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

We 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, w...

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Autores principales: Agudo, Rubén, Calvo, Patricia A., Martínez-Jiménez, María I., Blanco, Luis
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587808/
https://www.ncbi.nlm.nih.gov/pubmed/28911121
http://dx.doi.org/10.1093/nar/gkx633
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author Agudo, Rubén
Calvo, Patricia A.
Martínez-Jiménez, María I.
Blanco, Luis
author_facet Agudo, Rubén
Calvo, Patricia A.
Martínez-Jiménez, María I.
Blanco, Luis
author_sort Agudo, Rubén
collection PubMed
description We 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.
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spelling pubmed-55878082017-09-11 Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase Agudo, Rubén Calvo, Patricia A. Martínez-Jiménez, María I. Blanco, Luis Nucleic Acids Res Nucleic Acid Enzymes We 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. Oxford University Press 2017-09-06 2017-07-26 /pmc/articles/PMC5587808/ /pubmed/28911121 http://dx.doi.org/10.1093/nar/gkx633 Text en © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Nucleic Acid Enzymes
Agudo, Rubén
Calvo, Patricia A.
Martínez-Jiménez, María I.
Blanco, Luis
Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase
title Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase
title_full Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase
title_fullStr Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase
title_full_unstemmed Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase
title_short Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase
title_sort engineering human primpol into an efficient rna-dependent-dna primase/polymerase
topic Nucleic Acid Enzymes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587808/
https://www.ncbi.nlm.nih.gov/pubmed/28911121
http://dx.doi.org/10.1093/nar/gkx633
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