Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins

The Escherichia coli AlkB protein repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions in DNA and RNA by oxidative demethylation, a reaction requiring ferrous iron and 2-oxoglutarate as cofactor and co-substrate, respectively. Here, we have studied the activity of AlkB proteins on 3-methylthymine (3-meT) and 1-methylguanine (1-meG), two minor lesions which are structurally analogous to 1-meA and 3-meC. AlkB as well as the human AlkB homologues, hABH2 and hABH3, were all able to demethylate 3-meT in a DNA oligonucleotide containing a single 3-meT residue. Also, 1-meG lesions introduced by chemical methylation of tRNA were efficiently removed by AlkB. Unlike 1-meA and 3-meC, nucleosides or bases corresponding to 1-meG or 3-meT did not stimulate the uncoupled, AlkB-mediated decarboxylation of 2-oxoglutarate. Our data show that 3-meT and 1-meG are repaired by AlkB, but indicate that the recognition of these substrates is different from that in the case of 1-meA and 3-meC.     

Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO

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), alpha-ketoglutarate (alpha-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 single-stranded DNA (ssDNA) and 3-methyluracil (3-meU) in single-stranded 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.     

A nonradioactive restriction enzyme-mediated assay to detect DNA repair by Fe(II)/2-oxoglutarate-dependent dioxygenase

The Escherichia coli DNA repair enzyme AlkB belongs to the Fe(II)/2-oxoglutarate-dependent dioxygenase family. It removes methyl groups from 1-methyl adenine (1-meA) and 3-methyl cytosine (3-meC) lesions present in single-stranded DNA by oxidative decarboxylation. In the current article, we describe an in vitro assay that permits rapid detection of AlkB activity. To achieve this, we generated methylated oligonucleotide using methyl methanesulfonate and then monitored DNA repair using a methylation-sensitive restriction enzyme and novel agarose gel electrophoresis system capable of resolving small oligonucleotides. Our approach overcomes several drawbacks of NAD⁺-dependent formaldehyde dehydrogenase-coupled assay and radioisotope-based assay for determining AlkB DNA repair activity.     

Human AlkB homolog 1 is a mitochondrial protein that demethylates 3-methylcytosine in DNA and RNA

The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA.     

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N⁶-ethenoadenine (ɛA), 3,N⁴-ethenocytosine (ɛC) and 1,N²-ethenoguanine (1,N²-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate.Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N²-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N²-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.     

Interaction of human and bacterial AlkB proteins with DNA as probed through chemical cross-linking studies

The Escherichia coli AlkB protein was recently found to repair cytotoxic DNA lesions 1-methyladenine and 3-methylcytosine by using a novel iron-catalyzed oxidative demethylation mechanism. Three human homologs, ABH1, ABH2 and ABH3, have been identified, and two of them, ABH2 and ABH3, were shown to have similar repair activities to E.coli AlkB. However, ABH1 did not show any repair activity. It was suggested that ABH3 prefers single-stranded DNA and RNA substrates, whereas AlkB and ABH2 can repair damage in both single- and double-stranded DNA. We employed a chemical cross-linking approach to probe the structure and substrate preferences of AlkB and its three human homologs. The putative active site iron ligands in these proteins were mutated to cysteine residues. These mutant proteins were used to cross-link to different DNA probes bearing thiol-tethered bases. Disulfide-linked protein–DNA complexes can be trapped and analyzed by SDS–PAGE. Our results show that ABH2 and ABH3 have structural and functional similarities to E.coli AlkB. ABH3 shows preference for the single-stranded DNA probe. ABH1 failed to cross-link to the probes tested. This protein, unlike other AlkB proteins, does not seem to interact with DNA in its E.coli expressed form.     

Fanconi anemia complementation group A (FANCA) protein has intrinsic affinity for nucleic acids with preference for single-stranded forms

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ～60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ～30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5'-flap or 5'-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772-1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found.     

A real-time PCR-based quantitative assay for 3-methylcytosine demethylase activity of ALKBH3

Human AlkB homolog 3 (ALKBH3), a homolog of the Escherichia coli protein AlkB, demethylates 1-methyladenine and 3-methylcytosine (3-meC) in single-stranded DNA and RNA by oxidative demethylation. Immunohistochemical analyses on clinical cancer specimens and knockdown experiments using RNA interference in vitro and in vivo indicate that ALKBH3 is a promising molecular target for the treatment of prostate, pancreatic, and non-small cell lung cancer. Therefore, an inhibitor for ALKBH3 demethylase is expected to be a first-in-class molecular-targeted drug for cancer treatment. Here, we report the development of a novel, quantitative real-time PCR-based assay for ALKBH3 demethylase activity against 3-meC by highly active recombinant ALKBH3 protein using a silkworm expression system. This assay enables us to screen for inhibitors of ALKBH3 demethylase, which may result in the development of a novel molecular-targeted drug for cancer therapy.     

