Uracil appears in DNA as a result of cytosine deamination and by incorporation from the dUTP pool. As potentially mutagenic and deleterious for cell regulation, uracil must be removed from DNA. The major pathway of its repair is initiated by uracil-DNA glycosylases (UNG), ubiquitously found enzymes that hydrolyze the N-glycosidic bond of deoxyuridine in DNA. This review describes the current understanding of the mechanism of uracil search and recognition by UNG. The structure of UNG proteins from several species has been solved, revealing a specific uracil-binding pocket located in a DNA-binding groove. DNA in the complex with UNG is highly distorted to allow the extrahelical recognition of uracil. Thermodynamic studies suggest that UNG binds with appreciable affinity to any DNA, mainly due to the interactions with the charged backbone. The increase in the affinity for damaged DNA is insufficient to account for the exquisite specificity of UNG for uracil. This specificity is likely to result from multistep lesion recognition process, in which normal bases are rejected at one or several pre-excision stages of enzyme-substrate complex isomerization, and only uracil can proceed to enter the active site in a catalytically competent conformation. Search for the lesion by UNG involves random sliding along DNA alternating with dissociation-association events and partial eversion of undamaged bases for initial sampling.
Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination.
Uracil-DNA glycosylase (Ung) can quickly locate uracil bases in an excess of undamaged DNA. DNA glycosylases may use diffusion along DNA to facilitate lesion search, resulting in processivity, the ability of glycosylases to excise closely spaced lesions without dissociating from DNA. We propose a new assay for correlated cleavage and analyze the processivity of Ung. Ung conducted correlated cleavage on double-and single-stranded substrates; the correlation declined with increasing salt concentration. Proteins in cell extracts also decreased Ung processivity. The correlated cleavage was reduced by nicks in DNA, suggesting the intact phosphodiester backbone is important for Ung processivity.
Uracil-DNA glycosylase (Ung) is a DNA repair enzyme that excises uracil bases from DNA, where they appear through deamination of cytosine or incorporation from a cellular dUTP pool. DNA repair enzymes often use one-dimensional diffusion along DNA to accelerate target search; however, this mechanism remains poorly investigated mechanistically. We used oligonucleotide substrates containing two uracil residues in defined positions to characterize one-dimensional search of DNA by Escherichia coli Ung. Mg2+ ions suppressed the search in double-stranded DNA to a higher extent than K+ likely due to tight binding of Mg2+ to DNA phosphates. Ung was able to efficiently overcome short single-stranded gaps within double-stranded DNA. Varying the distance between the lesions and fitting the data to a theoretical model of DNA random walk, we estimated the characteristic one-dimensional search distance of ~100 nucleotides and translocation rate constant of ~2×106 s−1.
DNA glycosylases are enzymes that initiate the base excision repair pathway, a major biochemical process that protects the genomes of all living organisms from intrinsically and environmentally inflicted damage. Recently, base excision repair inhibition proved to be a viable strategy for the therapy of tumors that have lost alternative repair pathways, such as BRCA-deficient cancers sensitive to poly(ADP-ribose)polymerase inhibition. However, drugs targeting DNA glycosylases are still in development and so far have not advanced to clinical trials. In this review, we cover the attempts to validate DNA glycosylases as suitable targets for inhibition in the pharmacological treatment of cancer, neurodegenerative diseases, chronic inflammation, bacterial and viral infections. We discuss the glycosylase inhibitors described so far and survey the advances in the assays for DNA glycosylase reactions that may be used to screen pharmacological libraries for new active compounds.
An abundant oxidative lesion, 8‐oxo‐7,8‐dihydroguanine (8‐oxoG), often directs the misincorporation of dAMP during replication. To prevent mutations, cells possess an enzymatic system for the removal of 8‐oxoG. A key element of this system is 8‐oxoguanine‐DNA glycosylase (Fpg in bacteria, OGG1 in eukaryotes), which must excise 8‐oxoG from 8‐oxoG:C pairs but not from 8‐oxoG:A. We investigated the influence of various factors, including ionic strength, the presence of Mg2+ and organic anions, polyamides, crowding agents and two small heterocyclic compounds (biotin and caffeine) on the activity and opposite‐base specificity of Escherichia coli Fpg and human OGG1. The activity of both enzymes towards 8‐oxoG:A decreased sharply with increasing salt and Mg2+ concentration, whereas the activity on 8‐oxoG:C was much more stable, resulting in higher opposite‐base specificity when salt and Mg2+ were at near‐physiological concentrations. This tendency was observed with both Cl− and glutamate as the major anions in the reaction mixture. Kinetic and binding parameters for the processing of 8‐oxoG:C and 8‐oxoG:A by Fpg and OGG1 were determined under several different conditions. Polyamines, crowding agents, biotin and caffeine affected the activity and specificity of Fpg or OGG1 only marginally. We conclude that, in the intracellular environment, the specificity of Fpg and OGG1 for 8‐oxoG:C versus 8‐oxoG:A is mostly due to high ionic strength and Mg2+.
To perform their functions, many DNA-dependent proteins have to quickly locate specific targets against the vast excess of nonspecific DNA. Although this problem was first formulated over 40 years ago, the mechanism of such search remains one of the unsolved fundamental problems in the field of protein-DNA interactions. Several complementary mechanisms have been suggested: sliding, based on one-dimensional random diffusion along the DNA contour; hopping, in which the protein "jumps" between the closely located DNA fragments; macroscopic association-dissociation of the protein-DNA complex; and intersegmental transfer. This review covers the modern state of the problem of target DNA search, theoretical descriptions, and methods of research at the macroscopic (molecule ensembles) and microscopic (individual molecules) levels. Almost all studied DNA-dependent proteins search for specific targets by combined three-dimensional diffusion and one-dimensional diffusion along the DNA contour.
Uracil–DNA glycosylases are enzymes that excise uracil bases appearing in DNA as a result of cytosine deamination or accidental dUMP incorporation from the dUTP pool. The activity of Family 1 uracil–DNA glycosylase (UNG) activity limits the efficiency of antimetabolite drugs and is essential for virulence in some bacterial and viral infections. Thus, UNG is regarded as a promising target for antitumor, antiviral, antibacterial, and antiprotozoal drugs. Most UNG inhibitors presently developed are based on the uracil base linked to various substituents, yet new pharmacophores are wanted to target a wide range of UNGs. We have conducted virtual screening of a 1,027,767-ligand library and biochemically screened the best hits for the inhibitory activity against human and vaccinia virus UNG enzymes. Although even the best inhibitors had IC50 ≥ 100 μM, they were highly enriched in a common fragment, tetrahydro-2,4,6-trioxopyrimidinylidene (PyO3). In silico, PyO3 preferably docked into the enzyme’s active site, and in kinetic experiments, the inhibition was better consistent with the competitive mechanism. The toxicity of two best inhibitors for human cells was independent of the presence of methotrexate, which is consistent with the hypothesis that dUMP in genomic DNA is less toxic for the cell than strand breaks arising from the massive removal of uracil. We conclude that PyO3 may be a novel pharmacophore with the potential for development into UNG-targeting agents.
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