Site-specific DNA methylation plays an important role in epigenetic regulation of gene expression. Chemical methylation of DNA, including the formation of various methylated nitrogenous bases, leads to the formation of genotoxic modifications that impair DNA functions. Despite the fact that different pathways give rise to methyl groups in DNA, the main pathway for their removal is oxidative demethylation, which is catalyzed by nonheme Fe(II)/α-ketoglutarate–dependent DNA dioxygenases. DNA dioxygenases share a common catalytic mechanism of the oxidation of the alkyl groups on nitrogenous bases in nucleic acids. This review presents generalized data on the catalytic mechanism of action of DNA dioxygenases and on the participation of typical representatives of this superfamily, such as prokaryotic enzyme AlkB and eukaryotic enzymes ALKBH1–8 and TET1–3, in both processes of direct repair of alkylated DNA adducts and in the removal of an epigenetic mark (5-methylcytosine).

AlkB is a Fe(II)/α-ketoglutarate-dependent dioxygenase that repairs some alkylated bases of DNA and RNA in Escherichia coli. In the course of catalysis, oxidation of a co-substrate (α-ketoglutarate, αKG) leads to the formation of a highly reactive ‘oxyferryl’ enzyme-bound intermediate, Fe(IV) = O, ensuring hydroxylation of the alkyl nucleobase adducts. Previous studies have revealed that AlkB is a flexible protein and can adopt different conformations during interactions with cofactors and DNA. To assess the conformational dynamics of the enzyme in complex with single- or double-stranded DNA in real-time mode, we employed the stopped-flow fluorescence method. N¹-Methyladenine (m¹A) introduced into a sequence of 15-mer oligonucleotides was chosen as the specific damage. Single-turnover kinetics were monitored by means of intrinsic fluorescence of the protein’s Trp residues, fluorescent base analogue 2-aminopurine (2aPu), and a dye–quencher pair (FAM/BHQ1). For all the fluorescent labels, the fluorescent traces showed several phases of consistent conformational changes, which were assigned to specific steps of the enzymatic process. These data offer an overall picture of the structural dynamics of AlkB and DNA during their interaction.

The in vivo editing of genetic information necessitates tools of unprecedented accuracy. CRISPR-Cas-based systems have emerged as leading technologies for precisely targeting the genome. The Cas9 endonuclease derived from Streptococcus pyogenes is the most commonly used instrument for targeted DNA cleavage. The development of engineered and chimeric Cas9 variants with enhanced activity and specificity has enabled not only the simple knockout of target genes but also the sophisticated engineering of the epigenome. This advancement has broadened the potential applications of CRISPR-Cas9 technology for the treatment of various disorders characterized by a combination of mutations, deletions, and duplications in the coding and non-coding regions of the genome. The inherent simplicity and predictability of the CRISPR-Cas9 targeting mechanism have led to an explosive growth in the development of prototype gene-editing drugs. However, their therapeutic application is still challenged by potential off-target effects. The erroneous editing of tumor suppressors and oncogenes could lead to adverse outcomes that mitigate the benefits of CRISPR therapy. The evolution of DNA-targeting technologies requires a comprehensive understanding of the mechanisms underlying CRISPR-Cas9 off-target binding and cleavage. The use of massive libraries of DNA targets and guide RNAs, coupled with high-throughput sequencing, contributes significantly to the analysis of mismatch tolerance. Nevertheless, the detection of ultra-low levels of off-target activity is hindered by the sensitivity limitations of current technologies. This review focuses on the mechanisms responsible for off-target interactions during CRISPR-Cas9-mediated gene editing. We discuss the influence of various factors, including nucleotide context, enzyme concentration, guide RNA structure, and the energetics of the RNA–DNA hybrid on mismatch tolerance in vitro and in vivo. Recent advances in the development of technologies for predicting off-target effects are briefly summarized. Particular emphasis is placed on the role of the Cas9 protein structure in the allosteric regulation of the specific and non-specific activity of the Cas9–sgRNA complex.

The Cas9 endonuclease is an essential component of the CRISPR–Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR–Cas9 system. A deep understanding of the Cas9 mechanism and its structural-functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to undergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX-MS for the elucidation of mechanistic aspects of Cas9 functioning.

Fe2+/2-ketoglutarate-dependent DNA-dioxygenase AlkB from Escherichia coliis able to restore the native structure of alkylated DNA bases. The enzymatic process utilizes the molecular oxygen,...