Alkylation damage in DNA and RNA--repair mechanisms and medical significance

Alkylation lesions in DNA and RNA result from endogenous compounds, environmental agents and alkylating drugs. Simple methylating agents, e.g. methylnitrosourea, tobacco-specific nitrosamines and drugs like temozolomide or streptozotocin, form adducts at N- and O-atoms in DNA bases. These lesions are mainly repaired by direct base repair, base excision repair, and to some extent by nucleotide excision repair (NER). The identified carcinogenicity of O(6)-methylguanine (O(6)-meG) is largely caused by its miscoding properties. Mutations from this lesion are prevented by O(6)-alkylG-DNA alkyltransferase (MGMT or AGT) that repairs the base in one step. However, the genotoxicity and cytotoxicity of O(6)-meG is mainly due to recognition of O(6)-meG/T (or C) mispairs by the mismatch repair system (MMR) and induction of futile repair cycles, eventually resulting in cytotoxic double-strand breaks. Therefore, inactivation of the MMR system in an AGT-defective background causes resistance to the killing effects of O(6)-alkylating agents, but not to the mutagenic effect. Bifunctional alkylating agents, such as chlorambucil or carmustine (BCNU), are commonly used anti-cancer drugs. DNA lesions caused by these agents are complex and require complex repair mechanisms. Thus, primary chloroethyl adducts at O(6)-G are repaired by AGT, while the secondary highly cytotoxic interstrand cross-links (ICLs) require nucleotide excision repair factors (e.g. XPF-ERCC1) for incision and homologous recombination to complete repair. Recently, Escherichia coli protein AlkB and human homologues were shown to be oxidative demethylases that repair cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues. Numerous AlkB homologues are found in viruses, bacteria and eukaryotes, including eight human homologues (hABH1-8). These have distinct locations in subcellular compartments and their functions are only starting to become understood. Surprisingly, AlkB and hABH3 also repair RNA. An evaluation of the biological effects of environmental mutagens, as well as understanding the mechanism of action and resistance to alkylating drugs require a detailed understanding of DNA repair processes.     

Hybridizing ability and nuclease resistance profile of backbone modified cationic phosphorothioate oligonucleotides

Various stereochemically pure cationic phosphorothioate oligonucleotides bearing aminoalkyl moieties were synthesized, and their duplex-forming ability against single-stranded DNA (ssDNA), single-stranded RNA (ssRNA) and triplex-forming ability against double-stranded DNA (dsDNA) were evaluated by UV melting experiments. The cationic Rp stereoisomers showed improved duplex-forming ability against ssDNA, triplex-forming ability against dsDNA and nuclease stability.     

Methylation damage to RNA induced in vivo in Escherichia coli is repaired by endogenous AlkB as part of the adaptive response

Cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions induced in DNA and RNA in vitro and in pre-damaged DNA and RNA bacteriophages in vivo are repaired by the Escherichia coli (E. coli) protein AlkB and a human homolog, ALKBH3. However, it is not known whether endogenous RNA is repaired in vivo by repair proteins present at physiological concentrations. The concept of RNA repair as a biologically relevant process has therefore remained elusive. Here, we demonstrate AlkB-mediated repair of endogenous RNA in vivo by measuring differences in lesion-accumulation in two independent AlkB-proficient and deficient E. coli strains during exposure to methyl methanesulfonate (MMS). Repair was observed both in AlkB-overproducing strains and in the wild-type strains after AlkB induction. RNA repair appeared to be highest in RNA species below 200 nucleotides in size, mainly comprising tRNAs. Strikingly, at least 10-fold more lesions were repaired in RNA than in DNA. This may be a consequence of some 30-fold higher levels of aberrant methylation in RNA than in DNA after exposure to MMS. A high primary kinetic isotope effect (>10) was measured using a deuterated methylated RNA substrate, D₃-1me(rA), demonstrating that it is the catalytic step, and not the search step that is rate-limiting. Our results demonstrate that RNA repair by AlkB takes place in endogenous RNA as part of an adaptive response in wild-type E. coli cells.     

Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

Background

In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question.

Methodology/Principal Findings

We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate.

