate (2KG) and dioxygen to effect oxidative demethylation of the DNA base lesions 1-methyladenine (1-meA), 3-methylcytosine (3-meC), 1-methylguanine (1-meG), and 3-methylthymine (3-meT). ABH3, like Yi et al. Page 2 Acc Chem Res. Author manuscript; available in PMC 2010 August 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAlkB, works better on single-stranded DNA (ssDNA) and is capable of repairing damaged bases in RNA. Conversely, ABH2 primarily repairs lesions in double-stranded DNA (dsDNA); it is the main housekeeping enzyme that protects the mammalian genome from 1-meA base damage.The AlkB-family proteins have moderate affinities for their substrates and bind DNA in a nonsequence-specific manner. Knowing that these proteins flip the damaged base out from the duplex DNA and insert it into the active site for further processing, we first engineered a disulfide crosslink in the active site to stabilize the Michaelis complex. Based on the detailed structural information afforded by the active-site cross-linked structures, we can readily install a cross-link away from the active site to obtain the native-like structures of these complexes. The crystal structures show a distinct base-flipping feature in AlkB and establish ABH2 as a dsDNA repair protein. They also provide a molecular framework for understanding the demethylation reaction catalyzed by these proteins and help to explain their substrate preferences. The chemical crosslinking method demonstrated here can be applied to trap other labile protein-DNA interactions and can serve as a general strategy for exploring the structural and functional aspects of baseflipping proteins. IntroductionCellular DNA is constantly challenged by endogenous and exogenous chemicals, often resulting in cytotoxic or mutagenic covalent adducts. [1][2][3][4] Alkylating agents are one group of such chemicals widely used in clinical settings and prevalent in the environment that create cytotoxic DNA lesions. [5][6][7][8] Once damaged, cellular DNA must be promptly repaired.Organisms have evolved a variety of ...

PCA-1/ALKBH3 Contributes to Pancreatic Cancer by Supporting Apoptotic Resistance and Angiogenesis

“…In these proteins, it has been reported that AlkB and its human homologs play an important role in demethylation of DNA (13,14,19,20,22,23,34). Among human AlkB homologs, ALKBH3 is known as a unique member to demethylate RNA besides repairing methylated DNA (13,(35)(36)(37). Several previous studies suggest that ALKBH3 may play physiological key roles in humans.…”

DNA Repair by Reversal of DNA Damage

“…5B) (Sundheim et al 2006). Sequence alignments of ALKBH2 and ALKBH3 reveal a very hydrophobic Val101-Phe102-Gly103 motif for ALKBH2 and a heavily charged Arg122-Glu123-Asp124 sequence for ALKBH3 at the nucleotide-flipping region (Yi et al 2009). Swapping the two sequences surprisingly switches the ss/dsDNA preference of the two proteins (Chen et al 2010;Monsen et al 2010).…”

“…In the event of oxidative demethylation, the AlkB family proteins use an iron(II) site to activate the dioxygen molecule for oxidation of the aberrant alkyl groups. The hydroxylated alkyl groups, which are attached to the N 1 position of adenine or N 3 position of cytosine, then undergo facile C -N bond cleavage to yield the unmodified base and formaldehyde (Drablos et al 2004;Sedgwick 2004;Falnes et al 2007;Yi et al 2009). Such an oxidation mechanism is shared by a variety of enzymes within the nonheme iron-containing protein family, which has also inspired the discovery of the JmjC-domain-containing histone demethylases that mediate epigenetic histone demethylation (Tsukada et al 2006).…”

Mouse Tet1 Protein can Oxidize 5mC to 5hmC and 5caC on Single-stranded DNA

“…The Figure 1 The scheme of cytosine methylation and 5mC oxidation oxidation reaction accrues on Tet's C-terminal DNA binding and catalytic domain, which is similar to that of the AlkB family proteins and HIF prolyl-hydroxylases. [21][22][23] Although the three Tet proteins exhibit similar activity on 5mC oxidation in vitro, they were found to play different roles in vivo. For example, Tet1 maintains the stem cell properties of embryonic stem cells, [6,8] Tet2 significantly affects leukemia, [9][10][11][12][13] and Tet3 plays a key role in epigenetic reprogramming in zygote development.…”