alkB Mutant Research Articles

A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities.

AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

Selective Enzymatic Demethylation of N2,N2‐Dimethylguanosine in RNA and Its Application in High‐Throughput tRNA Sequencing

AbstractThe abundant Watson–Crick face methylations in biological RNAs such as N1‐methyladenosine (m1A), N1‐methylguanosine (m1G), N3‐methylcytosine (m3C), and N2,N2‐dimethylguanosine (m22G) cause significant obstacles for high‐throughput RNA sequencing by impairing cDNA synthesis. One strategy to overcome this obstacle is to remove the methyl group on these modified bases prior to cDNA synthesis using enzymes. The wild‐type E. coli AlkB and its D135S mutant can remove most of m1A, m1G, m3C modifications in transfer RNA (tRNA), but they work poorly on m22G. Here we report the design and evaluation of a series of AlkB mutants against m22G‐containing model RNA substrates that we synthesize using an improved synthetic method. We show that the AlkB D135S/L118V mutant efficiently and selectively converts m22G modification to N2‐methylguanosine (m2G). We also show that this new enzyme improves the efficiency of tRNA sequencing.

Selective Enzymatic Demethylation of N2 ,N2 -Dimethylguanosine in RNA and Its Application in High-Throughput tRNA Sequencing.

The abundant Watson-Crick face methylations in biological RNAs such as N1 -methyladenosine (m1 A), N1 -methylguanosine (m1 G), N3 -methylcytosine (m3 C), and N2 ,N2 -dimethylguanosine (m22 G) cause significant obstacles for high-throughput RNA sequencing by impairing cDNA synthesis. One strategy to overcome this obstacle is to remove the methyl group on these modified bases prior to cDNA synthesis using enzymes. The wild-type E. coli AlkB and its D135S mutant can remove most of m1 A, m1 G, m3 C modifications in transfer RNA (tRNA), but they work poorly on m22 G. Here we report the design and evaluation of a series of AlkB mutants against m22 G-containing model RNA substrates that we synthesize using an improved synthetic method. We show that the AlkB D135S/L118V mutant efficiently and selectively converts m22 G modification to N2 -methylguanosine (m2 G). We also show that this new enzyme improves the efficiency of tRNA sequencing.

Evaluation of the Escherichia coli HK82 and BS87 strains as tools for AlkB studies.

Within a decade the family of AlkB dioxygenases has been extensively studied as a one-protein DNA/RNA repair system in Escherichia coli but also as a group of proteins of much wider functions in eukaryotes. Two strains, HK82 and BS87, are the most commonly used E. coli strains for the alkB gene mutations. The aim of this study was to assess the usefulness of these alkB mutants in different aspects of research on AlkB dioxygenases that function not only in alkylated DNA repair but also in other metabolic processes in cells. Using of HK82 and BS87 strains, we found the following differences among these alkB(-) derivatives: (i) HK82 has shown more than 10-fold higher MMS-induced mutagenesis in comparison to BS87; (ii) different specificity of Arg(+) revertants; (iii) increased induction of SOS and Ada responses in HK82; (iv) the genome of HK82, in comparison to AB1157 and BS87, contains additional mutations: nalA, sbcC, and nuoC. We hypothesize that in HK82 these mutations, together with the non-functional AlkB protein, may result in much higher contents of ssDNA, thus higher in comparison to BS87 MMS-induced mutagenesis. In the light of our findings, we strongly recommend using BS87 strain in AlkB research as HK82, bearing several additional mutations in its genome, is not an exact derivative of the AB1157 strain, and shows additional features that may disturb proper interpretation of obtained results.

Effects of changes in intracellular iron pool on AlkB-dependent and AlkB-independent mechanisms protecting E.coli cells against mutagenic action of alkylating agent

An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH+ strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3→Arg+ reversion induction after λ>320nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg+ revertants in AB1157 alkB+hemH–/pMW1 strain was 40 and 26% reduced comparing to the alkB+hemH– and alkB+hemH+/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ>320nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg+ reversion was observed in irradiated and MMS-treated hemH–alkB– cells comparing to the hemH+alkB– strain. Also, the level of Arg+ revertants in the irradiated and MMS treated hemH– alkB– mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH– intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs.

