RNA Adenine Research Articles

Two dynamic, N-terminal regions are required for function in Ribosomal RNA Adenine Dimethylase family members.

Prominent members of the Ribosomal RNA Adenine Dimethylase (RRAD) family of enzymes facilitate ribosome maturation by dimethylating two nucleotides of small subunit rRNA including the human DIMT1 and bacterial KsgA enzymes. A sub-group of RRAD enzymes, named erythromycin resistance methyltransferases (Erm) dimethylate a specific nucleotide in large subunit rRNA to confer antibiotic resistance. How these enzymes regulate methylation so that it only occurs on the specific substrate is not fully understood. While performing random mutagenesis on the catalytic domain of ErmE, we discovered that mutants in an N-terminal region of the protein that is disordered in the ErmE crystal structure are associated with a loss of antibiotic resistance. By subjecting site-directed mutants of ErmE and KsgA to phenotypic and in vitro assays we found that the N-terminal region is critical for activity in RRAD enzymes: the N-terminal basic region promotes rRNA binding and the conserved motif likely assists in juxtaposing the adenosine substrate and the SAM cofactor. Our results and emerging structural data suggest this dynamic, N-terminal region of RRAD enzymes becomes ordered upon rRNA binding forming a cap on the active site required for methylation.

YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation

Prostate cancer is a leading cause of cancer-related mortality in males. Dysregulation of RNA Adenine N-6 methylation (m6A) contributes to cancer malignancy. m6A on mRNA may affect mRNA splicing, turnover, transportation and translation. m6A exerts these effects, at least partly, through dedicated m6A reader proteins, including YTHDF2. YTHDF2 is necessary for development while its dysregulation is seen in various cancers including prostate cancer. However, the mechanism underlying the dysregulation and function of YTHDF2 in cancer remains elusive. Here, we find that the deubiquitinase OTUB1 increases YTHDF2 protein stability by inhibiting its ubiquitination. With in vivo and in vitro ubiquitination assays, OTUB1 is shown to block ubiquitin transfer to YTHDF2 independent of its deubiquitinase activity. Furthermore, analysis of functional transcriptomic data and m6A-sequencing data identifies PRSS8 as a potential tumor suppressor gene. OTUB1 and YTHDF2 decrease mRNA and protein levels of PRSS8, which is a trypsin-like serine protease. Mechanistically, YTHDF2 binds PRSS8 mRNA and promotes its degradation in an m6A-dependent manner. Further functional study on cellular and mouse models reveals PRSS8 is a critical downstream effector of the OTUB1-YTHDF2 axis in prostate cancer. We find in prostate cancer cells, PRSS8 decreases nuclear β-catenin level through E-cadherin, which is independent of its protease activity. Collectively, our study uncovers a key regulator of YTHDF2 protein stability and establishes a functional OTUB1-YTHDF2-PRSS8 axis in prostate cancer.

Intracellular RNA Labeling Technologies for the Analysis of RNA Biology†

Comprehensive SummaryAs a cornerstone of the central dogma of molecular biology, RNA plays vital roles in living organisms. Over the past few decades, many RNA labeling technologies have been developed to elucidate the biological function of RNA. These technologies have significantly advanced our understanding of RNA secondary structure, localization, and turnover. Additionally, taking advantage of these innovative RNA labeling approaches, plenty of tool kits have been devised for the regulation of RNA‐related biological process, such as gene expression and gene editing. In this review, we primarily focus on an array of intracellular RNA labeling methods, encompassing chemical probes‐based labeling, metabolic labeling, and proximity‐dependent labeling. We also provide a brief overview of their applications in the research of RNA biology. Finally, the perspectives of RNA labeling are also discussed.Key Scientists4SU was first used in1978 as a safer way to directly examine RNA synthesis. However, the enrichment of 4SU‐labeled RNA seriously relied on mercurated cellulose columns, which are toxic. In 2005, Boothroyd et al. introduced a novel covalent‐modification method of 4SU based on thiol exchange reaction. Then, Salic et al. applied 5EU, which is clickable, for RNA metabolic labeling in animals. Later, Spitale et al. designed 5EC and 2’AzU, which enabled cell specific RNA metabolic labeling under the help of specific enzyme. In 2013, directly chemical RNA labeling in living cell for RNA structure mapping was realized by Chang and Kool et al. using SHAPE reagent, which was further modified with an azide handle in 2015. Weissman, Bevilacqua and Assmann et al. introduced DMS probe that targets adenine and cytosine of RNA for RNA structure mapping in 2014. Then, Chang et al. exploited psoralen for RNA duplex mapping in 2016. Later, He, Zhou and Zhang et al. described another clickable probe N3‐kethoxal targeting guanine for RNA secondary structure mapping. Besides, Chang and Ting et al. described a proximity‐dependent RNA labeling method based on APEX in 2019, revealing subcellular RNA localization. Meanwhile, Zou and Wang et al. achieved proximity‐dependent RNA labeling using miniSOG oxidase, which provided an alternative method to investigate the spatial transcriptome. Spitale et al. utilizing photosensitizer dibromofluorescein (DBF) to realize proximity‐dependent RNA labeling and sequencing.

