Living organisms protect the genome against external influences by recognizing and repairing damaged DNA. A common source of gene mutation is the oxidized guanine, which undergoes base excision repair through cleavage of the glycosidic bond between the ribose and the nucleobase of the lesion. We unravel the repair mechanism utilized by bacterial glycosylase, MutM, using quantum-chemical calculations involving more than 1000 atoms of the catalytic site. In contrast to the base-protonated pathway currently favored in the literature, we show that the initial protonation of the lesion's ribose paves the way for an almost barrier-free glycosidic cleavage. The combination of theoretical and experimental data provides further insight into the selectivity and discrimination of MutM's binding site toward various substrates.
5-Formylcytosine (fC or (5-CHO)dC) and 5-carboxylcytosine (caC or (5-COOH)dC) have recently been identified as constituents of mammalian DNA. The nucleosides are formed from 5-methylcytosine (mC or (5-Me)dC) via 5-hydroxymethylcytosine (hmC or (5-HOMe)dC) and are possible intermediates of an active DNA demethylation process. Here we show efficient syntheses of phosphoramidites which enable the synthesis of DNA strands containing these cytosine modifications based on Pd(0)-catalyzed functionalization of 5-iododeoxycytidine. The first crystal structure of fC reveals the existence of an intramolecular H-bond between the exocyclic amine and the formyl group, which controls the conformation of the formyl substituent. Using a newly designed in vitro mutagenicity assay we show that fC and caC are only marginally mutagenic, which is a prerequisite for the bases to function as epigenetic control units.
The common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) affects the efficiency and fidelity of transcription. We constructed plasmid substrates carrying single 8-oxo-G residues, specifically positioned in the transcribed or the non-transcribed DNA strands, to investigate their effects on the expression of an EGFP reporter gene and to explore the role of base excision repair in the mechanism of transcription inhibition. We report that 8-oxo-G does not directly block transcription in cells, since a single 8-oxo-G in the transcribed DNA strand did not reduce the EGFP expression levels in repair-deficient (OGG1-null) mouse embryonic fibroblast cell lines. Rather, inhibition of transcription by 8-oxo-G fully depends on 8-oxoguanine DNA glycosylase (OGG1) and, at the same time, does not require the localization of the lesion in the transcribed DNA strand. We propose that the interruption of transcription is induced by base excision repair intermediates and, therefore, could be a common consequence of various DNA base modifications. Concordantly, the non-blocking DNA modification uracil was also found to inhibit transcription, but in an OGG1-independent manner.
Living organisms protect the genome against external influences by recognizing and repairing damaged DNA. A common source of gene mutation is the oxidized guanine, which undergoes base excision repair through cleavage of the glycosidic bond between the ribose and the nucleobase of the lesion. We unravel the repair mechanism utilized by bacterial glycosylase, MutM, using quantumchemical calculations involving more than 1000 atoms of the catalytic site. In contrast to the base-protonated pathway currently favored in the literature, we show that the initial protonation of the lesions ribose paves the way for an almost barrier-free glycosidic cleavage. The combination of theoretical and experimental data provides further insight into the selectivity and discrimination of MutMs binding site toward various substrates.While DNA repair is of central importance for the stability of the genome of organisms, [1,2] the underlying processes such as lesion recognition and repair are still poorly understood. Repair enzymes have to recognize single lesions within a vast majority of undamaged bases. The most prominent endogenous DNA damage is the oxidized guanine, 7,8-dihydro-8-oxoguanine (8OG). 8OG has the ability to form a Hoogsteen base pair with adenine, which is the basis for the G:C!A:T transversion mutation (Figure 1). [3] Despite the existence of crystal structures showing 8OG-containing DNA in complex with the catalytically incompetent repair enzymes, one of the major unsolved questions is how 8OG is specifically recognized and excised in the genome. [4][5][6][7][8][9][10][11][12] It is still not clear how the enzymes are able to exquisitely select 8OG and avoid harmful excision of canonical guanidine (G). In bacteria, 8OG is repaired by MutM, also known as formamidopyrimidine glycosylase (Fpg). [13] MutM first recognizes 8OG and then guides it into the active site (lesion recognition complex, LRC).[14] The glycosidic bond is subsequently cleaved, creating an abasic intermediate. A crystal structure trapped after sodium borohydride treatment reveals that the enzyme, at some point, forms a covalent bond (Schiff base) between the anomeric center of 8OG and a proline residue in the active site.[15] However, the chemistry leading to this intermediate and contributing to the ability of the enzyme to discriminate G from 8OG is still unknown.The currently favored hypothesis is that MutM protonates the 8OG base, thereby converting it into a good leaving group, which facilitates the nucleophilic attack of the anomeric C1' carbon by the proline nucleophile.[16] The heterocycle of 8OG is more electron-rich than that of G, hence making the former a better leaving group. Using newly developed linear-scaling quantum-chemical methods we report a thorough computational investigation of the repair mechanism of MutM. We address MutMs exquisite 8OG selectivity as well as the specific catalytic role of central amino acids in the active site. The results of our theoretical study are in full agreement with the presented experimental...
Oxidative degradation of DNA is a major mutagenic process. Reactive oxygen species (ROS) produced in the course of oxidative phosphorylation or by exogenous factors are known to attack preferentially deoxyguanosine. The latter decomposes to give mutagenic lesions, which under physiological conditions are efficiently repaired by specialized maintenance systems in the cell. Although many intermediates of the degradation pathway are today well-known, we report in this study the discovery of a new intermediate with an interesting guanidinoformimine structure. The structure elucidation of the new lesion was possible by using HPLC-MS techniques and organic synthesis. Finally we report the mutagenic potential of the new lesion in comparison to the known lesions imidazolone and oxazolone using primer extension and pyrosequencing experiments.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.