Conclusions/Significance

A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a “searching” mode and “repair” mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.     

The DNase activity of RNase T and its application to DNA cloning.

RNase T is one of eight distinct 3'-->5' exoribonucleases present in Escherichia coli. The enzyme plays an important role in stable RNA metabolism, including tRNA end turnover and 3' maturation of most stable RNAs because it is the only RNase that can efficiently remove residues near a double-stranded (ds) stem. In the course of study of its specificity and mechanism, we found that RNase T also has single-strand-specific DNase activity. Purified RNase T degrades both single-stranded (ss)RNA and ssDNA in a non-processive manner. However, in contrast to its action on RNA, RNase T binds ssDNA much more tightly and shows less sequence specificity. As with RNA, DNA secondary structure strongly affects its degradation by RNase T. Thus, RNase T action on a dsDNA with a single-stranded 3'-extension efficiently generates blunt-ended DNA. This property of RNase T suggested that it might be a useful enzyme for blunt-ended DNA cloning. We show here that RNase T provides much higher cloning efficiency than the currently used mung bean nuclease.     

Recognition and processing of double-stranded DNA by ExoX,a distributive 3′–5′ exonuclease

Members of the DnaQ superfamily are major 3′–5′ exonucleases that degrade either only single-stranded DNA (ssDNA) or both ssDNA and double-stranded DNA (dsDNA). However, the mechanism by which dsDNA is recognized and digested remains unclear. Exonuclease X (ExoX) is a distributive DnaQ exonuclease that cleaves both ssDNA and dsDNA substrates. Here, we report the crystal structures of Escherichia coli ExoX in complex with three different dsDNA substrates: 3′ overhanging dsDNA, blunt-ended dsDNA and 3′ recessed mismatch-containing dsDNA. In these structures, ExoX binds to dsDNA via both a conserved substrate strand-interacting site and a previously uncharacterized complementary strand-interacting motif. When ExoX complexes with blunt-ended dsDNA or 5′ overhanging dsDNA, a ‘wedge’ composed of Leu12 and Gln13 penetrates between the first two base pairs to break the 3′ terminal base pair and facilitates precise feeding of the 3′ terminus of the substrate strand into the ExoX cleavage active site. Site-directed mutagenesis showed that the complementary strand-binding site and the wedge of ExoX are dsDNA specific. Together with the results of structural comparisons, our data support a mechanism by which normal and mismatched dsDNA are recognized and digested by E. coli ExoX. The crystal structures also provide insight into the structural framework of the different substrate specificities of the DnaQ family members.     

Mechanisms for RNA Capture by ssDNA Viruses: Grand Theft RNA

Viruses contain three common types of packaged genomes; double-stranded DNA (dsDNA), RNA (mostly single and occasionally double stranded) and single-stranded DNA (ssDNA). There are relatively straightforward explanations for the prevalence of viruses with dsDNA and RNA genomes, but the evolutionary basis for the apparent success of ssDNA viruses is less clear. The recent discovery of four ssDNA virus genomes that appear to have been formed by recombination between co-infecting RNA and ssDNA viruses, together with the high mutation rate of ssDNA viruses provide possible explanations. RNA–DNA recombination allows ssDNA viruses to access much broader sequence space than through nucleotide substitution and DNA–DNA recombination alone. Multiple non-exclusive mechanisms, all due to the unique replication of ssDNA viruses, are proposed for this unusual RNA capture. RNA capture provides an explanation for the evolutionary success of the ssDNA viruses and may help elucidate the mystery of integrated RNA viruses in viral and cellular DNA genomes.     

Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage

Methylating agents are ubiquitous in the environment, and central in cancer therapy. The 1-methyladenine and 3-methylcytosine lesions in DNA/RNA contribute to the cytotoxicity of such agents. These lesions are directly reversed by ABH3 (hABH3) in humans and AlkB in Escherichia coli. Here, we report the structure of the hABH3 catalytic core in complex with iron and 2-oxoglutarate (2OG) at 1.5 A resolution and analyse key site-directed mutants. The hABH3 structure reveals the beta-strand jelly-roll fold that coordinates a catalytically active iron centre by a conserved His1-X-Asp/Glu-X(n)-His2 motif. This experimentally establishes hABH3 as a structural member of the Fe(II)/2OG-dependent dioxygenase superfamily, which couples substrate oxidation to conversion of 2OG into succinate and CO2. A positively charged DNA/RNA binding groove indicates a distinct nucleic acid binding conformation different from that predicted in the AlkB structure with three nucleotides. These results uncover previously unassigned key catalytic residues, identify a flexible hairpin involved in nucleotide flipping and ss/ds-DNA discrimination, and reveal self-hydroxylation of an active site leucine that may protect against uncoupled generation of dangerous oxygen radicals.     