Emerging Roles for DNA 6mA and RNA m6A Methylation in Mammalian Genome.

Epigenetic methylation has been shown to play an important role in transcriptional regulation and disease pathogenesis. Recent advancements in detection techniques have identified DNA N6-methyldeoxyadenosine (6mA) and RNA N6-methyladenosine (m6A) as methylation modifications at the sixth position of adenine in DNA and RNA, respectively. While the distributions and functions of 6mA and m6A have been extensively studied in prokaryotes, their roles in the mammalian brain, where they are enriched, are still not fully understood. In this review, we provide a comprehensive summary of the current research progress on 6mA and m6A, as well as their associated writers, erasers, and readers at both DNA and RNA levels. Specifically, we focus on the potential roles of 6mA and m6A in the fundamental biological pathways of the mammalian genome and highlight the significant regulatory functions of 6mA in neurodegenerative diseases.

Demethylase-assisted site-specific detection of N1-methyladenosine in RNA

The dynamic RNA modifications have been viewed as new posttranscriptional regulator in modulating gene expression as well as in a broad range of physiological processes. N1-methyladenosine (m1A) is one of the most prevalent modifications existing in multiple types of RNAs. In-depth investigation of the functions of m1A requires the site-specific assessment of m1A stoichiometry in RNA. Herein, we established a demethylase-assisted method (DA-m1A) for the site-specific detection and quantification of m1A in RNA. N1-methyl group in m1A could result in the stalling of reverse transcription at m1A site, thus producing the truncated cDNA. E. coli AlkB is a demethylase that can demethylate m1A to produce adenine in RNA, thus generating full-length cDNA from AlkB-treated RNA. Evaluation of the produced amounts of full-length cDNA by quantitative real-time PCR can achieve the site-specific detection and quantification of m1A in RNA. With the DA-m1A method, we examined and successfully confirmed the previously well-characterized m1A sites in various types of RNAs with low false positive rate. In addition, we found that the level of m1A was significantly decreased at the bromodomain containing 2 (BRD2) mRNA position 1674 and CST telomere replication complex component 1 (CTC1) mRNA position 5643 in human hepatocellular carcinoma tissues. The results suggest that these two m1A sites in mRNA may be involved in liver tumorigenesis. Taken together, the DA-m1A method is simple and enables the rapid, cost-effective, and site-specific detection and quantification of m1A in RNA, which provides a valuable tool to decipher the functions of m1A in human diseases.

Targeting IGF2BP3 in Cancer.

RNA-binding proteins (RBPs) can regulate multiple pathways by binding to RNAs, playing a variety of functions, such as localization, stability, and immunity. In recent years, with the development of technology, researchers have discovered that RBPs play a key role in the N6-methyladenosine (m6A) modification process. M6A methylation is the most abundant form of RNA modification in eukaryotes, which is defined as methylation on the sixth N atom of adenine in RNA. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is one of the components of m6A binding proteins, which plays an important role in decoding m6A marks and performing various biological functions. IGF2BP3 is abnormally expressed in many human cancers, often associated with poor prognosis. Here, we summarize the physiological role of IGF2BP3 in organisms and describe its role and mechanism in tumors. These data suggest that IGF2BP3 may be a valuable therapeutic target and prognostic marker in the future.

