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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2017
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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. |
format | Online Article Text |
id | pubmed-5587808 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
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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