Calvo, Patricia A.Martínez Jiménez, María I.Blanco, LuisAgudo Torres, RubénGrupo: Parasitología e Inmunología molecular con aplicación biotecnológica, diagnóstica y terapéutica (PARINM)Universidad San Pablo-CEU. Departamento de Ciencias Farmacéuticas y de la Salud2024-03-212024-03-212017-07-26Agudo, R., Calvo, P. A., Martínez-Jiménez, M. I., & Blanco, L. (2017). Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase. Nucleic Acids Research, 45(15), 9046-9058–9058. https://doi.org/10.1093/nar/gkx6331362-4962http://hdl.handle.net/10637/15660We have developed a straightforward fluorometric assay to measure primase-polymerase activity of human PrimPol (HsPrimPol). The sensitivity of this procedure uncovered a novel RNA-dependent DNA priming-polymerization activity (RdDP) of this enzyme. In an attempt to enhance HsPrimPol RdDP activity, we constructed a smart mutant library guided by prior sequence-function analysis, and tested this library in an adapted screening platform of our fluorometric assay. After screening less than 500 variants, we found a specific HsPrimPol mutant, Y89R, which displays 10-fold higher RdDP activity than the wild-type enzyme. The improvement of RdDP activity in the Y89R variant was due mainly to an increased in the stabilization of the preternary complex (protein:template:incoming nucleotide), a specific step preceding dimer formation. Finally, in support of the biotechnological potential of PrimPol as a DNA primer maker during reverse transcription, mutant Y89R HsPrimPol rendered up to 17-fold more DNA than with random hexamer primers.enopen accessArtículo10.1093/nar/gkx633https://creativecommons.org/licenses/by-nc-nd/4.0/deed.es