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Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase

AuthorsAgudo, Rubén ; Calvo, Patricia A. ; Martínez-Jiménez, María I. ; Blanco, Luis
Issue Date12-Jul-2017
PublisherOxford University Press
CitationNucleic Acids Research 45: 9046- 9058 (2017)
AbstractWe 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.
Identifiersdoi: 10.1093/nar/gkx633
issn: 1362-4962
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