Transcriptome-wide analysis of RNA m6A methylation regulation of muscle development in Queshan Black pigs

BackgroundN6-methyladenosine (m6A) refers to the methylation modification of N6 position of RNA adenine, a dynamic reversible RNA epigenetic modification that plays an important regulatory role in a variety of life processes. In this study, we used MeRIP-Seq and RNA-Seq of the longissimus dorsi (LD) muscle of adult (QA) and newborn (QN) Queshan Black pigs to screen key genes with m6A modification involved in muscle growth by bioinformatics analysis.ResultsA total of 23,445 and 25,465 m6A peaks were found in the whole genomes of QA and QN, respectively. Among them, 613 methylation peaks were significantly different (DMPs) and 579 genes were defined as differentially methylated genes (DMGs). Compared with the QN group, there were 1,874 significantly differentially expressed genes (DEGs) in QA group, including 620 up-regulated and 1,254 down-regulated genes. In order to investigate the relationship between m6A and mRNA expression in the muscle of Queshan Black pigs at different periods, a combined analysis of MeRIP-Seq and RNA-Seq showed that 88 genes were significantly different at both levels. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes results showed that DEGs and DMGs were mainly involved in skeletal muscle tissue development, FoxO signaling pathway, MAPK signaling pathway, insulin signaling pathway, PI3K–Akt signaling pathway, and Wnt signaling pathway. Four DEGs (IGF1R, CCND2, MYOD1 and FOS) and four DMGs (CCND2, PHKB, BIN1 and FUT2), which are closely related to skeletal muscle development, were selected as candidate genes for verification, and the results were consistent with the sequencing results, which indicated the reliability of the sequencing results.ConclusionsThese results lay the foundation for understanding the specific regulatory mechanisms of growth in Queshan Black pigs, and provide theoretical references for further research on the role of m6A in muscle development and breed optimization selection.

RNA Editing Enzyme ADAR1 Suppresses the Mobility of Cancer Cells via ARPIN.

Deamination of adenine or cytosine in RNA, called RNA editing, is a constitutively active and common modification. The primary role of RNA editing is tagging RNA right after its synthesis so that the endogenous RNA is recognized as self and distinguished from exogenous RNA, such as viral RNA. In addition to this primary function, the direct or indirect effects on gene expression can be utilized in cancer where a high level of RNA editing activity persists. This report identified actin-related protein 2/3 complex inhibitor (ARPIN) as a target of ADAR1 in breast cancer cells. Our comparative RNA sequencing analysis in MCF7 cells revealed that the expression of ARPIN was decreased upon ADAR1 depletion with altered editing on its 3'UTR. However, the expression changes of ARPIN were not dependent on 3'UTR editing but relied on three microRNAs acting on ARPIN. As a result, we found that the migration and invasion of cancer cells were profoundly increased by ADAR1 depletion, and this cellular phenotype was reversed by the exogenous ARPIN expression. Altogether, our data suggest that ADAR1 suppresses breast cancer cell mobility via the upregulation of ARPIN.

Mini-dCas13X-mediated RNA editing restores dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy.

Approximately 10% of monogenic diseases are caused by nonsense point mutations that generate premature termination codons (PTCs), resulting in a truncated protein and nonsense-mediated decay of the mutant mRNAs. Here, we demonstrate a mini-dCas13X-mediated RNA adenine base editing (mxABE) strategy to treat nonsense mutation-related monogenic diseases via A-to-G editing in a genetically humanized mouse model of Duchenne muscular dystrophy (DMD). Initially, we identified a nonsense point mutation (c.4174C>T, p.Gln1392*) in the DMD gene of a patient and validated its pathogenicity in humanized mice. In this model, mxABE packaged in a single adeno-associated virus (AAV) reached A-to-G editing rates up to 84% in vivo, at least 20-fold greater than rates reported in previous studies using other RNA editing modalities. Furthermore, mxABE restored robust expression of dystrophin protein to over 50% of WT levels by enabling PTC read-through in multiple muscle tissues. Importantly, systemic delivery of mxABE by AAV also rescued dystrophin expression to averages of 37%, 6%, and 54% of WT levels in the diaphragm, tibialis anterior, and heart muscle, respectively, as well as rescued muscle function. Our data strongly suggest that mxABE-based strategies may be a viable new treatment modality for DMD and other monogenic diseases.

