Abstract
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.
© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
MeSH terms
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Amino Acid Substitution*
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Arginine / chemistry
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Arginine / metabolism
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Benzothiazoles
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Biological Assay*
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Cloning, Molecular
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DNA Primase / genetics*
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DNA Primase / metabolism
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DNA Primers / chemical synthesis
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DNA Primers / chemistry
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DNA-Directed DNA Polymerase / genetics*
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DNA-Directed DNA Polymerase / metabolism
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Diamines
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Fluorescent Dyes / chemistry
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Fluorometry / methods
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Gene Expression
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Gene Library
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Humans
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Multifunctional Enzymes / genetics*
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Multifunctional Enzymes / metabolism
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Mutation
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Oligonucleotides / chemistry
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Oligonucleotides / metabolism
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Organic Chemicals / chemistry
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Protein Binding
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Protein Engineering / methods*
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Protein Multimerization
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Quinolines
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RNA / genetics*
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RNA / metabolism
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RNA-Directed DNA Polymerase / genetics*
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RNA-Directed DNA Polymerase / metabolism
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Reverse Transcriptase Polymerase Chain Reaction / methods
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Tyrosine / chemistry
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Tyrosine / metabolism
Substances
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Benzothiazoles
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DNA Primers
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Diamines
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Fluorescent Dyes
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Multifunctional Enzymes
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Oligonucleotides
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Organic Chemicals
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Quinolines
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SYBR Green I
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Tyrosine
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RNA
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Arginine
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DNA Primase
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PrimPol protein, human
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RNA-Directed DNA Polymerase
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DNA-Directed DNA Polymerase