The DNA dioxygenase ALKBH2 protects Arabidopsis thaliana against methylation damage

The Escherichia coli AlkB protein (EcAlkB) is a DNA repair enzyme which reverses methylation damage such as 1-methyladenine (1-meA) and 3-methylcytosine (3-meC). The mammalian AlkB homologues ALKBH2 and ALKBH3 display EcAlkB-like repair activity in vitro, but their substrate specificities are different, and ALKBH2 is the main DNA repair enzyme for 1-meA in vivo. The genome of the model plant Arabidopsis thaliana encodes several AlkB homologues, including the yet uncharacterized protein AT2G22260, which displays sequence similarity to both ALKBH2 and ALKBH3. We have here characterized protein AT2G22260, by us denoted ALKBH2, as both our functional studies and bioinformatics analysis suggest it to be an orthologue of mammalian ALKBH2. The Arabidopsis ALKBH2 protein displayed in vitro repair activities towards methyl and etheno adducts in DNA, and was able to complement corresponding repair deficiencies of the E. coli alkB mutant. Interestingly, alkbh2 knock-out plants were sensitive to the methylating agent methylmethanesulphonate (MMS), and seedlings from these plants developed abnormally when grown in the presence of MMS. The present study establishes ALKBH2 as an important enzyme for protecting Arabidopsis against methylation damage in DNA, and suggests its homologues in other plants to have a similar function.

Novel AlkB Dioxygenases—Alternative Models for In Silico and In Vivo Studies

BackgroundALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1–8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes.Methodology and FindingsBioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB − mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment.ConclusionsOur in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis.

Lethal and mutagenic properties of MMS-generated DNA lesions in Escherichia coli cells deficient in BER and AlkB-directed DNA repair

Methylmethane sulphonate (MMS), an S(N)2-type alkylating agent, generates DNA methylated bases exhibiting cytotoxic and mutagenic properties. Such damaged bases can be removed by a system of base excision repair (BER) and by oxidative DNA demethylation catalysed by AlkB protein. Here, we have shown that the lack of the BER system and functional AlkB dioxygenase results in (i) increased sensitivity to MMS, (ii) elevated level of spontaneous and MMS-induced mutations (measured by argE3 --> Arg(+) reversion) and (iii) induction of the SOS response shown by visualization of filamentous growth of bacteria. In the xth nth nfo strain additionally mutated in alkB gene, all these effects were extreme and led to 'error catastrophe', resulting from the presence of unrepaired apurinic/apyrimidinic (AP) sites and 1-methyladenine (1meA)/3-methylcytosine (3meC) lesions caused by deficiency in, respectively, BER and AlkB dioxygenase. The decreased level of MMS-induced Arg(+) revertants in the strains deficient in polymerase V (PolV) (bearing the deletion of the umuDC operon), and the increased frequency of these revertants in bacteria overproducing PolV (harbouring the pRW134 plasmid) indicate the involvement of PolV in the error-prone repair of 1meA/3meC and AP sites. Comparison of the sensitivity to MMS and the induction of Arg(+) revertants in the double nfo alkB and xth alkB, and the quadruple xth nth nfo alkB mutants showed that the more AP sites there are in DNA, the stronger the effect of the lack of AlkB protein. Since the sum of MMS-induced Arg(+) revertants in xth, nfo and nth xth nfo and alkB mutants is smaller than the frequency of these revertants in the BER(-) alkB(-) strain, we consider two possibilities: (i) the presence of AP sites in DNA results in relaxation of its structure that facilitates methylation and (ii) additional AP sites are formed in the BER(-) alkB(-) mutants.

AlkB Homologue 2–Mediated Repair of Ethenoadenine Lesions in Mammalian DNA

Endogenous formation of the mutagenic DNA adduct 1,N(6)-ethenoadenine (epsilon A) originates from lipid peroxidation. Elevated levels of epsilon A in cancer-prone tissues suggest a role for this adduct in the development of some cancers. The base excision repair pathway has been considered the principal repair system for epsilon A lesions until recently, when it was shown that the Escherichia coli AlkB dioxygenase could directly reverse the damage. We report here kinetic analysis of the recombinant human AlkB homologue 2 (hABH2), which is able to repair epsilon A lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2(-/-) mice indicates that mABH2 is the principal dioxygenase for epsilon A repair in vivo. This is further substantiated by experiments showing that hABH2, but not hABH3, is able to complement the E. coli alkB mutant with respect to its defective repair of etheno adducts. We conclude that ABH2 is active in the direct reversal of epsilon A lesions, and that ABH2, together with the alkyl-N-adenine-DNA glycosylase, which is the most effective enzyme for the repair of epsilon A, comprise the cellular defense against epsilon A lesions.