Meteorites and the RNA World: Synthesis of Nucleobases in Carbonaceous Planetesimals and the Role of Initial Volatile Content

Prebiotic molecules, fundamental building blocks for the origin of life, have been found in carbonaceous chondrites. The exogenous delivery of these organic molecules onto the Hadean Earth could have sparked the polymerization of the first RNA molecules in Darwinian ponds during wet-dry cycles. Here, we investigate the formation of the RNA and DNA nucleobases adenine, uracil, cytosine, guanine, and thymine inside parent body planetesimals of carbonaceous chondrites. An up-to-date thermochemical equilibrium model coupled with a 1D thermodynamic planetesimal model is used to calculate the nucleobase concentrations. Different from previous studies, we assume the initial volatile concentrations more appropriate for the formation zone of carbonaceous chondrite parent bodies. This represents more accurately cosmochemical findings that these bodies have formed inside the inner, ∼2–5 au, warm region of the solar system. Due to these improvements, our model represents the concentrations of adenine and guanine measured in carbonaceous chondrites. Our model did not reproduce per se the measurements of uracil, cytosine, and thymine in these meteorites. This can be explained by transformation reactions between nucleobases and the potential decomposition of thymine. The synthesis of prebiotic organic matter in carbonaceous asteroids could be well explained by a combination of (i) radiogenic heating, (ii) aqueous chemistry involving a few key processes at a specific range of radii inside planetesimals where water can exist in the liquid phase, and (iii) a reduced initial volatile content (H2, CO, , and CH2O) of the protoplanetary disk material in the parent body region compared to the outer region of comets.

Abstract 2306: Two dynamic, N-terminal regions are required for function in Ribosomal RNA Adenine Dimethylase family members

Abstract 2306: Two dynamic, N-terminal regions are required for function in Ribosomal RNA Adenine Dimethylase family members

Systematic Design of Adenosine Analogs as Inhibitors of a Clostridioides difficile-Specific DNA Adenine Methyltransferase Required for Normal Sporulation and Persistence.

Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which makes the design of selective inhibitors more challenging. We explored the solvent-exposed edge of the SAM adenosine moiety and systematically designed 42 analogs of adenosine carrying substituents at the C6-amino group (N6) of adenosine. We compare the inhibitory properties and binding affinity of these diverse compounds and present the crystal structures of CamA in complex with 14 of them in the presence of substrate DNA. The most potent of these inhibitors, compound 39 (IC50 ∼ 0.4 μM and KD ∼ 0.2 μM), is selective for CamA against closely related bacterial and mammalian DNA and RNA adenine methyltransferases, protein lysine and arginine methyltransferases, and human adenosine receptors.

Interaction of 5-fluorouracil anticancer drug with nucleobases: insight from DFT, TD-DFT, and AIM calculations

In this study, the interaction of 5-fluorouracil (5FU) drug with adenine (A), guanine(G), cytosine(C), uracil (U), and thymine (T) nucleobases of DNA and RNA are surveyed at the ωB97XD/LANL2DZ, M06-2X/6-31G (d, p), MPW1PWQ1/6-31G(d, p), PBEPBE/6-31(d, p) and ωB97XD/6-31G(d, p) levels of density functional theory (DFT). The considered complexes of 5FU drug with nucleobases are optimized at the above level of theories. Max Force and RMS of optimization criteria are 0.00035 (Ha), and 0.0003 respectively. From optimized structures, the adsorption energy, thermodynamic parameters in gas and solvent media, quantum theory atom in molecule (QTAIM), electron localized function (ELF), and reduced density gradient (RDG) are calculated at ωB97XD/LANL2DZ and M06-2X/6-31G (d, p) level of DFT theory. The QTAIM, ELF, and RDG results confirm that the nature of bonding between 5FU drug with A, C, G, U, and T nucleobases is electrostatic or hydrogen bond type. The adsorption and thermodynamic energy results demonstrate that the interaction of the 5FU drug with C and G nucleobases is stronger than other nucleobases. The results of this study can be suggested the mechanism of interaction of the 5FU drug with nucleobases of DNA and RNA. Communicated by Ramaswamy H. Sarma

Population Genetic Structure and Diversity of Metaphire remanens (Oligochaeta: Megascolecidae) Based on Mitochondrial DNA Analysis, with a Note on a New Species of Metaphire remanens sp. nov.

