• deformylation

The regulation of transcription results from a complex interplay of molecular factors and markers that co-operate during all the stages of the gene expression. The instruction for the transcriptional expression is not only stored in the four canonical bases fashion but in the so-called epigenetic hallmarks, heritable modifications and chromatin rearrangements that are able to regulate the genome accessibility. 5-methyl-deoxycytidine(mdC) is an epigenetic modification able to mark a gene or an entire chromosome for the transcriptional silencing, therefore its deposition and removal is finely regulated by the cell. An active demethylation pathway has been demonstrated to occur through an oxidation cascade of mdC performed by the TET enzymes, leading to the formation of 5-hydroxymethyl-deoxycytidine(hmdC), 5-formyl-deoxycytidine(fdC) and 5-carboxy-deoxycytidine(cadC). The enzymes of the Base excision repair (BER) mechanism then catalyse the removal of the oxidised base, restoring the deoxycytidine(dC). The DNA-glycosylases are the first actors of this pathway as they recognize the modified base and cleave the N-glycosidic bond leaving an apyrimidinic site (AP). Nevertheless, this pathway creates single or double strand breaks which result detrimental for the genome stability. Different models have thus been proposed as alternative unharmful mechanisms for the mdC active removal. This project aims to investigate the role of different DNA-glycosylases as potential catalysts of the direct deformylation of fdC. To this extent, the deformylation assay was performed incubating the DNA-glycosylase in presence of an oligonucleotide carrying a 2’-Fluorinated-formyl-deoxycytidine(F-fdC). This modified base is fluorinated at the 2’ of the ribose ring thus it sterically impedes the DNA-glycosylase access to the N-glycosidic bond. The results were finally investigated using mass spectrometry analysis.