Repair of Methylation Damage in DNA and RNA by Mammalian AlkB Homologues

Human and Escherichia coli derivatives of AlkB enzymes remove methyl groups from 1-methyladenine and 3-methylcytosine in nucleic acids via an oxidative mechanism that releases the methyl group as formaldehyde. In this report, we demonstrate that the mouse homologues of the alpha-ketoglutarate Fe(II) oxygen-dependent enzymes mAbh2 and Abh3 have activities comparable to those of their human counterparts. The mAbh2 and mAbh3 release modified bases from both DNA and RNA. Comparison of the activities of the homogenous ABH2 and ABH3 enzymes demonstrate that these activities are shared by both sets of enzymes. An assay for the detection of alpha-ketoglutarate Fe(II) dioxygenase activity using an oligodeoxyribonucleotide with a unique modification shows activity for all four enzymes studied and a loss of activity for eight mutant proteins. Steady-state kinetics for removal of methyl groups from DNA substrates indicates that the reactions of the proteins are close to the diffusion limit. Moreover, mAbh2 or mAbh3 activity increases survival in a strain defective in alkB. The mRNAs of AHB2 and ABH3 are expressed most in testis for ABH2 and ABH3, whereas expression of the homologous mouse genes is different. The mAbh3 is strongly expressed in testis, whereas highest expression of mAbh2 is in heart. Other purified human AlkB homologue proteins ABH4, ABH6, and ABH7 do not manifest activity. The demonstration of mAbh2 and mAbh3 activities and their distributions provide data on these mammalian homologues of AlkB that can be used in animal studies.

Reversal of DNA alkylation damage by two human dioxygenases.

The Escherichia coli AlkB protein protects against the cytotoxicity of methylating agents by repair of the DNA lesions 1-methyladenine and 3-methylcytosine, which are generated in single-stranded stretches of DNA. AlkB is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase that oxidizes the relevant methyl groups and releases them as formaldehyde. Here, we identify two human AlkB homologs, ABH2 and ABH3, by sequence and fold similarity, functional assays, and complementation of the E. coli alkB mutant phenotype. The levels of their mRNAs do not appear to correlate with cell proliferation but tissue distributions are different. Both enzymes remove 1-methyladenine and 3-methylcytosine from methylated polynucleotides in an alpha-ketoglutarate-dependent reaction, and act by direct damage reversal with the regeneration of the unsubstituted bases. AlkB, ABH2, and ABH3 can also repair 1-ethyladenine residues in DNA with the release of acetaldehyde.

Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage.

Methylating agents generate cytotoxic and mutagenic DNA damage. Cells use 3-methyladenine-DNA glycosylases to excise some methylated bases from DNA, and suicidal O(6)-methylguanine-DNA methyltransferases to transfer alkyl groups from other lesions onto a cysteine residue. Here we report that the highly conserved AlkB protein repairs DNA alkylation damage by means of an unprecedented mechanism. AlkB has no detectable nuclease, DNA glycosylase or methyltransferase activity; however, Escherichia coli alkB mutants are defective in processing methylation damage generated in single-stranded DNA. Theoretical protein fold recognition had suggested that AlkB resembles the Fe(ii)- and alpha-ketoglutarate-dependent dioxygenases, which use iron-oxo intermediates to oxidize chemically inert compounds. We show here that purified AlkB repairs the cytotoxic lesions 1-methyladenine and 3-methylcytosine in single- and double-stranded DNA in a reaction that is dependent on oxygen, alpha-ketoglutarate and Fe(ii). The AlkB enzyme couples oxidative decarboxylation of alpha-ketoglutarate to the hydroxylation of these methylated bases in DNA, resulting in direct reversion to the unmodified base and the release of formaldehyde.

Defective processing of methylated single-stranded DNA by E. coli alkB mutants

Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated lambda phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by gamma irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. A recA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells.

Repair in Escherichia coli alkB mutants of abasic sites and 3-methyladenine residues in DNA

Escherichia coli alkB mutants are sensitive to methyl methanesulfonate and dimethylsulphate, and are defective in the processing of methylated DNA. The function of the AlkB protein has not been determined. Here, we show that alkB mutants are not defective in repairing several different types of potentially toxic DNA lesions that are known to be generated by MMS, including apyrimidinic and apurinic sites, and secondary lesions that could arise at these sites (DNA–protein cross-links and DNA interstrand cross-links). Also, alkB mutants were not sensitive to MeOSO2-(CH2)2-Lex, a compound that alkylates the minor groove of DNA generating primarily 3-methyladenine.

Molecular cloning and functional analysis of a human cDNA encoding an Escherichia coli AlkB homolog, a protein involved in DNA alkylation damage repair.

