Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.
Three new cytosine derived DNA modifications, 5-hydroxymethyl-2'-deoxycytidine (hmdC), 5-formyl-2'-deoxycytidine (fdC) and 5-carboxy-2'-deoxycytidine (cadC) were recently discovered in mammalian DNA, particularly in stem cell DNA. Their function is currently not clear, but it is assumed that in stem cells they might be intermediates of an active demethylation process. This process may involve base excision repair, C-C bond cleaving reactions or deamination of hmdC to 5-hydroxymethyl-2'-deoxyuridine (hmdU). Here we report chemical studies that enlighten the chemical reactivity of the new cytosine nucleobases. We investigated their sensitivity toward oxidation and deamination and we studied the C-C bond cleaving reactivity of hmdC, fdC, and cadC in the absence and presence of thiols as biologically relevant (organo)catalysts. We show that hmdC is in comparison to mdC rapidly oxidized to fdC already in the presence of air. In contrast, deamination reactions were found to occur only to a minor extent. The C-C bond cleavage reactions require the presence of high concentration of thiols and are acid catalyzed. While hmdC dehydroxymethylates very slowly, fdC and especially cadC react considerably faster to dC. Thiols are active site residues in many DNA modifiying enzymes indicating that such enzymes could play a role in an alternative active DNA demethylation mechanism via deformylation of fdC or decarboxylation of cadC. Quantum-chemical calculations support the catalytic influence of a thiol on the C-C bond cleavage.
A stable radical: The title compound 1 has been synthesized as the first example of a meso‐free variant and characterized as a strongly aromatic macrocycle with a spectacles‐like planar conformation. The incorporation of free meso positions is promising for the exploration of novel structural and electronic properties.
Direct repair of UV-induced DNA lesions represents an elegant method for many organisms to deal with these highly mutagenic and cytotoxic compounds. Although the participating proteins are structurally well investigated, the exact repair mechanism of the photolyase enzymes remains a vivid subject of current research. In this review, we summarize and highlight the recent contributions to this exciting field.
The dual function of runt-related transcriptional factor 1 (RUNX1) as an oncogene or oncosuppressor has been extensively studied in various malignancies, yet its role in gastric cancer remains elusive. Up-regulation of the ErbB2/HER2 signaling pathway is frequently-encountered in gastric cancer and contributes to the maintenance of these cancer cells. This signaling cascade is partly mediated by son of sevenless homolog (SOS) family, which function as adaptor proteins in the RTK cascades. Herein we report that RUNX1 regulates the ErbB2/HER2 signaling pathway in gastric cancer cells through transactivating SOS1 expression, rendering itself an ideal target in anti-tumor strategy toward this cancer. Mechanistically, RUNX1 interacts with the RUNX1 binding DNA sequence located in SOS1 promoter and positively regulates it. Knockdown of RUNX1 led to the decreased expression of SOS1 as well as dephosphorylation of ErbB2/HER2, subsequently suppressed the proliferation of gastric cancer cells. We also found that our novel RUNX inhibitor (Chb-M’) consistently led to the deactivation of the ErbB2/HER2 signaling pathway and was effective against several gastric cancer cell lines. Taken together, our work identified a novel interaction of RUNX1 and the ErbB2/HER2 signaling pathway in gastric cancer, which can potentially be exploited in the management of this malignancy.
The spore photoproduct lyase is a radical SAM enzyme, which repairs 5-(a-thyminyl)-5,6-dihydrothymidine. Here we show that the enzyme establishes a complex radical transfer cascade and creates a cysteine and a tyrosyl radical dyade to establish repair. This allows the enzyme to solve topological and energetic problems associated with the radical based repair reaction.UV irradiation causes the formation of a variety of dinucleotide lesions, which are typically formed between two pyrimidines. 1,2In cellular DNA, UV irradiation causes the formation of the well studied cyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4)-pyrimidone photoproducts (6-4PPs), and their Dewar-valence isomers.1 In bacterial endospores, in contrast, these bipyrimidine photoproducts are only formed in low amounts. Due to the unusual packing of the DNA in spores, UV irradiation was found to create selectively the (5R)-5-(a-thyminyl)-5,6-dihydrothymidine lesion (spore photoproduct or SP-lesion). [3][4][5] This dimeric thymidine-photoproduct is repaired during germination by the spore photoproduct lyase (SPL), which is a radical SAM enzyme. 6The SPL catalyzes the direct repair of the SP-lesion, utilizing a radical mediated mechanism (Fig. 1A) ( Fig. 1B) shows the topological problem associated with closing the catalytic cycle. While the allyl radical is situated and reduced at the 3 0 -side, transfer of the radical center back to the 5 0 -dAdoH requires moving the radical back to the 5 0 -part in the active side over a distance of roughly 10 Å. This creates next to a topological problem also an energetic obstacle, because regeneration of the adenosyl radical by the thiyl radical would be endothermic by 62 kJ mol À1. 16Here we provide first evidence that the enzyme uses a further tyrosyl radical intermediate to solve the energetic and topological problem. In the crystal structure the tyrosine bridges the conserved cysteine and the 5 0 -dAdoH. The structure shows that Tyr98 is located 3.6 Å and 5.1 Å away from both centers, respectively. In order to clarify the role of Cys140 and Tyr98 we prepared two mutants C140A and Y98F ( Fig. 2A) and determined their catalytic activity. To this end we quantified product formation after 3 h and after reaction overnight. The measured K m and V max values are listed in Table 1. The data show that both mutants have a strongly reduced catalytic activity. The Y98F mutant shows in addition a reduced substrate binding affinity, which indicates that the phenolic hydroxyl group is important to organize the substrate in the active site. Direct interactions with the lesion are, however, not observed in the crystal structure. For the catalytically competent wild type (wt) enzyme we measured a ratio of product/5 0 -dAdoH of greater than two indicating that the cleavage of one SAM molecule repairs more than one lesion (turnover). The mutants in contrast use more than one SAM per product showing that here the turnover is smaller than one product molecule (0.7 and 0.25, Fig. 2B) formed per SAM. This result shows that Cys1...
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