Preferential binding of Escherichia coli RecF protein to gapped DNA in the presence of adenosine (gamma-thio) triphosphate.

Escherichia coli RecF protein binds, but does not hydrolyze, ATP. To determine the role that ATP binding to RecF plays in RecF protein-mediated DNA binding, we have determined the interaction between RecF protein and single-stranded (ss)DNA, double-stranded (ds)DNA, and dsDNA containing ssDNA regions (gapped [g]DNA) either alone or in various combinations both in the presence and in the absence of adenosine (gamma-thio) triphosphate, gamma-S-ATP, a nonhydrolyzable ATP analog. Protein-DNA complexes were analyzed by electrophoresis on agarose gels and visualized by autoradiography. The type of protein-DNA complexes formed in the presence of gamma-S-ATP was different with each of the DNA substrates and from those formed in the absence of gamma-S-ATP. Competition experiments with various combinations of DNA substrates indicated that RecF protein preferentially bound gDNA in the presence of gamma-S-ATP, and the order of preference of binding was gDNA > dsDNA > ssDNA. Since gDNA has both ds- and ssDNA components, we suggest that the role for ATP in RecF protein-DNA interactions in vivo is to confer specificity of binding to dsDNA-ssDNA junctions, which is necessary for catalyzing DNA repair and recombination.     

Synthesis and properties of 3'-amino-2',4'-BNA, a bridged nucleic acid with a N3'-->P5' phosphoramidate linkage

The synthesis and properties of a bridged nucleic acid analogue containing a N3'-->P5' phosphoramidate linkage, 3'-amino-2',4'-BNA, is described. A heterodimer containing a 3'-amino-2',4'-BNA thymine monomer, and thymine and methylcytosine monomers of 3'-amino-2',4'-BNA and their 5'-phosphoramidites, were synthesized efficiently. The dimer and monomers were incorporated into oligonucleotides by conventional 3'-->5' assembly, and 5'-->3' reverse assembly phosphoramidite protocols, respectively. Compared to a natural DNA oligonucleotide, modified oligonucleotides containing the 3'-amino-2',4'-BNA residue formed highly stable duplexes and triplexes with single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded DNA (dsDNA) targets, with the average increase in melting temperature (T(m)) against ssDNA, ssRNA and dsDNA being +2.7 to +4.0 degrees C, +5.0 to +7.0 degrees C, and +5.0 to +11.0 degrees C, respectively. These increases are comparable to those observed for 2',4'-BNA-modified oligonucleotides. In addition, an oligonucleotide modified with a single 3'-amino-2',4'-BNA thymine residue showed extraordinarily high resistance to nuclease degradation, much higher than that of 2',4'-BNA and substantially higher even than that of 3'-amino-DNA and phosphorothioate oligonucleotides. The above properties indicate that 3'-amino-2',4'-BNA has significant potential for antisense and antigene applications.     

Methods for assessing DNA hybridization of peptide nucleic acid-titanium dioxide nanoconjugates

We describe the synthesis of peptide nucleic acid (PNA)-titanium dioxide (TiO₂) nanoconjugates and several novel methods developed to investigate the DNA hybridization behaviors of these constructs. PNAs are synthetic DNA analogs resistant to degradation by cellular enzymes that hybridize to single-stranded DNA (ssDNA) with higher affinity than DNA oligonucleotides, invade double-stranded DNA (dsDNA), and form different PNA/DNA complexes. Previously, we developed a DNA-TiO₂ nanoconjugate capable of hybridizing to target DNA intracellularly in a sequence-specific manner with the ability to cleave DNA when excited by electromagnetic radiation but susceptible to degradation that may lower its intracellular targeting efficiency and retention time. PNA-TiO₂ nanoconjugates described in the current article hybridize to target ssDNA, oligonucleotide dsDNA, and supercoiled plasmid DNA under physiological-like ionic and temperature conditions, enabling rapid, inexpensive, sequence-specific concentration of nucleic acids in vitro. When modified by the addition of imaging agents or peptides, hybridization capabilities of PNA-TiO₂ nanoconjugates are enhanced, providing essential benefits for numerous in vitro and in vivo applications. The series of experiments shown here could not be done with either TiO₂-DNA nanoconjugates or PNAs alone, and the novel methods developed will benefit studies of numerous other nanoconjugate systems.