Metaphire remanens sp. nov. is widely distributed throughout Hunan Province, China. We sequenced the mitochondrial DNA to investigate its population genetic structure and genetic diversity, including cytochrome c oxidase subunit I, cytochrome c oxidase subunit II, 12S ribosomal (r)RNA, 16S rRNA, and nicotinamide adenine dinucleotide dehydrogenase subunit 1, derived from 39 individuals from seven geographic locations in Hunan Province. The genetic diversity indices showed that populations of M. remanens have a strong genetic structure and obvious dispersal histories. M. remanens did not experience population expansion, except in Xiangtan City. This may be because of its evolution toward parthenogenesis. The divergence time estimates indicated that M. remanens originated at 19.2055 Ma and then generated two main lineages at 1.7334 Ma (Quaternary glaciation). These results indicate that glaciation, geographic isolation, and dispersal ability are significant factors that influence the differentiation and dispersal of M. remanens. In this study, we describe Metaphire remanens sp. nov. in morphology.

Global Landscape of m6A Methylation of Differently Expressed Genes in Muscle Tissue of Liaoyu White Cattle and Simmental Cattle.

Liaoyu white cattle (LYWC) is a local breed in Liaoning Province, China. It has the advantages of grow quickly, high slaughter ratew, high meat quality and strong anti-stress ability. N6 methyladenosine (m6A) is a methylation modification of N6 position of RNA adenine, which is an important modification mechanism affecting physiological phenomena. In this study, we used the longissimus dorsi muscle of LYWC and SIMC for m6A-seq and RNA-seq high-throughput sequencing, and identified the key genes involved in muscle growth and m6A modification development by bioinformatics analysis. There were 31532 m6A peaks in the whole genome of LYWC and 47217 m6A peaks in the whole genome of SIMC. Compared with Simmental cattle group, LYWC group had 17,351 differentially expressed genes: 10,697 genes were up-regulated, 6,654 genes were down regulated, 620 differentially expressed genes were significant, while 16,731 differentially expressed genes were not significant. Among the 620 significantly differentially expressed genes, 295 genes were up-regulated and 325 genes were down regulated. In order to explore the relationship between m6A and mRNA expression in the muscles of LYWC and SIMC, the combined analysis of MeRIP-seq and RNA-seq revealed that 316 genes were m6A modified with mRNA expression. To identify differentially methylated genes related to muscle growth, four related genes were selected for quantitative verification in LYWC and SIMC. GO enrichment and KEGG analysis showed that the differentially expressed genes modified by m6A are mainly involved in skeletal muscle contraction, steroid biosynthesis process, redox process, PPAR pathway and fatty acid metabolism, and galactose metabolism. These results provide a theoretical basis for further research on the role of m6A in muscle growth and development.

In silico Analysis of Ribosome-Inactivating Protein (Tritin) from Common Wheat Plants (Triticum aestivum L.)

Ribosome-inactivating proteins (RIPs) are one of the enzymes that inhibit protein synthesis after depurination of a specific adenine in ribosomal RNA. The tritin is one of type I RIPs that include RNA-N glycosidase domain from RIP family. In the present study, cDNA encoding tritin from leaves of wheat Kutluk-94 cultivar was isolated and cloned into pGEM-T Easy vector. The recombinant plasmid was sequenced. The different bioinformatics tools were used for assessment of tritin protein characteristics. A total of 38 tritin-like sequences were identified in some monocot plants. Results showed that tritin protein have conserved domain (Ricin-A) found in other RIPs associated with RNA N-glycosidase activity and shows chancing homology to the RIPs in other plant species. According to multiple sequence alignment, tritin has conserved amino acids which are crucial role in RNA N-glycosidase activity. Our study illustrates that results obtained from in silico analyses could provide a perspective to another researcher about molecular and structural properties of tritin protein.

