Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA

Ringvoll, Jeanette; Nordstrand, Line Mari; Vågbø, Cathrine Broberg; Talstad, Vivi; Reite, Karen; Arne, Per; Lauritzen, Knut H.; Liabakk, Nina Beate; Bjørk, Alexandra; Doughty, Richard William; Falnes, Pål Ø.; Krokan, Hans E.; Klungland, Arne

doi:10.1038/sj.emboj.7601109

Cited by 169 publications

(208 citation statements)

References 43 publications

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“…Likewise, in vivo experiments in humans, such as examination of the effect of gene polymorphisms [6] and nutritional status [6,7] on DNA metabolism, have used labeled one-carbon donors to label monocyte DNA. Nucleoside hydrolysates of DNA also have been used in determining the base composition of DNA, including investigation of epigenetic modification such as deoxycytosine methylation [8,9], oxidative damage [10,11], or unique DNA composition [12].…”

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DNA digestion to deoxyribonucleoside: A simplified one-step procedure

Quinlivan

Gregory

2008

Analytical Biochemistry

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We present a simple and inexpensive 'one-step' protocol for the hydrolysis of DNA to deoxyribonucleosides. Unlike the older DNA hydrolysis protocol which is cumbersome and labor intensive, thes new protocol is ideal for high-throughput assays and is suitable automation. Using this protocol we were able to hydrolyze several hundred samples within an 8-hour period. The new protocol is fully compatible with LC-MS/MS and gives similar recoveries for all five major deoxyribonucleosides when compared to the older protocol.The requirement to hydrolyze DNA to deoxyribonucleosides is a common component of several assays. To measure the turnover of DNA [1,2] or cells [3][4][5] in various tissues, DNA can be labeled with isotope-labeled precursor. In such studies, the DNA is extracted, hydrolyzed, and analyzed to determine label incorporation by mass spectrometry. Likewise, in vivo experiments in humans, such as examination of the effect of gene polymorphisms [6] and nutritional status [6,7] on DNA metabolism, have used labeled one-carbon donors to label monocyte DNA. Nucleoside hydrolysates of DNA also have been used in determining the base composition of DNA, including investigation of epigenetic modification such as deoxycytosine methylation [8,9], oxidative damage [10,11], or unique DNA composition [12].One of the most commonly used protocols for digesting DNA is the tri-enzyme protocol devised by Crain [13]. However, a major weakness of the Crain protocol is that the first enzyme in the reaction, nuclease P1 (E.C. 3.1.30.1), only can digest single stranded DNA and has a pH optimum (pH ~5) significantly lower and a reaction temperature significantly higher (~50°C) than the other two enzymes (pH >8 and ~37°C) in the reaction. This leads to an overly complex protocol [Fig 1], whereby the DNA must be boiled to denature (and rapidly cooled to prevent reversion), the pH of the sample is adjusted twice, and the sample undergoes three separate enzyme incubations. These complexities limit the number of samples that can be prepared. For logistical reasons (repetitive manipulations of tubes), we were typically [6] restricted to preparing ~60 samples per day when we used the Crain protocol. Furthermore, the Crain protocol uses high buffer concentrations (e.g., 50-100 mmol/L ammonium bicarbonate, pH 7.9) which can interfere with downstream reactions and assays. Therefore, we devised a simple *Corresponding Author: Email: epq@ufl.edu (E.P. Quinlivan). 1 Abbreviations Used: LC-MS/MS: liquid chromatography -tandem mass spectrometry; H-ESI: heated electrospray ionization; dCyt: deoxycytidine; MdCyt: 5-methyldeoxycytidine; dThy: thymidine; dAdn: deoxyadenosine; dGua: deoxyguanosine Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that dur...

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confidence: 99%

DNA digestion to deoxyribonucleoside: A simplified one-step procedure

Quinlivan

Gregory

2008

Analytical Biochemistry

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“…Bioinformatics analysis has identified a family of nine different mammalian AlkB homologues (ALKBH), including the obesity-associated protein FTO 13,14 . Although in vitro repair activities have been observed for some ALKBH proteins 10,13,15,16 , only ALKBH2 has been firmly established as a repair enzyme 17 , and non-repair functions have also been proposed [18][19][20] . We and others recently showed that ALKBH8 contains a MT domain, which is the functional mammalian Trm9 homologue, and requires a small accessory protein, TRM112, for activity (Fig.…”

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ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA

et al. 2011

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Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA (tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm 5 U) from its precursor 5-carboxymethyluridine (cm 5 U). In this study, we report that (R)-and (S)-5-methoxycarbonylhydroxymethyluridine (mchm 5 U), hydroxylated forms of mcm 5 U, are present in mammalian tRNA UCG Arg , and tRNA UCC Gly , respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm 5 U are generated from mcm 5 U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm 5 U into (S)-mchm 5 U in tRNA UCC Gly . These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis.

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“…A third one, FTO, has been recently shown to repair 3-meT in DNA, and it affects obesity in mammals through an unknown mechanism 17 . ABH2 acts as the primary housekeeping enzyme in mammals for repairing endogenously formed 1-meA lesions in duplex DNA 18 . The in vivo function of ABH3 is still unclear but it has been shown to repair base lesions in RNA as well as DNA 15 .…”

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Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA

Yang

Duguid

et al. 2008

Nature

238

405

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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 ....

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Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA

Cited by 169 publications

References 43 publications

DNA digestion to deoxyribonucleoside: A simplified one-step procedure

DNA digestion to deoxyribonucleoside: A simplified one-step procedure

ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA

Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA

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