Fiche publication
Date publication
octobre 2024
Journal
Angewandte Chemie (International ed. in English)
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MOTORINE Iouri
,
Dr MARCHAND Virginie
Tous les auteurs :
Henkel M, Fillbrunn A, Marchand V, Raghunathan G, Berthold MR, Motorin Y, Marx A
Lien Pubmed
Résumé
Dysregulation of DNA methylation is associated with human disease, particularly cancer, and the assessment of aberrant methylation patterns holds great promise for clinical diagnostics. However, DNA polymerases do not effectively discriminate between processing 5-methylcytosine (5 mC) and unmethylated cytosine, resulting in the silencing of methylation information during amplification or sequencing. As a result, current detection methods require multi-step DNA conversion treatments or careful analysis of sequencing data to decipher individual 5 mC bases. To overcome these challenges, we propose a novel DNA polymerase-mediated 5 mC detection approach. Here, we describe the engineering of a thermostable DNA polymerase variant derived from Thermus aquaticus with altered fidelity towards 5 mC. Using a screening-based evolutionary approach, we have identified a DNA polymerase that exhibits increased misincorporation towards 5 mC during DNA synthesis. This DNA polymerase generates mutation signatures at methylated CpG sites, allowing direct detection of 5 mC by reading an increased error rate after sequencing without prior treatment of the sample DNA.
Mots clés
5-methylcytosine, DNA methylation, DNA polymerase, next-generation sequencing, protein engineering
Référence
Angew Chem Int Ed Engl. 2024 10 24;:e202413304