The Escherichia coli AlkB protein is involved in protecting cells against mutation and cell death induced specifically by SN2-type alkylating agents such as methyl methanesulfonate (MMS). A human cDNA encoding a polypeptide homologous to E.coli AlkB was discovered by searching a database of expressed sequence tags (ESTs) derived from high throughput cDNA sequencing. The full-length human AlkB homolog (hABH) cDNA clone contains a 924 bp open reading frame encoding a 34 kDa protein which is 52% similar and 23% identical to E.coli AlkB. The hABH gene, which maps to chromosome 14q24, was ubiquitously expressed in 16 human tissues examined. When hABH was expressed in E.coli alkB mutant cells partial rescue of the cells from MMS-induced cell death occurred. Under the conditions used expression of hABH in skin fibroblasts was not regulated by treatment with MMS. Our findings show that the AlkB protein is structurally and functionally conserved from bacteria to human, but its regulation may have diverged during evolution.

Suppression of Escherichia coli alkB mutants by Saccharomyces cerevisiae genes

The alkB gene is one of a group of alkylation-inducible genes in Escherichia coli, and its product protects cells from SN2-type alkylating agents such as methyl methanesulfonate (MMS). However, the precise biochemical function of the AlkB protein remains unknown. Here, we describe the cloning, sequencing, and characterization of three Saccharomyces cerevisiae genes (YFW1, YFW12, and YFW16) that functionally complement E. coli alkB mutant cells. DNA sequence analysis showed that none of the three gene products have any amino acid sequence homology with the AlkB protein. The YFW1 and YFW12 proteins are highly serine and threonine rich, and YFW1 contains a stretch of 28 hydrophobic residues, indicating that it may be a membrane protein. The YFW16 gene turned out to be allelic with the S. cerevisiae STE11 gene. STE11 is a protein kinase known to be involved in pheromone signal transduction in S. cerevisiae; however, the kinase activity is not required for MMS resistance because mutant STE11 proteins lacking kinase activity could still complement E. coli alkB mutants. Despite the fact that YFW1, YFW12, and YFW16/STE11 each confer substantial MMS resistance upon E. coli alkB cells, S. cerevisiae null mutants for each gene were not MMS sensitive. Whether these three genes provide alkylation resistance in E. coli via an alkB-like mechanism remains to be determined, but protection appears to be specific for AlkB-deficient E. coli because none of the genes protect other alkylation-sensitive E. coli strains from killing by MMS.

Cloning and nucleotide sequence of the ada gene of Salmonella typhimurium encoding O6-methylguanine-DNA methyltransferase

Mutations induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) were strongly enhanced in the presence of o-vanillin in E. coli B. The enhancement was also observed in uvrA, umuC, recA, polA, or alkB mutants. This effect was lower in an alkA mutant, but was restored in an alk A umuC double mutant. By contrast, the enhancing effect was almost blocked in an ada and ada umuC double mutant. It was necessary to add simultaneously MNNG and o-vanillin to the growth medium.Further investigations were conducted on the induction of ada and umuC genes using ada′-lacZ′ and umuC′-lacZ′ plasmids. o-Vanillin suppressed the induction of the ada gene by MNNG treatment, but not that of the umuC gene. In fact expression of the umuC gene was induced by lower concentrations of MNNG in the presence of o-vanillin.The results suggest that o-vanillin inhibits induction of the adaptive response, and consequently, the MNNG-induced mutation frequency is increased due to unrepaired O6-methylguanine.

Inhibition of induction of adaptive response by o-vanillin in Escherichia coli B

Mutations induced by N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) were strongly enhanced in the presence of o-vanillin in E. coli B. The enhancement was also observed in uvrA, umuC, recA, polA, or alkB mutants. This effect was lower in an alkA mutant, but was restored in an alk A umuC double mutant. By contrast, the enhancing effect was almost blocked in an ada and ada umuC double mutant. It was necessary to add simultaneously MNNG and o-vanillin to the growth medium. Further investigations were conducted on the induction of ada and umuC genes using ada′-lacZ′ and umuC′-lacZ′ plasmids. o-Vanillin suppressed the induction of the ada gene by MNNG treatment, but not that of the umuC gene. In fact expression of the umuC gene was induced by lower concentrations of MNNG in the presence of o-vanillin. The results suggest that o-vanillin inhibits induction of the adaptive response, and consequently, the MNNG-induced mutation frequency is increased due to unrepaired O 6-methylguanine.

A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate.

Seven mutants of Escherichia coli were isolated that are sensitive to methyl methane sulfonate but not to UV light. They exhibited decreased host cell reactivation capacity for methyl methane sulfonate-treated phage lambda. Five of the mutations were mapped in the same region as alkA (previously called alk) and may indeed be identical to known mutations. Another mutation was found near nalA, and the gene responsible was named alkB. Its phenotype was different from that of ada, since the alkB mutant exhibited a normal adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine. A third type of mutation was mapped near polA, but this mutant contained an almost normal level of DNA polymerase I activity.