A non-heme iron-mediated chemical demethylation in DNA and RNA.
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 AlkB, 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 non-sequence-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 cross-link 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 cross-linking 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 base-flipping proteins.
Department of Chemistry, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
Chengqi
C
Yi
Cai-Guang
CG
Yang
Chuan
C
He
D000071499:AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D019175:DNA Methylation; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000737:chemistry*; D007656:Ketoglutaric Acids / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry* / Q000378:metabolism
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D007656:Ketoglutaric Acids; D012313:RNA; D004247:DNA; D007501:Iron; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C486439:ALKBH3 protein, human; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; C466427:AlkB protein, E coli; C486440:ALKBH2 protein, human; D000071499:AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; D045643:DNA Repair Enzymes