Three critical regions of the erythromycin resistance methyltransferase, ErmE, are required for function supporting a model for the interaction of Erm family enzymes with substrate rRNA.

6-Methyladenosine modification of DNA and RNA is widespread throughout the three domains of life and often accomplished by a Rossmann-fold methyltransferase domain which contains conserved sequence elements directing S-adenosylmethionine cofactor binding and placement of the target adenosine residue into the active site. Elaborations to the conserved Rossman-fold and appended domains direct methylation to diverse DNA and RNA sequences and structures. Recently, the first atomic-resolution structure of a ribosomal RNA adenine dimethylase (RRAD) family member bound to rRNA was solved, TFB1M bound to helix 45 of 12S rRNA. Since erythromycin resistance methyltransferases are also members of the RRAD family, and understanding how these enzymes recognize rRNA could be used to combat their role in antibiotic resistance, we constructed a model of ErmE bound to a 23S rRNA fragment based on the TFB1M-rRNA structure. We designed site-directed mutants of ErmE based on this model and assayed the mutants by in vivo phenotypic assays and in vitro assays with purified protein. Our results and additional bioinformatic analyses suggest our structural model captures key ErmE-rRNA interactions and indicate three regions of Erm proteins play a critical role in methylation: the target adenosine binding pocket, the basic ridge, and the α4-cleft.

Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA.

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3' to inosine in single-strand RNA, at a low reaction temperature of 20-25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.

Transcriptome Profiling of m6A mRNA Modification in Bovine Mammary Epithelial Cells Treated with Escherichia coli.

Mastitis is a common disease in dairy cows that is mostly caused by E. coli, and it brings massive losses to the dairy industry. N6-Methyladenosine (m6A), a methylation at the N6 position of RNA adenine, is a type of modification strongly associated with many diseases. However, the role of m6A in mastitis has not been investigated. In this study, we used MeRIP-seq to sequence the RNA of bovine mammary epithelial cells treated with inactivated E. coli for 24 h. In this in vitro infection model, there were 16,691 m6A peaks within 7066 mRNA transcripts in the Con group and 10,029 peaks within 4891 transcripts in the E. coli group. Compared with the Con group, 474 mRNAs were hypermethylated and 2101 mRNAs were hypomethylated in the E. coli group. Biological function analyses revealed differential m6A-modified genes mainly enriched in the MAPK, NF-κB, and TGF-β signaling pathways. In order to explore the relationship between m6A and mRNA expression, combined MeRIP-seq and mRNA-seq analyses revealed 212 genes with concomitant changes in the mRNA expression and m6A modification. This study is the first to present a map of RNA m6A modification in mastitis treated with E. coli, providing a basis for future research.

Human MettL3-MettL14 RNA adenine methyltransferase complex is active on double-stranded DNA containing lesions.

MettL3-MettL14 methyltransferase complex has been studied widely for its role in RNA adenine methylation. This complex is also recruited to UV- and X-ray exposed DNA damaged sites, and its methyltransfer activity is required for subsequent DNA repair, though in theory this could result from RNA methylation of short transcripts made at the site of damage. We report here that MettL3-MettL14 is active in vitro on double-stranded DNA containing a cyclopyrimidine dimer – a major lesion of UV radiation-induced products – or an abasic site or mismatches. Furthermore, N6-methyladenine (N6mA) decreases misincorporation of 8-oxo-guanine (8-oxoG) opposite to N6mA by repair DNA polymerases. When 8-oxoG is nevertheless incorporated opposite N6mA, the methylation inhibits N6mA excision from the template (correct) strand by the adenine DNA glycosylase (MYH), implying that the methylation decreases inappropriate misrepair. Finally, we observed that the N6mA reader domain of YTHDC1, which is also recruited to sites of DNA damage, binds N6mA that is located across from a single-base gap between two canonical DNA helices. This YTHDC1 complex with a gapped duplex is structurally similar to DNA complexes with FEN1 and GEN1 – two members of the nuclease family that act in nucleotide excision repair, mismatch repair and homologous recombination, and which incise distinct non-B DNA structures. Together, the parts of our study provide a plausible mechanism for N6mA writer and reader proteins acting directly on lesion-containing DNA, and suggest in vivo experiments to test the mechanisms involving methylation of adenine.